No title

Current Topics in Membranes and Transport Volume 17 Membrane Lipids of Prokaryotes

Advisory Board

M . P. Blaustein A . Essig

R. K . H . Kinne P. A . Knauf Sir H . L . Kornberg

P. Lauger C. A. Pasternak W . D.Stein W . Stoeckenius K. J . Ullrich

Contributors

John E. Cronmn, Jr. Marina A . Frcludenberg Chris Galanos H o Mtard Goldfine Th( I ina s A . La ng wort hy Volker Lehmann Otto Liideritz Ronald N . McElhanev

Donald L. Melchior Guy Ourisson Shmuel Razin Ernst Th. Rietschel Charles 0. Rock Michel Rohmer Shlomo Rottem Derek H . ShaM,

Current Topics in Membranes and Transport Edited by Felix Bronner

Arnost Kleinzeller

Department of Oral Biology University of Connecticut Health Center Farmington, Connecticut

Department qf Physiology University of Pennsylvania School of Medicine Philadelphia, Pennsylvania

VOLUME 17 Membrane lipids of Prokaryotes

Guest Editors Shrnuel Razin

Shlorno Rottem

Department of Membrane and Ulrrastructure Rearurch The Hebrew, Universit~~-Hadassai Medicul School Jeru.talem Israel

Department of Membrane and Ultrastructure Research The Hebrew University-Hadassah Medicul School Jerubulem, Israel

1982

ACADEMIC PRESS A Subsidiary o f Harcwurt Bract) Jovariovich, Publishers

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COPYRIGHT @ 1982, BY A C A D ~ MPRESS, IC INC. ALL RIGHTS RESERVED. N O PART OF THIS PUBLICATION MAY B E REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC O R MKHANICAL, INCLUDING P i i o rocow, HLCORDINC. OR ANY INFORMATION STORAGE AND RErRIEVAL SYSTEM, WITHOUT PERM15SION IN W R l l I N G FROM THE PUBLISHER.

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T o the Memory of Yuval Razin

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Contents List of Contributors, xi Foreword, xiii Preface, xvii Yale Membrane Transport Processes Volumes, xix Contents of Previous Volumes, xxi

Lipids of Prokaryotes-Structure

and Distribution

HOWARD GOLDFINE I. Introduction, 2 11. Structure of the Lipids of Prokaryotes, 2 111. Distribution of Lipids in Prokaryotes, 12 IV. F’rokaryotic Lipids and Phylogeny, 3 1

V. Conclusions, 34 References, 36

Lipids of Bacteria Living in Extreme Environments THOMAS A. LANGWORTHY I . Introduction, 45 Apolar Residues, 49 Ill. Neutral Lipids, 56 IV. Glycolipids, 62 V . Acidic Lipids, 66 VI. Overview, 69 References, 70 11.

Lipopolysaccharides of Gram-Negative Bacteria OTTO LUDERITZ, MARINA A. FREUDENBERG, CHRIS GALANOS, VOLKER LEHMANN, ERNST TH. RIETSCHEL, AND DEREK H. SHAW

I. Introduction, 79 Isolation, Structure, and Biosynthesis of Lipopolysaccharides, 82 I l l . Some Selected Aspects on the Biology of Lipopolysaccharides, 114 IV. Final Remarks, 130 References, 134 11.

vii

viii

CONTENTS

Prokaryotic Polyterpenes: Phylogenetic Precursors of Sterols GUY OURISSON AND MICHEL ROHMER I. Introduction, 154 11. The Sterols of Prokaryotes, 155

H I . The Polyterpenoids of Prokaryotes, 158 IV. The Prokaryotic Polyterpenoids as Phylogenetic Precursors of Sterols, 167 V. Addendum, 177 References, 178

Sterols in Mycoplasma Membranes SHMUEL RAZIN I.

Introduction, 183

11. Cholesterol Uptake, 185 111. Role of Sterols, 191

IV. Conclusions, 200 References, 201

Regulation of Bacterial Membrane Lipid Synthesis CHARLES 0. ROCK AND JOHN E. CRONAN, JR. 1. Introduction, 207 11. Regulation of Membrane Lipid Synthesis, 208 Ill. Conclusions, 226 References, 227

Transbilayer Distribution of Lipids in Microbial Membranes SHLOMO ROTTEM 1. Introduction, 235 11. Assessment of Transbilayer Distribution of Membrane Lipids, 236

111. Transbilayer Distribution of Outer Membrane Lipids, 239 TV. Transbilayer Distribution of Cytoplasmic Membrane Lipids, 244 V. How Lipid Asymmetry Is Maintained, 256 References. 256

Lipid Phase Transitions and Regulation of Membrane Fluidity in Prokaryotes DONALD L. MELCHIOR I. Introduction, 263 11. Lipid Phases, 264 111. Membrane Bilayer Transitions, 267

CONTENTS

IV. Fluidity-Modulating Lipids, 282 V. Patching, 284 VI. Biological Consequences of Membrane State, 292 VII. Biological Control, 299 References. 307

Effects of Membrane Lipids on Transport and Enzymic Activities RONALD N. McELHANEY I. 11. 111. 1V. V. VI.

Introduction, 317 Relevant Properties of Membrane Constituents, 318 Arrhenius Plots of Membrane Transport Systems and Enzymes, 320 Studies of Cells and Membranes, 323 Studies of Isolated Membrane-Bound Enzymes, 362 Conclusions, 369 References. 369

Index. 381

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List of Contributors Numbers in parentheses indicate the pages on which the authors’ contributions begin. John E. Cronan, Jr., Department of Microbiology. University of Illinois. Urbana, Illinois 61801 (207) Marina A. Freudenberg, Max-Planck-lnstitut fur Immunbiologie, D-78 Freiburg, Federal Republic of Germany (79) Chris Galanos, Max-Planck-Institut fur Immunbiologie, D-78 Freiburg, Federal Republic of Germany (79) Howard Goldfine, Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104 ( I ) Thomas A. Langworthy, Department of Microbiology, School of Medicine, University of South Dakota, Vermillion, South Dakota 57069 (45) Volker Lehmann, Max-Planck-lnstitut fur Immunbiologie, D-78 Freiburg, Federal Republic of Germany (79) Otto Luderitz, Max-Planck-Institut fur Immunbiologie. D-78 Freiburg, Federal Republic of Germany (79) Ronald N. McElhaney, Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada (3 17) Donald L. Melchior, Department of Biochemistry. University of Massachusetts Medical School, Worcester, Massachusetts 01605 (263) Guy Ourisson, Laboratoire de Chimie Organique des Substances Naturelles, Centre de Neurochimie-UniversitC Louis Pasteur, F 67008 Strasbourg, France (153) Shmuel Razin,* Department of Membrane and Ultrastructure Research, The Hebrew University-Hadassah Medical School, Jerusalem, Israel (183) Ernst Th. Rietschel, Max-Planck-lnstitut fur Immunbiologie, D-78 Freiburg, Federal Republic of Germany (79) Charles 0.Rock, Department of Biochemistry, St. Jude Children’s Research Hospital, Memphis, Tennessee 38101 (207)

*Address until September 1 , 1982: Mycoplasma Branch, Bureau of Biologics, Food and Drug Administration, 8800 Rockville Pike, Bethesda, Maryland 20205. xi

xii

LIST OF CONTRIBUTORS

Michel Rohrner, Ecole Nationale Suptrieure de Chimie de Mulhouse, F 68093 Mulhouse, France (153) Shlomo Rottem, Department of Membrane and Ultrastructure Research, The Hebrew University- Hadassah Medical School, Jerusalem, Israel (235) Derek H. Shaw, Northwest Atlantic Fisheries Centre, St. John’s, Newfoundland, Canada

(79)

Foreword Profligacy rather than economy characterizes the design of naturally occurring membrane lipids, or so it would appear from what we know today. If one were to define a structural denominator common to all components of membrane lipid bilayers, would it be more specific than the amphipathic nature or the inherent competence to form closed vesicles? Diversity seems to be the rule. In fact, and this is rarely mentioned, cell membranes containing a single phospholipid species do not seem to exist. It is true that one type usually predominates, e.g., phosphatidylcholine in eukaryotic membranes and phosphatidylethanolamine in bacteria, but they alone seem to be inadequate as matrices for the varied functions membranes are believed to perform. Is it possible that bilayer asymmetry is essential for biological function and that for this reason alone membrane phospholipids are not limited to a single species? Perhaps this intriguing question could be answered if it were possible to create viable bacterial mutants containing phospholipids of a single type. In view of the compositional complexity of natural membrane lipids, it is remarkable that chemically homogeneous liposomes mimic many natural membrane properties, including transport, phase transitions, or effects on membrane-associated enzymes. Clearly there must exist a wide variety of membrane-associated phenomena expressed only in cells which studies with single component model membranes cannot reveal. In the future, attention will have to be paid increasingly to this question, i.e., to the role of the minor membrane phospholipids and their involvement in regiospecific functions. Perhaps bulk phase properties as a function of phospholipid structure have been unduly emphasized. Prokaryotic membrane phospholipids, though by no means simple in composition, are nevertheless much less complex than their eukaryotic counterparts. As for fatty acyl structures, the CI6 and CIS saturated and monounsaturated fatty acids predominate, but with rare exceptions diand polyunsaturated fatty acids are absent. A departure from this general pattern may signify a specialized function. Olefin-derived cyclopropane acids are found in late log and stationary phase Escherichia coli cells. Yet the significance of these branched acids is by no means clear. Escherichia coli mutants lacking the requisite methyl transferase do not seem physiologically impaired. The need for relatively low-melting long-chain acids is met differently by Bacillus species. They produce iso- and anteiso acids xiii

xiv

FOREWORD

even though they have the mechanisms for introducing olefinic bonds into saturated acids. It is also worth noting that some bacteria generate olefink acids by the anaerobic dehydration pathway while others use oxidative desaturation, the universal eukaryotic pathway. A striking departure from the usual fatty acid patterns is found in cells living in extreme environments (temperature, acidity, ionic strength), e.g., extreme halophiles, thermophiles, and also the organisms recently classified as Archaebacteria. Instead of the common fatty acids, they employ as hydrophobic chains phytanyl residues in stable ether linkage. Even more remarkable, in the form of diphytanyl diglycerol tetrdethers they have the proper dimensions and therefore the potential for spanning the membrane bilayer. If they did they would in essence function as lipid monolayers. The exotic structures of the phytanylether lipids appear to represent alternative solutions to membrane rigidity or stability since their presence correlates with the absence of the peptidoglycan cell wall. It has been generally true for phospholipids from all sources that saturated Fdtty acids are esterified at C- I and unsaturated fatty acids at (2-2 of the glycerol moiety. Yet there are exceptions to this rule. Positional inversion in the phospholipids of clostridia has long been known and more recently observed in certain mycoplasmas. Physiological consequences of the nonrandom fatty acyl esterification sites and its inversion have not been recognized and therefore remain unexplained. In bacteria phosphatidylglycerol and phosphatidylethanolamine are the most common phospholipids, whereas phosphatidylcholine is only rarely found, a pattern which distinguishes prokaryotic membranes most strikingly from eukaryotes. Clearly, the bulk and the net charge of the polar head group cannot be trivial but must play a crucial role in the interaction between the cell envelope and the external milieu. If the subject of phospholipid specificity has remained almost entirely unexplored the reason is undoubtedly that it is rarely absolute and difficult to demonstrate. For the futher exploration of this important subject prokaryotes are clearly the cells of choice. Modulation and control of the environment and mutant selection are more readily realized than with eukaryotic cells. Phospholipid biosynthesis is reasonably well understood today, at least the chemistry of the pathways is. The respective enzymology is much less advanced since the component enzymes are membrane-associated and therefore more refractory to purification. For studies of prokaryotic phospholipid biosynthesis. E . coli has for obvious reasons been the organism of choice. However variations from the E . coli pathway are to be expected and have in fact been encountered earlier in clostridia. For mycoplasm a and acholeplasma phospholipid biosynthesis there is at best fragmentary information. Equally or even more uncharted territory is the regulation of phospho-

FOREWORD

xv

lipid biosynthesis in both prokaryotic and eukaryotic cells. It is perhaps not too unreasonable to predict that the control points and the identity of the modifier molecules for the two cell types will be unrelated. Certainly the physical environment and the stimuli to which the respective cells respond have little in common. How little we know in this area is illustrated by the fact that several decades after the discovery of the phenomenon proper, we still do not know how bacterial cells regulate the synthesis of more or less unsaturated phospholipid in response to temperature changes. Regulation may ocur at the stage of unsaturated fatty acid synthesis or glycerophosphate-acyl-CoA transacylation. Conceivably more than one of the component steps is under control and perhaps by the same controlling molecule. For microorganisms a compelling case can be made that phospholipid biosynthesis is coordinated with membrane assembly and macromolecular synthesis. Indeed, substantial evidence exists, at least from in vitro studies. that the magic spot nucleotides (ppGpp) are negative effectors for several of these processes. Sterols are rarely mentioned in conjunction with discussions of prokaryotic lipids, and understandably so. Sterol-producing or -requiring prokaryotes are exceedingly rare, and this fact seemed to support the view that molecules of this type were not invented prior to the appearance of eukaryotic cells along with intracellular membrane-bound organelles. This, as so many generalizations in biology, had to be abandoned even though sterols probably play, whenever they occur, a much more restricted and less specific role in bacteria than they do in higher cells. The formation of the sterol structure in amounts sufficient to affect membrane properties has been observed only in the instance of M r t h y l o c - o c ~ x cups sulatus. But even in this organism the sterol pathway stops short of full development. Equally unique among prokaryotes is the absolute sterol requirement of Mycoplasma species. Studies with these small bacteria have nevertheless provided useful information on sterol structure-function relationships that may be of more general significance even for eukaryotic systems. It has come as somewhat of a surprise that the sterol precursor squalene is quite widely found in prokaryotes including the anaerobic Archaebacteria. Moreover, squalene transformation to pentacyclic triterpenes of the hopane type, traditionally higher plant products, is not uncommon in these organisms. It appears that these early trials of nature to cyclize squalene-without intervention of oxygen-produced molecules that share certain structural and perhaps also functional features with the sterols. Evolutionary "tinkering" with squalene is in fact observable in Methylococurs ccipsulatns, an organism which produces both lanosterol derivatives from squalene epoxide and pentacyclic triterpenes from squalene.

xvi

FOREWORD

During its relatively short history prokaryotic lipid biochemistry has produced a wealth of novel and often unique information. This volume impressively demonstrates the viability and future promise of this field. The discovery of new structures is likely to continue and with less labor than in the past in view of the powerful analytical methods now available. Progress may come more slowly and may be less straightforward in the elucidation of membrane structure-function relationships. Yet this is the area of greatest challenge. Success, whenever it comes, should bring great rewards, including perhaps a better understanding and rationalization of bacterial systematics and phylogeny.

KONRADBLOCH Department of Chemistry Harvard University Cambridge, Massachusetts

Preface The relative simplicity of prokaryotic cells has made them useful in the study of numerous aspects of cellular biology, including membrane structure and function. Moreover, the availability of techniques for genetic manipulation has made possible the controlled alteration of membrane lipids and proteins in ways not yet possible in the case of eukaryotes. A striking example are the studies that provided the first direct demonstration of the bilayer organization of lipids in biological membranes, evidence for which was obtained by changing the fatty acid composition of the plasma membrane of Acholeplasma laidlawii and Escherichia coli. Other studies utilizing prokaryotes have elucidated the physical state and turnover of membrane lipids and their interrelationship with structural and catalytic membrane proteins. Similarly, our understanding of the pivotal role played by cholesterol and congeners in membrane structure and function-a major area of interest for eukaryotic cell membrane research-owes much to the studies of bacterial membranes. Prokaryotic membrane lipid research also has intrinsic interest. Examples are the lipopolysaccharides of gram-negative bacteria, complex molecules that exhibit a wide spectrum of biological properties. The unique lipids found in bacteria that live in extreme environments, such as the Dead Sea, constitute another area of recent research, since they may provide clues to the understanding of how living organisms have adapted to harsh environments. The wide interest in prokaryotic membrane lipids has given rise to many scientific reports and specialized reviews. This volume is the first to have assembled in one source descriptions of the significant advances made in prokaryotic lipid research during the past decade. In addition to providing systematic coverage, we hope the articles in this volume will also give rise to further research. Thus this work will not only serve as a reference source for scholars, teachers, and students, but will stimulate investigators to attempt solving the many problems that remain. The help of expert colleagues was indispensible in collating current knowledge covering such diverse fields as membrane and lipid biochemistry, microbiology, and cell biology. Our special thanks are due to the contributors for their willingness to help make this book a reality.

SHMUEL RAZIN SHLOMO ROTI-EM xvii

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Yale Membrane Transport Processes Volumes

Joseph F. Hoffman (ed.). (1978). “Membrane Transport Processes,” Vol. 1. Raven, New York. Daniel C. Tosteson, Yu. A. Ovchinnikov, and Ramon Latorre (eds.). (1978). “Membrane Transport Processes,” Vol. 2. Raven, New York. Charles F. Stevens and Richard W. Tsien (eds.). (1979). “Membrane Transport Processes ,” Vol. 3 : Ion Permeation through Membrane Channels. Raven, New York. Emile L. Boulpaep(ed.). (1980). “Cellular Mechanisms of Renal Tubular Ion Transport”: Volume 13 of Current Topics in Membranes and Transport (F. Bronner and A. Kleinzeller, eds.). Academic Press, New York. William H. Miller (ed.). (1981). “Molecular Mechanisms of Photoreceptor Transduction”: Volume 15 of Current Topics in Membranes and Transport (F. Bronner and A. Kleinzeller, eds.). Academic Press, New York. Clifford L. Slayman (ed.). (1982). “Electrogenic Ion Pumps”: Volume 16 of Current Topics in Membranes and Transport (A. Kleinzeller and F. Bronner, eds.). Academic Press, New York.

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Contents of Previous Volumes Volume 1

Volume 3

Some Considerations about the Structure of Cellular Membranes AND MAYNARD M. DEWEY LLOYDBARR The Transport of Sugars across Isolated Bacterial Membranes H. R. KABACK Galactoside Permease of Escherichia coli ADAMKEPES Sulfhydryl Groups in Membrane Structure and Function ASERROTHSTEIN Molecular Architecture of the Mitochondrion DAVIDH. MACLENNAN Author Index-Subject Index

The Na+, K+-ATPase Membrane Transport System: Importance in Cellular Function ARNOLDSCHWARTZ, GEORGEE. LINDENMAYER, AND JULIUSC. ALLEN Biochemical and Clinical Aspects of Sarcoplasmic Reticulum Function ANTHONY MARTONOSI The Role of Periaxonal and Perineuronal Spaces in Modifying Ionic Flow across Neural Membranes W. J. ADELMAN, JR. A N D Y. PALTI Properties of the Isolated Nerve Endings GEORGINA RODRiCUEZ DE LORES ARNAIZ A N D EDUARDO DE ROBERTIS Transport and Discharge of Exportable Proteins in Pancreatic Exocrine Cells: In Vitro Studies J . D. JAMIESON The Movement of Water across Vasopressin-Sensitive Epithelia RICHARD M. HAYS Active Transport of Potassium and Other Alkali Metals by the Isolated Midgut of the Silkworm R. HARVEY AND WILLIAM KARLZERAHN Author Index-Subject Index

Volume 2 The Molecular Basis of Simple Diffusion within Biological Membranes W. R. LIEBA N D W. D. STEIN The Transport of Water in Erythrocytes ROBERTE. FORSTER Ion-Translocation in Energy-Conserving Membrane Systems B. CHANCE A N D M. MONTAL Structure and Biosynthesis of the Membrane Adenosine Triphosphatase of Mitochondria ALEXANDER TZAGOLOFF Mitochondria1 Compartments: A Comparison of Two Models HENRYTEDESCHI Author Index-Subject Index

Volume 4 The Genetic Control of Membrane Transport W. SLAYMAN CAROLYN xxi

xxii Enzymic Hydrolysis of Various Components in Biomembranes and Related Systems MAHENDRA KUMAR JAIN Regulation of Sugar Transport in Eukaryotic Cells HOWAKD E. MORC~AN AND CAROLF. WHITFIELD Secretory Events in Gastric Mucosa RICHARD P. DURBIN Author Index-Subject Index

Volume 5 Cation Transport in Bacteria: K', Na+, and H+ FRANKLIN M. HAROLD AND KARLHEINZ ALTENDORF Pro and Contra Carrier Proteins: Sugar Transport via the Periplasmic GalactoseBinding Protein WINFRIED BOOS Coupling and Energy Transfer in Active Amino Acid Transport EKICH HEINZ The Means of Distinguishing between Hydrogen Secretion and Bicarbonate Reabsorption: Theory and Applications to the Reptilian Bladder and Mammalian Kidney WILLIAM A. BRODSKYA N D THEODORE P. SCHILB Sodium and Chloride Transport across Isolated Rabbit Ileum G. SCHULTZ AND STANLEY PErER F. CURRAN A Macromolecular Approach to Nerve Excitation ICHlJl TASAKl AND EMILIO CARBONE Suhjerr Index

Volume 6 Role of Cholesterol in Biomembranes and Related Systems MAHENDRA KUMAR JAIN Ionic Activities in Cells A. A, LEVA N D W. MCD. ARMSTRONG Active Calcium Transport and Ca*+-Activated ATPase in Human Red Cells H. J . SCHATZMANN The Effect of Insulin on Glucose Transport in Muscle Cells TORBEN CIAUSEN

CONTENTS OF PREVIOUS VOLUMES

Recognition Sites for Material Transport and Information Transfer HAI.VOR N . CHRISTENSEN Subject Index

Volume 7 Ion Transport in Plant Cells E. A. C. MACROBBIE H+ Ion Transport and Energy Transduction in Chloroplasts RICHARDA. DII-LEY AND ROBERT T. GIAQUINTA The Present State of the Carrier Hypothesis P A U IG. LEFEVRE Ion Transport and Short-circuit Technique S. REHM WARREN Sithjecr Index

Volume 8 Chemical and Physical Properties of Myelin Proteins M. A. MOSCARELLO The Distinction between Sequential and Simultaneous Models for Sodium and Potassium Transport P. J. GARRAHAN A N D R. P. GARAY Soluble and Membrane ATPase of Mitochondria, Chloroplasts, and Bacteria: Molecular Structure, Enzymatic Properties, and Functions RIVKAPANETA N D D. RAOSANADI Competition, Saturation, and lnhibitionIonic Interactions Shown by Membrane Ionic Currents in Nerve, Muscle, and Bilayer Systems RoRERr J. FRENCH AND WILLIAM J . A D ~ L M AJ RN. , Properties of the Glucose Transport System in the Renal Brush Border Membrane R. KINNE Suhjert Index

Volume 9 The State of Water and Alkali Cations within the lntracellular Fluids: The Contribution of NMR Spectroscopy ANL) MORDECHAI SHPORER MORTIMER M. CIVAN

xxiii

CONTENTS OF PREVIOUS VOLUMES

Electrostatic Potentials at Membrane-Solution Interfaces STUART MCLAUCHIJN A Thermodynamic Treatment of Active Sodium Transport S. ROYCAPLAN A N D A L V I NEssici Anaerobic Electron Transfer and Active Transport in Bacteria WIL N. KONINCS AND JOHANNES BOONSTRA Protein Kinases and Membrane Phosphorylation M. MARLENEH(ISF.YA N r ) MARIANO TAO Mechanism and Physiological Significance of Calcium Transport across Mammalian Mitochondria1 Membranes LRENAMEI.A Thyroidal Regulation of Active Sodium Transport F. ISMAIL-BEIGI Subject Index

Volume 10 Mechanochemical Properties of Membranes E. A. EVANSA N D R. M. HOCHMUTH Receptor-Mediated Protein Transport into Cells. Entry Mechanisms for Toxins, Hormones, Antibodies, Viruses, Lysosomal Hydrolases, Asialoglycoproteins, and Carrier Proteins DAVID M. NEVILLE, JR. A N D TA-MINCHANG The Regulation of Intracellular Calcium ERNESTO CARAFOI.1 A N D MARTIN CROMFTON Calcium Transport and the Properties of a Calcium-Sensitive Potasbium Channel in Red Cell Membranes VIRGIL10 L. L E W A N D HUGOG . FERREIRA Proton-Dependent Solute Transport in Microorganisms A. A. EDDY Subject Index

Volume 11 Cell Surface Glycoproteins: Structure, Biosynthesis, and Biological Functions

The Cell Membrane-A Short Historical Perspective ASERROTHSIEIN The Structure and Biosynthesis of Membrane Glycoproteins J E N N l F E R STURCESS, M A K I OMOSC.AKt.I.1~0,A N D HARRY SCHACHTER Techniques for the Analysis of Membrane Glycoproteins R. L. J U L I A N O Glycoprotein Membrane Enzymes J O H N R. RIORDAN AND GOKDON G. FOKSTNEK Membrane Glycoproteins of Enveloped Viruses RICHAKU C O M P A N S A N I ) MAURICE C. KEMP Erythrocyte Glycoproteins MICHAEL J. A . TA N N ER Biochemical Determinants of Cell Adhesion LLOYDA. CULP Proteolytic Modification of Cell Surface Macromolecules: Mode of Action in Stimulating Cell Growth KENNETH D. NOONAN Glycoprotein Antigens of Murine Lymphocytes MICHELLE LETAKTE Subject Index

w.

Volume 12 Carriers and Membrane Transport Proteins

Isolation of Integral Membrane Proteins and Criteria for Identifying Carrier Proteins MICHAEL J. A. TA N N ER The Carrier Mechanism S. B. HI-ADKY The Light-Driven Proton Pump of Hulobacterium halohium: Mechanism and Function MICHAEL EISENBACH AND S. ROYCAPLAN Erythrocyte Anion Exchange and the Band 3 Protein: Transport Kinetics and Molecular Structure PHILIPA. KNAUF

xxiv The Use of Fusion Methods for the Microinjection of Animal Cells R. G . KULKA A N D A. LOYTER Suhjecf Index

Volume 13 Cellular Mechanisms of Renal Tubular Ion Transport

PART I: ION ACTIVITY AND ELEMENTAL COMPOSITION O F INTRAEPITHELIAL COMPARTMENTS Intracellular pH Regulation WALTER F. BORON Reversal of the pHi-Regulating System in a Snail Neuron R. c . 'rHOMAS How to Make and Use Double-Barreled Ion-Selective Microelectrodes THOMAS ZUETHEN The Direct Measurement of K, CI, Na, and H Ions in Bullfrog Tubule Cells MAMORUFUJIMOTO, KUNIHIKO KOTERA,A N D YUTAKA MATSUMURA lntracellular Potassium Activity Measurements in Single Proximal Tubules of Necturuy Kidney TAKAHIKO KLIHOI'A. BRUCEBIAGI,A N D GERHARD GIEBISCH lntracellular Ion Activity Measurements in Kidney Tubules RAJA N. KHURI lntracellular Chemical Activity of Potassium in Toad Urinary Bladder JOE[. DELONGA N D MORTIMER M. ClVAN Quantitative Determination of Electrolyte Concentrations in Epithelial Tissues by Electron Microprobe Analysis ROGERRICK,ADOLFDORGE, RICHARD BAUER,FRANZ BECK, J U N E MASON, CHRISTIANE ROLOFF, A N D KLAUS THURAU PART 11: PROPERTIES O F INTRAEPITHELIAL MEMBRANE BARRIERS IN THE KIDNEY

CONTENTS OF PREVIOUS VOLUMES

Hormonal Modulation of Epithelial Structure JAMESB. WADE Changes in Cell Membrane Surfaces Associated with Alterations of Transepithelial Ion Movement MICHAEL KASHGARIAN The Dimensions of Membrane Barriers in Transepithelial Flow Pathways w. WELLING A N D LARRY DANJ. WELLING Electrical Analysis of Intraepithelial Barriers AND EM[I.EL. BOULPAEP HENRYSACKIN Membrane Selectivity and Ion Activities of Mammalian Tight Epithelia SIMON A . LEWIS,NANCYK. WILLS, A N D DOUGLAS C. EATON Ion Conductances and Electrochemical Potential Differences across Membranes of Gallbladder Epithelium LUISREUSS A Kinetic Model for Ion Fluxes in the Isolated Perfused Tubule BRUCEBIAGI,ERNESTO GONZALEZ, A N D GERHARD GIEBISCH The Effects of Voltage Clamping on Ion Transport Pathways in Tight Epithelia ARTHUR L. F I N N AND PAULA ROGENES Tubular Permeability to Buffer Components as a Determinant of Net H Ion Fluxes G . MALNIC, V . L. COSTASILVA,S. S. CAMPIGI.IA, M. DE MELLOAIRES,A N D G . GIEBISCH Ionic Conductance of the Cell Membranes and Shunts of Necturus Proximal Tubule AND GENJIROKIMURA KENNETH R. SPRING Luminal Sodium Phosphate Cotransport as the Site of Regulation for Tubular Phosphate Reabsorption: Studies with Isolated Membrane Vesicles HEIN] MURER,REINHARD STOLL, CARLAEVERS,ROLFKINNE, AND JEAN-PHILIPPE BONJOUR, HERBERT FLEISCH The Mechanism of Coupling between Glucose Transport and Electrical Potential in the Proximal Tubule: A Study of Potential-

xxv

CONTENTS OF PREVIOUS VOLUMES

Dependent Phlorizin Binding to Isolated Renal Microvillus Membranes PETERS. ARONSON Electrogenic and Electroneutral Na Gradient-Dependent Transport Systems in the Renal Brush Border Membrane Vesicle BERTRAM SACKTOR

Volume 14 Carriers and Membrane Transport Proteins

Interface between Two Immiscible Liquids as a Tool for Studying Membrane Enzyme Systems L. I. BOGUSLAVSKY Criteria for the Reconstitution of Ion PART 111: INTRAMEMBRANE Transport Systems CARRIERS AND ENZYMES IN AND ADILE. SHAMOO TRA NSEPITHELI AL TRANSPORT WILLIAM F. TIVOL The Role of Lipids in the Functioning of a Membrane Protein: The Sarcoplasmic ReSodium Cotransport Systems in the Proxiticulum Calcium Pump mal Tubule: Current Developments J . P. BENNETT, K. A. MCCILL,A N D R. K I N N EM. . BARAC,A N D H. M U R E R G. B. WARREN ATPases and Salt Transport in the Kidney The Asymmetry of the Hexose Transfer Tubule System in the Human Red Cell Membrane DE LA MARGARITA PEREZ-GONZALEZ W. F. WIDDAS MANNA,FULGENCIO PROVERRIO, AND Permeation of Nucleosides, Nucleic Acid GUILLERMO WHITEMBURY Bases, and Nucleotides in Animal Cells Further Studies on the Potential Role of an PETERG. W. PLAGEMANN AND Anion-Stimulated Mg-ATPase in Rat ProxROBERT M. WOHLHUETER imal Tubule Proton Transport Transmembrane Transport of Small E. KINNE-SAFFRAN A N D R. K I N N E Peptides Renal Na+- K+-ATPase: Localization and D. M. MATTHEWS AND J. W. PAYNE Quantitation by Means of Its K'-Depenof Epithelial Transport in Characteristics dent Phosphatase Activity Insect Malpighian Tubules REINIER BEEUWKES I11 A N D S. H . P. MADDRELL SEYMOUR ROSEN Subject Index Relationship between Localization of N+K+-ATPase, Cellular Fine Structure, and Volume 15 Reabsorptive and Secretory Electrolyte Transport Molecular Mechanisms of Photoreceptor STEPHEN A. EKNST, Transduction AND CLARAV. RIDDLE, KARLJ . KARNAKY, JR. Relevance of the Distribution of Na+ Pump PART I: T H E ROD PHYSIOLOGICAL RESPONSE Sites to Models of Fluid Transport across Epithelia The Photocurrent and Dark Current of JOHNW. MILLSA N D Retinal Rods DONALD R. DIBONA G. MATTHEWS A N D D. A. BAYLOR Cyclic AMP in Regulation of Renal TransSpread of Excitation and Background Adport: Some Basic Unsolved Questions aptation in the Rod Outer Segment THOMAS P. DOUSA K.-W. YAU,T . D. LAMB,A N D Distribution of Adenylate Cyclase Activity P. A. MCNAUGHTON in the Nephron F. MOREL,D. CHABARDES, Ionic Studies of Vertebrate Rods W. GEOFFREY OWENA N D A N D M. 1MBER.r-TEBOUL Subject Index VINCENT TORRE

xxvi Photoreceptor Coupling: Its Mechanism and Consequences GEOFFREY H. GOLD PART 11: THE CYCLIC NUCLEOTIDE ENZYMATIC CASCADE AND CALCIUM ION First Stage of Amplification in the CyclicNucleotide Cascade of Vision JAMESB. HURLEY, LUBERTSTRYER, A N D BERNARD K.-K. FUNC Rod Guanylate Cyclase Located in Axonemes FLEISCHMAN DARRELL Light Control of Cyclic-Nucleotide Concentration in the Retina THOMAS G. EBREY,PAUL KII.BRIDE, JAMES B. HURLEY. ROGERCALHOON, A N D MUIOYUKI TSUDA Cyclic-GMP Phosphodiesterase and Calmodulin in Early-Onset Inherited Retinal Degenerations G. J . CHADEK, Y. P. Liu, R. T. FLETCHER, G. AGUIRRE. R. SANTOS-ANDERSON, A N D M. T'so Control of Rod Disk Membrane Phosphodiesterase and a Model for Visual Transduction P. A. LIEBMAN ANI) E. N. PUGH,JR. lnteractions of Rod Cell Proteins with the Disk Membrane: Influence of Light, Ionic Strength, and Nucleotides HERMANN KUHN Biochemical Pathways Regulating Transduction in Frog Photoreceptor Membranes M. DERICBOWNDS The Use of Incubated Retinas in Investigating the Effects of Calcium and Other Ions on Cyclic-Nucleotide Levels in Photoreceptors I. COHEN ADOLPH Cyclic AMP: Enrichment in Retinal Cones DEBORA B. FARBER Cyclic-Nucleotide Metabolism in Vertebrate Photoreceptors: A Remarkable Analogy and an Unraveling Enigma G . L. WHEELER, M. W . BITENSKY, A. YAMAZAKI.M. M. RASENICK, AND P. J. STEIN

CONTENTS OF PREVIOUS VOLUMES

Guanosine Nucleotide Metabolism in the Bovine Rod Outer Segment: Distribution of Enzymes and a Role of GTP HITOSHISHICHI Calcium Tracer Exchange in the Rods of Excised Retinas ETE Z. Szurs The Regulation of Calcium in the Intact Retinal Rod: A Study of Light-Induced Calcium Release by the Outer Segment H. GOLD A N D GEOFFREY J U A N I . KORENBKOT Modulation of Sodium Conductance in Photoreceptor Membranes by Calcium Ions and cGMP ROwxr T. SOKBI PART 111: CALCIUM, CYCLIC NUCLEOTIDES, AND THE MEMBRANE POTENTIAL Calcium and the Mechanism of Light Adaptation in Rods BRUCEL. BASIIAN AND GORDON L. FAIN Effects of Cyclic Nucleotides and Calcium Ions on Bufo Rods JOEL E. BROWN AND GERALDINE WALOGA The Relation between Ca2+and Cyclic GMP in Rod Photoreceptors STUART A . LIETON A N D JOHNE. DOWLING Limits on the Role of Rhodopsin and cGMP in the Functioning of the Vertebrate Photoreceptor SANFORD E. OSTROY, EDWARD P. MEYERTHOI.EN, PETERJ. STEIN, ROBERTA A . SVOBODA. A N D MEECAN J . WILSON [Ca2+],Modulation of Membrane Sodium Conductance in Rod Outer Segments BURKSOAKLEY I1 A N D L A W R ~ NH. C EPINTO Cyclic-GMP-Induced Depolarization and Increased Response Latency of Rods: Antagonism by Light H . MILLERA N D WII.LIAM GRANTD. NICOL

xxvii

CONTENTS OF PREVIOUS VOLUMES

PART IV: AN EDITORIAL OVERVIEW Ca2+and cGMP WII I IAM H.

PART 111. REVERSIBILITY: ATP SYNTHESIS DRIVEN BY ELECTRIC FIELDS

MII.I.ER

Index

Volume 16

Electrogenic Ion Pumps PART I . DEMONSTRATION O F PUMP ELECTROGENICITY IN EUKARYOTIC CELLS Electrophysiology of the Sodium Pump in a Snail Neuron R. C . THOMAS Hyperpolarization of Frog Skeletal Muscle Fibers and of Canine Purkinje Fibers during Enhanced Na+-K+ Exchange: Extracellular K+ Depletion or Increased Pump Current? DAVID C. GADSBY The Electrogenic Pump in the Plasma Membrane of Nircllo ROGERM. SPANSWICK Control of Electrogenesis by ATP. Mg2+. H+, and Light in Perfused Cells of Clzuru MASASHITAZAWA AND TFRUO SHIMMF.N PART 11. THE EVIDENCE IN EPITHELIAL MEMBRANES An Electrogenic Sodium Pump in a Mammalian Tight Epithelium s. A. LEWIS A N D N . K. WILLS A Coupled Electrogenic Na+- K+ Pump for Mediating Transepithelial Sodium Transport in Frog Skin ROBIRT N I E I . S ~ N Transepithelial Potassium Transport in Insect Midgut by an Electrogenic Alkali Metal Ion Pump M I C H AG ~ L. WOLFF.RSBERG~R, WILLIAM R. HARVFY, AND MOIRACIOFFI The ATP-Dependent Component of Gastric Acid Secretion G. SACHS.B. WALLMARK. G . SACCOMANI. E. RABON, H. B. STEWART. D. R. DIBONA, AND T. BFRCLINDH

Effect of Electrochemical Gradients on Active H+ Transport in an Epithelium QAIS Ai.-AwQAri A N D TROY E. DIXON Coupling between H+ Entry and ATP Synthesis in Bacteria PI;.IERC. MALONEY Net ATP Synthesis by H+-ATPase Reconstituted into Liposornes YASUO KAGAWA Phosphorylation in Chloroplasts: ATP Synthesis Driven by A+ and by ApH of Artificial or Light-Generated Origin PETFRGRABFR PART 1V. SOME THEORETICAL QUESTIONS Response of the Proton Motive Force to the Pulse of an Electrogenic Proton Pump ERICHH E I N Z Reaction Kinetic Analysis of CurrentVoltage Relationships for Electrogenic Pumps in Neurosporu and A~~~tuhirluritr DETRIC‘HGRAUMANN, ULF-PETER HANSEN. AND CLIFFORD L. S L A Y M A N Some Physics of Ion Transport HAKOI.D J. M O R O W I ~ ~ PART V. MOLECULAR MECHANISMS OF CHARGE SEPARATION An H+-ATP Synthetase: A Substrate Translocation Concept I . A. Kozi.ov A N D V. P. SKULACHEV Proton Translocation by Cytochrome Oxidase MARI‘ENWIKSTROM Electrogenic Reactions of the Photochemical Reaction Center and the UbiquinoneCytochrome h / c pOxidoreductase P. LESLIEDUTI’ON,PAULMUELLER, DANIEL. P. O’KEEFE, NICELK. PACKHAM, ROGERC . PRINCE, AND DAVID M. TIEDE

xxviii Proton-Membrane Interactions in Chloroplast Bioenergetics R. A. DILLEY,L. J . PROCHASKA, G. M. BAKER.N . E. TANDY, A N D P. A. MILLNER Photochemical Charge Separation and Active Transport in the Purple Membrane BARRY HONlC Mitochondria1 Transhydrogenase: General Principles of Functioning 1. A. KOZLOV Membrane Vesicles, Electrochemical Ion Gradients, and Active Transport H. R. KABACK

CONTENTS OF PREVIOUS VOLUMES

PART VI. BIOLOGICAL SIGNIFICANCE OF ELECTROGENIC ION PUMPS The Role of Electrogenic Proton Translocation in Mitochondrial Oxidative Phosphorylation JANNA P. WEHRI.F. Electrogenic Reactions and Proton Purnping in Green Plant Photosynthesis WOIKANGJUNGE The Role of the Electrogenic Sodium Pump in Controlling Excitability in Nerve and Cardiac Fibers MARIOVASSALLE Pumps and Currents: A Biological Perspective FRANKLIN M. HAROI.D Index

.

CURRENT TOPICS IN MEMBRANES A N D TRANSPORT VOLUME 17

Lipids of Prokaryotes-Structure Distribution

and

HOWARD GOLDFINE Department of’ Microbinlog! School of Medicine University of Pennsylvuniu Philadelphia. Pennsyivuniu

I . tntroduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II . Structure of the Lipids of Prokaryotes . . . . . . . . . . . . . . . . . . . A . The Apolar Chains . . . . . . . . . . . . . . . . . . . . . . . . . B . Polar Lipids with a 1 . 2-Diradyl sn-Glycerol Backbone . . . . . . . . . . C . Other Polar Lipids . . . . . . . . . . . . . . . . . . . . . . . . . D . Nonpolar Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . E . Nonextractable Lipids . . . . . . . . . . . . . . . . . . . . . . . . Ill . Distribution of Lipids in Prokaryotes . . . . . . . . . . . . . . . . . . . . A . Cyanobacteria (Blue-Green Algae) . . . . . . . . . . . . . . . . . . . B . Phototrophic Bacteria . . . . . . . . . . . . . . . . . . . . . . . . C . The Gliding Bacteria and the Sheathed Bacteria . . . . . . . . . . . . . D . Budding and/or Appendaged Bacteria . . . . . . . . . . . . . . . . . E . The Spirochetes . . . . . . . . . . . . . . . . . . . . . . . . . . F. Spiral and Curved Bacteria . . . . . . . . . . . . . . . . . . . . . . G . Gram-Negative Aerobic Rods and Cocci . . . . . . . . . . . . . . . . H . Gram-Negative Facultatively Anaerobic Rods . . . . . . . . . . . . . . I . Gram-Negative Anaerobic Bacteria . . . . . . . . . . . . . . . . . . J . Gram-Negative Cocci and Coccobacilli . . . . . . . . . . . . . . . . . K . Gram-Negative Anaerobic Cocci . . . . . . . . . . . . . . . . . . . L . Gram-Negative Chemolithotrophic Bacteria . . . . . . . . . . . . . . . M . Methane-Producing Bacteria . . . . . . . . . . . . . . . . . . . . . N . Gram-Positive Cocci . . . . . . . . . . . . . . . . . . . . . . . . 0 . Endospore-Forming Bacteria . . . . . . . . . . . . . . . . . . . . . P . Gram-Positive, Non-spore-Forming Rods . . . . . . . . . . . . . . . . Q . Actinomycetes and Related Organisms . . . . . . . . . . . . . . . . . R . Rickettsia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S . Mycoplasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1V . Prokaryotic Lipids and Phylogeny . . . . . . . . . . . . . . . . . . . . . V . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 2 2 5 9 II 12 12 14 14 16

16 17 18 18 20 21 23 23 23 24 25 27 28 28 31 31 31 34 36

1 Copyright 0 1982 by Academic Press. lnc . All rights of reproduction In any form reserved ISBN 0-12-153317-4

2

HOWARD GOLDFINE

1.

INTRODUCTION

As background for the contributions to follow, this article will review the structures of the extractable lipids of prokaryotes and the distribution of these lipids in various groups of organisms. It follows the arrangement of the prokaryotes in the eighth edition of Bergey's Munual ofDeterminative Bucteriology. Since Bergey's Munual is arranged along pragmatic lines, and is not generally concerned with phylogenetic relationships among prokaryotes, an overview of the relationships of lipid compositions to recent synthesis of phylogeny will complete this article. The last two decades have revealed a wealth of information on the membranes of bacteria. The diversity of lipids seen earlier has continued to expand and yet some semblance of order is now becoming clearer. In the last decade the emphasis has shifted from descriptive to biophysical and functional aspects, but many gaps remain in our knowledge of the distribution of lipids in bacteria. The recent description of a new kingdom of prokaryotes, the Archaebacteriae, is based in part on a realization of the uniqueness of their membrane lipids (Fox et al., 1980; Langworthy, this volume). Although bacterial taxonomists are just beginning to use lipids as an aid in classification, it will hopefully be apparent to the reader that these membrane components provide a useful set of characteristics (Shaw, 1974; Lechevalier, 1977). The recent shift of emphasis among microbiologists from research on prokaryotes to eukaryotic unicellular species and to animal and plant cells in culture has lessened the intensity of work in this area. The even more recent rush toward molecular genetics and genetic engineering may perhaps serve to remind us of the importance of our prokaryotic roots.

II. STRUCTURE OF THE LIPIDS OF PROKARYOTES A. The Apolar Chains Since the structural organization of biological membranes depends on the presence of molecules containing from one to several nonpolar chains, linked either directly or indirectly to polar moieties, a description of the apolar moieties provides a useful starting point. Indeed, in the membranes of prokaryotes, as in those of higher organisms, the presence of molecules capable of hydrophobic associations is the essence of these biological structures.

PROKARYOTE LIPIDS: STRUCTURE AND DISTRIBUTION

3

I . FATTYACIDS

The most common apolar structures in prokaryotes are fatty acids, which may be linked to glycerol (most commonly as srz-l,2-di-O-acyl residues), to sugars, to amino acids, and rarely to the amino group of sphingosine. These fatty acids are usually 10 to 20 carbon atoms long with 15 to I8 carbon chains predominating. The major types found are straight chains, which may be even or odd numbered, saturated or monounsaturated; branched chains, predominantly of the isn and arzteiso types; and cyclopropane fatty acids. Examples of these fatty acid types are given in Table I. Although the table lists both oleic and cis-vaccenic acids as examples of 18:l found in prokaryotes, it should be noted that cisvaccenic is more common. This is a consequence of the predominant route o f synthesis of the monounsaturated fatty acids in bacteria, which introduces a double bond during the process of chain elongation, rather than by desaturation, usually between C-9 and C- 10, of preexisting long-chain fatty acids (see Rock and Cronan, this volume). Although polyunsaturated fatty acids occur in bacteria, they are rare; among the exceptions are 5,IO-hexadecadienoic acid in Bacillus /ichen/fnrnris (Fulco, 1974), and several methylene-interrupted polyunsaturated fatty acids in Flexihacter po/ymorphus (Johns and Perry, 1977). The blue-green algae are considered to be transitional between the bacteria and the higher protists such as algae and fungi, because some of them possess the ability to desaturate oleate to give di- and triunsaturated fatty acids (Kenyon et a / . , 1972, Kenyon, 1972). The iso- and witeiso-branched-chain fatty acids are found in many grampositive organisms, for example, Micrococcaceae, Bacillaceae, Corynebacteriurn species, and PropioNihucteriurn species. In these organisms they are often the predominant type of acyl chains. In gram-negative bacteria they are found among scattered groups of organisms (Shaw, 1974; Lechevalier, 1977). Similarly, the cyclopropane fatty acids, which are derived by C-l addition to monounsaturated fatty acyl chains (see Rock and Cronan, this volume), are widely distributed in both gram-negative and gram-positive organisms. The P-hydroxy fatty acids are important constituents of lipopolysaccharides (Luderitz ef al., this volume) and are not usually found in the phosopholipids and glycolipids of the bacterial cell membrane. 2. ALK-1-ENYL CHAINS Alk-1-enyl chains are present in phospholipids of the 1 -0-alk-1 ‘-enyl-2-Oacyl-sn-glycerol-3-P type. These are historically referred to as plasmalogens, since they yield a long-chain fatty aldehyde on acid hydrolysis (see Section II,B, I ) . Although the position on glycerol of the alkenyl moiety has been established in the plasmalogens of animal tissues (Hanahan, 1972), the position in bacteria, with one exception (Hagen and Goldfine, 1967), has not been studied. In general the alk-l-

4

HOWARD GOLDFINE

TABLE I SOMECOMMON FATTYACIDSO F PROKARYOTES Saturated n=

CH,-(CH,)n

COOH Common name

12

I2:O" I4:O

14 16

16:O 18:O

10

Monounsaturated m=

7 9

I

CE1:XCH,)n-CH=CH-(CH2)rn n= 5 5 7

Lauric acid Myristic acid Palmitic acid Stearic acid COOH

cis-9- 16 I cis- 1 1- 18: I cis-9- 18: 1

Palmitoleic acid cis-Vaccenic acid Oleic acid

Branched IS0

CH,- C H - (C H2)T,C OOH

CH,- CH,-

CH- (CH, ), C OOH

Cyciapropane I

n= 5

9

5

m=

Hydroxy a-

17:cycl"." I9:cycl

Lactobacillic acid

OH C H,-

I

(C H, )71-C H-C OOH

OH

8-

CH,-

n = 8

3-OH-

I (CH, ),,-CH-CH,-C

14 :O

OOH

P-Hydroxymyristic acid

Shonhand designations; alternative shorthand designations are given in the form C ,?:,, or C ,?. Shorthand designations; alternative shorthand designations are given in the form C,,:, or 16: 1 A 9 , and so on. Shorthand designations; alternative shorthand designations are 17:cy or I7:cyc. " Although no common name was suggested by the workers who originally described this fatty acid, the name colibacillic acid has been suggested in view of its initial discovery in E . coli (J. Asselineau, T. Kaneshiro, and W. M. O'Leary, personal correspondence). 'I

5

PROKARYOTE LIPIDS: STRUCTURE AND DISTRIBUTION

enyl chains have the same structures as the acyl chains of the organisms in which plasmalogens have been analyzed. Thus saturated, monounsaturated, cyclopropane, and iso- and anteiso-branched alk-I -enyl chains have been described (Goldfine and Hagen, 1972; Verkley et u l . , 1975).

3. O-Ai.wL CHAINS Extreme halophiles contain di-0-alkyl phospholipids with polyisopranoid side-chains, which are discussed more fully in the chapter by Langworthy , this volume. Alkyl glycerolipids have also been detected in very small amounts in the anaerobic bacteria that have plasmalogens (Kim et al., 1970; Hagen and Blank, 1970). The alkyl chains have only been examined in two species, and although they bear some qualitative resemblence to the acyl and alk-I-enyl chains, there are quantitative differences (Hagen and Blank, 1970; Kamio ef al., 1969). Tetraethers in which long-chain polyisopranoids are linked at both ends to glycerol or longer chain polyols have recently been found in methanogens and thermoacidophiles. These are described more fully by Langworthy (this volume).

6. Polar Lipids with a 1,P-Diradyl sn-Glycerol Backbone

1 . PHOSPHOLIPIDS The most widely occurring lipids in prokaryotes, as in eukaryotes, are phospholipids of the 1,2-diradyl-sn-glycerol-3-P type. The stereochemistry of these lipids is shown in Fig. 1 . The polar groups linked to phosphate are listed in Table 11. The various classes of diacylphosphatides are by no means equally common. The most widely distributed are phosphatidylglycerol, phosphatidylethanolamine, and cardiolipin. The products of N-methylation of phosphatidylethanolamine-

phosphatidyl-N-methylethanolamine, phosphatidyl-N,N‘-dimethylethanolamine, and phosphatidylcholine-are found mostly in gram-negative species. All of the 0 II

CHZO-C-R, 1 I O

R2C0

-

CH,O-C= O I1 R,CO+

I H CH, O P -X I

0

I H

C-R, I H

I 4

I::

H

CH,OP-X I

0

FIG. I . Structures of a 1 ,Z-diacyl glycerophospholipid (left) and a I-O-alk-l’-enyl-2-acyl glycerophospholipid (i.e., plasmalogen-right). See Table I1 for polar substituents.

6

HOWARD GOLDFINE

Poi

AR

TABLE I1 SUBSTITUENTS O F PROKARYOTIC PHOSPHOLIPIDS" Name of intact phospholipid

-OH + -O-CH,CH,YH, -O-CH,CH,YH(CH:,) --O-CH,CH,N(CH,), --O-CH,CH,N(CH:,):+ o -CH,CH -COO-

Phosphatidic acid Phosphdtidy lethanolamine Phosphatidyl-N-methylethanolamine Phosphatidy I-N.N'-dimethylethanolamine Phosphatidylcholine (lecithin) Phosphdtidykerine

I

NH3

-0 -CH,CHOHCH,OH

Phosphatid ylglycerol

-0-CH,CHOH-CH,

0 -Aminoacyl phosphatidylglycerol

t

i

F?

R-CH-C=O

-0-CH,CHOH-CH,OPO~-O

I R,OCH

Diphosphatidylglycerol (cardiolipin)

I

R,OCH,

I -O-~H-(CHOH)~~HOH

Phosphatidylinositol

CH, €I I I 0-CH-C-CH, I OH

Phosphatidylbutane-2.3-diol

'

Sec Fig. I for complete structures.

methylated ethanolamines are relatively rare, hence their occurrence has taxonomic and probably evolutionary significance. The 0-aminoacyl phosphatidylglycerols (Table 11) are found only in prokaryotes and are more common in gram-positive bacteria. Although they have been reported in a few gram-negative species, there i s still some doubt concerning the identity of these lipids in gram-negative organisms. The most common amino acid in 0-aminoacyl phosphatidylglycerols is lysine, but the occurrence of alanine and ornithine has also been recorded. An unusual lipid containing a glucosaminyl moiety rather than an amino acid has been found in Bacillus inegateriurn and Pseudomonas ovalis Chester (see Shaw, 1975). A butane-2,3-diol analogue of phosphatidylglycerol was found as a major phospholipid of Actinomyces (Streptomyces) olivaceus (see Batrakov and Bergelson, 1978). Other reports of phospholipids with this structure have not appeared.

PROKARYOTE LIPIDS: STRUCTURE AND DISTRIBUTION

7

Phosphatidic acid is a biosynthetic intermediate i n the pathways leading to the major phospholipids of bacteria (see Rock and Cronan, this volume), and, as such, is generally found only in trace amounts. Phosphatidylserine is also a biosynthetic intermediate in the pathway leading to phosphatidylethanolamine (see Rock and Cronan, this volume) and is generally not a major lipid of bacterial membranes. However, certain organisms have substantial amounts of phosphatidylserine. For example, van Golde et ul. (1975) have found relatively large amounts of this lipid in anaerobes that ferment lactic acid. Phosphatidylinositol is rarely found in gram-negative bacteria, uncommonly in gram-positive organisms, and most commonly in bacteria related to the actinomycetes, such as Arthrobacter and Corynebacterium, as well as in the actinomycetes themselves (Lechevalier, 1977). In these groups of bacteria, the major forms of inositide are members of a family of phosphatidylinositol mannosides, which may contain from one to five mannose units, the most common being the dimannoside. The first mannose unit is glycosidically linked to the C-2 of the inositol ring, and additional mannose units are added sequentially to the hydroxyl at C-6. There may be more than two acyl residues, but the locations of these are not known (Shaw, 1975). There are many reports of the presence of small amounts of monoacylated glycerophosphatides (lysophosphatides) in bacteria. Since these types of lipids may have a destabilizing effect on bilayers, it has generally been thought that their presence is artifactual and that they probably arise by degradation of the diacyl phospholipids. However, it is known that bacteria contain enzymes capable of forming lysophosphatides, and it is possible that small amounts of these lipids are naturally present and may in fact play important roles in the dynamics of bacterial membranes. Plasmalogens (Fig. 1 ) have been found in prokaryotes only in anaerobic species. These phospholipids are acid labile, readily yielding a long-chain fatty aldehyde on exposure to acid. In alkali, the alk-1-enyl ether is stable, but the acyl chain is cleaved, resulting in the formation of a I-0-alk-1'-enyl glycerol phosphoryl-X lipid. Since the diacyl phospholipids are stable in mild acid and alkali labile, these properties form the basis for one common method for analysis of these classes (Dawson ef al., 1962). The alkyl ether lipids are also alkali stable, and it is necessary to use other criteria, such as I, uptake (Gottfried and Rapport, 1962) or measures of the amount of aldehyde produced, to determine the amount of plasmalogen. The polar head groups on bacterial plasmalogens are somewhat more restricted than those found on the diacyl phosphatides. Plasmalogens with ethanolarnine, N-methylethanolamine, choline, glycerol, and serine have been reported (see Goldfine and Hagen, 1972; van Golde et al., 1975). Since relatively few species have been analyzed, other head groups may be found. In the recently approved nomenclature the ethanolamine-containing plasmalogen is called plas-

8

HOWARD GOLDFINE

menylethanolamine, the choline-containing plasmalogen, plasmenylcholine, and so forth (IUPAC-IUB Commission on Biochemical Nomenclature, 1978). A related lipid having the structure of a glycerol acetal of a plasmalogen has been found in Clostridium hutyricum, I F 0 3852 (Matsumoto et al., 1971) and ATCC 6015 (Khuller and Goldfine, 1974). The former strain has ethanolamine in the polar head group of this lipid, whereas the latter has N-methylethanolamine plus ethanolamine, with the former predominating. In this lipid the R, chain of the plasmalogen has a glycerol substituent: OCH,CHOHCH,OH H,CI

I

O-CH-CH,-RI

As previously noted (Section Il,A,3), 0-alkyl glyceryl ether lipids have also been detected in anaerobic bacteria. They usually represent less than 4% of the total phospholipids.

2. GLYCOSYLDIGLYCERIDES A thorough survey of bacterial glycolipids was published by Shaw (1 975), and some additional material will be found in Lechevalier (1 977). The most common type of glycolipid found in bacteria has the 1,2-diacyl sn-glycerol-3-(sugar)n structure in which n = 2 (Fig. 2). There is considerable variation in the structures of the sugar residues, and certain glycolipids are considered to be characteristic of a given genus. For example, streptococci have glycosyldiglycerides with the Glc(a 1+2)Glc(a I+) substituent; staphylococci and Bui1lu.s sp. have Glc(/3I+6)GIc(/31-+) (Fig. 2B); and lactobacilli and pneumococci have Gal(@I-+2)Glc(cu I+) substituents (Fig. 2A). These glycosyldiglycerides, digalactosyldiglycerides, and dimannosyldiglycerides are the most common bacterial diglycosyldiglycerides (Shaw, 1975). In addition to the diglycosyldigl ycerides , monoglycos yldiglycerides with Glc(a I +) , Glc@ I +), Gal@ 1+), Gal@ I -+I, GIcN@3I-), and glucuronic acid have been characterized. These are usually precursors to the diglycosyldiglycerides and as such do not accumulate, but in some species they do. Tri- and tetraglycosyldiglycerides have also been isolated from bacteria with glucose and galactose as the most frequent terminal sugars (Shaw, 1975). In view of the decreasing lipid solubility of compounds with more than three sugar residues, it is possible that compounds of this type with more than four sugars are present, but not extractable with lipid solvents. In many gram-positive bacteria the glycosyldiglycerides are substituted with a sn-glycerol-1-P moiety in which the glycerol may be acylated with one or two fatty acids (Fig. 2C). The relationship of these glycolipids to lipoteichoic acids is discussed in Section III,N.

PROKARYOTE LIPIDS: STRUCTURE AND DISTRIBUTION

9

A

CHOCOR CHZOCOR

HO

on on

CHOCOR I CHzOCOR

0

II CHZOCR

I P

C 0

CHO~R

I

II CH20POCH2

0 11

CHzOCR

G 0 A

HO

on

FIG.2 .

Structures of three representative bacterial glycosyldiglycerides. (A) Galactosyl ( a1 4 2 ) Glucosyl(ol1jdiglyceride). (B) Glucosyl(/31~6)Glucosyl(~ I+diglyceride). (C) Phosphatidylkojibiosyl diglyceride.

C. Other Polar Lipids 1 . ORNITHINE LIPIDS

Several nonphosphate-containing ornithine lipids have been found in bacteria. In one type both the carboxyl group and the amino group of ornithine are linked to fatty acids (Fig. 3A), the carboxyl group through a polyol, and the a-amino group directly in amide linkage. In the second type. which is zwitterionic, the carboxyl group of ornithine is unesterified, but there is a p-hydroxy fatty acid linked to the a-amino group to which other fatty acids are esterified at the hydroxyl group (Fig. 3B). These zwitterionic ornithine lipids are the predominant polar lipids in certain Streptoinyes species, which usually have little or no phosphatidylethanolamine, or in other species under conditions of phosphate limitation (Batrakov and Bergelson, 1978). A third type ofornithine lipid in which the carboxyl group of ornithine is

10

HOWARD GOLDFINE 0 0 II II C-0 (CH2)n-O-CR, I CHNH I

A NH,CH,(CH,),

c=o I

R2

0 II

B

0

c-0

I NH3CH ,(CH,),CHNH

0

I C=O I

0

CH2 II I RZCO-CH I Rl

FIG.3 . Structures of two omithine lipids found in bacteria. See explanation in text.

esterified to an a-hydroxy fatty acid and a 3-hydroxy fatty acid is linked to the a-amino group of ornithine has also been found in Actinomyces (Streptomyces) strain 660-15 (Batrakov and Bergelson, 1978).

2 . GLYCOLIPIDS

In addition to the glycosyldiglycerides and the phosphatidylinositol mannosides, there are a variety of glycolipids that are not readily classified. They are not widely distributed in bacteria and more complete details of their isolation and characterization in addition to primary references can be found in the reviews by Goren (1972), Shaw ( I 974, 1975), and Lechevalier (1977). Some examples are given in Fig. 4. The simplest is a polyacylated glucose (Fig. 4A). The triacylated form shown has been found in members of several groups of prokaryotes. Trehalose 6,6’ dimycolate (“cord factor”) is found in mycobacteria, corynebacteria, and nocardia (Fig. 4B). The R groups in this case are mycolic acids of the general structure OH I

R;-CH-CHCOOH I

R;

In mycobacteria, depending on the species, R’, is a linear alkane, C Z 2or C,,, and R‘?is a complex structure of approximately 60 carbon atoms with hydroxyl, methoxyl, carbonyl, carboxyl, cyclopropane, methyl branches, and carboncarbon double bonds. The intact mycolic acids from mycobacteria have recently been fractionated by high-performance liquid chromatography into homologous series (Quereshi er ul., 1978; Steck et ul., 1978). Trehalose may also be esterified by a series of polyunsaturated fatty acids called phleic acids, which have

PROKARYOTE LIPIDS: STRUCTURE AND DISTRIBUTION

A

11

0 CHzOCR I

OH

0

B

OH

c

0 H H II C H 3 C H ( C H ),3C-C-CH20POCH,CH,NH3 I I I I 0 CH3 HO YH 00

c=o (:Hz)fl CH3

FIG. 4.

(A) Triacylglucose. (B) Trehalose 6,6’-dimycolate. “cord-factor” from the C o r w r hactrrium~Mvc‘ohuc.rerium-Noc,ardiagroup. (C) Ethanolamine-containing sphingolipid from bacteroides.

been found in Mycobacrerium phlei. The principal phleic acid is hexatriaconta4,8,12,16,20-pentaenoic acid (Asselineau et a / . , 1972). Also characteristic of mycobacteria are the mycosides, in which 2-0-methylrhamnose is linked through phenol to a complex fatty group with both long-chain polyols and fatty acids (see Goren, 1972, for a review). Goren and his colleagues have also characterized a series of sulfolipids from M. tuherculnsis H37R, in which the 2’ hydroxyl of trehalose is sulfated and acyl groups are located at the 2,3,6, and 6’ positions (Goren, 1972). 3. S P H IN GO LI PI DS

Sphingolipids are rare in prokaryotes; however, they appear to be characteristic of some members of the genus Barteroides. These lipids contain various polar head groups such as phosphorylethanolamine, phosphorylglycerol, and in relatively small amounts, phosphorylglycerophosphate, which are esterified to ceramide (N-acylsphingosine) (Fig. 4C).

D. Nonpolar Lipids The nonpolar lipids of bacteria have often been neglected in general considerations of bacterial membranes. In part this is due to our scanty knowledge, the absence of clear-cut taxonomic correlations, and variation in the reporting of the quantitative analyses of this class of lipids. Most workers have simply stated that a given species has X % nonpolar among the total lipids. These may include

12

HOWARD GOLDFINE

varying amounts of monoacylglycerol , diacylglycerol, hydrocarbons, carotenoids, quinones, free fatty acids, fatty alcohols, waxes, and poly-P-hydroxybutyric acid. Triglycerides are generally not found, or are present in only trace amounts. Sterols, though generally either totally absent or present in very small amounts in most prokaryotes, are important for certain groups of organisms, which are discussed by Durisson and Rohmer and by Razin (this volume). Although certain nonpolar lipids such as the coenzymes Q, vitamins K, and carotenoids are known to play specialized roles in bacterial membranes-for example, in electron transport, oxidative phosphorylation, photosynthesis, and ion and solute transport-the functions, if any, of other neutral lipids remain unclear. Some may simply represent intermediates in pathways related to polar lipid catabolism or metabolism, others may represent a convenient pool of lipid components for eventual utilization; however, the possibility remains that small amounts of neutral lipids may be useful to ensure such membrane properties as stability, fluidity, or the provision of specific lipid structures for interaction with membrane proteins.

E. Nonextractable Lipids In many prokaryotes a portion of the total lipid is not extractable with the usual solvents such as mixtures of chloroform and methanol. However, on acid or alkaline hydrolysis of the nonextractable residue, further amounts of lipid, usually in the form of free fatty acid, are released. Among these bound forms are the lipopolysaccharides of gram-negative bacteria, which are described by Luderitz et al. (this volume), the lipoteichoic acids of gram-positive bacteria (see Section III,N), an acylated mannan found in Micrococcus lysodeikticus (Powell et al., 1975), and the complex waxes D of mycobacteria, corynebacteria, nocardia, and actinomycetes (reviewed by Goren, 1972). In the past decade lipids covalently linked to protein have been described. A major component of the outer membrane of enterobacteria is a lipoprotein of molecular weight 7500, which has a diacylglycerol linked to an N-terminal cysteine through a thioether (Braun and Hantke, 1974). With the recent discovery of protein-linked fatty acids in certain animal viruses (Schmidt et al., 1979), the possibility that this may represent a more general form of membrane organization should be considered.

111.

DISTRIBUTION OF LIPIDS IN PROKARYOTES

Several extensive reviews and compilations of microbial lipid compositions have appeared during the past decade (Goldfine, 1972; O’Leary, 1973; Shaw, 1974; Lechevalier, 1977). The last is the most complete; however, it and the

PROKARYOTE LIPIDS: STRUCTURE AND DISTRIBUTION

13

review by Shaw (1974) present qualitative rather than quantitative data. The review by Lechevalier (1977) also describes in some detail effects of pH, medium constituents, growth temperature, and age of cultures on bacterial lipid compositions. It is because of the variability induced by these factors that these authors have eschewed quantitative data. An understanding of the structure and organization of bacterial membranes, however, requires these quantitative relationships, information on the compositions of distinctive membrane fractions, and on the arrangement of lipids in the membranes. The last aspect will be discussed by Rottem in this volume. An unstated premise of this chapter has been that the lipids isolated from bacteria by extraction of whole cells are the lipids present in bacterial cell membranes. In general this conclusion has been supported by many studies on isolated bacterial membranes. Earlier work on gram-positive bacteria showed that membranes obtained from these organisms did contain the same lipids in approximately the same proportions as the whole cells (Vorbeck and Marinetti, 1965; Bishop et ul., 1967); however, specific associations of certain glycolipids with cell walls cannot be discounted (Shaw, 1975). In gram-negative organisms the problem is more complex because their outer membrane, which is part of their cell wall, contains lipids. Many species, especially those capable of photosynthesis, have internal membranes as well. The outer membranes of Escherichia coli and Salmonella typhirnuriurn contain the same phospholipids as the inner membrane, although the ratio of phosphatidylethanolamine to phosphatidylglycerol plus cardiolipin (Osborn et ul., 1972; Diedrich and CotaRobles, 1974; Rottem et ul., 1975) and the ratio of saturated to unsaturated fatty acids (White et al., 1972; Diedrich and Cota-Robles, 1974; Koplow and Goldfine, 1974; Rottem er a/., 1975; Lugtenberg and Peters, 1976) are somewhat higher in the outer than in the inner membrane. Kenyon (1978) has recently reviewed the lipids of photosynthetic bacteria. In several studies the phospholipid composition of the subcellular fractions, including the chromatophores, of these bacteria had similar phospholipid compositions to that of the whole cell (Gorchein, 1964, 1968; Haverkate er d.,1965; Takacs and Holt, 1971), whereas some quantitative differences in the chromatophores and crude membranes of Ectothiorhodospira halophila SL- 1 were observed in an unpublished study (Kenyon, 1978). It would indeed be surprising if the functionally differentiated membrane systems of prokaryotes did not show some differentiation in the compositions of their complex lipids. With these caveats in mind, the lipid compositions of the major groups of prokaryotes will be presented. I shall not duplicate recent reviews that have presented detailed quantitative data on bacterial lipid compositions (see previous discussion). Rather, the broader picture will hopefully emerge through the use of selected examples. However, it is important to realize that even within a bacterial genus, significant differences in lipid composition have been found.

14

HOWARD GOLDFINE

A. Cyanobacteria (Blue-Green Algae) The blue-green algae studied resemble green algae and the photosynthetic apparatus of higher plants in their complex lipid composition. The major lipids are phosphatidylglycerol, monogalactosyldiglyceride, digalactosyldiglyceride, and sulfoquinovosyldiglyceride [SQDG; 1,2-diacyl-sn-glycero-3-(6-sulfo-~u-oquinovopyranoside)]. They lack phosphatidylethanolamine and phosphatidylcholine (Nichols r t u l . , 1965). It is interesting to note that in fatty acid composition some of the blue-green algae resemble bacteria in the absence of polyunsaturated fatty acids, whereas others-especially , but not exclusively, the filamentous types-have polyunsaturated fatty acids (Kenyon and Stanier, 1970).

B. Phototrophic Bacteria The eighth edition of Bergey's Munuul of Determinative Bacteriology (Buchanan and Gibbons, 1974) divides the phototrophic bacteria (Rhodospirillales) into three families. Two of the best-studied members of the Rhodospirillaceae family, the purple nonsulfur bacteria, which are representative in terms of their lipid compositions, are Rhodospirillum ruhrurn and Rhodopseudomows sphueroides. Several of the more recent analyses of the former agree that the major polar lipids of light- and dark-grown cells are phosphatidylethanolamine, phosphatidylglycerol, cardiolipin, and a nonphosphate-containing ornithine lipid (Fig. 3B) (Hirayama, 1968; Depinto, 1967). Earlier reports of the presence of phosphatidylcholine were not confirmed (Brooks and Benson, 1972). Rp. sphueroides has phosphatidylethanolamine,phosphatidylglycerol, phosphatidylcholine, and an omithine lipid (Fig. 3B) as its major lipids. Small amounts of SQDG and cardiolipin were also reported. Fig. 5A presents the phospholipid composition of R p . sphueroides diagrammatically. The lipid composition of Rp. capsulatu is similar, and lecithin increases at the expense of the other major lipids in dark-grown cells (Steiner et al., 1970). There have been fewer studies of the purple sulfur bacteria (Chromatiaceae). The major lipids of Chromatium viiiosum are phosphatidylethanolamine and phosphatidylglycerol, with smaller amounts of cardiolipin and some glucose and mannose-containing lipids (see Kenyon, 1978, for references). Although the lipids of Thiocapsu roseopersirina and Ectothiorhodospira halophila, an extreme halophile, have been studied, no complete analyses are available. The latter organism does not have the d i - 0 alkylglycerol ether lipids characteristic of Halobacterium (see Langworthy, this volume). Its major lipid is phosphatidylglycerol, with smaller amounts of phosphatidylethanolamine and unknown phospholipids. Although total analyses of the green sulfur bacteria (Chlorobiaceae) are also lacking, it is of interest that several species have been found to have the monogalactolipid and SQDG charac-

15

PROKARYOTE LIPIDS: STRUCTURE AND DISTRIBUTION

w

0

C

rn Gal OG

Rhodopseudomonos sphaeroides a

Hyphornfcrobiurn vulgore N0-52Ib

Treponerno pollidum Kazon 5'

(light-grown)

@ CL Azofobacter

Pseudomonos

ieptospfro potocd

ogr1is (log)'

oerugfnosa'

G

I

H

@ CL

Aqroboclerium tumefociens flog)

'

Escherrchjo COll

Serratio rnorcescens'

(log)'

J

Proteus vulgaris

(Ioq)'

qonorrhoeoe

*

Megasphaera elsdenii

FIG. 5. Polar lipid compositions of gram-negative bacteria. Nonionic phospholipids are not shaded. Anionic phospholipids are stippled. Phosphatidylserine is shown with diagonal hatching. Unmarked areas of the diagrams represent unknown lipid components, mixtures of small amounts of known lipids, or a combination of the two. PE, Phosphatidylethanolamine; LPE, lysophosphatidylethanolamine; PlaE, plasmalogen form of PE; PME, phosphatidyl-N-methylethanolamine; PDME, phosphatidyl-N,N-dimethylethanolamine;PC, phosphatidylcholine; PG, phosphatidylglycerol; CL, cardiolipin; PS, phosphatidylserine; PlaS, plasmalogen form of PS; DG, diglyceride. References: "Gorchein. 1968; Russell and Harwood, 1979; "Goldfine and Hagen, 1968; Johnson or al., 1970b; "Johnson et ul., 1970a; '~Hancock and Meadow, 1969; 'Randle cf a / . , 1969; "Senff ef a / . , 1976: Beebe and Wlodkowski, 1976; "van Golde ef a / . , 1975. (

teristic of higher plants, in addition to phosphatidylglycerol. No phosphatidylethanolamine was found in pure cultures (Kenyon, 1978). The major fatty acids of photosynthetic bacteria are 16:0, 16:1, and 18:l. Substantial amounts of 14:O are found in the Chrornatiaceae (Kenyon, 1978).

16

HOWARD GOLDFINE

C. The Gliding Bacteria and the Sheathed Bacteria So little is known about the lipids of the Myxobacteriales that it would be premature to generalize about the membrane composition of this interesting group of organisms, which is generally capable of forming fruiting bodies. The phospholipids of a marine organism, tentatively identified as a Sporocytophaga sp., were shown to be typical of gram-negative bacteria, consisting principally of phosphatidylethanolamine (76%) and phosphatidylglycerol (20%) Oliver and Colwell, 1973). Oral isolates of Capnocyrophagu and the related Sporocytophuga were recently found to have considerable amounts of neutral lipids in addition to phospholipids. Phosphatidylethanolamine, lysophosphatidylethanolamine, and two unidentified phospholipids were the major polar lipid components of three species of Cupnocyrophaga. An ornithine lipid was also detected. In addition to phosphatidylethanolamine and the lyso analogue, phosphatidylserine was found in Sporocytophaga (Holt et al., 1979). An unusual class of lipids, based on the sulfonolipid capnine (2-amino-3-hydroxy15-methylhexadecane-I -sulfonic acid), has been found as a major constituent of the cell envelope of Capnocytophuga (Godchaux and Leadbetter, 1980). These lipids are similar to 1 -deoxyceramide-1 -sulfonic acid previously found as a minor component of the diatom Nirzschin alba (Anderson et a / . , 1978). Some species have the free form of capnine, but most have N-acylcapnines in which the acyl moieties are rich in 2- and 3-hydroxy groups, and have methyl branches (Godchaux and Leadbetter, 1981). Capnines have been found in a variety of other gliding bacteria including Cytophngu johnsonae, Vitreoscilh Jrercoruriu, Flexibacter, and Sporocytophaga myxococcoiries (W. Godchaux, personal communication). The structures of the capnines and N-acyl capnines are analogous to those of sphingosine and ceramide (see Fig. 4C). The principal phospholipids of the membranes of M\;xococcus xanthus were found to be phosphatidylethanolamine (76%), and phosphatidylglycerol (9%) (Omdorff and Dworkin, 1980). The lipid composition of the sheathed bacteria has received less attention.

D. Budding and/or Appendaged Bacteria Among this large group, data are available for Hyphornicrobiurri and Cuulobucter; the former divide by budding at the tips of their hyphae, whereas the latter produce adherent stalks, but divide by binary fission. Hyphomicrohiutn has an unusual mixture of phospholipids characterized by a high proportion of phosphatidyl-N,N’-dimethylethanolamine, which represented 36% of the phospholipids in strain NQ-521 and a similarly large proportion of the lipids in three other strains (Hagen et a / . , 1966). The other phospholipids in strain NQ-521 were phosphatidylcholine, phosphatidylethanolamine, and

17

PROKARYOTE LIPIDS: STRUCTURE AND DISTRIBUTION

polyglycerol phosphatides (Fig. 5B). The phospholipids are very rich in cis- 1 1 18:1 (cis-vaccenic acid) (Auran and Schmidt, 1972). Caulobacrer crrscrntiis has a phospholipid composition that is highly unusual for a gram-negative organism. The major phospholipid is phosphatidylglycerol (>80%). Cardiolipin, acylphosphatidylglycerol, and lysophosphatidylglycerol have been identified as minor phospholipids (Contreras et al., 1978; Jones and Smith, 1979; DeSiervo and Homola, 1980). A major proportion (-50%) of the polar lipids are glucosyldiglyceride and other glycolipids (DeSiervo and Homola, 1980).

E. The Spirochetes These flexible, helically coiled bacteria are divided into several genera on the basis of morphological and biochemical characteristics. Determinations of lipid composition were hampered by the difficulties encountered in the laboratory cultivation of these organisms. The major groups have now been studied and several generalizations have emerged. Spirochaeta are characterized by the presence of phosphatidylglycerol and cardiolipin, the absence of the N-containing phosphatides, and the presence of glycolipids of the monoglycosyldiglyceride type, which usually are close to 50% of the polar lipids. Thus their lipids resemble those of many gram-positive organisms. Treponeina have phosphatidylcholine and (usually) phosphatidylethanolamine, with minor amounts of polyglycerolphosphatides. They have the same type of glycolipids as the Spirochaeta, in similarly large amounts (Fig. SC) (Livermore and Johnson, 1974). Some of the treponemes have alk-1 -enyl acyl (plasmalogen) phospho- and glycolipids (Meyer and Meyer, 1971; Matthews et ul., 1979). Leptospira have neither glycosyldiglycerides nor phosphatidylcholine. The principal phospholipid is phosphatidylethanolamine, which may be accompanied by polyglycerolphosphatides (Fig. 5D) (Johnson et al., 1970a). Borrrlia hermsi has been shown to synthesize phosphatidylcholine, phosphatidylglycerol, monogalactosyldiglyceride, and cholesterylglucosides (Livennore et al., 1978). TABLE I11 CHARACTERI~TIC. POLARLiPrns OF T H E SPIROCHETES Lipid Monoglycosyldiglyceride Cholesterylglucosides Phosphatidylchol ine

Phocphatidylethanolamine ‘I

Spirochneru

Treponumu

+

+

~

~

Leptospiru

-

+ +

Except T . prillidum (Nichols virulent strain) (Matthews ef a l . , 1979)

Borreliii

+ + + +

18

HOWARD GOLDFINE

The lipid composition of B . hermsi is therefore similar to that of the treponemes, except that the latter do not have cholesterylglucosides. Table I11 presents a comparison of the lipid compositions of the spirochetes. These marked differences suggest that these groups diverged early in their evolution, which is consistent with other data (Fox et ul., 1980).

F. Spiral and Curved Bacteria This group has had little attention. A marine species, Spirillum linum, has a typical gram-negative phospholipid complement consisting of phosphatidylethanolamine (75%) and phosphatidylglycerol (23%) (Oliver and Colwell, 1973). The lipids of Campylobacter (Vihrio),fetus were reported to contain the same two major phospholipids plus small amounts of phosphatidylserine, phosphatidylinositol, and digalactosyldiglyceride (Tornabene and Ogg, 197 1). These cells had considerable amounts of neutral lipid, especially when in the coccoid form.

G. Gram-Negative Aerobic Rods and Cocci 1 . PSEUDOMONADACEAE Among the family Pseudomonadaceae, the genus Pseudomnnas has been studied most intensively. Most species have a phospholipid composition characteristic of gram-negative organisms, consisting of phosphatidylethanolamine, which is the most abundant lipid, phosphatidylglycerol, and cardiolipin (see Shaw, 1974, for references). The phospholipid composition of P . aeruginosu is shown in Fig. 5E. Some species, including P. ueruginosa, have been reported to contain small amounts of phosphatidylcholine (see Goldfine, 1972, for references), and a zwitterionic ornithine lipid (Fig. 3B) was found in several species sensitive to ethylenediaminetetraacetic acid when the cells were grown on nutrient agar (Wilkinson, 1970), but not when grown in nutrient broth (Wilkinson et ul., 1973). Dramatic increases in the ratio of the ornithine lipid to the phospholipids were shown in phosphate-limited cultures of P . jluorescens NCMB 129 (Minnikin and Abdolrahimzadeh, 1974a). Four species, P . diminuta, P . multophilia, P. vesicularis, and P . rubescens, have glycosyldiglycerides containing both glucose and glucuronic acid. Although still designated Pseudnmonas in the eighth edition of Bergey’s Manuul (Buchanan and Gibbons, 1974), the first three were placed in a distinct group based on their growth-factor requirements, and the last is no longer included in this genus, again strengthening the taxonomic value of lipid-compositional studies (Shaw, 1974; Lechevalier, 1977; Wilkinson and Galbraith, 1979). Gluconnbacter, another genus in the Pseudomonadaceae, has been reported to contain phosphatidylcholine and an

PROKARYOTE LIPIDS: STRUCTURE AND DISTRIBUTION

19

uncharacterized ornithine lipid in addition to the usual lipids of the genus Pseudomonas (Tahara et a / . , 1976; Heefner and Claus, 1978).

2. AZOTOBACTERACEAE Like the Pseudomonadaceae, the Azotobacteraceae are aerobic gram-negative rods, but they are capable of fixing nitrogen. The phospholipids of two species of Azotobucter, A . agilis and A . vinelundii, have been studied. The former has small amounts of phosphatidyl-N-methylethanolamineand phosphatidylcholine, in addition to major amounts of phosphatidylethanolamine and phosphatidylglycerol (Fig. 5F). The latter does not appear to possess the N-methylation system needed for the synthesis of phosphatidylcholine (Randle et ul., 1969; Jurtshuk and Schlech, 1969). When induced to encyst by addition of @-hydroxybutyrate to the medium, A . vinelundii accumulates 5-n-alkylrcsorcinols and their galactose derivatives. The alkyl chains are C,, and C 2 , (Reusch and Sadoff, 1979). These unusual lipids appear to become major membrane components during encystment because the phospholipids decrease to less than 10% of the total (Reusch and Sadoff, 1981). Little work appears to have been done on the membrane lipids of other genera in this family.

3 . RHIZOBIACEAE Rhizobia are capable of infecting the roots of leguminous plants to produce nodules, and agrobacteria infect diverse species of plants and produce gall hypertrophies. All rhizobia examined have phosphatidylcholine in addition to the usual lipids found in gram-negative bacteria. I n two strains examined by Gerson and Patel ( 1 9 7 3 , phosphatidylcholine represented 5 1 % and 21 % of total phospholipid in the free-living form, and 33% and 29% in the bacteroid isolated from root nodules. Phosphatidylinositol was also found in these strains, but not in R. juponicum (Bunn and Elkan, 1971) and R . leguminosururn (Faizova et al., I97 I ). All species of Agrobuc/eriutn likewise have phosphatidylcholine (Goldfine and Ellis, 1964), and A . fumefuciens (Fig. 5G) (Kaneshiro and Marr, 1962; Randle et ul., 1969) is typical. In this organism phosphatidylcholine increased to 28% of the phospholipid in stationary cells at the expense of its precursors (Randle e / ul., 1969).

4. METHYLOMONADACEAE A N D O T H E RMETHANE-UTILIZING BACTERIA Many of these organisms are characterized by an obligate requirement for one-carbon organic compounds as a source of carbon. Me/hvlomotzns methanolicu has a phospholipid composition similar to that of E . coli (Goldberg and Jensen, 1977) (see Fig. 5H),but Methylococcus cupsulutus has

20

HOWARD GOLDFINE

8% phosphatidylcholine and Methylosinus trichosporium is rich in N-methylethanolamine and N,N’-dimethylethanolamine, as well as choline phosphatides (Makula, 1978). However, Weaver ef NI. (1975) reported mainly phosphatidylglycerol and phosphatidylethanolamine in the latter organism. Unlike M . capsulatus and M . rnethanolica, which have intracytoplasmic membranes consisting of vesicular disks organized into bundles (type I), M . trichosporium has type I1 membranes, which are characteristically arranged either at the periphery of the cells or paired and extending throughout the cells. These two groups of methane-utilizing organisms also differ in the pathways utilized for 1-carbon assimilation (Makula, 1978). Two other strains of methane-utilizing bacteria, LaPaz and OBT, also had high levels of N,N’-dimethylethanolamine and choline- or N-monomethylethanolamine phosphatides (Makula, 1978). A facultative methylotrophic organism that has type I1 intracytoplasmic membranes when grown on methane, but none when grown on glucose or methanol, Methylobucterium orgunophiliurn, has phosphatidyl-N,N’-dimethylethanolamine, phosphatidylcholine, and phosphatidylethanolamine, with a somewhat higher proportion of the methylated bases in methanol- and glucose-grown cells than in methane-grown cells (Patt and Hanson, 1978).

5. HALOBACTERIACEAE These organisms, which require above 2 M sodium chloride for growth, have unusual polyisopranoid ether lipids that are described by Langworthy (this volume). 6. GRAM-NEGATIVE AEROBIC RODS A N D Cocci AFFILIAIION

OF

UNCERTAIN

Most of the organisms of this group, which includes Alculigenes, Acetobarter, Brucellu, and Bordetella, have typical gram-negative lipid compositions. Whereas Alculigenes (Lechevalier, 1977) and Bordetella pertussis do not have phosphatidylcholine, Brucella ubortus and Brucella melitensis are both rich in this lipid and have in addition the N-methylated intermediates between phosphatidylethanolamine and phosphatidylcholine (Thiele and Schwinn, 1973). Brucella and Bordetella also have ornithine lipids of the type illustrated in Fig. 3A.

H. Gram-Negative Facultatively Anaerobic Rods This group of organisms includes some of the most familiar prokaryotes, such as E . coli, Salmonella, Shigella, Klebsiella, Serratia, and Proteus, which with

PROKARYOTE LIPIDS: STRUCTURE AND DISTRIBUTION

21

several other genera are grouped together as the Enterobacteriaceae, and another family, Vibrionaceae, which includes Vihrio and Aeromonas with several other genera. Typical phospholipid patterns are illustrated in Fig. 5H and 51. Salrnonella is very similar to E . coli in having mainly phosphatidylethanolamine and lesser amounts of phosphatidylglycerol and cardiolipin (Ames, 1968). The ratios of phosphatidylglycerol and cardiolipin are often variable depending on growth phase, osmolarity, and carbon source, but such changes may be more directly related to growth rate (Merlie and Pizer, 1973). Some enteric organisms, such as Proreus (Goldfine and Ellis, 1964) and Yersinia species (Tornabene, 1973), have small amounts of the N-methylated ethanolamine phosphatides, but no lecithin (Fig. 5J). The phospholipids of a number of Vibrio species mainly of marine origin, but including V . cholerue, were analyzed by Oliver and Colwell (1973). With the exception of V . marinus, all vibrios had typical gram-negative phospholipid compositions: phosphatidylethanolamine (60-80%), phosphatidylglycerol ( 1530%), and small amounts of lysophosphatidylethanolamine and cardiolipin. V . marinus had more phosphatidylglycerol (54%) than phosphatidylethanolamine (23%). The phospholipid composition of Chrornobacterium violaceum (Randle et al., 1969) and Huemophilus influenzae Rd (Sutrina and Scocca, 1976) is very similar to that of E . coli log-phase cells (Fig. 5H), except that no cardiolipin was detected in the latter organism.

I. Gram-Negative Anaerobic Bacteria 1 . BACTEROIDACEAE

Many species of Bacteroides have been shown to contain sphingolipids, a unique finding among prokaryotes (White et a/., 1969; Fritsche and Thelen, 1973). Miyagawa et al. (1978) examined 15 species of Bacteroides and found evidence for sphingolipids in 10, but the other 5-B. succinogenes, B . furcosus, B. hypernegus, B. umylophilus, and B. multiacidus-did not have sphingolipids. On this basis it was proposed that the inclusion of these species among the bacteroides is in some doubt. The major sphingolipids in the bacteroides have been characterized as ceramide phosphorylethanolamine (Fig. 4C) and ceramide phosphorylglycerol. The major phosphoglycerides are phosphatidylethanolamine and smaller amounts of the polyglycerolphosphatides, which represent from one-third to one-half of the polar lipids (White et al., 1969; Stoffel et al., 1975). Even within a species, B . melaninogenicus, considerable variation was seen in the relative proportions of the sphingolipids and diacylphospholipids (Rizza el al., 1970). Among the organisms containing sphingolipids, there are branched chains in both the acyl groups and the long-chain bases.

22

HOWARD GOLDFINE

2 . Desirlfbvibrio

AND

Butyrivihrio

Two species of Desulfovibrio, D . clesulfuricuns Norway and D . vulgaris, have typical gram-negative phospholipid patterns consisting of 61 to 72% phosphatidylethanolamine, 20 to 21 5% phosphatidylglycerol, and smaller amounts of cardiolipin. D . desu~uricunshas, in addition, 1 I % phosphatidylserine (Makula and Finnerty, 1974). D . gigas differs considerably in having a phosphatidylethanolamine to phosphatidylglycerol ratio of 30:70, and in having an ornithine lipid of the zwitterionic type (Fig. 3B). The ornithine lipid represented 78% of the total lipid (Makula and Finerty, 1975). Butyrivibrio, obligately anaerobic bacteria of the rumen, appear to have. a unique group of lipids, which have been recently characterized by a group of workers at Babraham, Cambridge, England. Three unusual features are worth emphasizing. The polar lipids of these organisms have various short-chain fatty acids. For example, there are n-butyryl esters of phosphatidylglycerol, and lipids in which a galactose residue of a galactolipid is esterified with butyrate. In all of the species examined, alk-l -enyl acyl substituents are found on the diglyceride moieties (Clarke et ul., 1976). The most unusual feature of one such lipid is the cross-linking of two “diglyceride ” moieties with a long-chain dicarboxylic acid that has a vicinal dimethyl substitution at the center of the chain. These fatty acids have been named diabolic acids (Fig. 6) (Klein et ul., 1979). Thus the intact lipid can be thought of as a dimer of a plasmalogenic glycosyldiglyceride and a plasmalogenic phosphatidylglycerol in which the glycerol group is esterified with butyrate. The two plasmalogens are cross-linked by the diabolic acid. It is not known whether the lipid spans the membrane of these cells or is bent into a hairpin structure (Hazlewood et ul., 1980). Selenomonas ruminantium, another anaerobic rumen bacterium, contains ethanolamine phosphatides as the major polar lipid class in its cytoplasmic mem-

H,C [CH2],3CH=CH-O-hH,

n n

FIG. 6 . Structure of a diabolic acid-containing phospholipid isolated from Butyrivibrio S2 grown in the presence of palmitic acid. The R group esterified to the galactose is a butyroyl residue. The butyroyl group on the glycerol residue may be replaced by a palmitoyl group (Clarke er a / . , 1980). Two molecules of sn- 1 -alkenylglycero-3-pbospho-m1 ‘-glycerol butyroyl ester may also be linked through a diabolic acid.

PROKARYOTE LIPIDS: STRUCTURE AND DISTRIBUTION

23

brane. These exist in the diacyl, alk-I-enyl acyl, and alkyl acyl forms in the ratio 1:0.5:0.2 (Kamio and Takahashi, 1980). J. Gram-Negative Cocci and Coccobacilli Recent work on the membrane lipids of Neisseriu gonorrhoeae (Fig. 5K) and Brunhomellu caturrhalis has shown that gram-negative cocci, which like the gram-negative rods have inner and outer membranes, have phospholipids typical of gram-negative bacteria consisting largely of phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. Some lysophosphatidylethanolamine has been found, but this was largely attributed to the action of an endogenous phospholipase A (Senff et al., 1976; Beebe and Wlodkowski, 1976; see Lechevalier, 1977, for earlier references). Phosphatidylcholine was found in some strains of N . gonorrhoeue (Sud and Feingold, 1975) and in B . cuturrhalis (Beebe and Wlodkowski, 1976). Other gram-negative, cocci such as Acinetobucter sp. HO 1 -N (previously Micrococcus ceriJiccin.7) and Acirietobacter sp. MJT/F5/ 199A, similarly have typical gram-negative phospholipid patterns, but in strain HOI-N there is 12 to 3 1% phosphatidylcholine in addition to phosphatidylethanolamine and cardiolipin (Makula and Finerty, 1970; Thorne et ul.. 1973).

K. Gram-Negative Anaerobic Cocci The major membrane lipids of Veillonellu purvulu and Megusphaeru elsdenii have, like those of many anaerobes, a large proportion of alk-I-enyl acyl phospholipids. In addition, these organisms are unusual in that a major component of the phospholipids are serine phosphatides (Fig. 5L). This was found to be characteristic of anaerobes that ferment lactate (van Golde et al., 1975).

L. Gram-Negative Chemolithotrophic Bacteria This group of organisms is divided into those oxidizing ammonia or nitrite, those metabolizing sulfur, and those depositing iron or manganese oxide. Two of the nitrifying bacteria, which contain intracytoplasmic membrane systems, were found to have mainly phosphatidylethanolamine (67-78%) and polyglycerolphosphatides (17-1 8%) among their phospholipids. One of these, Nitrosococcus (Nitrosocystis) oceanus, has 3% phosphatidylcholine, whereas the other, Nitrosomonus europaeu, had no phosphatidylcholine (Hagen et al., 1966). Two species of Nitrobucter with internal membranes also have phosphatidylcholine (Auran and Schmidt, 1972).

24

HOWARD GOLDFINE

TABLE IV LIPIDSOF Thiobucillus

Group %GC I

56-57

I1 62-66:

111 SO-52

Fatty acids

Species

PE

PME

PC

C-14 and C-16 predominate C- I5 and C- 17 predominate

T . necipulitrrnu.7 7'..firmridtitis T . fhiopurus T . nowllus T . inti,rmedius" T . rhiooxicluns (log)

44' 20 65 25 58 20

23' 42

-

Most are C-14 or shorter

~~

Polyglycerol phosphatides 33 36 35 33 29 39

1.5

-

-

7

3s

14

-

36'

-

~

~~~

~

~~

%GC unknown. Values for phospholipids (% of lipid P) are the middle of the ranges given by Barridge and Shively (1968), Shively and Renson (1967). and Short r i nl. (1969). Increases in stationary phase (Shively and Benson, 1967; Agate and Vishniac. 1973). "

I

Among the organisms metabolizing sulfur, Thiobacillus and Sm/jo/~olohushave been studied intensively with respect to their membrane lipids. The latter group lives at high temperature and low pH. Its unusual lipids are described by Langworthy (this volume). In the eighth edition of Bergey's Manuul (Buchanan and Gibbons, 1974), the thiobacilli have been classified according to their lipid fatty acids and the percentage guanine-cytosine (CC) of their DNA. As can be seen in Table IV, the distribution of phospholipids does not follow any clear pattern. PhosphatidylN-methylethanolamine is found in members of each group. Phosphatidylcholinc is present in only one member of group 11, 7'.novellus, and one member of group 1 , T . (Ferrobucillus)ferrooxiduns. T . thiooxicluns has a zwitterionic ornithine lipid (Fig. 3B), but it is not known if this lipid is present in other thiobacilli.

M. Methane-Producing Bacteria The lipids of the methanogens, which are similar to those of the thermoacidophiles and related to those of the halobacteria, are discussed by Langworthy (this volume). They are characterized by branched chains (phytanyl) in ether linkage present in phospho- and glycolipids. It has recently been proposed that these organisms be grouped in a separate kingdom, the Archaebacteriae (Woese et d . , 1978).

25

PROKARYOTE LIPIDS: STRUCTURE AND DISTRIBUTION

N. Gram-Positive Cocci Differences in the cell wall composition and structure of the gram-positive and gram-negative groups of organisms are also reflected in major differences in the lipid compositions of their membranes. As noted above, most groups of gramnegative bacteria have phosphatidylethanolamine alone or together with the N-methylated ethanolamine phosphatides as the major nonionic group of polar lipids, which usually represent more than half of the total amphipathic lipid in these cells (Fig. 5). In gram-positive organisms the situation is reversed. Among the aerobic, facultatively anaerobic, and anaerobic cocci, for example, the major polar lipids are anionic phospholipids including phosphatidylglycerol, which is usually the most abundant, and cardiolipin. These may be accompanied by an 0-aminoacyl phosphatidylglycerol or phosphatidylinositol (Whiteside et ul., 1971; see Goldfine, 1972, and Lechevalier, 1977, for additional references). An early analysis of the lipids of Micrococcus lysodeiktici*s (lucteus) (Macfarlane, 1961a,b) is given in Fig. 7A. The glycolipid was identified as dimannosyldiglyceride (Lennarz and Talamo, 1966). The analyses of Whiteside e f al. (1971), based on 32P labeling, agree qualitatively, but the ratio of cardiolipin to phosphatidylglycerol is much higher (39:45). These authors found a similarly high ratio of cardiolipin to phosphatidylglycerol in M . refrugenus, other Micrococcus sp., and Surcinufluvu. It should also be noted that many of these organisms have

C

A

IGicDG

Muacoccus

lysodeikt,cus'

Slreptococcus pneurnonme t-192Ap

Slophylococcus aureus /logic

t?OCill"S

cereus

F

Baollus subti1,r'

80C~llUS

megateriurn'

L octabacillus pbntarumg

FIG. 7. Polar lipid compositions of gram-positive bacteria. PI, phosphatidylinositol. For other abbreviations, see legend to Fig. 5 . Lysyl-PG and ornithine-PG, which are cationic phospholipids, are shown with light stippling. Rcferences: ' I Macfarlane, 1961a.b; Brundish ef a/., 1967; Joyce r f d., 1970; Shaw, 1975; "Houtsmuller and van Deenen, 1963; "Bishop rt al., 1967; 'Bertsch af u l . . 1969; '' Exterkate t'r o l . , 107 I ; L . p l t r n t u r u t n contains an unknown proportion of galactosylglucosyldiglyceride (Shaw, 1975).

26

HOWARD GOLDFINE

a substantial amount of hydrocarbons ranging from 17 to 22% of total lipid (Tornabene er al., 1970). It is apparent from work with other gram-positive cocci that the lipid composition may vary considerably with growth stage and pH (Lechevalier, 1977). In Staphylococcus aureus, phosphatidylglycerol predominates during log phase (Fig. 7C), but the relative and absolute amount of lysylphosphatidylglycerol increases in stationary phase and/or at low external pH, whereas the absolute amount of phosphatidylglycerol decreases. Thus the ratio of the two lipids can reverse (Houtsmuller and van Deenen, 1965; Gould and Lennarz, 1970). In Planococcus, a group of motile, gram-positive cocci, some phosphatidylethanolamine is present along with cardiolipin and phosphatidylglycerol (Komura et ul., 1975a). In the streptococci the major phospholipids are also cardiolipin and phosphatidylglycerol, which may be accompanied by aminoacyl phosphatidylglycerol and glycolipids, predominantly diglucosyldiglyceride (Fig. 7B) (Goldfine, 1972; Shaw, 1975). The relative amounts of these lipids may vary with growth rates (Carson et al., 1979) and with growth phase (Chiu and Hung, 1979). Carson et al. (1979) noted that the predominant neutral lipid, diacylglycerol, and cardiolipin accumulated relative to cellular mass as the rate of growth decreased. At the shortest doubling time (30 minutes) the anionic phospholipids predominated. In addition to phospholipids and glycosyldiglycerides, many gram-positive cocci contain another class of lipids, which have been designated phosphoglycolipids (Pieringer and Ganfield, 1975). These are sn-glycerol- 1 -phosphate derivatives of diglycosyldiglycerides in which the glycerol-1 -phosphate may be acylated with fatty acids as in phosphatidylkojibiosyl diglyceride (Fig. 2C). These lipids may serve as a hydrophobic anchor for membrane teichoic acids in which there is a glycerol-P polymer linked to the disaccharide. The polymer usually has 20 to 40 glycerol-P units (Fischer et ul., 1980), which may be substituted with a variable number of disaccharide and alanine moieties (Pieringer and Ganfield, 1975). Many gram-positive cocci have an internal vesicular or tubular localized membrane system, the mesosome. It appears that the lipids of the mesosome and the plasma membrane are qualitatively similar; however, certain lipid fractions may be concentrated in the mesosome and the mesosome may have a higher content of lipid relative to dry weight than the plasma membrane (Thomas and Ellar, 1973; Beining et nl., 1975). With a few exceptions, most of the gram-negative organisms discussed earlier have mixtures of long-chain saturated and monounsaturated fatty acids. In many organisms, the monounsaturated fatty acids undergo conversion to cyclopropane fatty acids after they have been incorporated into membrane lipids (Law, 1971). In many gram-positive cocci, especially the Micrococcaceae and the anaerobic Peptococcaceae (Whiteside et ul., 1971; Shaw, 1974; Lambert and Armfield,

PROKARYOTE LIPIDS: STRUCTURE AND DISTRIBUTION

27

1979), unsaturated fatty acids are replaced by branched-chain fatty acids of the iso- and anteiso type.

0. Endospore-Forming Bacteria The aerobic and facultative genus Bncillits lies in phospholipid compositional terms between the aerobic gram-positive cocci and the gram-negative bacteria. In many species there is a considerable amount of phosphatidylethanolamine,which usually comprises from 20 to 45% of the total phospholipid (Fig. 7D-F) compared to the 55 to 80% phosphatidylethanolamine plus the N-methylated ethanolamine phosphatides found in many gram-negative bacteria. Conversely, the phosphatidyl-glycerol family occupies a more prominent place in the bacilli, sometimes including aminoacyl phosphatidylglycerol or a glucosaminyl phosphatidylglycerol, as in B. meguteriurn (Fig. 7F). As in many gram-positive cocci, the acyl chains of the lipids of Bacillus are usually branched (60 to 90% of the total fatty acids) (Kaneda, 1977), plus straight-chain saturated, and in a number of species these are accompanied by monounsaturated fatty acids. As in the gram-positive cocci, there may be considerable variation in lipid composition within a species, depending on environmental factors and stage of growth. Fatty acid desaturation is under temperature control in some Bucillus species (Fulco, 1974). Changes in the branched-chain amino acid composition of the medium affects the ratio of iso- to anteiso-acyl chains in the lipids (Kaneda, 1977). The effects of phosphate and magnesium limitation on the composition of the polar lipids of Bucillus can also be quite dramatic. In cheniostat cultures of B . sirhtilis (Marburg), phosphate limitation led to an increase in diglucosyldiglyceride at the expense of the phospholipids, especially phosphatidylethanolamine (Minnikin et ul., 1972). Less striking quantitative changes were noted in studies of B . sirbtilis var. niger, in which phosphate limitation led to an increased ratio of cardiolipin to phosphatidylglycerol at pH 7.0. Mg2’ -limited cultures had relatively more phosphatidylglycerol than phosphate-limited cultures, and phosphatidylethanolamine and lysyl-phosphatidylglycerol were not seen at pH 8.0 (Minnikin and Abdolrahimzadeh, 1974b). Although there has been considerable work on the acyl chains of clostridia (Goldfine, 1964; Moss and Lewis, 1967; Chan et a / . , 1971), and on the plasmalogen content of the lipids (Baumann et ul., 1965; Kamio et ul., 1969), there has not been sufficient work on the intact lipids to derive a general picture. However, it is already clear that there will be differences in the phospholipid cornposition of members of this genus when such a picture emerges. Baumann et al. ( 1965) found both diacyl and plasmalogen forms of phosphatidylethanolamine phosphatidyl-N-methylethanolamine, and phosphatidylglycerol in C.

28

HOWARD GOLDFINE

hutyricum, and a second ether lipid type in this organism was later characterized as a glycerol acetal of the ethanolamine and N-methylethanolamine plasmalogens (see Section II,B,l) (Matsumoto er al., 1971; Khuller and Goldfine, 1974). Macfarlane (1962) found a more gram-positive-like lipid pattern of C. welchii (perfringens), which had principally aminoacyl phosphatidylglycerol , phosphatidylglycerol , and cardiolipin. Interestingly, the presence of phosphatidylserine synthetase and decarboxylase in addition to phosphatidylglycero-P-synthetase was recently reported to this organism (Carman and Wieczorek, 1980).The relationship of these enzymes to the lipid composition of this organism remains to be clarified.

P. Gram-Positive, Non-spore-Forming Rods The lactobacilli are especially rich in phosphatidylglycerol and cardiolipin. As determined by :p2P,labeling, the range for 10 species was phosphatidylglycerol, 55-83% of lipid P; and cardiolipin, 3-15%. Eight of ten species also had lysylphosphatidylglycerol, representing 3 to 32% of lipid P. L . plantarum (Fig. 7G), therefore, presents a typical pattern (Exterkate et al., 1971). In addition to phospholipids, lactobacilli also have galactosylglucosyldiglycerides (Shaw, 1975). It should also be noted that lactobacilli lipids contain the saturated, monounsaturated, and cyclopropane fatty acids more typical of gram-negative than gram-positive bacteria. The related B$dobacterium, which is now classified with the actinomycetes (Buchanan and Gibbons, 1974), differs from the lactobacilli in having much more cardiolipin, less phosphatidylglycerol, a galactosyldiglyceride with an sn-glycerol-1-P substituted sugar, and alanylphosphatidylglycerol (Exterkate et al., 1971; Veerkamp and van Shaik, 1974).

Q. Actinomycetes and Related Organisms 1 . CORYNEBACTERIA The corynebacteria are a large and apparently heterogeneous group of organisms whose taxonomy is still in a state of flux. Recent work on the fatty acids, phospholipids, and mycolic acids of these organisms has provided information of taxonomic importance. One group of organisms, of which C . diphtheriae is an example, has 52-60% GC in its DNA, meso-diaminopimelic acid in its peptidoglycans, corynomycolic acids, and saturated and monounsaturated straight-chain fatty acids in their extractable lipids. There is a high cardiolipin to phosphatidylglycerol ratio, and phosphatidylinositol dimannosides (PIM) among the extractable polar lipids. They may in addition have phosphatidylinositol and phosphatidylethanolamine (Komura et al., 1975b; Lechevalier, 1977). A second

PROKARYOTE LIPIDS: STRUCTURE AND DISTRIBUTION

29

group has 69-70% GC in its DNA, diaminobutyric acid in its peptidoglycan, no mycolic acids, and mainly unteiso-15:0, unteiso-l7:0, and iso-16:O fatty acids. The polar lipids are largely cardiolipin, phosphatidylglycerol, and one to three uncharacterized, probably mannose-containing glycolipids. The latter are not of the PTM type (Komura et a l . , 197Sb; Collins and Jones, 1980). This group contains several plant pathogens such as C. tritici, C. imnicurn, C . sepedonicum, and C . rnichigutierrse. A third group, which has 66-71% GC, and ornithine in its cell wall, has an even higher proportion of a n t e i x - and iso-branched fatty acids, and the same phospholipids and several uncharacterized glycolipids as in the second group (Collins et a / . , 1980). These authors have suggested that on the basis of their cell wall and lipid compositions, C . fklccumfaciens, C. poinsettiae, C . bctue, and C . oortii should probably be included in the genus Curtobucterium. 2.

A R T HROBAC rER A N D PROPlONlBACTERl UM

These organisms also have large amounts of branched-chain fatty acids in addition to straight-chain saturated fatty acids. Arthrobacter phospholipids, like those of the other actinomycete-related groups, consist of a high proportion of cardiolipin, less phosphatidylglycerol, PIM, and in some organisms, phosphatidylinositol (Komura et a / . , 197Sb; Lechevalier, 1977). Three species were shown to have both galactosyldiglyceride and mannosyldiglyceride by Shaw and Stead ( 1 971). In A . crystallopoietes the glycolipids represented approximately 25% of the total lipid. The lipids of propionibacteria have not been extensively studied. P . shermunii was found to contain phosphatidylglycerol, phosphatidylinositol, and a diacylinositol mannoside. The presence of PIM in these organisms has not been established (Shaw, 1975). An isolated report of the presence of substantial amounts of aldehydogenic lipids, presumably plasmalogens, in anaerobic propionibacteria (Kamio e t a / . , 1969) has not been confirmed, as yet. 3 . ACTINOMYCEI ALES u . Mycobucrerium. The cell wall of mycobacteria has been an object of intense interest for many years because of its hydrophobic nature and its presumed role in the pathogenicity of these organisms. Underlying the wall is a typical 8-nm-thick cytoplasmic membrane (Ratledge, 1976). Since the cell wall is complex in structure and contains a number of unusual lipids of high molecular weight, it has not been easy to define the components of the membrane, the object of our interest, and distinguish them from the wall lipids. It is known that certain of the complex lipids such as the mycolic acids (see Section I,C,2), are covalently linked to the peptidoglycan through an arabinogalactan in the wax D structure (Goren, 1972;

30

HOWARD GOLDFINE

Ratledge, 1976). The mycosides, which are either phenolic glycolipids (mycosides A and B) or peptidoglycolipids (mycosides C ) , are also thought to be located peripherally (Goren, 1972). The major phospholipid, cardiolipin, is thought to be localized principally in the cell membrane, whereas the PIM are mostly associated with the cell wall (Akamatsu er ul., 1966). In this study phosphatidylethanolamine was found to be only slightly enriched in the cytoplasmic membrane fractions relative to the cell wall fraction. It is possible that, as in the outer membranes of gram-negative cells, certain phospholipids are associated with glycolipids in a wall fraction of the mycobacteria. Unlike the mycolic acids, with their very long-chain fatty acids, the phospholipids of mycobacteria are rich in acyl chains of ordinary length with C,, and C,, saturated and monounsaturated chains predominating (Goren, 1972). In addition, tuberculostearic acid (10-methylstearic acid) is widely distributed among actinomycetes and related groups of organisms (Lechevalier, 1977). Mycobacteria are relatively rich in triglycerides, and longer-chain (CZoto C Z 6 fatty ) acids are found on the 3-position of glycerol in these lipids (Ratledge, 1976). h. Nocurdiu. Like corynebacteria and mycobacteria, nocardia have complex nocardomycolic acids, which have chains of intermediate length between those of the longer eumycolates of mycobacteria and the shorter corynomycolates (Lechevalier, 1977). Their phospholipids are also similar, with cardiolipin and PIM predominating, along with somewhat less phosphatidylethanolamine (Khuller, 1977; Trana e t a / . . 1980; Lechevalier, 1977). In addition, small amounts of :v2Pi-labeledphosphatidylglycerol were found in all species of nocardia examined by Komura et ul. (1975b). One species, N . coefiucu, contains phosphatidylcholine as a major phospholipid (Yano e t u f . , 1969; Khuller and Brennan, 1972). Acylated trehaloses (cord factor) have also been found in nocardia (Lechevalier, 1977), strengthening the relationship of this group of organisms to the mycobacteria and corynebacteria. c. Strepiompces. The lipids of Streptomyces were recently reviewed (Batrakov and Bergelson, 1978). As in the related actinomycetes, cardiolipin, phosphatidylethanolamine, and PIM are the major phospholipids (Batrakov and Bergelson, 1978). In some species cardiolipin predominates; however, in S. griseus, phosphatidylethanolamine is 30-40% of total phospholipid, depending on the age of the culture (Talwar and Khuller, 1977). In this study PIM increased from 14.5 to 24% of total phospholipids as the cultures aged. The streptomyces may also contain several unusual polar lipids, such as the finding in one species of a butane-2,3-diol analogue of phosphatidylglycerol, in which the 4-carbon analogue replaces the unacylated glycerol (Table 11). An ornithine lipid of the zwitterionic type (Fig. 3B) has been found in two Streptomyces species, in one of which it appears to replace phosphatidylethanolamine (Batrakov and Bergelson, 1978). These authors have shown that in Actinomyces (Streptomyces) olivaceus the ornithine lipid can replace phosphatidylethanolamine when the cells are

PROKARYOTE LIPIDS: STRUCTURE AND DISTRIBUTION

31

grown in phosphate-deficient media. Some streptomyces have lysine lipids that resemble the ornithine lipids with substituents at both the carboxyl and a-amino groupings. The ratio of ornithine to lysine lipids varies in S. sioyuensis (Kimura and Otsuka, 1969).

R. Rickettsia These obligate, intracellular parasites have a cell envelope that resembles that of gram-negative species in its morphology. In addition, they have muramic acid and diaminopimelic acid, which are characteristic of prokaryotic cell walls (Buchanan and Gibbons, 1974). Rickertsiu prowazeki grown in chicken embryo yolk sacs has a typical gram-negative phospholipid composition consisting of phosphatidylethanolamine (60-70%), phosphatidylglycerol (20%), and phosphatidylcholine ( 15%). Small amounts of phosphatidylserine and cardiolipin were also detected (Winkler and Miller, 1978). Since these authors believe that the phosphatidylcholine is host derived based on its :"P-specific activity in labeling experiments, some caution must be exercised in the evaluation of the ratios of the other phospholipids.

S. Mycoplasma The polar lipids of the cell wall-less Mycoplasrna resemble those of grampositive bacteria in their high concentration of phosphatidylglycerol, cardiolipin, and glycosyldiglycerides. Phosphoglycolipids (Section 111,N; Fig. 2 ) have also been identified (Razin, 1978; Smith, 1979).

IV.

PROKARYOTIC LIPIDS AND PHYLOGENY

The outlines of prokaryotic membrane lipid compositions have emerged with increasing clarity during the past decade. The organization of Section 111 was based on the current arrangement of bacteria in Bergey's Manual (Buchanan and Gibbons, 1974), which is divided on pragmatic grounds into 19 groups and does not indicate the relatedness of the various groups of prokaryotes. It is, therefore, of some interest to examine these bacterial lipid compositions in the light of current work on bacterial phylogeny. The recent summary of the work of Fox, Woese, Wolfe and their colleagues, which was previously scattered in a number of papers, presents the opportunity to make such a comparison (Fox et ul., 1980). This phylogeny has been constructed on the basis of an extensive examination of 16 S ribosomal RNA sequences. It departs in several important ways from traditional taxonomies. For example, cell shape is shown not to be a

32

HOWARD GOLDFINE

workable criterion for relatedness; most spherical bacteria are seen to fall into groupings defined by nonspherical organisms. Mycoplasma, which had been assigned a distinct division in earlier phylogenies, are considered by Fox et (11. ( I 980) to be wall-less offshoots of the clostridial branch (see below). There are other recent phylogenies based on such molecular characteristics of prokaryotes as various protein sequences, cell wall analyses, 5 S rRNA, and DNA-RNA hybridization. Fox e1 ul. (1980) state that their scheme is in reasonable agreement with most of them. As can be seen in Fig. 8, the 16 S rRNA data indicate that most present-day gram-negative organisms have descended from a common ancestral group of purple photosynthetic bacteria. From this trunk there are three major branches. One includes Purucoccus and Rhizobi~ctn along with the purple nonsulfur Rhodopseudotrionus species. An examination of the lipids of these organisms (Section III,B) reveals that in addition to the usual phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin of most gram-negative organisms, many organisms in this subline also have phosphatidylcholine, which is sometimes accompanied by an ornithine lipid. In another major branch derived from the purple bacteria are grouped the enteric organisms, the pseudomonads, and the vibrios along with the purple sulfur bacteria. Of the two sublines of this branch, the enteric line has organisms that generally have the simplest gram-negative polar lipid composition consisting of phosphatidylethanolamine plus polyglycerolphosphatides, whereas the other major subline, which contains the pseudomonads and acinetobacter, includes a few organisms that have a small proportion of phosphatidylcholine or zwitterionic ornithine lipids in addition to the common gram-negative phospholipids. Descr@vibrio, a separate branch, contains species with the simplest gramnegative lipid pattern and one species, D. gigus, that has a large amount of a zwitterionic ornithine lipid. Thus we see that all the gram-negative bacteria presumably derived from purple bacterial ancestors have a common pattern of phosphatidylethanolamine plus polyglycerolphosphatides, to which have accreted in some branches and sub-branches the enzymatic capacities to N-methylate phosphatidylethanolamine and to form the ornithine lipids. According to Fox et (11. (1980) the cyanobacteria (blue-green algae) and the green sulfur bacteria form separate evolutionary groups, and their chloroplastlike lipid compositions reflect this. The major extractable polar lipids are galactosyldigl yceride, pol ygl ycerolphosphatides , and SQDG . Phosphatidylethanolamine and phosphatidylcholine have not been found in these organisms. The spirochaetes are also considered to be in a separate group. As noted above (Section III,E), the lipids of the Spirochaeta are similar to those of gram-positive bacteria. They contain polyglycerol phosphatides, glycosyldiglycerides, and no ethanolamine phosphatides. The Treponernu, which do not appear in this scheme, have both ethanolamine and choline phosphatides along with glycosyl-

33

PROKARYOTE LIPIDS: STRUCTURE AND DISTRIBUTION

Thcrmophilcr

1

ancestral state

FIG. R . The phylogeny of prokaryotes according to Fox et ul. (1980). Copyright 1980 by the American Association for the Advancement of Science.

diglycerides, and may, therefore, be more closely related to grani-negative organisms. Lcptospiru, as noted by Fox et u / . (1980), do not cluster with Spirochuetu, on the basis of their 16 S rRNA. They also differ in their lipid composition, which is much like that of gram-negative organisms (Fig. 5D, Table 111). The other major group of eubacteria in the scheme of Fox rt ul. (1980) includes all gram-positive eubacteria except for a sniall group of cocci. From this gram-positive trunk two major branches emerge. One includes the “actinomyces” group, which gives rise to one line that includes the arthrobacter group, a second that includes the Cor?.tiehacteriurn -M~cohuc.trriurii-Noc~urdiu (CMN) group, and a third, the streptomyces group. We have seen (Section JIJ,Q) how similar the extractable lipids of these three major groups of organisms are. They generally have a high proportion of cardiolipin and PIM. They may also have phosphatidylinositol in small amounts. Phosphatidylethanolamine appears in the mycobacteria, nocardia, and streptomyces. The CMN group is also distinguished by the presence of their unique mycolic acids.

34

HOWARD GOLDFINE

Another major branch of the gram-positive trunk is the ancestral “clostridial” line, which gives rise to one branch including both the genus Bacillus and the lactobacilli. The “intermediate” character of the lipids of the genus Bacillus was noted in Section 111,O. In composition they lie between the major groups of gram-negative and gram-positive organisms. In most of these organisms, there is a high proportion of both phosphatidylethanolamine and the polyglycerolphosphatides, characteristically there are diglucosyldiglycerides, and in some species either aminoacyl- or glucosaminyl-phosphatidylglycerol.It should be noted that Fox et al. ( 1980) also consider Staphylococcus epidermidis and Streptococcus luctis as close relatives of Bacillus. Streptococci generally have mainly polyglycerolphosphatides and glycosyldiglycerides, and in this regard resemble the lactobacilli. They also do not have the branched-chain fatty acids characteristic of Bacillus and of staphylococci. Both the bacilli and clostridia are relatively “deep” genera in that the association coefficients of the 16 S rRNA sequences indicate considerable evolutionary distances (Fox er al., 1980). The relatively incomplete information we currently have on the lipids of the clostridia similarly indicate considerable divergencies. Fox et al. (1980) consider the mycoplasma to be a subgroup of clostridia. Their gram-positive lipid composition, consisting largely of polyglycerolphosphatides plus glycolipids (Razin, 1978), is consistent with this grouping. The presence of large amounts of sterols in many mycoplasma is discussed by Razin (this volume). The “archaebacteria, ” originally separated from the main lines of bacterial descent by Woese, Fox, Wolfe, and their colleagues (Fox et al., 1980) on the basis of their cell walls, which contain no muramic acid; their tRNAs, which differ in the thymine, pseudouridine, cytidine loop; their distinctive RNA polymerases; and in the presence of several unusual coenzymes, also have unusual phospholipids. These are described by Langworthy (this volume). Thus, for all major prokaryotic lines of descent, membrane lipid compositions agree well with the phylogenetic tree proposed by Fox et al. (1980). The placement of the bacteroids with their unusual sphingolipids and the anaerobic gramnegative organisms with their alk-1-enyl acyl lipids, in this evolutionary scheme, will be of considerable future interest.

V. CONCLUSIONS The diversity of prokaryotic lipids stands as further testimony to the great age of this group of organisms. Similar conclusions have come forth from studies of other cellular macromolecules (see Fox et a l . , 1980, for references), the geological record, and from a consideration of biosynthetic pathways (Goldfine and Bloch, 1963). At the present time, it is more difficult to relate prokaryotic lipid composition

PROKARYOTE LIPIDS: STRUCTURE AND DISTRIBUTION

35

and membrane function. The presence of acyl and alk-I-enyl chains containing double bonds, methyl branches, or small rings is critical for the maintenance of membrane fluidity (Melchior, this volume), which in turn is important for the function of membrane enzymes and transport systems. Variation in polar head groups may also affect membrane fluidity and the association of certain lipid classes with these enzymes and transport systems, and in some cases the specificity of these systems for a particular lipid class may be of equal significance (McElhaney , this volume). In this chapter the location of membrane lipids with respect to intracellular membranes has been discussed briefly. It is now becoming evident that lipids are often distributed asymmetrically in the two leaflets of a given membrane. This topology is described in detail by Rottem (this volume). As organisms evolved, the need for elaboration of the cytoplasmic membranes into specialized intracytoplasmic membranes containing increased surface area for photoreception or greater electron-transport capacity may have required the evolution of lipids that permit or facilitate the required membrane infolding and pinching off. We have previously discussed the much greater frequency of occurrence of phosphatidylcholine in organisms with complex intracytoplasmic membranes (Hagen et al., 1966). A group of organisms that provides an exception to this correlation is the Rhizobiaceae (Section III,G,3). These organisms interact with their host plant cells in order to form tumors or root nodules. It is tempting to speculate that the evolution of the phosphatidylethanolamine methylation pathway in this group has served to promote these bacteria-host interactions. Goren (1977) has reviewed the evidence that the sulfatides of mycobacteria (Section II,C,2) aid in the intracellular growth of pathogenic strains by interfering with phagolysosome formation. As noted in Section 111, some organisms have evolved dual lipid-biosynthetic capacities, which allow them to substitute glycosyldiglycerides or ornithine lipids for phosphatidylethanolamine in the absence of phosphate. It is clear that within limits of size and charge, polar lipids may replace one another in biological membranes. Exogenous fatty acyl chains and alk-1-enyl chains may also be substituted for the naturally occurring chains in bacterial auxotrophs (chapters by Melchior and McElhaney, this volume). Much more work on membrane mutants will be needed before a more complete understanding of the multifaceted roles of prokaryotic lipids can be attained. Work on prokaryotic organisms has not only provided an abundance of new insights into their membrane structure and functions, it is also leading the way to a more complete understanding of eukaryotic cell membranes. ACKNOWLEDGMENTS

I should like to expresb my appreciation to Dr. G. P. Hazlewood for the use of material prior to publication, and to Roseann Femia for able assistance in the preparation of this manuscript.

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Clarke, N. G., Hazlewood, G . P., and Dawson, R. M. C. (1976). Novel lipids of Butyrivibrio spp. Chem. Phys. Lipids 17, 222-232. Clarke, N . G., Hazlewood, G. P . , and Dawson, R. M. C. (1980). Structure of diabolic acidcontaining phospholipids isolated from Butyrivibrio Sp. Biochem. J . 191, 561-569. Collins, M. D., and Jones, D. (1980). Lipids in the classification and identification of coryneform bacteria containing peptidoglycan based on 2,4-diaminobutyric acid. J . Appl. Bacteriol. 48, 459--470. Collins, M . D., Goodfellow, M., and Minnikin, D. E. (1980). Fatty acid, isoprenoid quinone, and polar lipid composition in the classification of Curtohacreriurn and related taxa. J . Gen. Microhiol. 118, 29-37. Contreras, I . , Shapiro, L., and Henry, S . ( 1 978). Membrane phospholipid composition of Caulohacter crcsceiztus. J . Bucteriol. 135, 1130-1 136. Dawson, R. M. C., Hemington, N., and Davenport, J . B. (1962). Improvements in the method of determining individual phospholipids in a complex mixture by successive chemical hydrolyses. Biochem. J . 84, 497-501. Depinto, J . A. (1967). Omithine-containing lipid in Rhodospirillum rubrum. Biochim. Biophys. Actu 144, 113-117. DeSiervo, A. J . , and Homola, A. D. (1980). Analysis of Cuulubacter crescenrus lipids. J . Bucteriol. 143, 1215-1222. Diedrich, D. L., and Cota-Robles, E. H. (1974). Heterogeneity in lipid composition of the outer membrane and cytoplasmic membrane of Pseutlomonas BAL-31. J . Barterid. 119, 10061018. Exterkate, F. A , . Otten, B. J . , Wassenberg, H. W . , and Veerkamp, J. H. (1971). Comparison ofthe phospholipid composition of Bifidohurrerium and Lactobacillus strains. J . Bucteriol. 106, 824-829. Faizova, G. K., Borodulina, Y . S . , and Samsonova, S . P. (1971). Lipids in nodular bacteria (Rhizohium leguminosurum). Microbiology ( E n g l . Trunsl.) 40, 41 1-413. Fischer, W . , Koch, H. U . , Rosel, P. Fiedler, F., and Schmuck, L. (1980). Structural requirementsof lipoteichoic acid carrier for recognition by the poly (ribitol phosphate) polymerase from Stuphy/ococcus uureus H. A study of various lipoteichoic acids, derivatives, and related compounds. J . B i d . Chem. 255, 4550-4556. Fox, G. E . , Stackebrandt, E . . Hespell. R. B., Gibson, J.. Maniloff, J . , Dyer, T. A., Wolfe. R . S . , Balch, W. E., Tanner, R . S . , Magrum, L. J . , Zablem, L. B., Blakemore, R., Gupta, R., Bonen, L., Lewis, B. J . , Stahl, D. A . , Luehrsen. K . R., Chen, K. N., and Woese, C. R . (1980). The phylogeny of prokaryotes. Science 209,457-463. Fritsche, D . , and Thelen, A. (1973). Die Abgrenzung der Genera Bacteroides and Sphaerophorus auf komplexen Lipoide. Zentrulhl. Bakteriol., Hyg. Parusitenkd. Infectionkr. Abt. I : Orig., Reihr A 223, 356-365. Fulco, A. J. (1974). Metabolic alterations of fatty acids. Annu. Rev. Biochem. 43, 215-241. Gerson, T . , and Patcl, J . J . (1975). Neutral lipids and phospholipids of free-living and bacteroid forms of two strains of Rhiiohirrm infective on Lorus peduneularus. A p p l . Microbid. 30, 193- 198. Godchaux, W . , 111, and Leadbetter, E. R . (1980). Capnocytophaga spp. contain sulfano-lipids that are novel in procaryotes. J . Barterid. 144, 592-602. Godchaux, W . , I l l , and Leadbetter, E. R. (1981). Sulfonolipids of gliding bacteria: Structure of N-acylcapnine. Fed. Proc.. Fed. A m . So(,. E x p . B i d . 40, 1845. Goldberg, I . , and Jensen, A . P. (1977). Phospholipid and fatty acid composition of methanolutilizing bacteria. J . Bacteriol. 130, 535-537. Goldfine, H. ( 1964). Composition of the aldehydes of Clostridium butyricum plasmalogens. Cyclopropane aldehydes. J . B i d . Chem. 239, 2130-2134. I

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Nichols, B . W., Harris, R. V . , and James, A. T. (1965). The lipid metabolism of blue-green algae. Biochem. Biophys. Res. Comtiruti. 20, 256-262. O’Leary, W. M. (1973). Lipoidal contents of specific microorganisms. I n “Handbook of Microbiology” (A. I. Laskin and H. A. Lechevalier, eds.), Val. 11, pp. 275-327. CRC Press, Cleveland, Ohio. Oliver, J . D., and Colwcll, R. K. (1973). Extractable lipids of‘ gram-negative marine bacteria: Phospholipid composition. J . Buctrriol. 114, 897-908. Omdorff, P. E., and Dworkin. M . (1980). Separation and properties of the cytoplasmic and outer membranes of vegatative cells of M~xococcrrsxcrirfhus. J . Buctrriol. 141, 914-927. Osborn, M. J . , Gander, J . E . , Parisi, E . , and Carson, J . (1972). Mechanism of assembly of the outer membrane of Salmonellu tvphimurium. J . B i d . Chrm. 247, 3962-3972. Patt, T . E . , and Hanson, R. S. (1978). lntracytoplasmic membrane, phospholipid and sterol content of Methylobac.ierium or,qatii~phi~rrrn cells grown tinder different conditions. J. Bac,frriol. 134, 636-644. Pieringer, R . A., and Ganfield, M.-C. W. (1975). Phosphatidylkojibiosyl diglyceride: Metabolisin and function as an anchor in bacterial cell membranes. Lipids 10, 421-426. Powell, D. A., Duckworth, M., and Baddiley, J . (1975). A membrane-associated lipomannan in micrococci. Biochem. J . 151, 387-397. Qureshi, N., Takayama, K . , Jordi, H. C . , and Schnoes, H. K . (1978). Characterization of the purifieti components of a new homologous series of a-mycolic acids from Mycvbacterium trrhercrrlosis H37Ra. J . Bin/. Chem. 253, 541 1-5417. Randle, C. L . , Albro, P. W . , and Dittmer, J. C. (1969). The phospholipid composition of gramnegative bacteria and the changes in composition during growth. Biochim. Biophys. Acta 187, 214-220. Ratledge, C. (1976). The physiology of the mycobacteria. Adv. Microb. Physiol. 13, 115-234. Razin, S. (1978). The mycoplasmas. Microbiol. Rev. 42, 414-470. Reusch. R. N . , and Sadoff, H. L. (1979). 5-n-Alkylresorcinols from encysting Azotobacter vinelundii: Isolation and characterization. J . Bactrriol. 144, 448-453 Reusch, R. N . , and Sadoff. H. L. (1981). Unique lipids in membranes of Aiofnhacter virielundii cysts. Absfr., Annu. Meer. Am. Soc. Micruhiol. p. 166. Rizza, V . , Tucker, A. N., and White, D. C. (1970). Lipids of Bacteruides meluninogenicus. J . Bucferiol. 101, 84-91. Rottem, S . , Hasin, M . , and Razin, S. (1975). The outer membrane of Proteus mirubilis. 11. The extractable lipid fraction and electron paramagnetic resonance analysis of the outer and cytoplasmic membranes. Biochim. Biophy. Acra 375, 395-405. Russell, N. J . , and Harwood, J . L. (1979). Changes in the acyl lipid composition of photosynthetic bacteria grown under photosynthetic and non-photosynthetic conditions. Biochem. J . 181, 339-345. Schmidt, M . F. G . , Brancha, M . , and Schlesinger, M. J . (1979). Evidence for covalent attachment of fatty acids to Sindbis Virus glycoproteins. Proc. Natl. Acad. Sci. U.S.A. 76, 1687-1691, Senff, L. M., Wegener, W . S., Brooks, G. F., Finnerty, W. R . , and Makula, R . A. (1976). Phospholipid composition and phospholipase A activity o f Neisseriu gonorrhoeae. J . Bacteriol. 127, 874-880. Shaw, N. (1974). Lipid composition as a guide to the classification of bacteria. Adv. Appl. Microbiol. 17, 63-108. Shaw, N. (1975). Bacterial glycolipids and gtycophospholipids. Adv. Microb. Physiot. 12, 141- 167. Shaw, N . , and Stead, D. (1971). Lipid composition of some species of Arthrobacter. J . Bacreriol. 107, 130-133. Shively, J . M., and Benson, A. A. (1967). Phospholipids of Thiobacillus thiooxidans. J . Bacferiol. 94, 1679-1683.

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Short, S . A.. White. D. C . , and Aleem, M. I . H. (1969). Phospholipid inetabolisni in Ferrohuci//u.\ /erromiduti.\. J . Buctcriol. 99, 142- I 50. Smith, P. F. (1979). The composition of membrane lipids and lipopolysaccharides. I n "The Mycoplasmas" (M. F. Barile and S . Razin. eds.), Vol. I , pp. 23 1-259. Academic Press. New York. Steck, P. A . , Schwartz. €3 A., Rosendahl, M. S . , and Gray. G. R . (1978). Mycolic acids. A reinvestigation. J . Biol. Chem. 253, 5625-5629. Steiner, S . , Sojka, C. A,, Conti. S . F., Geat, H., and Lester, R . L. (1970). Modification of membrane composition in growing photosynthetic bacteria. Biochirn. Biophys. Actu 203, 571 -574 Stoffel, W., Dittmar, K . , and Wilmes, R. (197% Sphingolipid metabolism in Bocteroideacear. HO{J{Jr-Sry/u,-'s z. Phxsiol. Chem. 356, 71 5-725. Sud. I. J . , and Feingold, D. S . (1975). Phospholipids and fatty acids of NtJissrritr gonorrhorue. J . Bucteriol. 124, 713-717. Sutrina, S . L.. and Scocca, J . J . (1976). Phospholipids of Harmophilus influrnzar Rd. during exponential growth and following the development of competence lor genetic transformation. J . & t i . Mictohrol. 92, 410-412. Tahara. Y . , Yamada, Y ., and Kondo, K . (1976). Phospholipid composition of G/uc.oriohncrur crrirrriA. A , y r w B i d . Chr/rt. 40, 2355-2360. 'l'akacs, B. J . . and Holt, S . C. (1971). Thioctrpscr /7orit/crnu; a cytological physical, and chemical characterization. I I . Physical and chemical characteristics of isolated and reconstituted chrornatophores. Biochim. Biophys. Actu 233, 278-295. Talwar, P., and Khuller. G. K . (1977). Effect of age on the major phospholipids of Streptotnwrs griseu.i. fndiun J . Bioc.hcw. Biophys. 14, 85-86. Thiele, 0. W., and Schwinn, G. (1973). The free lipids of Brucellu wic/ireii.sis and Bor-&rr//u /irr/rcssis. E u r . J . Bioc,hewt. 34, 333-344. Thomas. T . D., and Ellar, D. J. (1973). Properties of plasma and mesosomal membranes isolated from Microcacws /ysodrikticrts: Rates of synthesis and characterization of lipids Biochirn. Biophyr. Acru 316, 180-195. Thome. K . J . I . , Thornley. M. J . , and Glauert, A. M. (1973). Chemical analysis of the outer membrane and other layers of the cell envelope of Acirrerobuctrr sp. J . Bncteriol. 116, 41 0-41 7. Tornabene, T. C. (1973). Lipid composition of selected strains of Yersiriicr pestis and Yersinitr p,irudor~rhei-ert/oSiS.Biochirn. B i o p h w . Acttr 306, I 73- 185. Tornabene, T. G.. and Ogg. J . E. (1971). Chromatographic studies of the lipid components of Vihrio ,fetus. Biochir~t.Biophys. Actu 239, 133-141. Tornahene, T. G., Morrison, S. J . , and Kloos, W. E. (1970). Aliphatic hydrocarbon contents of various members of the family Micrococraceue. Lipids 5, 929-937. Trana, A. K . , Khuller, G. K . , and Subrahmanyam, D. (1980). Metabolism of phospholipids in Nocardia pcJ/yc~hmino~ene.r. J . Gen. Microhid. 116, 89-92. van Golde, L. M. G . , Akkermans-Kryawijk, J . , Franklin-Klein, W . , Lankhorst, A , , and Prins, R. A . (1975). Accumulation of phosphatidylserine in strictly anaerobic lactate fermenting bacteria. FEES Lett 53, 57-60. Veerkamp, J . H.. and vim Sheik, F. W . (1974). Biochemical changes in Brfidohuc,rcriurn hifidus var. penn.r$i~trnicits after cell wall inhibition. VII. Structure of the galactosyldiglycerides. Biochirr?. B i o p h n . Actu 348, 370-387. Verkley, A. J., Ververgaert, P. H. J. T., Prins, R. A., and van Golde, L. M. G. (1975). Lipid-phase transitions of the strictly anaerobic bacteria Veillonclla parvula and Anaeroiihrio ripolytica. J . B u c t ~ r i ~124, l . 1522-1528. Vorheck. M. L., and Marinetti, G. V . (1965). Intracellular distribution and characterization of the lipids of Srreptocuccit.s fiec~ulis (ATCC 9790). Biochemistry 4, 296-305.

PROKARYOTE LIPIDS: STRUCTURE AND DISTRIBUTION

43

Weaver. T . L., Patrick. M. A , . and Dugan, P. R. (1975). Whole-cell and membrane lipids o f t h e methylotrophic bacterium Mrt/?\/o.siurc.\ tri[./ro.s/’~~riri,,r. J . Hrrcter-fol. 124, 602-605 White. D. A , , Lennari, W . J . , and Schnaitman, C . A . (1971). Distribution of lipids in the wall and cytoplasmic mernbrane subfractions of the cell envelope of E.sc/rrric,/rftr< d . J . Bocrcriol. 109, 686-690. White, D. C., Tucker, A. N . , and Sweeley. C. C . (1969). Characterization of the iso-branched sphinganines IroiTi the ceramide phospholipids of Buc~teroit/~.s f ~ f ~ , / ~ f J f ; r r ~ ~ ~ ~ , Jh’ioc,/i;r?i. ii(,i~.\. Bio/)/r,ys. Actti 187, 527 532 Whiteside, 7 L., deSiervo, A . J . , and Salton, M . R . (1971). Use of antibody to membrane adenosine triphosphalase in the study of bacterial relatiomhipa. J . Bucterrd. 105, 957-967. Wilkinson. S . G . (1970).Cell walk of P.\c’rrc/oJ,?o,rtr.s species sensitive to ethylenediaminetetraacelic acid. J . Bercteriol. 104, 1035-1044. Wilkinson, S . G . , and Galbraith, L. ( l Y 7 9 ) . Polar lipid of P.\rtrc/orrrori~rsi,r.sic.rt/uri.s.Presence of a ~i. Actu 575, 244-254. heptosyldiacylglycerol. B i o ~ h i ~Riophys. Wilkinson. S . G . , Galbraith, L . , and Lightfool, G . A . (1973). Cell walls. lipids, and lipopolysaccharides of P.~rrrc/o~rrr~~ro.s species. E i ~ r ../. H r o c h P r , r . 33, 158- 174 Winkler, H. H., and Miller, E. T. (197X). Phospholipid composition of Rtrkmrici prowcreki grown i n chicken embryo yolk sacs. J . Buc.rr,-io/. 136, 175-178. Woese. C. R . . Magruin. 1.. J . . and Fox, G . E. (1978). Archaebacteria. J . Mn/. E i d . 11. 245-252. Yano, I . , Furukawa, Y ., and Kuaunose. M. (1969). Phospholipids of Noc.urdiu w e / i c i ~ ~Ju. . Beic.teriol. 98, 124- 130.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 17

Lipids of Bacteria Living in Extreme Environments THOMAS A . LANGWORTHY Department of Microbiologv School qf Medicine Uriiversit~of Sourh Dakota Vermrllion. South Dakoro

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Organisms and Environments . . . . . . . . . . . . . . . . . . . . . I1 . Apolar Residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . FattyAcids . . . . . . . . . . . . . . . . . . . . . . . . . . . . B . Isopranyl Glycerol Ethers . . . . . . . . . . . . . . . . . . . . . . 111. Neutral Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . Isoprenoid Derivatives . . . . . . . . . . . . . . . . . . . . . . . B . Other Neutral Lipid Components . . . . . . . . . . . . . . . . . . . IV . Glycolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . Glycosyldiacylglycerols . . . . . . . . . . . . . . . . . . . . . . . B . Tetrahydroxyhacteriohopane Glycosides . . . . . . . . . . . . . . . . C . Isopranyl Glycerol Ether Glycosides . . . . . . . . . . . . . . . . . . D . Other Polar Lipids . . . . . . . . . . . . . . . . . . . . . . . . . V . Acidic Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . Phospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . B . Phosphoglycolipids . . . . . . . . . . . . . . . . . . . . . . . . . C . Sulfolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . O v e r v i e w . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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45 46 49 49 51

56 56 62 62 62 63 64 65 66 66 67 68 69 70

INTRODUCTION

The isolation of new and metabolically diverse species of bacteria during the past decade has renewed a fundamental interest in the ecology. biogeochemistry. physiology. and evolution of bacteria from extreme environments (Heinrich. 1976; Brock. 1978; Brierley. 1978; Kushner. 1978a; Shilo. 1979). Considerable 45

Copyrlghf @ 1982 by Academic Press. Inc All right5 of reproduction in any form rmerved ISBN 0-12-153317-4

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interest has developed in the membrane structure of these bacteria, since their membranes must carry out normal physiological processes, yet must intercede against such hostile environmental parameters as extreme temperatures, pH, salinity, radiation, pressure, and dryness. To be sure, the lipids of a vast number of bacteria from extreme environments have not been investigated. Yet it is becoming clear that although the membrane lipids of many of the bacteria thus far examined are quite ordinary, the lipid structures of some of these organisms-notably the isopranyl ether lipids of thermoacidophilic, halophilic, and methanogenic bacteria-are not only unusual, but are changing our perceptions of the supramolecular lipid bilayer model as the universal membrane lipid matrix (Langworthy, 1Y77a,b, 1979a,b). These lipids are additionally promoting our understanding of biochemical, biogeochemical, and cellular evolution (Rohmer et ul., 1979; Tornabene et ul., 1979; Holzer et ul., 1979; Fox et ul., 1980). In view of the variable information between different groups of organisms, this article is necessarily incomplete and can only provide in broad outline the nature of the membrane lipids of bacteria from extreme environments. It is the intent of this article to present a descriptive summary of the more unusual lipid structures from obligately thermophilic, psychrophilic, acidophilic, thermoacidophilic, halophilic, and methanogenic bacteria.

The Organisms and Environments Microbial populations in naturally occurring extreme environments are quite limited, yet a fairly large number and physiological variety of bacteria have been isolated. Although a considerable number of species are able to survive or tolerate exposure to extreme environmental parameters, this article is restricted to the lipids of those bacteria that have an obligatory requirement for their extreme or unusual condition. A brief description follows of the major genera of bacteria in which some aspects of the lipids have been investigated. Thermophilic bacteria have been isolated from such natural habitats as volcanic regions, geothermal soils, and hot springs where temperatures may reach 90"C, as well as from mining waste dumps and soil (Brock, 1978; Tansey and Brock, 1978; Castenholz, 1979; Zeikus, 1979). Moderate thermophiles that have been examined include spore-forming, gram-positive, aerobic Bucillus species, principally B . steurotheniiophilus, which grow optimally at 50-65°C and between 37 and 70°C (Allen, 1953) and several anaerobic Clostridium species, with optimum growth including C. turturivorurn and C. thcrmosarchurol~tici~~n, at 55°C (Chan et ul., 1971). Extreme thermophiles include the Bucillus species B . culdolyticus, B . culdovelox, and B. culdotenux, which grow between 70 and 85°C (Heinen and Heinen, 1972). Members of the genus Thermus, which are

LIPIDS OF BACTERIA IN EXTREME ENVIRONMENTS

47

gram-negative, non-spore-forming rods, include T . uquuricus (Brock and Freeze, 1969) and T . jlaviis (Saiki et ul., 1972), which grow between 40 and 80°C with an optimum at 70°C, whereas T . thermophilus (Oshima and Imahori, 1974) grows between 47 and 85°C and has an optimum at 75°C. Some species of Thermovnicrobiurn (Jackson et ul., 1973; Phillips and Perry, 1976) have also been investigated. These are gram-negative, pleomorphic rods growing between 45 and 85°C and optimally at 60-70°C. Methanobacterium thermouutotrophirum is an example of an obligately anaerobic, thermophilic methanogen that grows between 40 and 75°C with an optimum at 70°C (Zeikus and Wolfe, 1972). Several recent reviews, in addition to those just mentioned, concerning the biochemistry of thermophily and including aspects of thennophile lipids have appeared (Zuber, 1976: Friedman, 1978; Amelunxen and Murdock, 1978; Ljungdahl, 1979). Psychrophilic bacteria populate polar regions and arctic waters where temperatures approach 0°C (Morita, 1975; Baross and Morita, 1978; lnniss and Ingraham, 1978). The lipids of some psychrotrophic bacteria, which will grow below 20°C but have growth optima at higher temperatures, have been examined (Cullen er ul., 1971; Gill and Suisted, 1978). Studies on truly psychrophilic bacteria, which grow only between 0 and 20"C, have been limited primarily to several psychrophilic marine pseudomonads (Brown and Minnikin, 1973), and Vibrio sp. (Bhakoo and Herbert, 1979), Serrutiu sp. (Kates and Hagen, 1964), anaerobic clostridia (Sinclar and Stokes, I964), and Micrococcus cqophitus (Russell, 1971 ). Acidophilic bacteria, represented by the chemolithotrophic Thiobaciilus, are found associated with acidic regions such as acid mine drainage and acidic mining waste dumps (Lundgren et ul., 1964, 1974; Langworthy, 1978a; Tuovinen and Kelly, 1978). The lipids of two species that have been investigated in some detail include T . thiooxicluns and T .jierrooxickrns, which grow optimally at pH 2 and between pH 1 and 4. They are obligately autotrophic, non-sporeforming, gram-negative rods that obtain energy from the oxidation of iron and sulfur with the concomitant production of sulfuric acid. Several thermophilic Thiohuc.illcis species have been reported (Brierley, 1978; Brock, 1978), as well as alkalophilic Bacillus species (Langworthy, 1978a), which grow optimally at pH 10, but the lipids of these organisms have not been detailed. Thermoacidophilic bacteria have been isolated from acid hot springs, solfotara soils, and self-heating coal refuse piles where temperatures and acidity range from 55 to 85°C and pH 1-3. These organisms must thus contend with high temperature and low pH simultaneously. Thermoacidophiles are composed of three morphologically and physiologically distinct types. Bucillus ucidocdduriiis is a spore-forming rod that grows within the range of 40-70°C and pH 2-6 and optimally at 60-65°C and pH 3 (Darland and Brock, 1971). Sulfolobiis ucio'oculrlurius, a facultative autotroph capable of growth on sulfur and iron,

48

THOMAS A. LANGWORTHY

possesses an atypical cell wall and grows within the limits of 55-85°C and pH 2-5 and optimally at 75°C and pH 3 depending on strains (Brock et ul., 1972; de Rosa et al., 1975a). Perhaps the most unusual organism is Thermuplusmu acidophilurn, a wall-less mycoplasma, whose membrane is directly exposed to its hot acid environment (Belly et ul., 1973; Langworthy, 1979a). It grows within the limits of40-62°C and pH 1-4 and optimally at 59°C and pH 2. Additionally, hydrogen ions are specifically required for maintaining cellular integrity, as Thrrrnopla.smu is lysed by neutrality (Smith et d., 1973). Both 7hermoplusmu and Su/fo/obus are characterized by the possession of isopranyl ether lipids. The lipids of thermoacidophilic bacteria have been the subject of several recent reviews (Langworthy 1978b, 1979b, 1980a, 1981). Halophilic bacteria inhabit solar salt flats, brine, and hypersaline lakes where salt concentrations approach saturation (Dundas, 1977; Bayley and Morton, 1978; Kushner, 1978b; Lanyi, 1979). The extreme halophiles comprise the rodshaped Hulobacteriuni species H. cutirubrum, H . Izulobium, H . salinarium, and H . rnurisrnortui, as well as the coccal forms Surcinu litoralis and S. morrhuue. These organisms grow optimally in 20-25% NaCl and between salt concentrations of 15 and 3 0 8 . Like Sulfolobus, the halophiles possess an atypical cell wall structure. Like Thermoplasmu, which requires protons, halophiles require sodium ions for structural integrity, being lysed by low salt concentrations. In addition, the discovery (Oesterhelt and Stoeckenius, 1973) that H . halobium contains bacteriorhodopsin, a photosensitive purple pigment that converts light to chemical energy, has generated considerable interest in the bioenergetics of halophilic bacteria (Caplan and Ginzburg, 1978). The halophilic bacteria possess isopranyl ether lipids, which have been the most fully established of any of the bacteria from extreme environments, principally by Kates and associates. The chemistry of these lipids has been extensively reviewed by Kates (1972, 1978), Kates and Kushwaha (1976), and Kates and Kushwaha (1978). Methanogenic bacteria are strictly anaerobic organisms whose metabolism is based on the formation of methane from carbon dioxide and hydrogen, formate, acetate, or ethanol. They are found in sewage, bogs, and sediments, and they comprise a variety of Methunobacteriurn, Methanosarcina, Methunospiritlum, and Merhanococcus species (Zeikus, 1977; Balch et al., 1979). They, too, possess an atypical cell wall structure and isopranyl ether lipids (Tornabene and Langworthy, 1979). Although they live in more of an unusual than an extreme environment, the methanogenic bacteria are included in this review because of the recent realization of the close phyletic relationship between Thermoplasma, Sulfolobus, halophiles, and methanogens. Based on 16 S rRNA sequence analyses, the presence of ether lipids, and the absence of typical cell walls, Woese and associates have proposed that this group of bacteria be given the name Archaebacteria. According to their view this group represents a line of evolutionary

49

LIPIDS OF BACTERIA IN EXTREME ENVIRONMENTS

descent different from either prokaryotic or eukaryotic cells (Woese and Fox, 1977; Woese et al., 1978; Fox et al., 1980).

II. APOLAR RESIDUES

A. Fatty Acids Influence of temperature on the physical state of membrane lipids has focused considerable attention on the fatty acyl moieties of the acylglycerol residues in thermophilic and psychrophilic bacteria. In general, the fatty acid profiles extend the trends that are reflected in mesophilic bacteria, such as Escherichia coli, which have been subjected to short-term shifts in temperature (Marr and Ingraham, 1962). Psychrophiles are characterized by high proportions of unsaturated and shorter saturated fatty acids, whereas thermophiles possess a high content of longer saturated and predominantly isobranched acids. This trend is in general concert with the anhydrous melting points of the fatty acids, although the actual melting points of the ester-linked acids in the membrane are certainly influenced by cooperative interactions with other molecules. Examination of extremely thermophilic Thermus species reveal that isoC,, fatty acid is most abundant (50-61%), followed by isoc,,, and together the isoC,, and isoC,, pair accounts for about 7 5 4 5 % of the fatty acid content (Heinen et al., 1970; Ray et a l . , 1971a; Oshima and Miyagawa, 1974; Oshima, 1978). Oshima and Miyagawa (1974) found that the ratio of isoC,, to isoC,, acids increased in T . thermophilus grown at increasing temperatures from 49 to 82"C, indicative of chain elongation at higher temperatures. Ray el al. (1971a), however, observed a slight decrease in isoC and concomitant increase in the nC,, and isoC,, fatty acid content with increasing temperatures of T . aquaticus grown between 50 and 75°C. In the extreme thermophiles B . caldotenax, B . caldovelox, and B . caldolyticus, branched fatty acids represent about 80% of the total, consisting mainly of isoc,,, isoC,,, and isoC,, acids. A pronounced shift from isoC,, to isoC,,, and also isoC,, to nC,,, was demonstrated on increasing growth temperatures from 45 to 80°C (Heinen and Heinen, 1972; Weerkamp and Heinen, 1972; Hasegawa er a / . , 1980). The fatty acids of several hydrocarbonutilizing thermophiles, apparently closely related to species of Thermomicrobium, were examined by Merkel and Perry (1977). The fatty acid distribution varied depending on growth substrate, but consisted mostly of CIS-,c16-7 and C,,-branched and nC,, fatty acids. The branched acids were reported to have the anteiso rather than the iso configuration. The most dramatic change resulted after growth on n-heptadecane, which caused a large shift from the branched fatty acids to nC,, and nC,,, which together represented 50-70% of the total fatty

,,

50

THOMAS A. LANGWORTHY

acids. Among moderate thermophiles, R. stearotherinophilirs contains isoC,, and isoC,,acids, but the total of this pair (34-64%) is substantially less than in extreme thermophiles, and considerable amounts of tic,,, is0 and unteisoc,,, and unteisoC,, are present as well (Cho and Salton, 1966; Daron, 1970;Yao et ul., 1970;Shen et al., 1970;00 and Lee, 1971;Oshima and Miyagawa, 1974). Similar trends are apparent in the moderately thermophilic Closfridiuin examined by Chan etul. (1971).The main fatty acids were nC,,, iiC,,, and predominantly isoc,,. Small quantities (8-10%)of a new unsaturated, C,,,cyclopropane fatty acid, identified as 12,13-methylene-9-tetradecenoicacid, were found. This is the first reported occurrence of an unsaturated cyclopropane fatty acid in bacteria. Psychrophiles, in contrast to thermophiles, are distinguished by large quantities of monoenoic acids, mainly nC,,:, and HC,,:,. The psychrophilic marine pseudomonads examined by Brown and Minnikin (1973)were grown at 10°C and contained a simple fatty acid profile consisting of nC,,. tic,,:,,and small amounts of nC,,,,. When grown between 10 and 20"C,the fatty acid profiles remained unchanged, suggesting a lack of a mechanism whereby fluidity may be controlled in these organisms. Micrococcus cryphilus, grown at either 20°C or O"C,contained 95% nC,,,, and nC,,:,, but Russell (1971)observed a 4-fold increase in the nC,,:, to nC,,:, ratio when growth temperature was changed from 20°C to O"C, indicative of chain shortening. Kates and Hagen (1964)reported that a Serrutiu-like psychrophile, grown at 5°C or 10"C, had large amounts of tic,, and nC,,,, but not j7Cl,:l or cyclopropane fatty acids as in its psychrotrophic counterpart, S. niarcescens. Bhakoo and Herbert (1979)investigated the fatty acids of four different marine vibrios grown between 0 and 15°C.The isolates contained no fatty acids longer than 17 carbons. Two of the isolates increased the proportions of nC,,,, , nC,,:, , and nC,,,, on lowering growth temperatures. One responded by chain-length shortening by increasing the amount of nC,,,, , but one isolate contained 60% nC,,,,, which did not change at all in response to temperature. The psychrophilic Clostridium examined by Chan et a / . (1971) contained nC and nC (40%) and a large quantity (45%) of unsaturated cyclopropane fatty acids, mainly 12,13-methylene-9-tetradecanoicacid. Acidophilic Thiobacillus species are characterized by a high content (nearly 50%) of C,,-cyclopropane fatty acids (Levin, 1971, 1972). A C,, P-hydroxy fatty acid, 3-hydroxyhexadecanoate (Knoche and Shively, 1972) and a CIS,cyclopropane hydroxy acid, cis- 1 I ,12-methylene-2-hydroxyoctadecanoicacid (Knoche and Shively, 1969) are also found in covalent linkage to the ornithinecontaining lipid of these organisms (see Section IV,D). The thermoacidophile, B . acidoculdarius, contains both C,,-branched fatty acids, like thermophiles, and a prevalence of cyclized fatty acids like acidophiles, However, the major fatty acids (50-90%) are composed of the alicyclic, w-cyclohexyl, C,, and C19acids, 1 1-cyclohexylundecanoate and 13cyclohexyltridecanoate, which are biosynthesized from glucose via the shikimate

LIPIDS OF BACTERIA IN EXTREME ENVIRONMENTS

51

pathway (de Rosa et ai.,1972; Oshima and Ariga, 1975; Oshima et al., 1978). Although de Rosa et crl. (1974) discuss a complex relationship between the fatty acid composition, temperature, pH, and metabolism, Oshima and Ariga ( I 975) could find no significant alterations in the fatty acid composition in cells grown at different temperatures and pH values. The function of cyclohexyl fatty acids becomes even less clear, since a mutant of B . suhtilis, which is able to synthesize cyclohexyl fatty acids on addition of appropriate precursors, is not provided with any special advantage for growth at high temperature or low pH. In fact, these fatty acids caused a decrease in the transition temperature of the lipids (Blume Pt ui., 1978). It has been suggested (Blume ot ui., 1978) that the bulkiercyclohexyl rings in the interior of the membrane may provide optimal packing and orientation with the numerous triterpene derivatives present in B . ac.itlocalr1r~riu.s(see Section III,A,3,4 and Ourisson and Rohmer, this volume). Cyclohexyl fatty acids are not restricted to B . acidocaldurius but also occur in hydrocarbonutilizing Mycohucterium and Nocardii species grown on n-alkyl-substituted cycloparaffins (Beam and Perry, 1974).

6. lsopranyl Glycerol Ethers Unlike any other organisms, diacylglycerol residues are absent in the halophilic, methanogenic, and thermoacidophilic archaebacteria, Thertnoplusrna and Su(fo1obu.s. The apolar residues consist either of two C,, or two C,,, fully saturated and isopranoid-branched hydrocarbons in ether linkages to glycerol as either di-0-phytanyl glycerol (1) or tetra-0-di(biphytany1) diglycerol derivatives (2). Halophiles possess the diether (Kates, 1972, 1978), whereas Ther~noplastna (Langworthy, 1977a) and Sulfolohirs (de Rosa et N I . , 1977b) contain tetraethers, and methanogens possess both diether and tetraethers (Tornabene and Langworthy, 1979).

Di-0-phytanyl glycerol was first recognized to constitute the sole apolar residue in halophilic bacteria through the extensive studies of Kates and associates (reviewed by Kates, 1972, 1978). The 0-alkyl groups were found to consist of

52

THOMAS A. LANGWORTHY

the C,,-hydrocarbon, phytane, and the glycerol to have the sn-2,3 configuration opposite that of naturally occurring diacylglycerols. The di-0-phytanyl glycerol (1) was thus shown to be 2,3-di-0-(3R, 7R, 1 l R , 15-tetramethylhexadecyl)-snglycerol. Initial studies on Thermoplasrna and Sulfolobus (Langworthy et al., 1972, 1974) revealed the sole presence of ether lipids. These contained glycerol but instead had C ,,,-hydrocarbon chains. Their structural assembly as diglycerol tetraethers (2) has been only recently established and confirmed (Langworthy , 1977a; de Rosa ef al., 1977b, 1980e; Yang and Haug, 1979). The diglycerol tetraether structure consists of two sn-2,3-glycerol molecules bridged through ether linkages by two identical pairs of C,,-terminal diols with the resultant primary hydroxyl groups of the glycerols in the trans configuration. The C,,hydrocarbon chains have been shown by de Rosa et al. (1977a,b, 1980e) to have the w,w-biphytanyl skeleton made up of two C B,rphytanyl units joined “head to head” at the 16,16‘-geminal ends. The diglycerol tetraethers (MW 1300) are therefore the structural equivalent of two molecules of di-0-phytanyl glycerol (MW 650) that have been condensed by covalent linkage through the 16,16’terminal ends of their 0-phytanyl side-chains. The C,,-biphytanyl chains also differ in the additional feature that they may contain up to four cyclopentane rings (de Rosa et ul., 1977a,b, 1980e). The series of diols constituting the ether linkages to glycerol may be the acyclic biphytane, C4,HR202(3), as already noted; the monocyclic-C,,H,,O, (4); bicyclic-C,,H,,O, ( 5 ) ; tricyclicC,,H,,O, (6);or tetracyclic-C,,H,,O, (7) biphytane derivatives. There are H0.

OH

HO

OH

LIPIDS OF BACTERIA IN EXTREME ENVIRONMENTS

53

HO

% % H O

(7)

therefore five different molecular species of tetraethers that are theoretically ,;, MW 1300- 1 194, depending on cyclization in the identipossible, C Jl 172-ll,R0 cal pair of biphytane chains. However, only (3), (4), and ( 5 ) are the main biphytnne constituents of diglycerol tetraethers in Thermoplasma and Sulfolobus. The more cyclized biphytanes (6) and (7)are primarily associated with the second specialized class of tetraether peculiar to Suljolobus. This more polar tetraether, representing 50-75% of the total ethers in Sulfolobus-depending on heterotrophic or autotrophic growth-contains a pol yo1 substituted for one of the glycerol molecules in the tetraether assembly (Langworthy et a l . , 1974; Langworthy, 1977b, 1978b; de Rosa et al., 1980a). De Rosa et al. (1980a) have shown the polyol to be a C,-branched nonitol, called by them calditol, giving rise to a Cg2H l,ixO12, MW 1472-1464, calditol glycerol tetraether (8). The extent to which pentacyclic rings do occur appears dependent on the strains involved, as

well as the growth temperature (Langworthy et ol., 1972, 1974; de Rosa et a l . , 1976a, 1980d; Yang and Haug, 1979; Furuya et al., 1980). In Thermoplasma, acyclic (3) and monocyclic (4) biphytanes are predominant, whereas in Sulfolobus, which grows at much higher temperatures, the monocyclic (4), bicyclic (3,tricyclic (6), and tetracyclic (7)biphytanes are most pronounced. De Rosa et al. (1980d) and Furuya et a l . (1980) have also shown that Sulfolobus increases the amount of cyclization in the biphytanyl chains with increasing temperatures from 50 to 85°C; suggesting a role in membrane stabilization at high temperatures. Thus the apolar residues of Thermoplasma are composed of diglycerol tetraethers, whereas Sulfolobus contains approximately equal amounts of more highly cyclized diglycerol tetraethers and calditol glycerol tetraethers. However, both organisms do possess small quantities of di-0-

54

THOMAS A. LANGWORTHY

phytanyl glycerol associated with the polar lipids (Langworthy , I979b, and unpublished). A11 methanogenic bacteria so far investigated contain di-0-phytanyl glycerol, but depending on genera, contain diglycerol tetraethers as well. Of nine different species representing four different genera examined (Tornabene ct ul., 1978; Makula and Singer, 1978; Tornabene and Langworthy, 1979). the coccal forms, Methanococcus and Methatiosarcinu, contain only di-0-phytanyl glycerol, whereas the rod- and spiral-shaped methanogens, MethntinbaL.tL.riiirn and Methnrios~~irilht~i, possess di-0-phytanyl glycerol (38-72%) and diglycerol tetraether (28-62%). Cyclization is absent and the tetraether contains only acyclic biphytanyl chains (3). Di(biphytany1) diglycerol tetraether (2) is the only molecular species of tetraether so far detected in methanogens. Thus the occurrence of both ethers in methanogens, which grow under normal physiological conditions (albeit anaerobically), indicates that the ethers of halophiles and thermoacidophiles cannot be viewed as an adaptation for survival in hypersaline or hot acid environments. These lipids are however, well suited for such purpose (Kates, 1972, 1978; Langworthy, 1977a, 1979a). Rather, these lipids reflect a phyletic relationship and a more profound evolutionary development in which these organisms share a common evolutionary episode distinctly different from other cells (Fox et ul., 1980). The distribution of diethers and tetraethers among halophilic, methanogenic, and thermoacidophilic archaebacteria is summarized in the table. The discovery of tetraether lipids within the archaebacteria is of considerable interest in terms of molecular organization and membrane biogenesis. The di0-phytanyl glycerol residues of halophiles allow for the formation of a typical membrane lipid bilayer through interaction of separate and opposite phytanyl residues, the only constraint being that the chain length is invariably fixed at 20 carbons. Tetraethers, however, accounting for the majority of the membrane hydrocarbon of Thermnplusinu and Sulfolobus, approximate 45-75 in length depending on cyclization in the hydrocarbon chains and span the membranes, which average about 70 8, in width (Langworthy, 1977a, 1978b, 1979a,b, 1980a). Therefore, these two organisms, along with regions within the membranes of those methanogens containing tetraether, can be considered to possess a covalently cross-linked, or sealed, membrane bilayer created by virtue of the extension of the C,,-hydrocarbon chains across the membrane in covalent linkage to glycerol residues on the inner and outer membrane faces. These membranes cannot, therefore, be considered to comprise a lipid bilayer in the strict sense of the word, but are structural equivalents of an amphiphilic monolayer that has been condensed at the center joining both halves of the bilayer together. Correlating well with a monolayer membrane, Thermoplasrna and Sulfolobus fail to freeze-fracture tangentially to yield inner and outer membrane faces, but instead characteristically cross-fracture perpendicularly through the membrane as ex-

55

LIPIDS OF BACTERIA IN EXTREME ENVIRONMENTS D I P H Y T A NGI.YCEROI. YL ETHER A N D DICI.YCEROL TETRAETHER DISTRIEIJTION I N HAI.OPHII.IC. METHANOGENK, A N D THI:RMOACIDOPHILIC BACTERIA" Organism

Diethcr (%)

I00 I00

Hulohucterium cutiruhrum Hulnhuc~trriumhtilohiitm Holohwterium sulinrrrium Hulohuctrriurn mcirismortui Surc,rnu literulis Surt,inu morrhuur

100 1 00

Mrthunosurcinci burkeri Mrthanor~oc~c~u.~ strain PS Mcjthrinococcus vunnirllr Mr~thunoha~.terium ruminutiuni M- I Methonohuc~tc,riumruminritiunr PS Methanobacterium thrrmoti utntrophic.rtm Methunobwtrrium strain M.v.H. Me~thuno.spirrllirtnhungutiG Methunospirillum strain AZ

I00 99.9 71.8 44.7 44.5 43.5 40.5

Thrrmoplasmu uc~idophilum Su!/olobus cic~idc~caldurtus "

I00 1 00

I 00

Tetraether (47.) 0 0 0 0

0 0 0

37.5

0 0.I 28.2 55.3 55.5 56.5 59.5 62.4

10.0 5 .O

90.0 95.0

Data from Kates (197X); Tornabene and Langworthy (1979); Langworthy (1979b. IY80a, 19x1).

pected of a monolayer assembly (Langworthy, 1979a). In light of a monolaycr membrane, the role of cyclization in the tetraethers of Therrnoplustnu and SU/,folohus might also be explained. Since the hydrocarbon chains comprising the tetraethers are fixed at 40 carbon atoms linked to glycerol on each end of the chain, cyclization would reduce rotational freedom within the chains and thereby the interior of the membrane. In addition, cyclization provides an effective means of controlling chain length and simultaneously tetraether and membrane width. Thus cyclization may provide a mechanism of controlling internal mernbrane viscosity and condensing the membrane in response to temperature and permeability in a fashion similar to cholesterol (see Razin, this volume). Cyclization is absent in the tetraethers of methanogens, but perhaps membrane fluidity and permeability may be controlled by altering proportions of diether and tetraethers, although the effect of temperature and ions has not been reported. The question of membrane asymmetry is also an interesting one since the tetraethers are themselves symmetrical molecules. Substitution of either one or both of the primary hydroxyl groups on opposite ends of the tetraether molecule will determine asymmetry. First indications (Kuswaha et ui., 1981a,b) are that both types of substitution exist, wherein glycolipids contain carbohydrate linked to only one side, whereas the acidic phosphoglycolipids contain carbohydrate attached to one

56

THOMAS A. LANGWORTHY

side and the phosphate radical to the opposite end of the tetraether (see Sections IV,C and V,B). Furthermore, diether and tetraether biosynthesis clearly involves the isopentenyl pyrophosphate pathway, at least to the C,,-geranylgeraniol pyrophosphate intermediate (Kates, 1978; Langworthy, 1979a; de Rosa et ul., I980b; see Fig. 15 in the article by Ourisson and Rohmer, this volume). From this point the biosynthetic steps are unknown. However, the biphytane chains of tetraethers are condensed “head to head” through the geminal ends of two C,,, residues rather than tail to tail through the terminal phosphates of two C2,geranylgeraniol pyrophosphates as in carotenoid synthesis. Thus, combined with the fact that tetraethers are the structural equivalents of two covalently linked diether molecules, tetraether biosynthesis could occur via “head to head” condensation between the two diether molecules, or in fact two polar lipid derivatives of diethers to yield unsubstituted tetraethers or tetraether complex polar lipids (Langworthy, 1979a, 1980a; de Rosa et al., 1980e; Kushwaha e f al., 1981a,b). The rapid turnover of the small quantity of di-0-phytanyl glycerol in Thennoplusmu indirectly adds support to this hypothesis (Langworthy, 1980b). Tetraether biosynthesis is clearly unusual and its elucidation should provide a new route of hydrocarbon biosynthesis.

111.

NEUTRAL LIPIDS

A. lsoprenoid Derivatives The neutral lipids of halophilic, thermoacidophilic, and methanogenic archaebacteria have been the most fully elucidated among the bacteria from extreme environments. They represent approximately 10-30% of the total lipids and are composed almost exclusively of isoprenoid derivatives. The neutral lipids of other bacteria have been largely ignored, but several constituents have been identified in the thermoacidophile B . acidocaldarius, the acidophile T . ferrooxidans, and the thermophile T . aquaticus. Neutral lipids of these organisms range respectively from about 16% in B . acidocaldarius to 60% in the extreme thermophile T . aquaticus. The neutral lipids that have been identified can be broadly grouped into seven major classes based on chain length as follows: C,,-isoprenoids (geranylgeraniol, phytanes, phytanyl ethers, retinal), C,,-isoprenoids (pentaisoprenalogues), C,,isoprenoids (squalene, hopanes), C,,-isoprenoids (tetrahydroxybacteriohopane), C,,,-isoprenoids (carotenes), and C,,-isoprenoids (bacteriorubrins, polyprenols). I . C,,,-~SOPRENOIDS

a. Geranylgeruniol. Geranylgeraniol(9), containing one cis double bond, constitutes the main C2, component of the halophilic bacteria (Kushwaha et ul.,

57

LIPIDS OF BACTERIA IN EXTREME ENVIRONMENTS

1975a; Kushwaha and Kates, I978b). All-trans-geranylgeraniolhas been reported as a minor component in Sulfolobus (de Rosa et al., 1980b). Since geranylgeraniol may be an intermediate in diether and tetraether biosynthesis, it is likely to occur in Thermoplasma and methanogenic archaebacteria as well.

b. Phytanes. Although halophilic bacteria contain trace amounts of phytanol (Kushwaha and Kates, 1978b), phytane and its unsaturated homologues dominate the C,,-isoprenoids of Thermoplasma, Sulfolobus, and methanogenic bacteria. Methanogens contain mainly phytane and phytaene, Sulfolobus phytadiene and phytatriene, whereas phytatetraene predominates in Thermoplusma (Tornabene e t a / . , 1979; Holzer ef al., 1979). c. Di-0-Phytunyl Glycerol. Primarily associated with the polar lipids, the diether is present in the free form amounting to about 8% of the neutral lipid fraction of halophilic bacteria (Kushwaha e f al., 1975a; Kushwaha and Kates, 1978b). The small quantities of di-0-phytanyl glycerol in Thermoplasma and Sulfolobus are associated with the polar lipids and have not been detected free in the neutral lipid fraction (Langworthy, 1979b, 1980a, also unpublished). De Rosa et al. (l976b) reported the occurrence of an unusual tri-0-phytanyl glycerol ether containing partially or fully saturated phytanyl chains as a minor component of Sulfolobus neutral lipids. d. Retinal. All-trans-retinal (10) as a minor component has only been reported in the neutral lipids of pigmented halophiles (Kushwaha and Kates, 1973; Kushwaha et al., 1974; Kates, 1978). Its presence is dependent on growth conditions being produced anaerobically in the presence of light. It is associated principally with the purple membrane in the retinal-protein complex, bacteriorhodopsin. Of considerable interest is whether retinal or an analogue may occur in other arc hae bac teria .

2. C

2

,

-

I

~

~

~

~

~

~

~

~

~

~

Acyclic pentaisoprenes with a continuous range of hydropentaisoprene derivatives are relatively major neutral lipid species in Thermoplasma, Sulfolobus, and various strains of methanogenic archaebacteria (Tomabene et al., 1979; Holzer

58

THOMAS A. LANGWORTHY

ul., 1979). In Thet-rnoplasma the C,,H,, pentaene is predominant, whereas fully saturated C,,H,, is the major pentaisoprene in Sulfolobus. The full range of C 2;,H3 2 - - 1 2 pentaisoprenes is found among different species of methanogens.

CI

3 . C:~O1SO PKE N 0 11)s

u . Squalenes. The presence of squalenes and hydrosqualene derivatives as the major acyclic isoprenoid neutral lipids is a feature that distinguishes archaebacteria. Squalenes, representing about 36% of the neutral lipids of halophiles grown aerobically, have been identified as C,,H,,,, all-trans-squalene (11); C ,JI ;,2r all-trans-dihydrosqualene (12); C :,,$I ;,4, all-trans-tetrahydro squalene (13); and C:,,,H 4 X , dehydrosqualene (14) (Tornabene et ul., 1969; Kramer et al., 1972; Kushwaha et al., 1972). The relative proportions vary among halophiles (Kushwaha et al., 1974), and the ratio of squalene to dihydro- and tetrahydrosqualene decreased proportionately when cells are grown anaerobically in the light (Kushwaha ef al., 1975b) or under microaerobic conditions (Tornabene, 1978).

The squalenes of methanogens represent between 64 and 95% of the total neutral lipids (Tornebene et al., 1978, 1979). These are composed of a continuous range of C & 32-,(i,, hydrosqualenes from dihydrosqualene up to and including

LIPIDS OF BACTERIA IN EXTREME ENVIRONMENTS

59

decahydrosqualene, but squalene. dihydro-, and tetrahydrosqualene are the predominant species. Squalene itself is predominant in Thc.i-r,io~ltr.c./~ltr, but octaand decahydrosqualene are the major species in Sulfi~lobiis(Tornabene ~t a [ . , 1979). Dehydrosqualene is absent in methanogens, Tlwrmoplu.smc~, and Sirl,folohu.s. Although characteristic, squalenes are not restricted to archaebacteria. De Rosa e/ u l . ( 1973) identified squalene in the therinoacidophile R . acicloculclurius, which is the likely precursor to the pentacyclic hopanoid triterpenes that characterize this organism (Ourisson rt ul.. 1979). Squalenes have now been 1978; detected in a few aerobic and anaerobic eubacteria as well (Amdur e/ d., Mercer P / al.. 1979). h. Hopunes. Pentacyclic triterpenes of the hopane family are now known to have a widespread occurrence in a variety of microorganisms (Ourisson c/ ul., 1979; Rohmer rt u l . , 1979). The function of this class of lipids, which may be the structural equivalent and phylogenetic precursors of sterols, is considered in detail by Ourisson and Kohmer (this volume). Among the bacteria from extreme environments considered herein, only one, B . uciclocultlarius, possesses hopanes in the neutral lipids. De Rosa ~t ul. (1973) demonstrated that free hopanes account for about 0.3% of the cell dry weight of this bacterium and consist of about 86% hop-22(29)-ene (15), trace amounts of hop-17(21)-ene (16), and 4% hopane (17).

60

4. C

THOMAS A. LANGWORTHY

3

5

-

I

~

~

~

~

~

~

~

~

~

~

In addition to squalene and hopanes, B . acidocaldarius possesses a third triterpene derivative, tetrahydroxybacteriohopane (18). This polar compound contains the hopane nucleus but is substituted at C-29 with n-l,2,3,4-tetrahydroxypentane (Langworthy and Mayberry, 1976). It equals nearly 1.5% of the cell dry weight but only a small amount exists in the free form. It serves primarily as a major new type of aglycone in the glycolipids of the organism (Langworthy et al., 1976; see Section IV,B). O H OH

5. C&OPRENOIDS With the exception of the pigmented halophiles, carotenoids of bacteria from extreme environments have not been well defined. Halophiles possess low concentrations of lycopersene, cis- and trans-phytoene, cis- and trans-phytofluene, lycopene, neo-a-carotene, and p- and neo-p-carotene (Kushwaha et al., 1972; Kushwaha and Kates, 1973). The low concentrations of carotenoids suggest that they might serve as biosynthetic precursors to retinal or bacteriorubrins (Kates, 1978). The neutral lipids of the extreme thermophile T . uquaticus were shown by Ray et al. (1971b) to be composed of about 8% phytoene, 7% A-carotene, and 75% very polar carotenoids, which were not identified. Although the distribution remained the same. the total carotenoid content increased I .8-fold on increasing the growth temperature from 50 to 75"C, suggesting a role in membrane stabilization at high temperatures. Yellow and orange carotenoids were also noted in the high neutral lipid content of the acidophile T . ferrooxidans by Short et al. (1969), but were not investigated. 6. LIPOQUINONES Menaquinones-7, -8, and -9, containing C3sr C4,,, and C4,-prenyl chains, respectively (19), have been demonstrated in several aerobic bacteria from extreme

61

LIPIDS OF BACTERIA IN EXTREME ENVIRONMENTS

habitats. Thermoplasma contains menaquinone-7 (Langworthy et a / . , 1972); halophiles, menaquinone-8 (Tornabene et al., 1969; Kushwaha rt al., 1974, 1975a); and B . acidocaldurius contains menaquinone-9 (de Rosa ef a / . , 1973). Ray et ul. (1971b) found that T . uquuticus contained all three menaquinones, 7-9. Menaquinones were found to be absent in T . ferrooxidans (Short et al., 1969), but it contained coenzyme 4-8 as the sole lipoquinone. De Rosa et al. (1975b) found that Surolobus contains an unusual triterpenoid 4,7thianaphthenequinone, which is the first occasion for this compound to be detected in a natural source. 7. C ,,,-ISOPRENOIDS Polyprenols containing more than 45 carbons have only been described in halophilic bacteria and in B . acidoculdarius. Pigmented halophiles contain, in decreasing proportions, the tetrahydroxy , C,,,-noncyclic carotenoid, bacteriorubrin (20); the C,,-triol, mono-anhydrobacteriorubrin (21); and the C,,-diol, bisanhydrobacteriorubrin (22); these are associated primarily with the red membrane fraction (Kushwaha et al., 1974, 1975a,b). The prenol fraction from B . acidoculdarius was shown by de Rosa et al. (1973) to contain a series of polyprenols between 9 and 12 isoprene units. The C,, and C,, species were predominant, consisting of a-cis-, all-rrans-, and a-tert-prenyl derivatives. OH

OH

OH

OH

62

THOMAS A. LANGWORTHY

B. Other Neutral Lipid Components I n addition to the major isoprenoids just discussed, Holzer et a / . ( 1 979) have described a series of methyl-branched isoprenes and isopranes (C,,-C30) and n-alkanes (C,9-C32)that are present in small quantities in Thermoplasmu, Sulfolobus, and methanogenic bacteria. Kushwaha and Kates (1978a) showed low levels of mevalonic acid in a number of halophiles but this is not surprising because the lipids of these bacteria contain exclusively isoprenoid chains. The neutral lipids of halophiles, however, do contain significant amounts of a nonisoprenoid compound, indole (Kushwaha et ul., 1977). The physiological significance of indole and its intracellular presence is unknown.

IV. GLYCOLIPIDS Considering the fairly large variety of bacteria from extreme habitats, glycolipids have only been investigated in a few thermophilic and thermoacidophilic bacteria, with most attention centered on the archaebacteria. However, it is becoming evident that within the thermophilic eubacteria and thermophilic archaebacteria, carbohydrate-derived lipids constitute the major lipid class.

A. Glycosyldiacylglycerols Although Short et al. (1969) detected no glycolipids at all in the mesophilic acidophile T . Jerrooxiduns, the thermophilic acidophile B . acidocalclarius has a glycolipid content of about 64% (Langworthy ef al., 1976). The major glycolipids are glucosyl-glucosamidyl-diacylglycerol derivatives (23),and comprise about 70% of the total glycolipid fraction. They consist of approximately 25% Glcp(P I +4)GlcNacyl(P I +1 )diacylglycerol, 41 9% of Glcp(p 1-+4)GlcNacyl(p I + I)monoacylglycerol, and trace amounts of Glcp(@1 -+4)GlcNacyl(P 1- 1)glycerol. The configuration of the glycerol residue is uncertain but the ester- and amide-linked fatty acids have a similar distribution. HOCHv

HOCH:!

OH

NH I

c =o I

R

(23)

HC- 0 - C - R I 0 HpC-0-C-R

LIPIDS OF BACTERIA IN EXTREME ENVIRONMENTS

63

Extreme thermophiles of the genus Thermus, including T . thermophilus, T . Juvus, and T . uquuticus, contain an unusual tetraglycosyldiacylglycerol that constitutes 50-70% of the total lipids in the organisms (Oshima and Yamakawa, 1974; Oshima and Ariga, 1976; Oshima, 1978; Pask-Hughes et a/., 1977). Oshima and Yamakawa (1974) found the compound to be a galactosyl- galactosyl-glucosamidyl-glucosyl-diacylglycerol with a terminal galactofuranose residue (24). The structure has now been established as Gal,f(P1-2)Galp ((-~1+6)GlcN(15- methylhexadecanoyl) (pl-2)Glcp ( a l ~ l ) d i a c y I g l y c e r o l al, though the glycerol configuration is uncertain (Oshima and Ariga, 1976). In this lipid, isoC,, is the sole fatty acid in amide linkage to glucosamine. Additionally, the glycolipid content of a number of Thhrrmus species increases almost 2-fold at temperatures increasing from 50 to 80°C (Ray et ul., 1971b; Oshima, 1978). This correlates with the observations of Pask-Hughes c2t al. (1977) and Sharom et ul. (1976) that increasing glycolipid concentrations or glycolipids with an increasing number of sugar residues renders phospholipid bilayers increasingly rigid. Thus the high glycolipid content, as well as the presence of tetraglycosyldiacylglycerol, and, in fact, glycosylglycerols in general, may play a major role in stabilizing membranes to thermal or other environmental stresses.

B. TetrahydroxybacteriohopaneGlycosides

B . acidocald~irhscontains, in addition to glucosyl glucosamidyldiacylglycerols, an N-acylglucosaminyl-tetrahydroxybacteriohopane,which constitutes about 25% of the glycolipid fraction of cells grown at pH 3 and 60°C (Langworthy et al., 1976; Langworthy and Mayberry, 1976). N-acylglucosamine, which is joined by a P-glycosidic linkage to the primary -OH of the tetrahydroxybacteriohopane aglycone, has been established as 1-( O-~-N-acylglucosaminy1)-2,3.4tetrahydroxypentane-29-hopane (25). Poralla and associates ( 1980; Kannenberg et a/.. 1980), demonstrated that the glycolipid as well as the free aglycone produced a condensing effect similar to cholesterol in synthetic monolayer membranes, suggested that the lipid may function in diminishing diffusion of H ’ ions through the membrane.

64

THOMAS A. LANGWORTHY

NH I

c=o

C. lsopranyl Glycerol Ether Glycosides 1 . DIET'HER DERIVATIVES

Di-0-phytanylglycerol ether glycolipids have been identified in halophilic and methanogenic archaebacteria. Most extreme halophiles encountered contain the tri gl ycosyldiether Galp ( p1+6)Manp ( a1-2)Glcp ( a1+1 )2,3, - di- 0 - phytanylsn-glycerol (Kates and Deroo, 1973; Kates, 1978). The glycolipid per se occurs in lesser amounts, but the acidic sulfate derivative is one of the major lipids in these organisms (see Section V,C). However, the lipids of one halophile from the Dead Sea, H . mirismortui, contain about 1 1 % of a novel triglycosyldiether, Glcp (pi-6)Manp (a14 2 ) G l c p ( a l b1)2,3-di- 0-phytanyl- sn-glycerol (Evans et ul., 1980). In the first report on the nature of the complex lipid structures in methanogenic bacteria that contain both diethers and tetraethers, Kushwaha et ul. (1981a,b) have shown that M . hungutei possesses two new galactofuranosyl-containing diglycosyldiethers: Calf@ 1+6)Galf(p I +1)2,3-di-O-phytanyl-sn-glycerol and Glcp ( a1 +2)Gal,f(a 1- 1)2,3-di-O-phytanyl-sn-glycerol. These accounted for 2 and 17% of the total lipids, respectively. 2. TETRAETHER DERIVATIVES Tetraether glycolipids, identified thus far in archaebacteria, include those of M . hungatei, Sulfolobus, and Thermoplasma. The methanogen M . hungatei contains two diglycosyltetraether glycolipids, which equal less than 1% of the total lipids (Kushwaha et al., 1981a,b). The same disaccharides as in the diether analogues are glycosidically linked to one -OH of the diglycerol tetraether, with the other -OH radical remaining free, existing as Ga!f'(P 1 +6)Galf(p 1- 1)O-[diglyceryltetraetherl-OH and Glcp ( a1+2)Galf(pl- 1)-0-[diglyceryltetraetherl-OH. The two glycolipids representing 68% of Sulfolobus lipids are based on the two types of diglycerol and calditol glycerol tetraether species (Langworthy et al., 1974; de Rosa et at., 1980~). A diglycosyl diglycerol tetraether and glycosyl

65

LIPIDS OF BACTERIA IN EXTREME ENVIRONMENTS

calditol glycerol tetraether are present in about equal proportions in heterotrophically grown cells, but the calditol glycerol tetraether glycolipid is the major derivative in cells grown autotrophically (Langworthy, 1977b). The two glycolipids have been partially characterized as Glcp(P+)Galp(P-+)-O-[diglyceryltetraetherl-OH and Glcp(P-+)-0-[caldityIglyceryltetraether]lOH. The glucosyl-galactosyl disaccharide is linked to one side of the diglycerol tetraether, whereas glucose is linked to one of the -OH groups of the calditol radical in the calditol glycerol tetraether. The glycolipid fraction of Thennc~plusmcirepresents 25% of the lipids but none of the six diglycerol tetraether glycolipids have been identified (Langworthy et ai., 1972). However, an unusual linear lipoglycan (MW 5300) containing 24 mannose, one glucose, and terminating in a diglycerol tetraether has been isolated from Thermoplusrnu (Mayberry-Carson et ul., 1974). The molecule, which can be considered to be a glycolipid with an extended 25-sugar chain, accounts for 3% of the cell dry weight. Its structure (26) has been fully established by Smith ( 1980) to be [ Manp ( a1+2)Manp ( a1-4)Manp ( a1-+3)],-Glcp (al-+l)-O-[diglyceryltetraetherl-OH in which the sugar chain is attached to one side of the diglycerol tetraether molecule. The lipoglycan has physical properties similar to gram-negative lipopolysaccharides (Maybemy-Carson et ul., 1975) and is located on the cell surface (Mayberry-Carson et al., 1978). Its finding in Thermoplasma has led to the isolation of similar diacylglycerol lipoglycans in the Acholeplasrnu species (Smith et ul., 1976). CH,OH

CH,OH

SH,OH

FH,OH

1

CH,OH

D. Other Polar Lipids An ornithine-containing lipid was found by Shively and Knoche ( 1 969) among the lipids of the acidophile T . thiooxiduns. Its structure (27), in which 3-hydroxyhexadecanoic acid is amide-linked to the amino group of omithine and cis- 1 I , 12-methylene-2-hydroxyoctadecanoicacid is ester-linked to the 3-OH group, has been established (Knoche and Shively, 1969, 1972; Hilker et ul., 1978). The biological significance of this lipid is unknown. O=C-CH-(CH, ),-CH-CH-(CH,);-CH, COOH

0 OH H, N-(cH,),-cH-NH-c-cH,-~H-(cH,),IcH, II

I

\

I

CH,

66

THOMAS A. LANGWORTHY

V.

ACIDIC LIPIDS

Acidic lipids of eubacteria from extreme environments are composed largely of ordinary phospholipids including either diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (Pi), phosphatidic acid (PA), or phosphatidyl-N-monomethylethanolamine (PME) as major constituents. Within the halophilic and methanogenic archaebacteria, the diether-derived phospholipids occur as analogues of the respective diacylglycerol phosphatides. However, in the thermoacidophilic and methanogenic archaebacteria possessing tetraethers, the tetraether phospholipids established thus far exist almost exclusively as phosphoglycolipids in which the carbohydrate and phosphoryl radicals are attached asymmetrically to opposite ends of the tetraether molecule. Additionally, acidic sulfolipids occur in several eubacterial and archaebacterial species.

A. Phospholipids 1 . PHOSPHATIDES Among the thermophilic Bacillus species, B . steurothermophilus (Card ef d., 1969; Card, 1973) and B . caldotenux (Hasegawa et d.,1980), phospholipids composed mainly of DPG, PG, and PE constitute 60-90% of the total lipids. Hasegawa et u / . (1980) noted a substantial increase in lower melting PG and a decrease in the higher melting PE content when the growth temperature of B. cu/do/yticus was lowered from 65 to 45°C. The extreme thermophile T . apaticus contains DPG, PG, PI, and PA, representing only 20% of the phospholipid fraction (Ray et a / . , 1971b). It contained, in addition, a major unidentified phospholipid having a minimum molecular weight of 1800, which possessed phosphate, three fatty acids, one glycerol, and a long-chain unsaturated amine. It was also noted that the phospholipid content increased 2-fold in cells grown from 50 to 75°C. The four psychrophilic Vibrio species examined by Bhakoo and Herbert (1979) all contained DPG, PG, and PE, but two of the isolates possessed significant quantities of PS. Changes in the phospholipid distribution of some isolates were noted at 20"C, the upper temperature limit for growth, suggesting a thermal-sensitive impairment of phospholipid synthesis. It was also shown that total phospholipid levels increased markedly on decreasing the growth temperature to 0"C, but Cullen et (I/. (1971) found no change in the phospholipid composition of a psychrotrophic Pseudotnonasfluorescens species on decreasing temperatures. The mesophilic acidophiles, T . thiooxidans and T . ferrooxidans, have been shown by Shively and Benson (1967) and Short et a/. (1969) to have phospholipids made up mostly of DPG, PG, PE, and DME. The presence of PS

LIPIDS OF BACTERIA IN EXTREME ENVIRONMENTS

67

reported by Korczynski et ( I / . (1967) could not be confirmed. The phospholipids of T . ,ferrooxiiluti.s were found to have a slow rate of tnetabolism and no differences in proportions or turnover were found during growth at either pH I .5 or 3.5 (Short (’t u l . , 1969). Of the total lipids from the thermoacidophile B . Nc,iik,ctildurii,.s, 20% are acidic lipids, of which DPG, PG, and traces of PA and PE account for 57% (Langworthy et ( I / . , 1976). The remaining 43% consists of a sulfonolipid (see Section V,C,2). 2. DIETHER ANAI.OGI~ES The phospholipids of extremely halophilic bacteria are composed of the di0-phytanyl glycerol ether analogues of phosphatidyl glycerolphosphate (PCP), PG, and phosphatidylglyceryl- 1 ‘-sulfate (PGS), which constitute about 65 and 4% of the total acidic lipids, respectively (Kates, 1972, 1978; Kates and Kushwaha, 1976). The PGS is exclusively associated with the purple membrane fraction (Kushwaha r t ul.. 1975b). The Dead Sea halophile H . rriarisrirortiri contains the same phospholipids but differs from the other halophiles examined by having a significantly higher amount ( 1 7%) of PGS, perhaps compensating 1980; see for the deficit of any glycolipid sulfate in this organism (Evans et d., Section V , C , I ) . In M . hungutei, a small amount of PG (5%) was found, and this is the only di-0-phytanyl glycerol ether analogue to be reported thus far in methanogens (Kushwaha rt ul., I9Xla,b).

B. Phosphoglycolipids Phosphoglycolipids are the major constituents of archaebacteria that contain tetraether residues. In M . hungutc’i the phosphoglycolipids occur as glyceryl phosphoryl derivatives of the two diglycerol tetraether glycolipids in which sn3-glyceryl phosphate is linked to the free -OH group of the tetraether moiety (Kushwaha et u l . , I98 1a.b). The two phosphoglycolipids have been established as Galfw 1 -6)GalfG 1- 1)-0-[diglyceryltetraetherl-0-PO,-glycerol and Glcp(cu l b 2 ) G a l f w l-+ 1)-0-[diglyceryltetraether]-0-P03-glycerol.They represent 14 and 50% of the total lipids, respectively. These compounds are clearly the structural analogues of the di-0-phytanyl glycerol glycolipids that have been covalently condensed “head to head” with the di-0-phytanyl phosphatidylglycerol present in the organism. This lends strong support to the view that tetraether lipids are derived biogenically via condensation through the geminal ends of the C,,,-phytanyl chains of either free diethers or the complex lipids themselves. The three phospholipids of Su(fo/obirs are inositol phosphate derivatives, representing about 21% of the total lipids (Langworthy et al., 1974; de Rosa et ul.,

68

THOMAS A. IANGWORTHY

1 9 8 0 ~ ) .Present in close proportion are the tetraether analogue of phosphatidylinositol, inositol-OP0,-[diglyceryltetraetherl-OH,and the inositolphosphoryl derivatives of the two partially characterized glycolipids; Glcp(P+)Galp(P-)- 0-[diglyceryltetraetherl-OH and Glcp(P-)-O- [calditylglyceryltetraetherl-OH. The location of inositol phosphate residues on the latter two glycolipids has not been established. Phosphoglycolipids constitute about 57% of the total lipids from Thrrmoplusmu, all containing glycerol phosphate residues (Langworthy et ul., 1972). Although only partly characterized at a time prior to the recognition of tetraethers (Langworthy, 1977a), Thermoplusmu can be described as containing a glycerylphosphoryl monoglycosyl diglycerol tetraether that accounts for 80% of the lipid phosphorus and nearly half of the entire lipids of the organism. Four minor components include amine-containing diglycerol tetraether phosphoglycolipids. Thus, among archaebacteria containing tetraethers, essentially the total complex lipids (glycolipids plus acidic lipids) contain carbohydrate residues.

C. Sulfolipids 1 . SULFATIDES

Sulfate-containing glycolipids occur in Sulfolobus and most halophilic archaebacteria. About 6% of the acidic lipids of Su(fo1obus are composed of the sulfate derivative of its partially characterized tetraether glycolipid, Glcp(P--+)-0-[calditylglyceryltetraetherl-OH,but the location of the sulfate residue has not been determined (Langworthy rt ul., 1974; Langworthy, 1977b; de Rosa et ul., 1 9 8 0 ~ ) Most . halophiles examined, with the exception of H . mcirismortui, contain the triglycosyl diether sulfate (28) identified as -O:, SO3-Galpp 1+6)-Manp(a 1-+2)Glcp(a 1- 1)2,3-di-O-phytanyI-sn-glycerol(Kates and Deroo, 1973; Kates, 1978). It represents about 25% of the lipids, and thus combined with the phospholipids makes essentially all of the polar lipids of extreme halophiles acidic. Its function is unknown, but it is associated with the purple membrane (Kushwaha rt ul., 1975b), and it has been speculated that it might serve as a proton donor for the functioning of the purple membrane as a light-driven proton pump (Kates and Kushwaha, 1978). CH*OH

qojq

OH

o&

CH,-O-C,,H,,

I

I

CH-O-C2,JH41

69

LIPIDS OF BACTERIA IN EXTREME ENVIRONMENTS

2.

SULFONOLIPrDS

Sulfonolipids, containing the C-SO, bond, rarely occur in nonphotosynthetic bacteria (Haines, 197 1). However, the thermoacidophile B . ucidocaldarius contains a sulfonolipid (29) that appears identical to the plant sulfonoquinovosyldiacylglycerol, -03S-6-quinovosyl(a 1+1) 1,2-di-0-acyl-sn-glycerol (Langworthy et al., 1976). It represents nearly half (43%) of the acidic lipids and 8% of the total lipids of the organism. Its function is unknown, but since sulfonolipids are the most acidic lipids known, being ionized at all pH values, it was speculated (Langworthy et al., 1976) that it might serve in H+-ion exclusion or perhaps as a cation exchanger in the acidic environment. 0

I1

CH,SOJ

CH2-O-C--R I CH -0- C--R

I

0-CHz

II

0

OH

(29)

VI.

OVERVIEW

Although studies are necessarily incomplete, it is clear that bacteria from extreme environments contain many ordinary as well as quite unusual lipid structures. Perhaps the most significant class of lipids found to occur are the isopranyl ether lipids. Although of interest in their own right in terms of their individual biochemistry and how they might function in membrane stabilization in hostile environments, they also extend significant insight into our perceptions of taxonomy, evolution, biogeochemistry, and the molecular organization of membranes. The isopranyl ether lipids serve not only as a chemical marker for identifying archaebacteria, but also strongly support the taxonomic relationship of these organisms, which appear to have evolved through a line of descent different from either eukaryotic or prokaryotic cells. Moreover, the same range of archaebacterial isoprenoids, including phytane and “head to head” linked biphytanes, have recently been found in sediments (Anderson et al., 1977), kerogen (Michaelis and Albrecht, 1979), shale (Chappe et al., 1979), and petroleum (Moldowan and Seifert, 1979). Thus the fact that Thermoplasma, Sulfolobus, halophiles, and methanogens live in environments presumably dominating earlier periods of the Earth’s geological evolution suggests that the isoprenoids found in sediments and petroleum could have been synthesized directly by archaebacteria. Perhaps some of these organisms are actively involved in petrogenesis. Simi-

70

THOMAS A. LANGWORTHY

larly, the geochemical occurrence of hopanoids can now be ascribed to a bacterial origin (Rohmer rt (41.. 1979). In terms of molecular organization, the elevated content of carbohydratecontaining lipids in many of the bacteria from extreme environments indicates that the often neglected glycolipid class may have a significant function in controlling membrane stability. In addition, the molecular organization of membrane proteins, energy transduction, and transport of the tetraether-derived monolayer membranes will be of considerable interest, and will also provide a useful model for assessing our ideas of normal lipid bilayer systems. The currently recognized and yet to be discovered bacteria from extreme or unusual environments should continue to provide a rich source of material for study. New lipids will surely be uncovered to test the ingenuity and patience of the investigator. ACKNOWLEDGMENTS The author thanks R. Uecker for structural illustrations and J . Ratzlaff for editorial assistance. Portions of the author’s work described herein were supported by a grant from the National Science Foundation (PCM-7809351). REFERENCES Allen, M. B. (1953). The thermophilic aerobic sporeforming bacteria. Bacteriol. R e v . 17, 125-173. Amdur, R . H., Szabo. E. I . , and Socransky, S . S . (1978). Presence of squalene in gram-positive bacteria. J . Bacreriol. 135, 161-163. Amelunxen, R . E., and Murdock. A. L. (1978). Mechanisms of thermophily. CRC Crir. Rev. Microhiol. 6 . 343-393. Anderson, R . , Kales, M. , Baedeckcr, M . J .. Kaplan, I. R . , and Ackman, K.G . ( 1977). The stereoisomeric compositionof phytiinyl chains in lipids of Dead Sea sediments. Gf,o~.him.Cosmoc.him. A&l 41, 1381-1390. Balch, W. E., Fox, G. E., Magrum, L. J . , Woese. C. R., and Wolfe, R. S . (1979). Methanogens: Reevaluation of a unique biological group. Microhid. Re,,. 43, 260-296. Baross, J . A , , and Morita, R. Y . (1978). Life at low temperatures: Ecological aspects. I n “Microbial Life in Extreme Environments” (D. J . Kushner, ed.), pp. 9-71, Academic Press, New York. Bayley, S. T . , and Morton, R. A. (1978). Recent developments in the molecular biology of extremely halophilic bacteria. CRC Crit. Rev. Mirrohiol. 6 , 151-205. Beam, H . W., and Perry, J . J . (1974). Microbial degradation and assimilation of ti-alkyl-substituted cycloparaffins. J . Butterid. 118, 394-399. Belly, R. T., Bohlool. B. B., and Brock, T . D. (1973). The genus 7 h u r m o p l u s m . Ariri. N . Y . Acud. Sri. 225, 94-107. Bhakoo, M., and Herbert, R. A. (1979). The effects of temperature on the fatty acid and phospholipid composition of four obligately psychrophilic Vihrio spp. A r r h . Microbiol. 121, 121-127. Blume, A , , Dreher, R . . and Poralla, K . (1978). The intluence of branched-chain and o-alicyclic fatty acida on the transition temperature of Bucillrr.~suhrilis lipids. Biochim. Biophvs. Acru 512, 489-494. Brierley, C. L. (1978). Bacterial leaching. CRC Crir. Re),. Microbiol. 6 , 207-262. Brock, T. D. (1978). “Thermophilic Microorganisms and Life at High Temperatures. ’’ SpringerVerlag, Berlin and New York.

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Brock, T. D., and Freeze. H. (1969). Theritirrs uqiruricirs gen. n . and sp. n . , a nonsporulating extreme thermophile. J . Bucteriol. 98, 289-297. Brock, T. D., Brock, K . M . , Belly, R. T . , and Weiss, R. L. (1972). Su/folohu.s: A new genus of sulfur-oxidizing bacteria living at low pH and high temperature. Arch. Mierohiol. 84, 54-68. Brown, C . M., and Minnikin, D. E. (1973). The effect of growth temperature on the fatty acid composition of some psychrophilic marine pseudomonads. J . G m . Microhiol. 75, IX. Caplan, S. R., and Ginzburg, M., eds. (1978). “Energetics and Structure of Halophilic Microorganisms. ElsevieriNorth-Holland Publ., Amsterdam. Card, G. L. (1973). Metabolism of phosphatidylglycerol, phoaphatidylethanolamine, and cardiolipin of Btrcillrrs stecirothermophilus. J . Bucteriol. 114, I 1 25- I 137. Card, G. L., Georgi, C. E . , and Militzer. W . E. (1969). Phospholipids from Bacillirs .rtearotherinophilus. J . Bacteriol. 97, I 86- 192. Castenholz, R. W. (1979). Evolution and ecology of thermophilic microorganisms. In “Strategies of Microbial Life in Extreme Environments” (M. Shilo, ed.), pp. 373-392. Verlag Chemie, Weinheim. Chan, M . , Himes, R . H.. and Akagi, J . M . (1971). Fatty acid composition of thermophilic, mesophilic and psychrophilic cloatridia. J . Bacteriol. 106, 876-881. Chappe, B. (Chap Sim). Michaelis, W., Albrecht, and Ourisson, G. (1979). Fossil evidence for a novel series of archaebacterial lipids. Naturwissenschuften 66, 522-523. Cho, K. Y.,and Salton, M . R. J . (1966). Fatty acid composition of bacterial membrane and wall lipids. Bioehiin. Biophys. Acta 116, 73-79. Cullen, J . , Phillips, M. C.. and Shipley, G . G. (1971). The effectsoftemperature on the composition and physical properties of the lipids of Psriidotnonasfluorescens.Biochein. J . 125, 733-742. Darland. G . , and Brock. T. D. (1971 ). Bucillu.~trcirloc.alrlcrriIrs sp. nov., an acidophilic thermophilic spore-forming bacterium. J . Gen. Microhiol. 67, 9- 15. Daron, H. H. (1970). Fatty acid composition of lipid extracts of a thermophilic Bacillus species. J . Bacteriol. 101, 145-151. de Rosa, M., Gambacorta, A , , Minale, L., and Bu’Lock, J. D. (1972). The formation of w-cyclohexyl-fatty acids from shikimate in an acidophilic thermophilic bacillus. Biocheni. J . 128, 751-754. de Rosa, M., Gambacorta, A , , Minale, L.. and Bu’Lock J . D. (1973). Isoprenoids of Bacillrts ucidoculdurirrs. Phytocheniistry 12, 1 1 17- 1 123. de Rosa, M., Gambacorta, A , , and Bu’Lock, J . D. (1974). EffectsofpH and temperature on the fatty acid composition of Bacillus ricidoc~uldarirts.J . Bacteriol. 117, 212-214. de Rosa, M., Gambacorta, A., and Bu’Lock, J . D. (1975a).Extremely thermophilic acidophilic bacteria convergent with Sulfolohirs acidocaldurirts. J . Gen. Microbiol. 86, 156- 164. de Rosa, M., Gambacorta. A , , and Minale, L. (197%). A terpenoid 4.7-thianaphthenequinone from an extremely thermophilic and acidophilic micro-organism. J . Chem. Soc., Chem. Coiiirnun. pp. 392-393. de Rosa, M., Gambacorta. A , , and Bu’Lock, J . D. (1976a).The Caldariella group of extreme thermoacidophile bacteria: Direct comparison of lipids in Sulfi,lohus, Thermoplasmu, and the MT strains. Phyrochemistry 15, 143-145. de Rosa, M . , de Rosa, S . , Gambacorta, A , , and Bu’Lock, J. D. (1976b). Isoprenoid triether lipids from Caldariella. Phytocheiiiistry 15, 1996-1997. de Rosa, M., de Rosa, S . , and Gambacorta, A . (1977a). ‘:’C-NMR assignments and biosynthetic data for the ether lipids of Caldariellu. Phyrochcmistg 16, 1909-1912. de Rosa, M., de Rosa, S . , Gambacorta, A . , Minale, L., and Bu’Lock, J. D. (1977b). Chemical structure of the ether lipids of thermophilic acidophilic bacteria of the Calrluriella group. Phytochemistry 16, 1961-1965. de Rosa, M., de Rosa, S . , Gambacorta, A . , and Bu’Lock, J . D. (1980a). Structure of calditol, a new ”

72

THOMAS A. LANGWORTHY

branched-chain nonitol. and of the derived tetraether lipids in thermoacidophile archaebacteria of the Culduriellu group. Phytochernistp 19, 249-254. de Rosa, M., Gambacorta, A,, and Nicolaus, B. (1980b). Regularity of isoprenoid biosynthesis in the ether lipids of archaebacteria. Phytochernistry 19, 791 -793. de Rosa, M., Esposito, E., Gambacona, A,, Nicolaus, B., and Bu’Lock, J. D. (1980~).Complex lipids of Culdurieflu ucidophilu, a thermoacidophile archaebacterium. Phytochernistv 19, 821 -825. de Rosa, M., Eposito, E., Gambacorta, A,, Nicolaus, B., and Bu’Lock, J . D. (1980d). Effects of temperature on ether lipid composition of Culduriello ucidophilu. Phytochemistry 19, 827831. de Rosa, M., Gambacorta, A.. Nicolaus, B., Sodano, S . , and Bu’Lock, J . D. (1980e). Structural regularities in tetraether lipids of Culluriello and their biosynthetic and phyletic implications. P hytochernistry 19, 833-836. Dundas. 1. E. D. (1977). Physiology of the Hulubucteriaceue. Adv. Microh. Physiol. 15, 85-120. Evans, R. W . , Kushwaha, S . C., and Kates, M. (1980). The lipids of Hulobacteriurn rnurisrnorrui. an extremely halophilic bacterium in the Dead Sea. Eiochim. BiophJs. Arra 619, 533-544. Fox, G. E., Stackenbrandt, E., Hespell, R. B., Gibson, J., Maniloff, J., Dyer, T. A,, Wolfe, R . S., Balch, W. E., Tanner. R. S . . Magrum, L. J., Zablen, L. B., Blakemore, R., Gupta, R., Bonen, L., Lewis, B. J . , Stahl, D. A., Luehrsen. K . R., Chen. K . N., and Woese, C. R. (1980). The phylogeny of prokaryotes. Science 209, 457-463. Friedman, S . M., ed. (1978). “Biochemistry of Thermophily.” Academic Press, New York. Furuya, T . , Nagumo, T.. Itoh, T . . and Kaneko, H. (1980). The effect of growth temperature on the lipids in an extremely thermoacidophilic bacterium, TA-I. Agric. B i d . Chern. 44,517-521. Gill, C. 0 . .and Suisted. J . R. (1978). The effects of temperature and growth rate on the proportion of unsaturated fatty acids in bacterial lipids. J . Gen. Microhiol. 104, 31-36. Haines, T. H. (1971). The chemistry of the sulfolipids. Prog. Chern. Furs Other Lipids 2,297-345. Hasegawa. Y.. Kawada, N., and Nosoh, Y,(1980). Change in chemical composition of membrane of Eaciltus ctrltlotmux after shifting the growth temperature. Arch. Microhiol. 126, 103- 108. Heinen, U. J . , and Heinen, W. (1972). Characteristics and properties of a caldo-active bacterium producing extracellular enzymes and two related strains. Arch. Mikrubiul. 82, 1-23. Heinen, W.. Klein, H. P., and Volkmann, C. M. (1970). Fatty acid composition of Tlterrnrrs tiquuticu.~at different growth temperatures. Arch. Mikrohiol. 72, 199-202. of Microbial Adaptation. ” Heinrich. M. R . , ed. (1976). “Extreme Environments-Mechanisms Academic Press, New York. Hilker. D. R . , Gross. M. L.. and Knocke, H. W . (1978). The Interpretation ofthc mass spectrum of an ornithine-containing lipid from Thiohacillus thiooxidnns. Hiurned. Muss Spectrum. 5 , 64-7 I . Holzer, G . , Orb, J., and Tornabene, T. G. (1979). Gas chromatographic-mass spectrometric analysis of neutral lipids from methanogenic and thermoacidophilic bacteria. J . Chrornarogr. 186, 795-809. Inniss. W . E.. and Ingraham. J . L. (1978). Microbial life at low temperatures: Mechanisms and molecular aspects. In “Microbial Life in Extreme Environments” (D. J. Kushner, ed.), pp. 73-104. Academic Press, New York. Jackson, T . J . , Ramalcy. R . F., and Meinschein, W. G. (1973). Thcrrnornicrubiurn, a new genus of extremely thermophilic bacteria. Inr. J . Syst. Eac~trriol.2.1.28-36. Kannenbcrg. E . . Poralla, K.. and Blume, A. (1980). A hopanoid from thermo-acidophilic Eucillus ucidoculdurius condenses membranes. Nuturwissenchuften 67,458-459. Katcs, M. (1972). Ether-linked lipids in extremely halophilic bacteria. I n “Ether Lipids: Chemistry and Biology” (F. Snyder, ed.). pp. 351-398. Academic Press, New York.

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Kates, M. (1978). The phytanyl ether-linked polar lipids and isoprenoid neutral lipids of extremely halophilic bacteria. f r o g . Chtm. Futs Othor Lipids 15, 301-342. Kates, M . , and Deroo, P. W. (1973). Structure determination of the glycolipid sulfate from the extreme halophile Hu/ohoctrrium c.utiruhrum. J . Lipid Res. 14,438-445. Kates, M., and Hagen, P . - 0 . (1964). Influence of temperature on fatty acid composition of psychrophilic and mesophilic Srrrutiu species. Cun. J . Biochrm. 42, 481 -488. Kates. M., and Kushwaha, S. C. (1976). The diphytanyl glycerol ether analogues of phospholipids and glycolipids in membranes of Hulohrrcterium cutiruhrum. In “Lipids” (R. Paoletti, G . Procellati, and G. Jacini, eds.), Vol. I , pp. 267-275. Raven, New York. Kates, M., and Kushwaha. S . C. (1978). Biochemistry of the lipids of extremely halophilic bacteria, I n “Energetics and Structure of Halophilic Microorganisms” ( S . R. Caplan and M. Ginzburg, eds.), pp. 461-480. ElaevieriNorth-Holland Publ., Amsterdam. Knoche, H. W . , and Shively. J . M. (1969). The identification of cis-l I , 12-methylene-2hydroxyoctadecanoic acid from Thiolmcillus rhiooxid(rns. J . B i d . Chem. 244,4773-4778. Knoche, H. W.. and Shively. J . M. (1972). The structure of an ornithine-containing lipid from Thiob(1cillu.s thi[)[).~i(/~ln.s. J . Bioi. Cht~m.247, 170- 178. Korczynski, M. S . , Agate. A . D., and Lundgren, D. G . (1967). Phospholipids from thechemoautotroph Ferrohtrc.i//rrsfi.rroo.ri(/uns. Bioc.hc,m. BiophyJ, Ros. Commun. 29, 457-462. Kramer, J. K. C.. Kushwaka, S . C.. and Kates. M. (1972). Structure determination of squalene. dihydrosqualene and tctrahydrosqualene in Ha/ohucteriurn cutiruhrum. B i ( ~ ~ h i rBiophys. n. Actrr 270, 103-1 10. Kushner. D. J . , ed. (1978a). “Microbial Life in Extreme Environments.” Academic Press, New York. Kushner, D. J . (1978b). Life in high salt and solute concentrations: Halophilic bacteria. In “Microbial Life in Extreme Environments” (D.J . Kushner, ed.), pp. 3 17-368. Academic Press, New York. Kushwaha, S. C . , and Kates, M. (1973). Isolation and identification of “bacteriorhodopsin” and minor C ,,,carotenoids in HalohrrctcvYum curiruhrum. Riochim. Biopliy.~.Ac.tn 316, 235-243. Kushwaha, S . C., and Kates, M. (1978a). Mevalonic acid concentrations in halophilic bacteria. Phyrochrmisrry 17, 1793. Kushwaha, S. C., and Kates, M. (lg78h). 2.3-Di-0-phytanyl-sn-glyceroland prenols from extremely halophilic bacteria. Phvtocliernistry 17, 2029-2030. Kushwaha, S . C . , Pugh. E. L.. Kramer, J . K . G . . and Kates, M. (1972). Isolation and identification of dehydrosqualene and C ,,,-carotenoid pigments in Hulohuctrrium cutirubrum. Biochirn. Biophys. A m 260, 492-506. Kushwaha, S . C . , Gochnauer, M . B . . Kushner, D. J . , and Kates, M. (1974). Pigments and isoprenoid compounds in extremely and moderately halophilic bacteria. Can. J . Mic.robio/. 20, 24 I-245. Kushwaha, S. C., Kramer, J . K . C . . and Kates, M. (1975a). Isolation and characterization of C,,,carotenoid pigments and othcr polar isoprenoids from Hulohuc,tc,rium cutiruhrum. Biorhim. Biophy.~.Ai.ru 398, 303-3 14. Kushwaha, S. C., Kates, M., and Martin, W. G. (197%). Characterization and composition of the purple and red membrane from ~ ( i ~ ~ ) ~ r r ( .cutit-uhrum. ~ e r i ~ ~ , nCun. J . Biochrm. 53, 284-292. Kushwaha, S. C., Kates, M.. and Kramer, J . K . G. (1977). Occurrence of indole in cells of extremely halophilic bacteria. C‘un. J . Mic.rohiol. 23, 826-828. Kushwaha, S. C.. Kates. M.. Sprott, G . D., and Smith, I . C. P. ( I 98 la). Novel complex polar lipids froin the methanogen Mc,rhrrno.c/Jtril/umhungatei. S(,ienc,r 21 1 , I 163-1 164. Kushwaha, S. C., Kates. M . , Sprott. G . D.. and Smith, I . C. P. (I981b). Novel polar lipids from the methanogen Mc~thufiospirillu,,~ hungutei GPI. Bioc,him. Bioph,~.~. Acta 664, 156- 173.

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Langworthy, T. A. ( 1977a). Long-chain diglycerol tetraethers from Thermoplasmu ucidophilum. Bioclrim. Biophys. Acru 487, 37-50. Langworthy, T. A. (l977b). Comparative lipid composition of heterotrophically and autotrophically grown Sulfolobus acidocaldurius. J . Bacteriol. 130, 1326- 1332. Langworthy, T. A . (1978a). Microbial life in extreme pH values. In “Microbial Life in Extreme Environments” (D. J. Kushner, ed.), pp. 279-315. Academic Press, New York. Langworthy, T. A. ( I 978b). Membranes and lipids of extremely thermoacidophilic microorganisms. In “Biochemistry of Thermophily” (S. M. Friedman, ed.), pp. 11-30. Academic Press, New York . Langworthy, T . A. (1979a). Special features of thennoplasmas. In “The Mycoplasmas” (M. F. Barile and S. Razin, eds.), Vol. I , pp. 495-513. Academic Press, New York. Langworthy, T. A . (l979b). Membrane structure of thermoacidophilic bacteria. In “Strategies of Microbial Life in Extreme Environments” (M. Shilo, ed.), pp. 417-432. Verlag Chemie, Weinheim. Langworthy, T . A. ( 1 980a). Archaebacterial membrane assembly. i n “Dissipative Structures and Spatistemporal Organization Studies in Biomedical Research” (G. P. Scott and J. M. McMillin, eds.), pp. 82-102. Iowa State Univ. Press, Ames. Langworthy, T . A. (1980b). Turnover of di-0-phytanyl glycerol in Thermoplasma. Absrr. Conf. In?. Org. Mycoplasmol. 3rd, 1980, p. 151. Langworthy, T. A. (1981). Diglyceryl tetraether lipids. In “Ether Lipids: Biomedical Aspects” (H. K . Mangold and F. Paltauf. eds.). Academic Press, New York (in press). Langworthy, T. A.. and Mayberry, W. R . (1976). A I ,2,3,4-tetrahydroxy pentane-substituted pentacylcic triterpene from Bacillus acidocddarius. Biuchirn. Biophys. Actu 431, 570-577. Langworthy, T. A., Smith, P. F., and Mayberry, W. R. (1972). Lipids of Thermoplusmcc ucidophilum. J . Racreriol. 112, 1 193-1200. Langwonhy, T . A., Mayberry, W. R . , and Smith, P. F. (1974). Long chain diether and polyol dialkyl glycerol triether lipids of Sulfolohus acidocaldurius. J . Bacteriol. 119, 106- I 16. Langworthy, T. A., Mayberry, W. R . , and Smith, P. F. (1976). A sulfonolipid and novel glucosamidyl glycolipids from the extreme tbermoacidophile Bucillus acidocaldarius. Biochim. Biophys. Actu 431, 550-569. Lanyi. J . K . (1979). Physiochemical aspects of salt-dependence in halobacteria. In “Strategies of Microbial Life in Extreme Environments” (M. Shilo, ed.), pp. 93-107. Verlag Chemie, Weinheim. Levin, R. A. (1971). Fatty acids o f Thir~liacillusthiooxidans. J . Bucreriol. 108, 992-995. Levin, R. A. (1972). Effect of cultural conditions on the fatty acid composition of Thiobacillus novellus. J . Bacreriol. 112, 903-909. Ljungdahl, L. G . (1979). Physiology of thermophilic bacteria. Adv. Microb. Physiol. 19, 149-243. Lundgren, D. G., Andersen, K. J., Remsen, C. C., and Mahoney, R. P. (1964). Culture, structure and physiology of the chcmoautotroph Ferrohacillus ferrooxidans. Drv. I d . Microbial. 6 , 250-259. Lundgren, D. G . , Vestal, J . R . , and Tabita, F. R. (1974). The iron oxidizing bacteria. In “Microbial Iron Metabolism” (J. B. Neilands, ed.), pp. 457-473. Academic Press, New York. Makula. R. A., and Singer, M. E. (1978). Ether-containing lipids of methanogenic bacteria. Bioclzrm. Biophys. Res. Commun. 82,716-722. Marr, A. G., and Ingraham, J. L. (1962). Effect of temperature on the composition of fatty acids in Esc-herichia c d i . J . Brwreriol. 84, 1260- 1267. Mayberry-Carson, K. J . , Langworthy, T. A , , Mayberry, W. R., and Smith, P. F. (1974). A new class of lipopolysaccharide from Thrrmoplasma ucidophilum. Biochim. Biophy,r. Acta 360, 2 17-229.

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Mayberry-Carson, K . J . . Roth, 1. L . , and Smith, P. F. (1975). Ultrastructure of lipopolysaccharide isolated from Thrrmopkrsmti trcidophihm. J . Bncteriol. 121, 700-703. Mayberry-Carson, K . J . , Jewell. M. J . , and Smith, P. F. (1978). Ultrastructural location of Thermop/osmcr trcidophilum surface carbohydrate by using concanavalin A. J . Bac~teriol. 133, 15 10-15 13. Mercer, E. I . , Modi, N., Clarke, D. J . . and Morris. J. C. (1979). The occurrence and location of squalene in Clostridium pusrcrtn‘unum. J . Gon. Miuohiol. 1 1 1, 437-440. Merkel, G . J . , and Perry, J. J . (1977). Effect of growth substrate on thermal death of thermophilic bacteria. Appl. Environ. Microhiol. 34, 626-629. Michaelis, W . , and Albrecht. P. (1979). Molecular fossils of Archaebacteria in kerogen. Naturwissenshufrcn 66, 420-422. Moldowan, J . M., and Seifert, W. K . (1979). Head to head linked hydrocarbons in petroleum. S c i r n w 204, 169- 17 1 Morita. R. Y. (1975). Psychrophilic bacteria. Bacterial. R t v , 39, 144-167. Oesterhelt, D . , and Stoeckenius, W . (1973). Functions of new photoreceptor memhranc. Proc. N u t / . Aeud. Sci, U . S . A . 70, 2853-2857. 00,K. C . , and Lee. K . L. ( 1971). The lipid content of RociMus .Ffearothrrrnophifusat 37“and at 55”. J . G m . Microhiol. 69. 287-289. Oshima, M . (1978). Structure and function of membrane lipids in thermophilic bacteria. I n “Biochemistry of Thermophily” ( S . M. Friedman. ed.). pp. 1-10. Academic Press, New Y ork . Oshima, M., and Ariga, T . (1975). w-Cyclohexyl fatty acids in acidophilic thermophllic bacteria. J . B i d . Chem. 250, 6963-6968. Oshima, M . , and Ariga, T. (1976). Analysis of the anomeric configuration of a galactofuranose containing glycolipid from an extreme thermophile. FEBS Lrtt. 64, 440-442. Oshima, M., and Miyagawa, A. (1974). Comparative studies on the fatty acid composition of moderately and extremely thermophilic bacteria. Lipids 9 , 476-480. Oshima, M., and Yamakawa, T . (1974). Chemical structure of a novel glycolipid from an extreme thermophile, Flnv~htrc~terium thc~rmophilun7.Biochrmistry 13, I 140- I 145. Oshima, M . , Sakaki, Y . . and Oshima. T . (1978). w-Cyclohexyl fatty acids in acido-thermophilic bacterial membranes and phage capsids. In “Biochemistry of Thermophily” ( S . M. Friedman. ed.), pp. 31-44. Academic Press, New York. Oshima, T.. and Imahori, K. (1974). Description of Thmnus /h<wmophi/rr.s(Yoshida and Oshima) Comb. nov.. a non-sporulating thermophilic bacterium from a Japanese thermal spa. Ini. J. Syst. Bac,tc,riol. 24, 102- I 12. Ourisson, G . , Albrecht. P , and Rohmer. M. (1979). The hopanoids. Pure Appl. Chcm. 51, 709729. Pask-Hughes. R. A . , Mozaffdry, H . , and Shaw, N. (1977). Glycolipids in procaryotic cells. Bioc,hrm. Soc.. T r m r . 5 , 1675-1677. Phillips, W. E., Jr.. and Perry, J . J. (1976). Thmnomicwhium fi).sfrri sp. nov. a hydrocarbonutilizing obligate thermophile. IN/. J . Sjw. Boc.;eriol. 26, 220-225. Pordk+, K . , Kannenberg, E . , and Blume, A. (1980). A glycolipid containing hopanc isolated from the acidophilic, thermophilic Bac~illu.rnc,idoccildrrriu.s, has a cholesterol-like function in mcmbranes. FEBS Lcrt. 113, 107-1 10. Ray, P. H., White, D. C.. and Brock. T. D. (1971a). Effect of temperature on the fatty acid composition of Therrnu.\ ciquuticu. J . Bnctrriol. 106. 25-30. Ray, P. H . , White, D. C., and Brock, T . D . (1971b). Effect of growth temperature on the lipid composition of Therrnus tiqictrtic~rrs.J . Btrc./c,riol. 108, 227-235. Rohmer, M., Bovier. P . . and Ourisson. C. (1979). Molecular evolution of biomembranes: Structural

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cquivalents and phylogenetic precursors of sterols. Prnc. N u t / . Accrd. P i . U.S.A. 76, 847851. Russell. N. J (1971). Alteration in fatty acid chain length in MicrocYJc.cus c,ryophilu.s grown at different temperatures. Biochim. B i o p h y . A&i 231, 254-256. Saiki. T . . Kimura. R., and Arima, K. (1972). Isolation and characterization of extremely thermophilic bacteria from hot springs. A g r i c . B i d . Chem. 36, 2357-2366. Sharom. F., Barratt, D. G . , Thede, A. E., and Grant, C. W. M. (1976). Glycolipids in model membranes; spin label and freeze-etch studies. Bioc-him. Biophys. Acra 455, 485-492. Shen. P. Y.. Coles, E . , Foote, J . L., and Stenesh, J . (1970). Fatty acid distribution in mesophilic and thermophilic strains of the genus Burillus. J. Rucreriol. 103, 479-48 1. Shilo, M., ed. ( I 979). “Strategies of Microbial Life in Extreme Environments.” Verlag Chemie, Weinheim. Shively, J . M.. and Benson, A. A . (1967). Phospholipids of Thiohacillus rhiooxiduns. J. Bac’furicd. Y4, 1679- 1683. Shively, J . M., and Knoche. H. W. (1969). Isolation of an ornithine-containing lipid from Thiohuc~il/us rhiotzriilrrn.s. J. Bocteriol. 98, 829-830. Short, S . A., White, D. C., and Aleem, M. I. H. (1969). Phospholipid metabolism in Ferrohuri/lirs ferrooxiduns. J. Brrc,terio/. 99, 142-1 50. Sinclair. N. A . , and Stokes, J. L. (1964). Isolation of obligately anaerobic psychrophilic bacteria. J. Bucrrriol. 87, 562-565. Smith. P. F. (1980). Sequence and glycosidic bond arrangement of sugars in lipvpulysaccharide from Tliermoplasmu acidophilum. Biochim. Biuphys. Actti 619, 367-373. Smith, P. F., Langworthy, T. A , , Maybeny, W. R., and Houghland, A. E. (1973). Characterization of the membranes of Thermopkusmci uckhphilitm. J. Barrerid. 116, 1019-1028. Smith, P. F., Langworthy, T . A , , and Mayberry, W. R. (1976). Distribution and composition of lipopolysaccharides from mycoplasmas. J. Bacferiol. 125, 9 16-922. Tansey, M. R . , and Brock, T. D. (1978). Microbial life at high temperatures: Ecological aspects. In “Microbial Life in Extreme Environments”(D. J . Kushner, ed.). pp. 160-216. Academic Press, New York. Tornabene. T . G . (1978). Non-aerated cultivation of Halohac~reriumcutirrthrum and its effect on cellular squalenes. J . M o l . Evol. 11, 253-257. Tornabene, T . G . , and Langworthy, T . A . (1979). Diphytanyl and dibiphytanyl glycerol ether lipids of methanogenic archaehacteria. Science 203, 5 1-53. Tornahene, T . G.. Katcs, M . , Gelpi, E., and Oro, J . (1969). Occurrence of squalene, di-, and tetrahydrosqualenes and vitamin MK, in an extremely halophilic bacteriuni, Halobacterium cmriru/wurn. J. Lipid Res. 10, 294-303. Tomabene, T . G . , Wolfe, R. S . , Balch, W. E., Holzer, G., Fox, G. E., and Orb, J . (1978). Phytanyl-glycerol ethers and squalene in the archaebacterium Methrmohucteriurn thtrmotrutorrophicum. J . M u / . Evol. 1 1 , 259-256. Tornabene, T . G . , Langworthy, T. A.. Holzer, G., and Orb, J . (1979). Squalenes, phytanes and other isopranoids as major neutral lipids of methanogenic and thermoacidophilic “archaebacteria.” J . M i d . Evol. 13, 73-83. Tuovinen, O . , and Kelly. D. P. (1978). Metabolic transitions in cultures of acidophilic thiobacilli. In “Metallurgical Applications of Bacterial Leaching and Related Microbiological Phenomena” (L. E. Murr, A . E. Torma, and J . A . Brierley, eds.), pp. 61-81. Academic Press, New York. Weerkamp, A,. and Heinen, W . (1972). Effect of temperature on the fatty acid composition of the extreme thermophiles. Bucillus c.rrl&J/-vticus and 3uci//irs culdormur. J . Burteriiil. 109, 443446. Woese, C. R., and Fox, G. E. (1977). Phylogenetic structure of the prokaryotic domain: The primary kingdoms. Proc. Nut/. Acnd. Sci. U.S.A. 74, 5088-5090.

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Woese. C. R . , Magrum, L. J . , and Fox, G. E. (1978). Archaebacteria. J . M o l . Evol. 11, 245-252. Yang, L. L., and Haug, A . (1979). Structure of membrane lipids and physico-biochemical properties of the plasma membrane from Thermoplusmu ui,idophi/um. adapted to growth at 37°C. Biochim. Biophys. Acta 513, 308-320. Yao, M., Walker, H. H., and Lillard, D. A . (1970). Fatty acids from vegetative cells and spores of Bac,i/lus stL.arothermt,philus. J . Bacteriol. 102, 877-878. Zeikus, J . G. (1977). The biology of methanogenic bacteria. Bucteriul. Rev. 41, 514-541. Zeikus, J . G.( 1979). Thermophilic bacteria: Ecology, physiology and technology. Enzyme M i i w h . Ti,chno/. I , 243-251. Zeikus, J . G., and Wolfe, R . S. (1972). Mrth~inobai,teriumthermoautotrophicum sp. n., an anaerobic. autotrophic extreme thermophile. J . Bacteriol. 109, 707-7 13. Zuber, H., ed. (1976). “Enzymes and Proteins from Thermophilic Microorganisms.” Birkhaeuser, Basel.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 17

Lipo polysacc harides of G ram-Neg ative Bacteria OTTO LUDERITZ, MARINA A . FREUDENBERG, CHRIS GALANOS, VOLKER LEHMANN, ERNST TH. RIETSCHEL, A N D DEREK H . SHAW* Mux-PIunc,k-lnstitut f u r Irnmunhiologic, Freihurg, Federul Repuhlic of' Gertnrrn~ und

*Northwesf Atlantic Fisheries Centre John's, Newfoundlund, Cunudu

St.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . Isolation, Structure, and Biosynthesis of Lipopolysaccharides . . . . . . . A. Isolation and Purification of Lipopolysaccharides . . . . . . . . . . B. Structure and Biosynthesis of the 0 Chains . . . . . . . . . . . . C . Structure and Biosynthesis of the Core . . . . . . . . . . . . . D. Structure and Biosynthesis of Lipid A . . . . . . . . . . . . . . 111. Some Selected Aspects on the Biology of Lipopolysaccharides . . . . . . A. Endotoxic and Immunogenic Properties of Lipid A . . . . . . . . . B. Physicochemical and Structural Prerequisites for Biological Activities of Lipopolysaccharide . . . . . . . . . . . . . . . . . . . . . . . C . The Fate of Lipopolysaccharides (Lipid A) in Experimental Animals . . IV. Final Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . I.

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INTRODUCTION

Lipopolysaccharides (LPS) form a large, unique class of macromolecules representing a characteristic attribute of gram-negative bacteria. Associated with proteins, they are located in the outer leaflet of the outer membrane of the bacterial cell (Nikaido and Nakae, 1979). In this exposed position on the cell surface, lipopolysaccharides are involved in the interaction of the cell with the environment. Thus contact of the bacterium with the immune system leads to the stimulation of specific antibodies directed predominantly against determinant 79

Copyright 0 1982 hy Academic Press, lnc All rights of reproduction in any form reserved ISBN 0-12-153317-4

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structures of the lipopolysaccharide. Hence, lipopolysaccharides represent the main surface antigens of gram-negative bacteria; these antigens have for many years been known as the 0 antigens (Morgan and Partridge, 1941; Tal and Goebel, 1950; Luderitz ei al., 1966b). A synonymously used term for lipopolysaccharide is endotoxin. The injection of lipopolysaccharide-containingbacteria or of purified lipopolysaccharide into experimental animals causes a wide spectrum of so-called endotoxic reactions. In contrast to the specific, delayed immune response, these effects are, in general, nonspecific and acute, and would include such phenomena as fever, changes in the white cell counts, shock, and death after high doses of lipopolysaccharide (Kadis et al., 1971; Kass and Wolff, 1973; Schlessinger, 1977). Lipopolysaccharides have attracted scientific interest in the past mainly because of their 0-antigenic and endotoxic properties. Lipopolysaccharides often act as specific receptor sites for bacteriophages, and this system has been intensively studied as a model for phage-bacterium interaction (Lindberg, 1977; Braun and Hantke, 1981). The important physiological role of lipopolysaccharide itself has recently been recognized with the demonstration that the outer membrane of the cell acts as a barrier for passive penetration of various compounds (Nikaido, 1979; Nikaido and Nakae, 1979). Low-molecular-weight nutrients and excretion products (< 600 daltons) may pass through the barrier but products of higher molecular weight (such as antibiotics and other poisons) are prevented from crossing the membrane. Lipopolysaccharides are essential for maintaining the integrity of the cell wall (Henning, 1975). Bacteria that, due to a mutational defect, are not able to synthesize lipopolysaccharide, are no longer able to reproduce, and cease growing. Another quality of lipopolysaccharides has been elucidated. It appears that lipopolysaccharides are of importance in the normal physiological bacterium-host relationship. Lipopolysaccharides have the ability to activate and suppress lymphocyte functions (Melchers, 1980; Koenig and Hoffmann, 1979; McGhee et d.,1980). They stimulate polyclonal B lymphocytes to differentiation, proliferation, and secretion of immunoglobulin. This may have special significance, when the omnipresence of gram-negative bacteria in the gut and in the environment is considered. Humans and animals must deal with lipopolysaccharides throughout their lives. The fact that in germ-free animals the immune system is poorly developed indicates that the host requires this continuous exposure for the development of vital physiological systems, such as the immune apparatus (see Schwab, 1977). In the interrelationship of plants and gram-negative bacteria the specific role of rhizobia lipopolysaccharides is presently under active discussion (Wolpert and Albersheim, 1976). There is evidence that the binding of the bacterial cells to the roots is mediated by specific interactions of the lipopolysaccharides with lectins of the plants (see Section Il,B,4). In summary, the lipopolysaccharides of gram-

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negative bacteria exhibit a plurality of effects and functions. They represent the 0 antigens, they are the endotoxins expressing their harmful activities during infection, they have indispensable functions for the bacterium and, possiblywhen symbiosis with animals and some plants is considered-also for the host. Just how this class of macromolecules is capable of inducing such a wide variety of effects has been the subject of considerable investigations and speculation. Now, since the structural principles of many lipopolysaccharides have been evaluated, more sophisticated attempts can be undertaken, both to identify biologically active moieties in the molecule, and to study the mode of action of lipopolysaccharide, when it acts as an antigen, an adjuvant, a toxin, and a factor indispensable for !he life of bacteria, and possibly also for the life of other organisms. Of the various lipopolysaccharides studied to date, those of Sulrnonellu are probably the most thoroughly investigated. The results obtained with Salmonetlu lipopolysaccharides may serve as the base to which results on lipopolysaccharides from other bacterial families may be compared. Figure 1 shows the schematic representation of the structure of Salmonella lipopolysaccharides. It contains a lipid region, the lipid A, and a long, covalently linked heteropolysaccharide that, according to composition, structure, and mode of biosynthesis, can be subdivided into the core and the 0-specific chain. These three regions are not only distinct in their chemical structure, but also in their biological and functional properties. In the course of this chapter, we will illustrate that the viability of the cells is dependent on a minimal core-lipid A structure, the 0-antigenic specificity is determined by structures of the 0 chain, and the endotoxic principle of the molecule is expressed by lipid A . The first part of this chapter (Section 11) describes general aspects of the

1

0 Monosaccharide,

---

P h o s p h a t e , ru

Ethanolamine

Long C h a i n l H y d r o x y l F a t t y Acid

FIG. I. Schematic structure of Sulmonelllr lipopolysaccharides. The number of nonhydroxylated and hydroxylated fatty acids given in this scheme is arbitrary.

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0-specific chains and the core, and principles of their biosynthesis; the structure of Salmonella lipid A will then be discussed, together with the present knowledge of its biosynthesis; and, finally, lipid A's of other gram-negative bacteria, which have recently been investigated will be described. The second part of the chapter (Section 111) will deal with some selected aspects of the biological properties of lipopolysaccharides.

11.

ISOLATION, STRUCTURE, AND BIOSYNTHESIS OF LIPOPOLYSACCHARIDES

A. Isolation and Purification of Llpopolysaccharides A number of extraction procedures for the isolation of lipopolysaccharides or lipopolysaccharide-protein complexes have been described (for a summary, see Galanos er al., 197713; Wilkinson, 1977). The method of choice is usually the phenoUwater procedure (Westphal et al., 1952), which yields a water-soluble extract that is subsequently purified by high-speed centrifugation. These lipopolysaccharide preparations in general contain small amounts of contaminants, such as protein (about 1%). It has occasionally been observed that lipopolysaccharides, which are more lipophilic in nature, partition mainly into the phenol phase during phenol/water extraction. This is also true for lipopolysaccharides from R(rough-)-form bacteria. For the extraction of R mutants, the phenol/chloroform/petroleum ether procedure has been proved to be highly efficient and specific (Galanos et al., 1969; Galanos and Liideritz, 1982). Water-soluble preparations of high purity are thus obtained. Because of the presence of carboxyl, phosphoryl, and ethanolamine residues in the molecule, lipopolysaccharides are amphoteric. The overall charge is negative. In the original lipopolysaccharide preparation, negatively charged groups are neutralized by Na+, K + , Mg2+, and Ca2+, the mixture of cations being variable depending on the culture medium used. In addition, polyamines synthesized by the bacterium are present. It has been found that the nature of cations present in lipopolysaccharides greatly influences their state of aggregation and, as a consequence, their biological activities (see Section III,B, 1). For special purposes, therefore, i t IS useful to convert lipopolysaccharides into a uniform salt form. This is achieved by electrodialysis of the lipopolysaccharide, whereby cations are removed, and the acidic form of the lipopolysaccharide is obtained. Neutralization with base then leads to a defined salt form of the lipopolysaccharide (Galanos and Luderitz, 1975). Recently, the purified lipopolysaccharide of Salmonella abortus equi has been prepared through a number of specific purification steps, including electrodialysis and conversion to the sodium salt form (Galanos et al., 1 9 7 9 ~ ) .This highly purified S . abortus eyui lipopoly-

LIPOPOLYSACCHARIDES OF GRAM-NEGATIVE BACTERIA

83

saccharide is available as a standard prepartion in the form of ampules containing 1.0 and 0.1 pg/ml of LPS under the name of Novo-Pyrexal (Henna1 Chemie, K . Hermann. Reinbeck B. Hamburg). It will also soon be available in solid form. It should be kept in mind that in nature lipopolysaccharides d o not occur in pure form. Lysis of bacteria leads to either water-soluble or insoluble complexes containing lipopolysaccharide, proteins, phospholipids, and other components of the bacterial cell. Such complex mixtures are representative of natural endotoxin. Since all the components may exhibit their own endotoxin or endotoxin-like effects, and, moreover, may act synergistically or inhibitory, it is of prime importance to use clearly defined preparations when the mechanisms of endotoxin effects or relationships between structure and biology are to be studied. Lipopolysaccharide must be free of contaminants, proteins, murein, and nucleic acids or, alternatively. defined mixtures of the components should be used. This is a prerequisite for obtaining reproducible results in biological investigations.

B. Structure and Biosynthesis of the 0 Chains 1 . STRUCTURE OF 0 CHAINS

As indicated in Fig. I , the 0-specific chains of lipopolysaccharides are made up of repeating units of identical oligosaccharides (Robbins and Uchida, 1962). These units usually contain different constituents, thus the 0 chain represents a heteropolysaccharide. In some cases, however, the repeating units may contain an oligomer of a single sugar type though i n a distinct linkage sequence, hence repeating units can also be recognized. In these cases the 0 chain represents a homopolysaccharide, for instance, a mannan in Escherichia coli 09 (Prehm et al., 1976b), and a galactan in Klebsiellu 08 (Curvall et ul., 1973). It should be noted that 0 chains have also been identified containing a homopolymer with only one type of linkage such as the PI ,2-linked poly 6-deoxy-~-altropyanose isolated from Yersiniu rnterocolifica (Hoffman et u / . , 1980a). The 0 chains contain the irnmunodeterminant structures against which the anti-0 antibodies formed during infection or on immunization are directed (Luderitz e f ul., 1966b, 1971). Each bacterial serotype synthesizes a unique lipopolysaccharide, characterized by a specific composition and structure of the 0 chain, and by an individual 0 antigenicity. There consequently exist in nature as many distinct lipopolysaccharides as there exist bacterial serotypes. This number is certainly very high. But as far as we know today, irrespective of their individual detailed composition and structure, all lipopolysaccharides are built up according to the structural principle illustrated in Fig. I , with lipid A , core, and 0 chain. Figure 2 shows, as an example, the structure of the lipopolysaccharide of S. fyphirnuriurn. As indicated, lipid A, core, and 0 chain are interlinked by

84

I- -

OTTO LUDERITZ ET AL.

- -

-

-

--

- - - - I Core

Poiysocchrrde (kgron

i7)

1- - - - - - -

- -

- -I

/

8 I‘

D-GlcN- ( F A )

D-GlcNp- ( F A )

k

-

Lipid A

(Region

z)1- -1

FIG. 2. Structure of the lipopolysaccharide of Sa/tnnnellu fvphimurium (0-antigen factors 4,5.12), as derived from studies by the groups of A . M. Staub, M . J . Oshorn, H. Nikaido, P. W . Robbins, B. Lindberg, and the authors of this chapter. Dotted linkages, incomplete substitutions;_p, pyranose form; L-cr-D-Hep, L-glycero-rr-u-munnoheptose-equivalent to p-L-o-Hep or p-Hep; KDO, 2-keto-3-deoxyoctonate: P, phosphate; FA, fatty acids.

covalent linkages. The 0 chain contains repeating units of a pentasaccharide with mannosyl, rhamnosyl, and galactosyl residues in the main chain, and acetylabequosyl and glucosyl residues as branches. The oligosaccharide (I), which is formed and polymerized during biosynthesis, represents the “biological” repeating unit. Due to the acid lability of the rhamnosyl (and abequosyl) linkage, mild acid hydrolysis of this lipopolysaccharide yields the “chemical” repeating unit (2) with a different sequence (Luderitz et ul., 1966b). Chemical analysis of lipopolysaccharides in general will reveal chemical repeating units, and only by chance are they identical with the biological ones. A be

1

Man

Glc

1

Gal

Glc +

1

Rha (1)

.--f

Gal

(Abe) +

1

Man (2 )

+

Rha

85

LIPOPOLYSACCHARIDES OF GRAM-NEGATIVE BACTERIA

The S. typhimuriurn 0-antigen factors 4.5, and 12 of the Kauffmann-White Salmonella classification scheme are determined primarily by the immunodominant sugars D-abequose , 2- 0-acetyl-D-abequose and D-glUCOSe, respectively. The complete antibody-combining sites (0 factors) comprise the immunodominant sugars together with the adjacent 3 to 4 sugar units in their distinct conformations (Luderitz ef al., I97 1 ; Nghiem and Staub, 1975). In recent years, an increasing number of lipopolysaccharides from various bacterial families have been studied with respect to their 0 chains, and a great diversity of constituents, linkage types, and structural peculiarities has become apparent. During this time, many new sugar classes and sugar derivatives have been discovered, together with sugar alcohols, acidic compounds, and even amino acids (Wilkinson, 1977; Jann and Westphal, 1975; Jann and Jann, 1977; Weckesser el al., 1979). In the past, more than a decade was required for the complete analysis of one 0 chain (e.g., S. rjphirnuriurn). Thanks to the advances made in carbohydrate chemistry and particularly in structural analysis, these studies are now accomplished much more efficiently. B. Lindberg in Stockholm and N. Kochetkov in Moscow and their schools have elaborated new methods of polysaccharide analyses, especially “methylation analysis’’ and nuclear magnetic resonance spectrometry (Lindberg e f a l . , 1975; Lindberg, 1979; Kochetkov and Chizhov, 1966; Jennings and Smith, 1980). There are few laboratories in the world performing structural studies on lipopolysaccharides that have not cooperated with one of these laboratories, and an increasing number of lipopolysaccharide structures will be available in the future. Table I lists about 70 gram-negative serotypes, which have had the detailed structure of their 0 chains determined mainly during the past 10 years (reviewed by Wilkinson, 1977; Orskov erul.. 1977; Jann and Jann, 1977, 1978). Similarly, a great number of bacterial capsular antigens have been investigated (Jann and Jann, 1977). ~

2 . HETEROGENEITY O F 0 CHAINS The true repeating units (Abe-Man-Rha-Gal of Fig. 2 ) in 0 chains are strictly constant. In contrast, modifying substitiients (e.g., acetyl groups), and sidechain sugars (e.g., glucose) added in later steps of biosynthesis (see Section 11,B,3), are often not present in equimolar ratios (Wright and Kanegasaki, 1971). Furthermore, their presence or absence is often subject to variation (e.g., antigenic conversion by phages). This incomplete substitution of 0 chains is one of the causes for microheterogeneity of lipopolysaccharides. Another important type of‘ heterogeneity of 0 chains has recently been revealed. Hurlbert, Makela, and Leive and their co-workers (Hurlbert and Hurlbert, 1977; Palva and Makela, 1980; Goldman and Leive, 1980; see also Jann ef al., 1975) have independently shown that radiolabeled lipopolysaccharide separates on SDS-polyacrylamide electrophoresis into a number of de-

o n 0 LUDERITZ ET AL.

86

TABLE I BACTERIAL SEROTYPES WHOSE0 - C H A I N STRUCTURE HASBEENEVALUATED 0 Chains Derived from

Salmonella S . purutvphi A S . typhimurium

S. ryphimuriitrn Col 11 S . bredenev

S. rnreritidis S . strusbourg S . zuerich S . muenstrr S . anntum

S. newingroti S . illinois S . simftenherg

S.friedrnau S . minnc~sotu S . goclrsherg S . mil ~ a u k e e

Escherichia E . coli 08 09 020 032 058 069 075 086 0100 0111 0124 0141

References

Hellerqvist et a / . (1971~) Hellerqvist i’t ul. (1968, 1969~); Liideritz et crl. (1966b) Hoffmann et ul. ( I980b) Bagdian et d.(1966); Hellerqvist et d. ( I969b) Hellerqvist r t d . (1970h. 1971a. 1 9 7 2 ~ ) Hellerqvist e t a / . (1970b, 1971a. 1972~) Tinelli and Staub (1960); Bagdian et ul. ( 1967) Hellerqvist et ( I / . (1969a, 1971d) Hellerqvist et al. (1970a. 1971d) Nghiem and Staub ( I 975) Hellerqvist et i d . ( I97 I e) Robbins and Uchida (1962); Hellerqvist OI d.(1971b) Robbins and Uchida (1962); Hellerqvist et c d . ( 197 1 b) Robbins and Uchida (1962) Staub and Girard (1965); Hellerqvist et u / . (19710 Simmons et N / . (1965a) Liideritz rt a / . ( I 966a) Simmons et ul. (1 96%) Liideritz et id. (1965)

Reske and Jann ( I 972) Prehm et a / . ( 1 976b) Vasiliev and Zakharova (1976) Jann et ul. (1971) Dmitriev et a / . ( 1 9 7 7 ~ ) Erbing et al. (1977a) Erbing et ul. (1978) Springer (1971) Jann et a / . (1970) Edstrom and Heath (1967) Dmitriev et al. (1976a) Jann i’t ul. (1966)

Shigella Sh. dysenteriae Type 1

Dmitriev et al. (1976b) (continued)

87

LIPOPOLYSACCHARIDESOF GRAM-NEGATIVE BACTERIA

TABLE I (Continued) 0 Chains Derived from

Type 4 Type 5 Type 6 Type 8 Type 9 Type 10 Sh.jlexneri All known serotypes and aubserotypes Variant X Variant Y

References Dmitriev rt a / . (1977a) Dmitriev rt ul. (1977d); Kochetkov et n l . (1977) Dmitriev et ul. ( 1 977e) Dmitriev et ul. (l977b) Dmitriev et ul. (1975h) Dmitriev et ul. (1978a) Dmitriev er (11. (1978h) Dmitriev et ul. (19770 Lindberg rt ul. ( 1973) Kenne et ul. (1977a,b, 1978a)

Sh. hoydii

Type 4 Sh. boydii Type 6 Sh. sonnei Phase I Phase I1 Sh. newcastle

Lvov et nl. (1980) Dmitriev rt ul. ( I 9753) Kenne et ul. (1980) Kontrohr and Kocsis (1978) Dmitriev et crl. (1979)

Citrobacter Cirrobacter sp. 396

Jann et ul. (1978)

Klebsiella All known 0 groups 1 - I2

Erhing et ul. (1977h)

Serratia S.murcescens 08 Bizio

Tarcsay et ul. (1973) Wang and Alaupovic (1973)

Proteus P . mirabilis (D52)

Gmeiner ( 1977)

Yersinia Y . pseudotuberculosis All 10 known sero groups and subgroups (I-1V) Type 111 (pathogenic strain) Y . enterocoliticu Ye 128

Samuelsson et 01. ( 1974) Hellerqvist et u l . (1972a,h) Gorshkova et a / . (1980) Hoffman rt ul. ( 1980a)

Vibrio V . cholerue (Inabe)

Redmond (1979); Kenne et ul. (1978b)

Pseudomonas Ps. ueruginosu group 7 Pr. maltophilia P s . cepuciu

Dmitriev et a / . (1980) Neal and Wilkinson (1979) Knirel et a / . (1980)

88

OTTO LUDERITZET AL.

fined (double) bands. From their investigations, these authors conclude that a lipopolysaccharide preparation consists of a family of molecules differing in the length of the 0 chain, that is, in the number of repeating units. This method reveals the presence of free core-lipid A (i.e., lipopolysaccharide devoid of repeating units) and a spectrum of species with up to 40 repeat units. In the lipopolysaccharide of a S. t ~ ~ ~ strain ~ ~(Palva i and ( ~Makela, ~ ~ 1980), ~ ~ for? instance, there exists an accumulation of lipopolysaccharide species with 20 to 35 repeat units (30%), but also a large proportion of lipopolysaccharide with unsubstituted core (60%); double bands are assumed to be due to differing phosphate substitutions in the core or in lipid A . The distribution of lipopolysaccharide species is dependent on the strain and on growth conditions. This method has disclosed new aspects of the microheterogeneity of 0 chains and certainly will be used for revealing new structure-function relationships, e.g., the role of 0-chain length in virulence. 3. PRINCIPLES O F 'THE BIOSYNTHESIS OF 0 CHAINS

0-chain biosynthesis is catalyzed by a multienzyme system that utilizes a membrane-bound polyisoprenoid compound as a carrier for the glycosyl residues. In Sufrnoneliu groups A , D, E (Robbins and Wright, 1971; Wright and Kanegasaki, 1971) and C,, C, (Shibaev e t u l . , 1979), the synthesis, according to the now classical pathway, begins with the reversible transfer of galactose I -phosphate from UDPgalactose to the phosphorylated carrier lipid. Sequential transfer of additional sugars to the lipid-linked intermediate leads to the formation of the 0-chain repeating unit linked through a pyrophosphate bridge to the carrier molecule. The repeating units are then polymerized and eventually modified by substituents. Polymerization occurs by constantly transferring the growing chain to a newly activated repeating unit (growth at the reducing end). This results in a long-chain polymeric intermediate that is still linked at the reducing end to the carrier lipid. In a final step the 0 chains are transferred from the lipid carrier to the independently synthesized core lipid A to form the completed molecule. Recently, a second pattern of 0-chain synthesis has been shown to occur in Sulrnonella groups C, and L (Makela and Stocker, 19811, and in E . coli 08 and 09 (Schmidt et ul., 1976; Kanegasaki and Jann, 1979; Jann et u l . , 1979). In this case the synthesis appears to proceed by sequential addition of the sugars to the nonreducing end of the growing chain, which again is linked to a carrier lipid. I t has further been demonstrated that in E . coli 08 and 09, the polymannose 0-chain synthesis starts in fact with a glucose unit at the reducing end, and in SuOno/iellu group C, glyceraldehyde (Gmeiner, 1975) or Mannose (Heasley , 1981) has been identified at the reducing end of the 0 chain (Gmeiner, 1975), which is assumed to play an analogous starter role to the aforementioned

LIPOPOLYSACCHARIDES OF GRAM-NEGATIVE BACTERIA

89

glucose unit. In these systems, in contrast to the “classical” system, a gene of the rfe locus is required for 0-chain synthesis (as well as for the synthesis of TI antigen and enterobacterial common antigen (ECA) (Schmidt et al., 1976; see Section II,B,4), whereas the gene of the rfc locus, which codes for 0-chain polymerase, is nor needed. The genetics of lipopolysaccharide synthesis have been reviewed recently (Makela and Stocker, 1981). Acquisition of an understanding of the mechanisms of lipopolysaccharide biosynthesis and the underlying genetics have made it feasible to synthesize congenic strains differing only in one precisely known lipopolysaccharide feature. Structure-function relationships of parent and altered strains and their 1ipopoIysaccharides can now be studied, for example the effect of repeat-unit structure on virulence or its role in protection.

4. CHANGES IN

AND

REPLACEMENT OF 0 C H A I N S

a . Conversions Due to Extrachromosomal Elements. Lipopolysaccharide modification can occur through genes not linked to the chromosome, but to phages or plasmids. In their classical work, Robbins and Uchida (1962) and Robbins et al. (1965) elucidated the mechanisms underlying antigenic conversion by phage. When bacteria of Salmonella group E, are lysogenized by the converting phage the bacteria acquire the serological specificity of group E,; that is, the lipopolysaccharide 0 chain is converted from a-linked to P-linked repeat units. Genes of the phage code for a protein to act as inhibitor of the old a-polymerase, and also for a new P-polymerase. Many conversion systems have been studied in Salmonellu by A. M. Staub and her group, but lysogenic conversion has also been observed in other Entcrobacteriaceae (reviewed by Luderitz rt al. , 1966b, 1971; Nghiem and Staub, 1975; Makela and Stocker, 1981). Plasmid-linked conversions have only recently been recognized. A most dramatic plasmid effect has been described by Hoffman et al. (1980b). S . typhimurium (as well as Salmonella group D and E species) infected with the plasmid Col Ib drd 2 (a mutant Col Ib derepressed in colicin production) was shown to synthesize a lipopolysaccharide different in its 0 chain from the wild type (see Fig. 2), containing a new repeat unit (3) Rha-Glc-Man-tGlcNAc

t Gal

(3)

Plasmid-controlled expression of Migella sorinei phase I antigen has recently been recognized (Kopeck0 rt ul.. 1980). Rhizobia have the ability to infect leguminous plants, and in some cases, lipopolysaccharide seems to play a decisive role in the infection. Nodulating bacteria of Rhizobium trifolii have recently been shown to contain two different “Nod” plasmids. Nod-negative mutants

90

OTTO LUOERITZ ET AL.

have lost the capability of inducing nodulation and their lipopolysaccharide exhibits a changed composition (Zurkowski and Lorkievicz, 1979; R . Russa and E. Rietschel, unpublished data). b. SIT Variation. Salmonellu T forms were identified by Kauffmann (1956); they had been isolated from clinical cases and were then recognized as mutants of the infecting Salmonella bacteria of groups B and D. T1 and T2 forms were identified. They are characterized by a lipopolysaccharide carrying, instead of 0 chain, polymers of ribose and galactose (partly in the furanose form) in the case of T1 (Berst et a / . , 1969), and substituted glucosamine in the case of T2 (Bruneteau et al., 1974). T I - and T2-determining genes (rft and rfn, respectively) can be transferred experimentally to other strains, which then express T specificity (Sarvas, 1967; Valtonen et al., 1976). c. Lipopolysaccharide-Bound Enterobacterial Common Antigen (ECA). The haptenic ECA is a glycolipid present in all members of Enterobacteriaceae and responsible for the common serological cross-reactions among them. Its structure has been determined as a polymer of the disaccharide N-acetylglucosamine-Nacetylmannosaminuronic acid, containing small amounts of fatty acids. In its immunogenic form, which rarely occurs naturally, ECA is linked to the core of lipopolysaccharide, replacing 0 chains. Only those core types not containing terminal N-acetylglucosamine were found to function as acceptors for ECA (i.e., the core types R 1 , R4, and to some extent K-12 of Fig. 3). Genetic manipulation allows the construction of strains lacking ECA, or expressing haptenic or immunogenic ECA (for reviews, see Makelii and Mayer, 1976; Mayer and Schmidt, 1979). d . Lipopolysaccharide-Bound Capsular Polysaccharide. The possibility that capsular polysaccharides may replace 0 chains in E . coli has been suggested and discussed (Galanos ef al., 1977a; qrskov e f al., 1977).

C. Structure and Biosynthesis of the Core 1 . STRUCTURE O F CORETYPES

Diversity in lipopolysaccharide structures is usually associated with the 0 chains; core structures are more uniform. The core of S. typhimuriurn (as shown in Fig. 2) is, for instance, common to all Sulmonella species and also occurs in other enterobacterial lipopolysaccharides. The Salmonella core contains a lipid A-distal hexose oligosaccharide consisting of D-glucosamine, D-glucose, and D-galactose, and an inner lipid A-proximal region consisting of an oligosaccharide of the core-specific sugars, L-glycero-D-rnanno-heptose (L-D-Hep) and 2-keto-3-deoxy-~-manno-octonate (KDO or dOclA), each forrning a branched trisaccharide (Liideritz et al., 1966a; Osborn, 1966; Hellerqvist and Lindberg, 1971). As indicated in Fig. 2, the exact linkages in the KDO

LIPOPOLYSACCHARIDES OF GRAM-NEGATIVE BACTERIA

91

trisaccharide region have not been definitely established (Droge et ul., 1970; chemistry and biology of KDO have been reviewed by Unger, 1981). KDO I links the polysaccharide to lipid A in a relatively acid-labile (ketosidic) linkage (for designation of the core sugar units see legend of Fig. 4). The 0 chains are attached to the subterminal glucose I1 unit of the core. A characteristic feature of the core is its substitution by such polar groups as phosphate and ethanolamine. Furthermore, the carboxyl groups of KDO contribute to the acidic character of lipopolysaccharides. This negative change is believed to be physiologically important. Lipopolysaccharides may be interlinked to other components of the cell wall through divalent cations and polyamines in ionic linkages, thus providing integrity and stability to the cell wall. These cations and amines are also usually found in lipopolysaccharide preparations associated with the core and lipid A . As with the 0 chains and lipid A, the core region of lipopolysaccharides exhibits a high degree of heterogeneity. Substituents of the main chain of the inner core (Hep 111, P, EtN, and possibly N-acetylglucosamine and galactose IT) are not necessarily present in molar amounts. Furthermore, alkali-labile substituents on the core such as glycine have occasionally been detected (Gentner and Berg, 1971; see also Hellerqvist and Lindberg, 1971). Recent evidence indicates the presence of an acid-labile substituent linked to the branched galactose (Fumahara and Nikaido, 1980). The nature of these substituents is unknown. In recent years five other core types have been identified in Enterobacteriaceae, the core types coli R1LR4 and K-12. As seen in Fig. 3, all differ in the hexose region of the core but are similar in the Hep-KDO region (Janssen e r a / . , 1981). The overall structural similarities are obvious. As indicated in Table 11, these cores have been identified in serotypes of E . c d i , Shigellu, Arizoriu, and others. Table 111 lists strains whose core structures have been studied; some of them are R mutants with incomplete biosynthesis of the core. Compositional analyses of the core from various bacterial species have revealed the presence of such unusual constituents as u-glycero-D-nrannoheptose. uronic acids (Kotelko ei al., 1974), or amino acids (Drewry et ul., 1975). and, in the case of Vibrio choler~ie strains, fructose (Redrnond r f ul., 1973; Jann et ul., 1973; Raziuddin, 1980) or seduheptulose ( K . W. Broady, unpublished data) may be present. These probably replace KDO in linking the core to lipid A . Lipopolysaccharides containing a core devoid of heptose and/or KDO have also been identified (summarized by Galanos et ul., 1977b; Wilkinson, 1977). The analysis of core structures in S-form lipopolysaccharides is hampered by the fact that the core represents only a relatively small part of the whole molecule. Recognition of the presence of nonsubstituted core stubs in S-form lipopolysaccharides (see Section If,B,2) offers the possibility of studying core oligosaccharide devoid of 0 chains in cases where Ra mutants (see next paragraph) are not available. Treatment of S-form

92

OTTO LUDERITZ ET AL

Salmonella

Ra

Gal

GlcNAc

@-@-EtN

HIP

KDO-?**@-EtN

R1

R2 R3 E.coli

R4

014

Gal

Gal

t2la

141p

HIP

,J?la_-

Gal $b Glc -!$~Glc*Hep

K-12

G!cNAc Gic ' ' 2 ' G k

-p]

t - E t N $-EtN

E coli K12 161

t$p'$%KW-(1(00)

Gal

Hep & H i p

pl-3 %GIC+H~~&H~~-(KDO)~% 1.64

Rha . @-EtN .

i ;

L--6--J FIG 3 Structures of enterobacterial core types identifed thub far Ra Osbom (1966). Ludentz et ul. (1966a); Hellerqvist and Lindberg (1971). R1: Feige and Stirm (1976); Feige (1977); Jansson ef al. (1981); Katzenellenbogen and Romanowska (1980). R2: Hammerling etal. (1971). R3: Johnston el al. (1967); Jansson et a / . (1979). R4: Feige ef u l . (1977). E . coli K-12: Mayer et u / . (1976); Prehm e/ al. (1976a). For designation of sugars, see Fig. 2 . P-Hep, 1-a-D-Hep (according to IUPAC-IUB rules).

93

LIPOPOLYSACCHARIDES OF GRAM-NEGATIVE BACTERIA

COKF

coli R I

Ra Salmonella Arizona

TABLE I1 TYPI:S~ D l ~ . N T l F l f IN ~ l ) ENT~~KOHACTERIACEAE" coli R4

coli R 3

coli R2

E . coli 01 1 I Citr(JbU&'r 9b. 10

E . coli 08 E . C Y J / ~09 E . c d i 08 E . c w f i 0100 E. cdi C Sh. sonnri phase II Sh. hoydii type 3 S h . jlcxneri type 6

coli K-12

E . coli 014 E . u d i K-12 Sh. dysenteriac type I

Sh. ,/lr.rneri type 4 b

" The classification is based on serological relationships. reactivity with Con A . comparison of phage patterns. and chemical analyses. For references see Galanos P I ul. (1977b).

Strains

Structure''

References

( P , E t N ) KDO Hep I I Glc - Glc - Hep - Hep KDO KDO

E . culi B E . coli K-12

-

Ter-Mutants CR 3 4 E . coli 0 I 1 I

X I

Glc- Hep--Hep-KDO X = H, Hep, or Glc - GlcN Gal - Glc

I

E . coli ATCC 12408

(Hep - Hep),

Prehm ef al. (1975) Ohkawa (1980) Blache ef a / . (1981)

-

Fuller rt d . ( I 973)

KDO

-

KDO

-

A0

Morton and Stewart ( 1972)

Hep- P

P rofrus rnirahilis

Glc - H!?p- KDO

Radziejewska-Lebrecht rt ul. (1980)

GlC - G!c

Pseudomonus arruginosci

Glc

-

Rha - Glc - - - GhlN - - -Hep - Hep - KDO - KDO I (P,EtN)

ma11 2

Drewry rt ctl ( 1 975)

Gtc NAc,

Borderella pertussis

Xunthomonas wwn.si.y

,Hep G~CUA" Man Man Glc - Man - KDO ~

I

P I

GalUA - Amide "

Details of linkages are omitted

Chaby et d.(1977)

W. A. Volk (unpublished data)

94

Om0 LUDERITZ ET AL.

lipopolysaccharide with mild acid will release a mixture of polysaccharides, which subsequently is separated on Sephadex G-50 into 0 chain-substituted and nonsubstituted core (Miiller-Seitz e t ul., 1968). The core is thus obtained in a pure form, though it is probable that degradation takes place under the acidic conditions needed to separate lipid A from polysaccharide i n lipopolysaccharides that do not contain KDO (Shaw, 1982). 2. LIPOPOLYSACCHAKIDES O F R MUTANTS

Evaluation of the core structure of Salmonella lipopolysaccharides was greatly facilitated when it was found that so-called R mutants are defective in lipopolysaccharide biosynthesis. Depending on the defect, these mutants synthesize complete or incomplete core structures, still linked to lipid A , but all devoid of the 0 chain. Figure 4 shows a series of R-form lipopolysaccharides derived from Salmonella mutants defective in different steps of the biosynthetic pathway. Similar series of R mutants have also been isolated from E . coli, Shigrllu, Proteus, and others. Ra mutants are defective in 0-chain synthesis, and produce lipopolysaccharides with complete core. Rb through Re mutants are core defective and synthesize lipopolysaccharides with incomplete core. Re lipopolysaccharides (Re glycolipids) are the most defective and contain only KDO and lipid A . The different R mutants and their respective R lipopolysaccharides can be differentiated and identified by means of lectins (Ahamed et ul., 1980), and antibodies (Ruschmann and Niebuhr, 1972; Nixdorff and Schlecht, 1972), as the terminal sugar sequences, which are different, are recognized by these tools. Recently developed immunoadsorbants make the isolation of pure monospecific R antibodies possible (see Section III,A,2). As well as being recognized by chemical analysis of their lipopolysaccharides, the mutants can also be identified by their phage pattern (Wilkinson et ul., 1972). Since the linkage of KDO to lipid A is not affected by mutation, and since this linkage is acid labile, mild acid

FIG. 4. The structures of 0-chain-defective (chemotype Ra) and core-defective (chemotypes Rb-Re) lipopolysaccharides of Salmonella R mutants, as derived from chemical, biosynthetic, and genetic investigations of the groups of M. J . Osbom, H. Nikaido, B . A . D. Stocker, P. H. Makela, and the present authors. For designation of sugars. see Fig. 2. Hep. 1.-glycero-o-mannoheptosc; DHep. o-glyccro-o-mannoheptose. The Rb, stmcmre seems to occur in some Rb, lipopolysaccharides (Hellerqvist and Lindberg, 1971). R, represents an unusual chemotype and is not an intermediate of lipopolysaccharide biosynthesis. The Re, glycolipid has been detecied in a mutant of E . coli B . For simplicity, these products of R mutants are often called lipopolysaccharides, although they are rather lipooligosaccharides. being termed sometimes R glycolipids according to Liideritz rt u / . ( 1 969). Designation of the sugar units: Glc N

Gal I1 H e p 111

I

I

KDO 111 I

Glc 11- Gal I- Glc I- H e p 11- H e p I- KDO 11- KDO I- Lipid A

LIPOPOLYSACCHARIDES OF GRAM-NEGATIVE BACTERIA

95

96

OTTO LuDERlTZ ET AL.

hydrolysis will liberate lipid A from all these lipopolysaccharides. The Re lipopolysaccharide is of course the richest source of free lipid A . In recent investigations, Gmeiner and Schlecht (1979, 1980) have analyzed the cell walls of S . typhimurium S - and R-form mutants comparatively for peptidoglycan, lipopolysaccharide, phospholipids, proteins, and covalently linked lipoprotein. The results showed that with increasing defect the strains incorporated increasing amounts of R lipopolysaccharide into their cell walls. In Re mutants, the lipopolysaccharide content of the cell wall was about four times higher than that of the wild type. Also, the content of phospholipid increased, whereas that of protein decreased. Based on their findings, the authors discuss the occurrence of different molecular organizations of the outer membrane in the various strains, this being reflected in their different properties in biological systems (e.g., sensitivity to detergent and antibiotics; for a controversial viewpoint regarding the amounts of lipopolysaccharide, see Nikaido and Nakae, 1979). Occurrence of R forms is rare in nature. Although they can be cultivated under laboratory conditions, they appear to be less resistant to in vivo situations. They are generally nonvirulent, are phagocytized without opsonization by macrophages (Roantree, 1971) and amebas (Gerisch et al., 1967), and are sensitive to toxic agents. They have been found, however, in urinary tract infections (Ryan e t a / . , 1973; Westenfelder et a l . , 1977). 3. BIOSYNTHESIS O F 'I H E

CORE

It is evident that the lipopolysaccharide structures of Fig. 4 represent intermediates in the pathway of core-lipid A synthesis, which would proceed from Re to Ra by sequential addition of the constituents. That this mechanism is valid in r + \ v is concluded from experiments where many of the transfer reactions have already been accomplished in ivirro (Wright and Kanegasaki, 1971; Osborn and Rothfield, 1971). In some cases, the respective sugar transferases have been purified and, recently, also cloned (Creeger et al.. 1979). The activated forms of the sugars are known. In similar manner to 0-chain biosynthesis, a lipid carrier is involved. In this case, the carrier is lipid A , which remains linked to the core. Thc genetics of core biosynthesis have also been evaluated (for review, see Make18 and Stocker, 1981). 0-chain synthesis proceeds independently of the synthesis of the core and therefore core-defective mutants also make 0 chains. Incomplete core does not, however, act as receptor for 0 chains, and thus the synthesized 0 chains are not transferred and remain bound to the polyisoprenoid carrier. The 0 chains can then be isolated by phenol/water extraction, because the pyrophosphate linkage to the lipid carrier is cleaved by this treatment (Beckmann et d.,1964; Kent and Osborn, 1968). An R mutant has been identified whose defect results in the synthesis of an

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97

R-form lipopolysaccharide not representing a natural precursor of the parent lipopolysaccharide (Rx of Fig. 4). This R-form lipopolysaccharide contains D-glycero-D-manno-heptose (D-D-Hep) linked to KDO-lipid A (Lehmann et a / ., 1973). According to a proposed sequence of reactions, sedoheptulose-7phosphate is an intermediate of heptose synthesis in the bacterial cell. It is isomerized in the bacterial cell to ~-~-Hep-7-phosphate and subsequently converted to the activated nucleotide-diphospho derivative, NDP-D-D-Hep, which is epimerized in position 6 to form the activated form of L-D-Hep. L-D-Hep is then transferred to KDO. The diphosphonucleotide has been identified as ADP (Kontrohr and Kocsis, 1981). The above mutant is presumably defective in the synthesis of the enzyme, NDP-D-D-heptose 6-epimerase. The reaction sequence stops at the level of NDP-D-D-Hep. Hep I transferase may recognize the D-I?IUII/ZO configuration of this “unnatural” heptose. D-D-Hep is, under these conditions, transferred to KDO. D-D-Hep, however, does not act as acceptor for glucose I transfer. The original mutant was leaky, and also synthesized in addition the correct lipopolysaccharide. Recently, a nonleaky E . coli K-12 mutant of this type has been isolated (Coleman and Leive, 1979). It is interesting to note that strains of the photosynthetic bacterium Rhodopsrudornonus gelutinosu synthesizes naturally a lipopolysaccharide containing D-D-Hep, KDO, and lipid A as the only constituents (Drews e f a / . , 1978; Weckesser et ul., 1979). Furthermore, polysaccharides resembling 0 chains have been isolated from these organisms by phenol/water extraction. As the authors discuss, these lipopolysaccharides .d polysaccharides could represent ancient forms of lipopolysaccharide and capsules, respectively, or, alternatively, these strains are results of mutations (i.e., core-defective R forms) that could survive. Another example of the occurrence of R-form bacteria in nature are Yersinia pesfis organisms, whose lipopolysaccharide contains glucose, L-glycero- and D-glycero-D-manrw-heptose, KDO, and lipid A (Hartley rt ul., 1974).

D. Structure and Biosynthesis of Lipid A Historically, structural investigations of Sulmonella lipopolysaccharides started with studies on the 0 chains, which then led to identification and investigation of the core, and only recently have detailed structural studies on lipid A been performed, even though the presence of a covalently linked lipid component in lipopolysaccharides had long been recognized (Boivin et ul., 1933; Morgan and Partridge, 1941; Tal and Goebel, 1950; Ikawa et ul., 1953; Westphal and Liideritz, 1954). This delay was caused mainly by technical difficulties existing at that time in the field of lipid analysis and in particular by difficulties regarding this lipid species. The resumption of structural work on lipid A in the last decade was greatly stimulated by the recognition of lipid A as the endotoxic principle in the lipopolysaccharide molecule.

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Due to the acid lability of the KDO linkage, lipid A can be liberated from lipopolysaccharides containing KDO by mild acid treatment. Free lipid A thus obtained is a water-insoluble precipitate, soluble in organic solvents such as chloroform. Similar conditions of cleaving lipid A are applicable to lipopolysaccharides in which ketoses (fructose and sedoheptulose in V . cholerae) mediate the linkage to lipid A (Redmond et al., 1973; K . W. Broady, personal cornmunication). Great difficulties, however, are encountered with lipopolysaccharides containing a relatively acid-stable linkage to lipid A, and in these cases either degraded free lipid A or lipid A still linked by polysaccharide fragments is obtained. 1. STRUCTURE O F Salmonella LIPIDA

Figure 5 shows the structure of Salmonella lipid A with the actual positions of the ester-bound acyl residues left open. Lipid A represents an unusual phospholipid in that it contains a central disaccharide of two u-glucosamine residues linked /ill .6. Both glucosamine residues are substituted by a phosphate group: one is bound in ester linkage to C-4 of glucosamine I1 (the nonreducing glucosamine); the other one is bound to C-1 of glucosamine I (the reducing glucosamine), thus occupying the glycosidic hydroxyl group and rendering the

- ...-.

.

.

FIG.5. Structure of lipid A from Salmondla. Dotted linkages indicate incomplete substitutions

LIPOPOLYSACCHARIDES OF GRAM-NEGATIVE BACTERIA

99

molecule nonreducing. In the lipopolysaccharide, the terminal KDO unit of the core is attached to C-3 of glucosamine I1 (Gmeiner et al., 1969, 1971; Rietschel et a l . , 1977). The P-GlcN-GlcN-P region of lipid A is termed the lipid A backbone. It represents a phosphorylated polyol and therefore resembles the hydrophilic region found in normal phospholipids (e.g., glycerophosphate). In common with other phospholipids, the backbone of lipid A is also substituted by acyl residues. Salmonella lipid A contains the following 7 moles of long-chain fatty acids: 4 moles of D-3-hydroxytetradecanoic acid (3-OH-14:0, P-hydroxymyristic acid) and about 1 mole each of dodecanoic (12:0, lauric acid), tetradecanoic (14:0, myristic acid), and hexadecanoic acid ( I 6 9 , palmitic acid). Recently additional small amounts of L-2-hydrpxytetradecanoic acid (2-OH-14:0, a-hydroxymyristic acid) and 3-hydroxydodecanoic acid (3-OH- 12:0, P-hydroxylauric acid) were identified. Careful analysis has shown that in many preparations tested the sum of 14:O and 2-OH-14:0 equals 1 mole equivalent (Rietschel et ul., 1972; Bryn and Rietschel, 1978). T w o moles of 3-OH-14:0 are linked to the amino groups of the disaccharide. The remaining 5 moles of fatty acids are ester linked. The exact positions of the ester-bound fatty acids in lipid A can be identified only partly by the methods presently available. There is strong evidence that only the two ester-bound P-hydroxymyristoyl residues are linked directly to the lipid A backbone, where 3 hydroxyl groups are available; one hydroxyl group, therefore, would remain free. It has also been shown that 14:O plus 2-OH-14:O either substitute one of the two ester-bound 3-OH- l4:O residues, or, alternatively, are distributed on both (Rietschel et u l . , 1972). Finally, there is preliminary evidence (Rietschel ct a l . , 1981; Wollenweber d a / . , 1981) that 12:O and 16:O are bound to the 3-hydroxyl groups of the two amide-linked 3-OH-14:O fatty acids in a still-unknown distribution (not shown in Fig. 5 ) . If these findings prove to be correct, all of the 3-hydroxylated fatty acids would substitute the backbone glucosamines directly, two of them in amide, and two in ester linkage as would have been predicted from the lipid A precursor structure (see Section ll,D,6, Fig. 10). The nochydroxylated fatty acids (and 2-OH-14:O) would then be linked to the hydroxy groups of the hydroxy acyl residues. Similarly to 0 chains and core, lipid A also exhibits structural heterogeneity (Nowotny, 1971; Chang and Nowotny, 1975; Banerji and Alving, 1979), one reason being a partial substitution of the phosphate groups by amino compounds, the so-called polar head groups (Muhlradt et ul., 1977). In approximately 50% of the lipid A molecules of Sultnonella, the phosphate group linked to glucosamine I is substituted by a phosphorylethanolamine residue with free amino group. In about 30 to 60% of the lipid A molecules, the phosphate bound to glucosamine I1 is substituted by 4-amino-4-deoxy-L-arabinose (4-AraN), the linkage being through C - l . The amino group and the hydroxyl groups of 4-AraN appear to be

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nonsubstituted. This rare sugar had been previously identified as a component of Sultnonella lipopolysaccharides (Volk et al., 1970; Volk and Luderitz, 1981), but it was shown only recently that it is a constituent of the lipid A component of the molecule (Miihlradt et al., 1977; Rietschel et al., 1977; Hase and Rietschel, 1977). The formula in Fig. 5 contains some details that are still under investigation. Thus the pyranose form of glucosamine I is not proved, neither is its anomeric configuration known. The same applies for 4-AraN. Earlier determinations of fatty acids in lipid A had indicated the presence of three equivalents of 3-OH-14:O. New methods of analysis, however, have led to the identification of an additional mole (for a detailed discussion, see Rietschel et a l . , 1981). It was previously anticipated that lipid A units were interlinked by pyrophosphate bridges (Luderitz et al., 1973; Rosner et ul., 1976); this has recently been shown by "P-NMR spectrometry to be incorrect (Muhlradt et al., 1977; Rosner et al., 1979a,b). This may also be concluded from the fact that the phosphate groups of lipid A are in many cases quantitatively substituted by polar head groups. Most lipid A analyses have been performed with a lipopolysaccharide from a Salmonella tninnesota Re mutant, but sufficient data have been accumulated to conclude that all serotypes of this genus contain an identical lipid A . Apart from the uncertainties already mentioned, the lipid A formula seems to be correct, and in several laboratories lipid A's of other Enterobacteriaceae have been studied with analogous results (see Section Il,D,4). In most cases, however, the applied analytical methods were the same as those originally used for Salmorrellu lipid A. Recently, however, the structure of E . cofi K-12 lipid A has been investigated with quite different methods of analysis, including chemical and enzymatic degradations as well as :j'P-NMR spectroscopy. The results of this work of Khorana and his co-workers (Rosner et al., 1979a,b,c) agree excellently with those on Salmonella lipid A-apart from details specific to E . coli. This is satisfying. It is suggested that when new lipid A's are studied, the Salmonella lipid A should always be analyzed in parallel, in order that new findings with other lipid A's are recognized as specific for the strain or lead to a modification of the original model of lipid A structure. I N T H E STRUCTURE O F LIPIDA 2. C O N D I T I O NVARIATION AL

Under normal physiological conditions of growth, the composition and structure of lipid A seems to be relatively constant, but effects of extreme conditions in bacterial growth, for instance, have not been systematically studied. Changes hitherto observed concerned the lipid A head groups. When comparing lipid A isolated from different bacterial batches of the same strain, Muhlradt et al. (1977) could detect variations in the degree of substitution by head groups. These authors conclude that gram-negative bacteria-as it is known for grampositives-are able to modify their surface charge by addition or omission of

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101

substituents in order to adapt to the ionic environment. An analogous mechanism of adaptation may apply to polar core substituents. Characteristic changes in the pattern of ester-bound fatty acids have been observed when the bacteria were grown at low temperature. In Proteus rnirabilis, grown at 15"C, the hexadecanoic (16:O) acid content of lipid A was markedly decreased, whereas the unsaturated analogue, hexadecenoic acid (16:l ) , appeared (Rottem et al., 1978). A change in the composition of ester-linked fatty acids was also observed in E . coli K-12. When grown at 1 2 T , the normal dodecanoic acid was decreased and replaced by the unsaturated palrnitoleic acid (van Alphen et ul., 1979). Identical changes have recently been observed with lipopolysaccharides from Salmonefla S and R forms grown at 12°C (H. Wollenweber, S. Schlecht, and E. T. Rietschel, unpublished data). Unsaturated fatty acids are rarely found in lipid A's. The appearance of unsaturated fatty acids in lipid A probably influences its fluidity, as was demonstrated in the following system. Lipopolysaccharide of E . rofi K-12 represents a cofactor for protein d of E . coli K-12, which functions as the receptor for phage K3 (in the case of E . coli K-12 and phage T4 the lipopolysaccharide acts as the receptor, but it requires protein Ib to exhibit activity; Henning and Jann, 1979; Datta et al., 1977). When complexed with lipopolysaccharide this protein d inactivates the phage. It was shown that complexes with lipopolysaccharide from bacteria grown at 37°C inactivated the phage only above 20"C, whereas complexes with lipopolysaccharide from 12°C growth were active at above 10°C (van Alphen et al., 1979). The lack of activity below these temperatures is attributed to the influence of the respective lipopolysaccharide on fluidity. Analogous results were obtained with corresponding whole cells. This system demonstrates in a most elegant way the influence of lipid environment on the function of an outer membrane protein. 3. CONFORMATION OF Sulmonella LIPIDA Empirical force-field calculations are now being applied to obtain information on the conformation of lipid A disaccharide. The approach includes calculations using glucopyranoses, p-maltose, /3-cellobiose, and P-gentiobiose as models (Melberg, 1979; Melberg and Rasmussen, 1979; Rasmussen, 1980). Using the methods of Ramachandran, computer calculations have been performed on the (KDO),-lipid A molecule (Formanek, 1978; Formanek and Weidner, 1980). The permitted torsion angles of the ketosidic linkages in the trisaccharide, and of the linkage to lipid A, as well as the angles of the phosphate residue bound to glucosamine 11, have been determined and proved to be highly restrictive. Only one terminal KDO residue would exhibit free rotation. Furthermore, X-ray diffraction studies indicated a very compact molecule with a lattice periodicity of about 4.1 A and a hexagonal order of the fatty acids, much like other phospholipids (Wawra et ul., 1979; Ueki et al., 1979; Rottem and Leive, 1977). On the basis of these data, an atomic model has been built (Fig. 6). It repre-

102

OTTO LUDERITZ ET AL.

LIPOPOLYSACCHARIDES OF GRAM-NEGATIVE BACTERIA

103

sents the Sulmonellu Re-form lipopolysaccharide containing KDO and lipid A (see Figs. 4 and 5 ) . The lipid A backbone is substituted by 4-aminoarabinose, phosphorylethanolaniine, and 4 moles of P-hydroxyniyristic acid, esterified by 1 mole each of lauric, myristic, and palmitic acid (see Section II,D,l). The fatty acids are oriented in parallel and exhibit together with the lipid A backbone a length of about 22 A; the KDO trisaccharide is about 5 A in length, in accordance with X-ray diffraction data (Formanek and Weidner, 1980; Veki pf ul., 1979). Recently two classes of binding sites for divalent cations have been identified in lipopolysaccharides (Schindler and Osborn, 1979). The first class, of low binding affinity, was attributed to phosphate groups of the KDO-lipid A region; the second class, of higher affinity, was related to the KDO trisaccharide representing a specific site of interaction with divalent cations. In accordance with the experimental findings, the orientation of the carboxyl groups of the KDO residues in the model allows the binding of Ca2+ and Mg2+. The model clearly shows the amphipathic nature of the molecule. In addition, with an accumulation of polar groups it also exhibits amphoteric properties. Its integration in the bacterial outer membrane can be visualized, as can its reactivity with artificial and natural membranes. 4. STRUCTURAI. FEATURES O F LIPIDA’s T H A N Salmonella

FROM

BACTERIA OTHER

Although detailed structures have been completely evaluated for only a few lipid A’s, many results are available regarding constituents and partial structures (reviewed by Galanos er ul., 1977b; Rietschel rt u l . , 1981), and they allow first conclusions to be drawn on the structural constancy or variability of lipid A’s from different bacterial families. In order to study lipid A structures in a comparative manner, standard procedures have been worked out that allow screening of different lipid A’s (Rietschel, 1982). Amide- and ester-bound fatty esters as well as ester-bound 3 - 0 acylhydroxy fatty acids, are sequentially released under specified differentiated hydrolysis or transesterification conditions and subsequently identified by gas-liquid chromatography (GLC) and mass spectrometry (MS). The optically isomeric form of hydroxy fatty acids is determined by GLC after their conversion into diastereomeric derivatives. For studying the lipid A backbone and head-group substituents, classical degradation pathways have been worked out, starting from the corresponding lipopolysaccharide, the final product being the central glucosamine disaccharide FIG. 6 . Atomic model of Srrlmor~rlla lipid A . This model was built according to the structure given in Fig. 5 and to the results on the conformation of lipid A (Forrnanek, 1978; Formanek and Weidner. 1980). All nonhydroxylated fatty acids have been attached in ester linkages to the Thydroxy groups of the P-hydroxymyristoyl residues (see text).

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in the reduced form (Hase, 1982; Volk and Luderitz, 1982). The intermediates of degradation are purified by electrophoresis and analyzed for their respective constituents. In the following, results of these investigations are summarized. They have been reviewed in more detail by Galanos et ul. (1977a) and by Rietschel et al. (1981). The lipid A backbone (P-GlcN-GlcN-P, see Fig. 5 ) is a common structure. Besides being identified in all Enterobacteriaceae investigated thus far (six genera), it has also been found in two plant pathogens, in two anaerobes, and in two photosynthetic bacteria (Rietschel et al., 1977). Some exceptions will be discussed later (Section 11,DS). In most cases the backbone is substituted by at least one head group. There is no variation regarding the substituent of the phosphate on glucosamine 11: either 4-aminoarabinose (Salmonellu, P . mirahilis, Y . enterocoliticu, Chrotnobucterium violaceum, Rhodospirillum tenue), or no head group ( E . c d i , V . cholerae) is present. In contrast, variation does occur regarding the substituent of the phosphate on glucosamine I: phosphate ( E . coli), phosphorylethanolamine (Su/motzella. V . cholerue), D-glUCOSamine (Chr. violureum), and D-arabinofuranose (Rhsp. tenue) have been found, as well as no substituent ( P . rnirubilis, Y . enterocolitica). Partial as well as quantitative substitutions occur, this leading again to microheterogeneity in the lipid A molecule (and to lipid A species devoid of head groups as in the case of E . coli). The head groups are not acylated and the amino groups where they occur are free. It was found, without exception, that the amide-bound fatty acids are saturated, 3-hydroxylated, and of the D form (for a new specific chemical synthesis of D-3-hydroxy fatty acids, see Tai el ul., 1980). In some rare cases a mixture of two, three, or four types of ~ - 3 - 0 Hfatty acids have been found in amide linkage. The following N-acyl residues have been identified: 3-OH- 1 O:O, 3-OH- 12:0, 3-OH-14:0, 3-OH-16:0, 3-OH-18:0, 3-OH-I I-Me-12:0, 3-OH-15-Me-16:0, 3-OH-17:O. A unique pattern of amide-bound fatty acids is present in the lipid A components of Vihrio anguillarum (D. H . Shaw, unpublished data) and Rhodopseudomorias sphueroides ATCC I7023 (W. Strittmatter, unpublished data), which both contain 3-oxotetradecanoic acid (besides 3-OH-14:0). Since 3-hydroxy fatty acids are rarely found in other lipids of gram-negative bacteria and in biological fluids, they can serve as a specific marker for lipid A and endotoxin. A great variation regarding ester-linked fatty acids is seen in different lipid A's (for references, see Wilkinson, 1977; Galanos et ul., 1977a; Drews e.'a/., 1978; Weckesser et d., 1979). Nonhydroxylated, 2-, and 3-hydroxylated nonbranched, iso-, and unteiso fatty acids of chain lengths varying from 10 to 20 carbon atoms have been identified. 3-Hydroxy fatty acids are of the 11 form, and 2-hydroxy fatty acids are of the I. form. It appears as though nonsaturated fatty acids may also occur (Broady et al., 1981; Raziuddin, 1980a,b). Ester-bound 3-acyloxyacyl residues as present in Salmonellu lipid A (see Fig. 5 ) , are frequently but not

LIPOPOLYSACCHARIDES OF GRAM-NEGATIVE BACTERIA

105

generally found; when they do occur, however, the substituted acyl ester is always a 3-hydroxy fatty acid, whereas the substituting fatty acid may be non-, 2-, or 3-hydroxylated. That hydroxy fatty acids are exclusively linked directly to the lipid A backbone (as in Salmonellu lipid A) is therefore not a general phenomenon. Whether nonhydroxylated fatty acids are linked exclusively to hydroxylated fatty acids (as proposed for Salmonella lipid A) is presently being investigated. Since the overall fatty acid spectrum of lipid A’s is, within certain limits, constant for members of a genus or a family, this may prove to be an additional tool in taxonomic classification of the bacteria (Nikaido, 1970; Jantzen et ul., 1975; 1976; Rietschel and Liideritz, 1980; Wilkinson and Caudwell, 1980).

5. PRESENTLY KNOWN LIPIDA STRUCTURES Aside from Salmonella and closely related enterobacterial lipid A’s, detailed structures are presently available for the lipid A’s of Chr. violuceum, Rhsp. tenue, Rhodopseudomonus viridis, and Rhps. palustris (Liideritz et al., 1978). Lipid A of Chr. violuceum (Fig. 7) contains a backbone identical to that of Sulmonellu (P-GN-PI ,6-GN-P), and this is substituted by ~-3-OH-12:0in amide linkage, and ~-3-OH-10:0,~-2-OH-12:0,12:0, and 16:O in ester linkage. The head groups, 4-amino-~-arabinoseand D-glucosamine, are present in molar ratios and are neither 0- nor N-substituted (Hase and Rietschel, 1977). This lipid A, therefore, represents an unusual acylated tetrasaccharide with four amino sugars linked together either glycosidically or by phosphate bridges to give a nonreducing molecule. The glucosamine I I-P I-glucosamine is reminiscent of a trehalose type of structure. Lipid A of Rhsp. tenue (Fig. 8) also contains a backbone identical to that of Salmonella, which is acylated by amide-linked ~-3-OH-10:0 and ester-linked ~-3-OH-10:0,14:0, and 16:O. The phosphate groups are completely substituted by 4-amino-~-arabinose and D-arabinose, respectively, the latter being present in the furanose form. A third substituent, D-glucosamine, is linked to glucosamine I at position 4 (Tharanathan et ul., 1978; Weckesser et

L-L-AraN 1 @--_D-GlcNp

-

FIG. 7.

a

D-GlcN -1@ l Q - G k N

Structure of lipid A from Chrornohacterium violuceum

O n 0 LUDERITZ ET AL.

106

FIG. 8. Structure

of lipid A from Riiodospirillitrrr t m w .

al., 1979). The structure of this lipid A resembles a pentasaccharide with glycosidic linkages present in an inner-branched glucosamine trisaccharide, and two phosphate bridges linking the remaining two monosaccharides in such a way that the molecule is nonreducing. Lipid A of Rhps. viridis (Fig. 9) has a structure very different from other lipid A's. I t is devoid of phosphate and glucosamine, and instead contains a 2,3-diamino-2,3-dideoxy-~-glucose@-aminoglucosamine derivative, glucosamine), which is substituted by ~-3-OH-14:0and by acetyl groups. A lipid A structurally indistinguishable from that of Rhps. viridis is present in Rhps. pulustris (and probably in Rhps. sulfiviridis). As will be shown (Section IIl,B,3), these types of unusual lipid A's are neither endotoxically active, nor do they serologically cross-react with Sulrnorzellu lipid A (Galanos et u l . , 1 9 7 7 ~ ; Weckesser et uf.,1979). 2,3-Diaminoglucose has also been found in the lipid A of Pseudominus dirninuta and Ps. vesicirluris (Wilkinson and Taylor, 1978). Lipid A components devoid of phosphate have been identified in Chrornatiurri

107

LIPOPOLYSACCHARIDES OF GRAM-NEGATIVE BACTERIA

vinosum, Thiocupsu roseopersicinu, and Rhodomicrohiurn vunniclii (Weckesser et ul., 1979). 6. F I N A L STEPSI N

THE

BIOSYNTHESIS ot, Sulmonellci LIPIDA

As lipid A is the endotoxic principle of lipopolysaccharides, its study represents a key area of research. However, the only source of free lipid A is lipopolysaccharide, from which it has to be cleaved by acid hydrolysis with possible undetermined minor degradation. In order to circumvent this possibility, the search for mutants defective in the synthesis of the KDO was of obvious prime interest, as it was expected that such mutants would synthesize only the lipid A portion of the molecule. Conventional phage-selection techniques, which had been employed successfully for the isolation of mutants defective in more distal regions of the polymer, failed to yield such mutants, suggesting that KDO-lipid A might be essential for the maintenance of the structural or functional integrity of the cell. Accordingly two approaches have been devised to isolate conditional Sulmotiellu mutants with defects in this part of the molecule. One approach was based on the dependence of the desired mutant on an exogenous source of KDO, both for synthesis of complete lipopolysaccharide and for growth (Rick and Osborn, 1972; Osborn r t d.,1974). The mutant from S. fyphimurium obtained in this manner contained an altered KDO-8-P synthetase that catalyzes the reaction i)-arabinose-5-P

+

phoaphoenolpyruvate + KDO-X-P

+ P,

This enzyme had a K , value for arabinose-5-P 35 times higher than the wild-type enzyme, and because of this the normal pool of arabinose-5-P was not sufficient for KDO synthesis when the reaction occurred at 37°C. Exogenous D-arabinose-5-P was required to maintain an internal concentration of this substrate sufficient to support both KDO synthesis and growth. It was subsequently found that the mutation was also temperature sensitive and at 42°C the mutant phenotype was expressed even in the presence of arabinose-5-P. In a second approach, KDO-lipid A mutants were selected for temperature sensitivity, both in synthesis of lipopolysaccharide and in growth (Osborn et ul., 1974; Lehmann et u l . , 1977; Osborn, 1979). Most of these mutants proved to be defective in KDO metabolisni. At the permissive temperature they synthesized KDO and lipopolysaccharide and grew normally, but when such cultures were shifted to the nonpermissive temperature, KDO synthesis ceased. As a consequence growth continued in these mutants for one to two generations at a normal rate but then ceased. Analysis of KDO-deficient mutants after incubation at the nonpermissive temperature led to the identification of new products synthesized by the cells during the absence of KDO (Rick rt ul., 1977; Lehmann, 1977). These substances were subsequently demonstrated to be incomplete lipid A .

108

a

C

d

O n 0 LUDERITZ ET AL.

G-3-OH-U:O D-3-OH-1L:O

I

D-3W-lL:0 D3OH-lL:O 16:O 16:O

1D-3-OH-1LD D-3-OH-M:O

e

1

D-3QH-14 0

FIG. 10. Incomplete lipid A molecules isolated from Salmonella and E . coli mutants. Products (a). (b). and (d) accumulate in KDO-defective Sulmonella mutants, products (a) and (c) in phosphatidylglycerol-defective E . roli K-12 mutants. The underacylated lipopolysaccharide (e) is formed from product a by KDO-defective Salmonellu mutants following a shift from nonpermissive to permissive conditions in the presence of cerulenin, which inhibits de novo synthesis of fatty acids.

LIPOPOLYSACCHARIDES OF GRAM-NEGATIVE BACTERIA

109

Structural analysis showed that the lipid A precursor I molecule (Fig. 10a) contained the diphosphorylated PI ,6-diglucosamine backbone, substituted by 3-OH-14:O residues, two in amide linkage and two (Lehmann, 1977) in ester linkage (only one was found by Rick et a / . , 1977), and additionally by small amounts of ester-linked 16:O. As was expected, the precursor I , due to the defective KDO synthesis, does not contain KDO, but it was also shown to lack the saturated fatty acids 12:0, 14:0, and most of 16:O present in complete lipid A . More recently it was found that similar incomplete lipid A molecules were accumulated in a temperature-sensitive E . coli K-12 mutant unable to synthesize phosphatidylglycerol at the nonpermissive temperature (Nishijima, 1980; Nishijima and Raetz, 1979; Nishijima et al., 1981). Formation of phosphatidylglycerol in this mutant was prevented by two mutations. One mutation in the pgsA gene reduced the activity of the phosphatidylglycerol synthetase to less than 5% of the wild-type level but still permitted normal growth of the mutant. A second mutation in the pgsB gene rendered the pgsA strain temperature sensitive both in synthesis of residual phosphatidylglycerol and in growth. The incomplete lipid A isolated from this E . coli mutant could be separated into two fractions. Fraction I contained a phosphorylated glucosamine backbone substituted by 4 moles of 3-OH-14-0 (Fig. lOa), and fraction II contained in addition 2 moles of esterified 16:O (Fig. 10c). It should be noted that palmitic acid is a minor component of E . coli K-12 derived lipid A (van Alphen er af., 1979; Boman and Monnez, 1975). Genetic studies have revealed that fraction I accumulation is primarily associated with the pgsB mutation, whereas fraction I1 builds up when both mutations, pgsA and pgsB, are expressed. These results suggest that the incorporation of 16:O into fraction 1, and phospholipid biosynthesis are somehow coordinated. Furthermore, since these lipid A fractions are similar to those isolated from KDO-deficient mutants and lack KDO residues, it is possible that incorporation of KDO requires an appropriate phospholipid composition in the membrane. The incomplete lipid A produced by Salmonella mutants conditionally defective in the synthesis of KDO was shown to represent an intermediate in the synthesis of the KDO-lipid A region of lipopolysaccharides, and it acts as an efficient acceptor of KDO residues from cytidine monophosphate (CMP)-KDO in virro (Munson et a l . , 1978). Using particulate cell envelope fractions or a partially purified detergent-soluble fraction as source of the enzyme, a single reaction product was obtained containing two KDO residues. Since lipopolysac-

During conversion of (a) to (e) there is a transient accumulation of (d). Complete lipid A contains in addition to 3-OH-14:0 the nonhydroxylated fatty acids 12:0, 14:0, and 16:O in the case of Salmonella, and 12:0, 14:0, and traces of 16:O in the case of E . coli K-12. References: (a) Rick et al., 1977; Lehmann, 1977; Nishijima, 1980. (b) Jxhmann e / a / . , 1978. (c) Nishijima, 1980. (d) Walenga and Osborn, 1980a. (e) Walenga and Osborn, 1980b.

110

OTTO LUDERITZ ET AL.

charide has been shown to contain three KDO residues in a branched trisaccharide structure (Fig. 2), the enzymatic product may correspond to lipid A precursor with either the main-chain KDO disaccharide, the branch unit, or a mixture of both. It is not known why the in vztro system failed to add the third KDO residue, but it is possible that the third KDO unit is transferred only after incorporation of the nonhydroxylated fatty acids. It has been known for many years from the work of Heath et ul. (1966) that KDO is transferred from CMPKDO to a chemically 0-deacylated lipid A preparation from E . coli, a product resembling the lipid A precursor in structure. I n vivo, the incomplete lipid A molecules are rapidly converted to lipopolysaccharide when the cultures of the KDO-defective mutants are shifted from nonpermissive to permissive conditions. Other intermediate products that accumulate transiently during this conversion have been isolated and identified. One of these products, lipid A precursor 11 (Fig. lob), resembled precursor I , but carried, in similar manner to complete lipid A , the two polar head groups 4-aminoarabinose and phosphotylethanolamine (Lehmann et u l . , 1978). Substantial amounts of precursor I1 have also been found when the mutants were incubated at intermediate growth temperature (Lehmann and Rupprecht, 1977). Another intermediate product was a derivative of lipid A precursor 1 containing two residues of KDO (Fig. 10d). It was indistinguishable in composition and chromatogaphic properties from the product obtained by enzymatic addition of KDO to isolated lipid A precursor (Walenga and Osborn, 1980a). This intermediate was produced transiently in vivo after shift of the culture to permissive conditions. It could be completed to lipopolysaccharide when the culture was returned to the nonpermissive temperature, where its continued formation is interrupted (Walenga and Osborn, 1980a). From pulse-chase experiments the early steps of lipopolysaccharide biosynthesis can be visualized (Fig. 11). Precursor I consisting of the phosphorylated diglucosamine backbone substituted by four P-hydroxymyristic acids is the acceptor for polar groups of the lipid A region, including 4-aminoarabinose, phosphorylethanolamine, and the KDO residues (Osborn, 1979). Since the glycolipid of Re mutants comprises the KDO-lipid A portion of lipopolysaccharides and contains the full complement of 0-acyl chains (Liideritz er ul., 1973), it was anticipated that the transfer of lauric, myristic, and palmitic acid would be the next step after the addition of KDO. Furthermore, it was assumed that full acylation would be obligatory prior to subsequent extension of the core polysaccharide chain. Recent results, however, have shown (Walenga and Osborn, 1980b) that the latter assumption is incorrect by studying the effect of cerulenin on the conversion of the acyl-deficient lipid A precursor to lipopolysaccharide. Concentrations of cerulenin that caused greater than 95% inhibition in the de novo synthesis of fatty acids and lipopolysaccharide had no

LIPOPOLYSACCHARIDES OF GRAM-NEGATIVE BACTERIA

111

FIG. 1 1 . Possible pathways (I. II. 111) of bio5yntheais of Solrrrorrrlki lipopolysaccharide (LPS). Products a , b, and d have been isolated from Salrtiotic~llamutants (Fig. 10). KD0,-lipid A has been isolated from an E . ( o l i B mutant (Prehm ('t ( I / . , 1975). It is possible that product d could be isolated because the parent strain of the KDO-defective mutant had an additional mutation affecting the transfer of the third KDO residue (Walenga and Osborn, 19ROa). Whether product d and KD0,-lipid A (pathway I) or the theoretical product KDO,,-a (pathway 11) are the natural inlerniediates cannot be decided. Pathways I and I1 lead to lipopolysaccharide molecules devoid of the polar head groups in lipid A; pathway 111 proceeds via product b and leads to lipopolysaccharide carrying these groups.

effect on the rate or extent of the conversion of the preformed lipid A precursor to lipopolysaccharide (devoid of nonhydroxylated fatty acids, Fig. I Oe). These results indicate that incorporation of the nonhydroxylated 0-acyl residues of lipid A is not necessary for the extension of the core and the 0 chains, and that under certain conditions lipopolysaccharide lacking these fatty acids can be synthesized (Fig. IOe). These fatty acids may, however, have an essential function for the survival of the cells. If this is true, the Re lipopolysaccharide represents the minimal structure required to maintain bacterial viability. The pathway responsible for the biosynthesis of the v-3-hydroxy fatty acids of lipid A remains an unsolved problem. Humphreys et al. (1972) have shown that after incubation of particulate fractions of Pseudomonas aeruginosa with radioactive decanoic or dodecanoic acyl-CoA, labeled 3-OH-1O:O o r 3-OH- I2:O, respectively, were found in the lipopolysaccharide. Whether hydroxylation of the fatty acids occurs prior to or after transfer, remains open to discussion. If the hydroxy fatty acids are formed by the usual @-oxidation pathway, the initial 3-hydroxy acids should have the L configuration, but since the lipid A-linked 3-hydroxy fatty acids are of the D form, the involvement of an epimerase such as that already identified (Overath rt a / . , 1967) is indicated. Kawahara ef al. (1979) have studied the biosynthesis of bacterial 2-hydroxy fatty acids and have demonstrated that in Pseudomonas ovulis, cultivated in the presence of IxO,, dodecanoic acid incorporates lXOto form the 2-OH-12:O present in the lipid A of the Iipopolysaccharide. 3-OH-12:0, which is also a constituent of this lipid A, was found to be unlabeled. Although oxygen incorporation into lipid A-bound fatty acids has not been proved, this finding could explain the identical position of 14:O and 2-OH-14:O on ester-bound 3-OH-14:O in Salinonella lipid A , and may also explain the phenomenon wherein different strains

112

O n 0 LoDERITZ ET AL.

containing different ratios of 14:O and 2-OH-14:0, always have the sum of both fatty acids equal to approximately 1 equivalent (Bryn and Rietschel, 1978). A N D INTEGRATION 01.LIPOPOLYSACCHARIDES 7. TRANSLOCATION I N 1 0 T H E O U T E R MEMBRANE

Major emphasis has recently been placed on lipopolysaccharide translocation and insertion into the outer membrane. The development of techniques for separation of this outer membrane from the underlying cytoplasmic membrane has offered a new approach to investigations of these processes (Osborn et ul., 1972a). Lipopolysaccharides pulse-labeled in vivn appeared initially in the cytoplasmic membrane, but were rapidly transferred to the outer membrane during a subsequent chase. This was true for both core sugars and 0 chains (Osborn e t a / . , 1972b). The enzymes of 0-chain biosynthesis were shown to be entirely in the cytoplasmic membrane, and the enzymes that synthesize the core region of lipopolysaccharides were probably in the same location, though they tended to redistribute during fractionation (Osborn et a / ., 1972a). From these experiments Osborn et u l . concluded that lipopolysaccharides are synthesized on the inner membrane and subsequently translocated to the other membrane. Furthermore, in contrast to translocation of phospholipids, which appears to be readily reversible (Jones and Osborn, 1977a,b), the overall process of lipopolysaccharide translocation is unidirectional-that is, lipopolysaccharide molecules that are integrated into the outer membrane are no longer accessible to the inner-membrane enzymes (Osborn et al., 1972a,b). It is not known whether this apparent unidirectionality is imposed by the mechanism of inter- or transmembrane translocation per se, or by subsequent lipopolysaccharide-protein or lipopolysaccharide-phospholipid interaction within the outer leaflet of the outer membrane. Lipopolysaccharide-protein interactions in the outer membrane have been clearly demonstrated (Yu and Mizushima, 1977; Yamada and Mizushima, 1980; Schindler and Rosenbusch, 1978; Schweizer et ul., 1978; Henning and Jann, 1979). The current understanding of translocation of lipopolysaccharides postulates a transfer from the inner to the outer membrane at sites of contact between the two, the so-called zones of adhesion initially described by Bayer ( I 975) and Muhlradt et a / . (1973, 1974). However, the detailed molecular mechanisms of translocation and integration are still only poorly understood (for models see Osborn, 1979). As the carbohydrate portion of the lipopolysaccharide must for biosynthetic reasons face inward i n t o the cytoplasm initially, but outward after translocation, the translocation process may require transmembrane movement (flip-flop) as well as intermembrane transfer. Mutants in lipopolysaccharide synthesis have facilitated studies directed to the question of the possible role of the lipopolysaccharide structure in biosynthetic

LIPOPOLYSACCHARIDES OF GRAM-NEGATIVE BACTERIA

113

processes. Clearly, neither completion of the core nor of the 0 chains is a necessary prerequisite for translocation and integration in the outer membrane, since such lipopolysaccharide translocation occurs in a variety of mutants including Re mutants. The KDO-deficient lipid A precursors accumulated in the mutants are, however, only incorporated very slowly into the outer membrane (Osborn et ul., 1980), providing strong evidence that the structural features of the internal region of the lipopolysaccharide molecules are indeed important (Osborn, 1979). Slow incorporation of lipid A intermediates into the outer membrane does not give a definitive answer to the question of whether the primary defect in assembly lies at the level of translocation or arises from failure of subsequent interactions required for stable integration. The latter possibility appears, however, unlikely on the basis of experiments on liposome-mediated incorporation of exogenous lipid A precursor into the outer membrane of intact cells. No significant transfer to the inner membrane and no detectable conversion to lipopolysaccharide occurred under conditions where extensive intermembrane translocation of liposome-derived phospholipids took place (Jones and Osbo’rn, 1977b). In order to define the smallest translocation unit, lipopolysaccharide intermediates were tested for their translocation properties. Lipid A precursor substituted by two KDO residues accumulates in the inner membrane and is not translocated (Walenga and Osborn, 1980a). In contrast, the KDO-lipid A structure of Re mutants does not accumulate and is translocated at near-normal rates into the outer membrane (Osborn et ul., 1980). Re lipopolysaccharides contain the nonhydroxylated fatty acids lauric, myristic, and palmitic acid and a third KDO residue, which are missing in the structure of the KDO lipid A precursor, though lauric, myristic, and palmitic acid have been demonstrated to be unnecessary for normal rates of translocation (Walenga and Osborn, 1980b). Lipopolysaccharides formed in the presence of cerulenin lack these fatty acids and are nevertheless translocated into the outer membrane. The head groups of lipid A, such as 4-aminoarabinose or phosphorylethanolamine, which may be present in the intermediates, are found in variable and nonstoichiometric amounts in lipopolysaccharides (Lehmann and Rupprecht, 1977; Muhlradt et al., 1977), and are therefore also unlikely to be essential. Therefore, a hypothetical molecule containing KDO trisaccharide linked to lipid A precursor appears to be the smallest unit capable of being translocated at normal rate into the outer membrane. The KDO residues have been shown to provide a high-affinity binding site for divalent cations (Schindler and Osborn, 1979), but the exact relationship of this site to translocation or integration remains unknown. Lipopolysaccharides inserted into the outer membrane are known to diffuse laterally within this membrane, from the insertion loci, to cover the entire surface. This diffusion appears to be dependent on the structure of lipopolysaccharide. Thus the translational diffusion coefficient of lipopolysaccharides of the

114

OTTO LUDERITZ ET AL.

chemotype Rc is similar to that of phospholipids ( D = 10 !' cm2 SS') as measured 1980). In by fluorescence photobleaching recovery techniques (Schindler et d., contrast, S-form lipopolysaccharides are more restricted (Miihlradt ef ul., 1974; Rottem and Leive, 1977; Leive, 1977), and appear to be organized at least partially in domains.

111.

SOME SELECTED ASPECTS ON THE BIOLOGY OF LIPOPOLYSACCHAIDES

There is a vast and still expanding literature on the biological effects of lipopolysaccharides, and a comprehensive review on this topic is outside of the scope of this article. The reader is referred to recent reviews (Galanos et a / . , 1977a; Berry, 1977; Rietschel ef d . , 1980b, 1981; Morrison and Ulevitch, 1978; Jirillo and Fumarola, 1979; Bradley, 1979; Agarwal, 1980). and monographs (edited by Kadis et ul., 1971; Kass and Wolff, 1973; Schlessinger, 1977, 1980). Only selected aspects of the topic will be discussed here.

A. Endotoxic and Immunogenic Properties of Lipid A PRINCIP1.E 1. LIPID A , T H E ENDOTOXIC

Ob

LIPOPOLYSACCHARIDES

Lipid A is the endotoxic principle of lipopolysaccharides. This was initially postulated in the early 1950s as a conclusion from the fact that lipid A has been found as the only component common to all lipopolysaccharides from the Enterobacteriaceae (Westphal and Liideritz, 1954). A further strong indication was suggested by the observation that R-mutant lipopolysaccharides, lacking most of the polysaccharide chain (Fig. 4), and even the Re lipopolysaccharide, containing only KDO and lipid A , were all equally active as endotoxins (Liideritz et ul., 1966a). However, only recently could direct and definitive proof be offered that lipid A, devoid of 0 chain and core, represents the endotoxic moiety of lipopolysaccharides (Galanos et al., 1971a, 1977a,b). Free lipid A , obtained by mild acid hydrolysis of lipopolysaccharide and rendered water soluble by electrodialysis and subsequent neutralization, was shown to exhibit all the endotoxic reactions displayed by the complete parent lipopolysaccharide with the exceptions to be noted. This has been confirmed in various laboratories for many biological test systems, and is now generally accepted. It has been found, however, that in contrast to lipopolysaccharide, free lipid A does not induce necrosis and regression of tumors in mice (Wasilauskas and Cameron, 197 1 ; Tanamoto et al., 1979; Westphal et al., 1979, 1981). Since the elucidation of the structure of lipid A , several teams have started a

115

LIPOPOLYSACCHARIDES OF GRAM-NEGATIVE BACTERIA

TABLE 1V SYNTHETIC LIPIDA MODEISUBSTANCES WITH ENDOTOXIN ACTIVITIES Compound

N-Palmitoyl-o-glucosamine Myristic acid-bovine serum albumin complex

N-Palmitoyl- and N-myristuyl-pglucosamine O-Palmitoyl-dextranphosphate Maltose tetrapalmitate 3-hydroxymyristoyl hydroxamate N-Myristoyl- and N-o.1 -3hydroxymyristoyl-o-glucosamine 6-phosphate

Activity B-Cell mitogen Lethality for mice. induction of reciprocal endotoxin tolerance Adjuvant effect, protection against radiation Antitumor activity Antitumor activity Serological crossreactivity with lipid A Serological crossreactivity with lipid A

Reference Rosenstreich er ul. ( 1 974) Bradley ( I 976)

Behling et d.(1976)

Suzuki et ul. (1977) V. Nigam (personal communication) Lugowski and Romanowska (1974) Gorbach et ul. (1979)

chemical synthesis of this component. Some groups have taken the approach of synthesizing model compounds resembling lipid A partial structures or compounds being endowed with physicochemical properties characteristic of lipid A . These products exhibit endotoxin-like activities and they are listed in Table I V . 2 . IMMUNOGENICITY O F L I P I DA ANTI-LIPID A ANTISERA

AND

PROPER^ I E S O F

In contrast to lipopolysaccharide-bound lipid A , free lipid A complexed or linked to a suitable carrier is immunogenic. I n the original approach, bacterial cells carrying lipid A on the surface were used for immunization (Galanos et u l . , 1971b, 1977a,b). For the preparation of these immunogens, the gram-negative bacterial cell was treated with acetic acid in order to remove the polysaccharide portion of the lipopolysaccharide, thus unmasking lipid A and exposing it on the surface. These cells were then coated with an excess of external free lipid A . Immunization of experimental animals with such immunogens leads to the formation of specific high-titer antisera, and this procedure has been widely adopted. It must be emphasized, though, that these antisera also contain antibodies evoked by other antigens exposed after the acid treatment (e.g., lipoprotein). Therefore, other forms of immunogenic lipid A have been developed. Lipid A complexed with bovine serum albumin (Gorbach et ul., 1979) or coated on erythrocytes (Mattsby-Baltzer and Kaijser, 1979) has been used for obtaining

116

OTTO LUDERITZ ET AL.

anti-lipid A antisera. Liposomes with actively incorporated free lipid A proved to represent a valuable immunogen (Schuster et ul., 1979; Banerji and Alving, 1979, 1981; C. Galanos, unpublished data); these antisera, however, also contained antibodies against phospholipids of the liposomes. Such antibodies were not induced by lipid A-free liposomes, but reacted with them (Schuster et ul., 1979). The possibility of conjugating antigenically active lipid A fragments to protein carriers will be discussed later. Anti-lipid A antibodies are detected and measured by the passive-hemolysis test, using sheep erythrocytes coated with lipid A or alkali-treated lipid A (Galanos et al., 197 I b). An enzyme-linked immunosorbent assay (ELISA) has also been published (Jay, 1978; Mattsby-Baltzer and Kaijser, 1979; Fink and Kozak, 1980; Fink and Galanos, 1981). Lipopolysaccharide does not evoke anti-lipid A antibodies, nor do these react with lipopolysaccharide. Due to the close structural relationships between lipid A’s of Enterobacteriaceae and other gram-negative bacteria, complete serological cross reactions are seen among free lipid A’s of Enterobacteriaceae and other families (Galanos et a l . , 1977b; Rietschel et al., 1981; for exceptions see Section III,B,3). The immunodominant structure of lipid A comprises the linkage region of glucosamine and amide-bound hydroxy fatty acid (Liideritz et al., 1973). This was concluded from the observations that free lipid A and 0-deacylated free lipid A exhibit equal serological activity, and furthermore that a fragment of lipid A (obtained from Re lipopolysaccharide by mild hydrazine treatment and subsequent hydrolysis), containing the glucosamine-disaccharide devoid of all but one amide-bound hydroxy fatty acid (linked probably to glucosamine I, see Fig. 12), is still antigenically highly active (Liideritz et al., 1973; Galanos et al., 1977a). This agrees with the observation that Salmonella free lipid A and precursor lipid A show complete cross-reaction (see later), and that some synthetic compounds exhibit lipid A specificity (Table IV). Thus Lugowski and Romanowska (1974) have shown that 3-hydroxymyristoyl hydroxamate acts as an inhibitor of a lipid A/anti-lipid A system. In analogy, Gorbach et u l . (1979) tested the following compounds for their inhibitory activity: (1) N-acetylglucosamine; (2) N-acetylglucosamine 6-phosphate; (3) N-myristoyl-, and (4) N-o,L-P-hydroxymyristoylglucosamine 6-phosphate. Whereas compounds ( I ) and (2) proved to be inactive, an almost equal inhibitory activity could be demonstrated with compounds (3) and (4). Unfortunately, the two optical antipodes present in (4)-that is, the natural (D) and the unnatural (L) hydroxy acid-have not been tested separately; therefore, it is, not possible to define the contribution of the stereochemistry of the hydroxyl group of the amide-bound fatty acid to antigenic specificity. Further, these results do not permit a conclusion regarding the role of the anomeric configuration of glucosamine in specificity. In all these cases, the synthetic lipid A analogues

117

LIPOPOLYSACCHARIDES OF GRAM-NEGATIVE BACTERIA

exhibited an activity approximately 100 to 1000 times less than expected for free lipid A. The aforementioned lipid A product of mild hydrazine treatment contains a free amino group on one of the two glucosamine units. This amino group has been used as a functional group to couple, with the aid of glutaraldehyde, this hapten to suitable carriers (Fig. 12). With edestin o r other proteins, very efficient, nontoxic immunogens are obtained that on immunization lead to highly specific anti-lipid A antibodies. Such vaccines may be valuable for clinical use. Binding of the fragment to a solid carrier has afforded an immunoadsorbant, which made possible the isolation of anti-lipid A antibodies (see also Lugowski and Romanowska, 1974). The efficiency of this procedure has been demonstrated and pure lipid A antibodies can now be produced for use in biological investigations (C. Galanos and D. Nerkar, unpublished data). A similar strategy has been applied to S- and R-form lipopolysaccharides for obtaining nontoxic 0 and R haptens. In this case, complete hydrazinolysis was performed and water-soluble preparations were obtained containing the respective 0 and/or R region linked to the lipid A backbone with free amino groups on the glucosamine residues available for coupling. Artificial immunogens and immunoadsorbants have been prepared from, for instance, hydrazine-treated lipopolysaccharides of different R chemotypes (Nixdorff et ul., 1975; C. Galanos, unpublished data). In this way pure monospecific R antibodies have been obtained after adsorption on different columns. Many animal species respond readily to primary or booster injections of acidtreated and lipid A-coated bacterial cells with the production of anti-lipid A antibodies (rabbits, goats, dogs, rats, vervets, baboons) (Galanos ef ul., 1971b). These animal species and humans often contain natural antibodies with lipid A

LPS

Hydrazine 1 h, 60'

acet. acid

A PLGlcN-pl.6-GlcN-!-P

I

0.5 h, 100'

Q

Glutaraldehyde Carrier

0

CH

C.0

p 2 $H2

p 2 H$-OH

FH2

HC=N-Corricr

[$"Z)lO CH3

FIG.12. Preparation of lipid A-specific immunogen (camer. protein) and immunoadsorbant (carrier, AH-Sepharose).

118

OTTO LUDERITZ ET AL.

specificity in addition to a large variety of anti-0 specificities. In mice, however, naturally occurring antibodies with lipid A specificity have never been detected, although their sera also contain various anti-0 specificities. Moreover, mice fail to show a primary anti-lipid A antibody response, even after booster injections. Under specified conditions, however, mice will respond, and high anti-lipid A titers have been obtained using two injections of lipid A-coated bacteria with a time interval between the injections of longer than 3 weeks (Freudenberg, 1975; Galanos et al., 1971b). The presence of anti-lipid A antibodies in normal animals and humans and their increase in patients with gram-negative infections indicate that under certain natural conditions, lipid A becomes sufficiently exposed to express its immunogenicity . Biological activities of anti-lipid A antisera and their possible clinical role and application have been discussed recently (Rietschel and Galanos, 1977; Galanos et a l . , 1977a; Blake ct d.,1980; Westenfelder et al., 1977).

B. Physicochemical and Structural Prerequisites for Biological Activities of Lipopolysaccharide 1 . PHYSICAL S T A T E A N D BIOLOGICAL ACTIVITY OF LIPO PO L Y sACC H A R I DE s

Isolated lipopolysaccharide in solution forms aggregates (micelles) due to nonpolar interactions of the lipid A component. Recently, an additional cause of aggregation in which divalent cations and polyamines form intermolecular ionic linkages with the acidic groups of the molecule (phosphate, carboxyl groups of KDO) has been identified. The conversion of lipopolysaccharides into uniform salt forms by electrodialysis (see Section II,A) offered the possibility of investigating the influence of different cations on the degree of aggregation and, furthermore, of studying the effect of aggregation on the activity of lipopolysaccharide in various in vitro and in vivo biological tests (Galanos, 1975; Galanos et a / . , 1979a). The results of these investigations are summarized in Fig. 13. S. ubortus eyui lipopolysaccharide was converted into different salt forms and various parameters were rested. The state of aggregation (represented by sedimentation coefficient) and the solubility of the preparations were characteristic of the respective cation. Further, individual biological activities changed with increasing aggregation. Thus lethal toxicity for mice and pyrogenicity in rabbits decrease with increasing sedimentation coefficient, whereas lethality for rats, clearance from the blood, reactivity toward complement, and affinity to mammalian cells show a reverse dependence. Mitogenicity and activity in the Limulus lysate test, on the other hand, are not influenced by the state of aggregation. It is obvious that the distinctive physical state of a lipopolysaccharide influ-

S abortus --

LPS

Triet hylomine

(TEN1 Pyridine Ethanolomine No

K Putrescine Ca

Roteddcarcm Interaction with C ' from the Mood invivo and in vitro

jedirnentoticm Coefficient

1I

irnulus lysate gelation

I

230

partly insol

FIG. 13. Physicocheniical properties and biological activities of the lipopolysaccharide of Soltnorirllr ubortus c y r r i in different salt forms. Influence )f the nature of the cation neutralizing the acidic groups of the lipopolysaccharide (after Galanos et d., 197Ya). Arrows indicate direction of increasing stivity; (a) TEN form is completely inactive: (b) the different salt forms are of equal activity.

120

OTTO LUOERITZ ET AL.

TABLE V BIOLOGICAL ACTIVITIES OF COMPLtTt ~u/rnonc//u LIPID A. LIPIDA PRECURSOR 1 A N D CHFMtCAl.-DEGRADATION PRODUCTS" 0-

Type of activity

Free lipid A

Cross-reaction with anti-Su/rnonc//a lipid A anti serum Complement reactivity Mitogenicity Pyrogenicity Lethal toxicity Limulus activity

+ + + + + +

"

Lipid A precursor

Deacylated free lipid A

+

+

5

-

+ '' + +

0 , NDeacylated free lipid A

+

After Luderitz cf a / . 1978; R.G.McKenzie and C. Galanos, unpublished data.

ences the endotoxic activity. It is not surprising, therefore, that the same lipopolysaccharide, obtained by different methods, may differ in activity according to the cations present in the preparation, and certainly, for specific investigations it is important to know what kind of preparation is used with regard to the cations and amines present. The standardized S. aborrus equi lipopolysaccharide mentioned in Section II,A represents the sodium salt form.

2. BIOLOGICAL ACTIVITIES O F LIPIDA FRAGMENTS Complete free Salmonella lipid A, precursor I , and two chemically degraded Salmonella lipid A preparations were investigated comparatively for biological activity (Luderitz er al., 1978). The precursor lacks the head groups and the nonhydroxylated fatty acids (see Fig. IOa), whereas the 0-deacylated preparation obtained by alkali treatment contains only the amide-linked P-hydroxymyristoyl residues (and some phosphoryl and pyrophosphorylethanolamine units). The 0,N-deacylated product prepared by hydrazinolysis represents the glucosamine disaccharide partially substituted by phosphate groups. These preparations were tested in the following systems: cross-reactivity with anti-Salmonella lipid A antiserum, complement and Lirnulus reactivity, mitogenicity to B lymphocytes, pyrogenicity (rabbits), and lethality (adrenalectomized mice). As seen from Table V , the completely deacylated product is inactive in all tests, whereas the 0-deacylated lipid A and the precursor, both containing amide-bound fatty acids, cross-react with the lipid A antiserum, are mitogenic, and react in the Limulus gelation test. The precursor, in addition, exhibits lethal

LIPOPOLYSACCHARIDES OF GRAM-NEGATIVE BACTERIA

121

toxicity, but is a weak pyrogen and shows decreased complement reactivity. A clear separation of the different endotoxic activities is thus apparent. 3 . BIOLOGICAL A C T I V I T IOEFSL I P O P O I . Y S A ~ C H A ARNI DI ) LIPID ES FAMILIES A’S FROM DIFFERENT BACTERIAL

In the past it was generally accepted that lipopolysaccharides, independent of their source, were equally active endotoxins, and this is still valid for lipopolysaccharides of Enterobacteriaceae and related families. This is of course due to the great structural similarities of their lipid A components. A few exceptions were known, however; for example, lipopolysaccharides from Brucellu had been found to exhibit very low toxicity in experimental animals (see Kreutzer et a l . , 1979). In recent years, lipid A’s derived from lipopolysaccharides of bacterial families remote from Enterobacteriaceae have been evaluated, and their structures have been discussed in Section 1I,D,5. In view of their structural differences when compared with Salmonella lipid A , it was not surprising lo find that differences also existed in the biological activities of these lipid A’s and of the corresponding lipopolysaccharides. Table VI shows some of the results obtained in various test systems with lipopolysaccharide and free lipid A from Chr. violaceurn, Rhsp. tenue, and Rhps. viridis and Rhps. pulustris, in comparison ; et a l . , 1978). with those from Sulrnonellu (Galanos et u l . , 1 9 7 7 ~ Luderitz Neither the lipopolysaccharide nor the free lipid A from Rhps. viridis and Rhps. palusrris exhibit any cross reaction with anti-Salmonellu lipid A antiserum. They are nontoxic and nonpyrogenic but react strongly with complement. In the Limulus gelation test, Rhps. pulustris is highly active. In this case pyrogenicity and Limulus activity do not run parallel. Chr. violaceurn and Rhsp. tenue free lipid A cross-react in the Salmonella lipid A system, whereas the corresponding lipopolysaccharides d o not. These preparations are inactive toward complement. The Chr. violaceurn lipopolysaccharide and free lipid A exhibit lethal toxicity and pyrogenicity, the free lipid A from Rhsp. tenue is toxic, but only a weak pyrogen; the lipopolysaccharide is also weakly pyrogenic, but of low lethality to mice. With our present knowledge, it is not possible to explain the different biological activities of the preparations on the basis of particular structural features. Differences in the serological behavior of free lipid A and the parent lipopolysaccharide may indicate a masking of the determinants of the Iipopolysaccharidebound lipid A by the 0 chains or by the polar head groups. These are usually removed (the head groups only partially) during the acid-catalyzed liberation of these lipid A ’ s . A similar reasoning may explain why even though the Rhsp. tenue free lipid A is toxic, the lipopolysaccharide is not. Chemical and biological

TABLE V1 B I O L O C I CACTILITIES ~L O F LIPOFOLYSACCHARIDES A N D CORRESFONDINC FREELIPIDA'S

Sulmonella Type of activity ~

LPS ~

Immunological cross-reactivity with anti-Solmonellu lipid A antiserum Reactivity with complement Pyrogenicity Lethal toxicity Limulus activity

~

Lipid A

Chromobacrerium violareurn LPS

Lipid A

-

-

FROM

DIFFERENT BACTERIAL GROUPS"

Rhodospirillum tenue LPS

Lipid A

-

+

Rhodopseudomonas viridus LPS

Rhodopseudomonas palustris

Lipid A

LPS

ND

~

+

+

+

+ +

+

+ + + +

+ + +

+

+ +

NDb

I

-

+

-

-

-

+

+

2

-

ND

-

+

ND

+

-

+

" After Liideritz et (11.. 1978; Galanos et al. 1977c; R. G . McKenzie and C. Galanos. unpublished data. Cross-reactivity of LPS with anti-lipid A antiserum was determined in vivo according to Rietschel and Galanos (1977). " N D . not determined.

123

LIPOPOLYSACCHARIDES OF GRAM-NEGATIVE BACTERIA

studies must be conducted on further unusual lipid A systems before it will be possible to identify substructures in lipid A.

C. The Fate of Lipopolysaccharides (Lipid A) in Experimental Animals I . T H EROLEOF HIGH-DENSITY ~ I P O I ’ R O T E I N(HDL) LIPO PO L Y SAC c H A R I DE T R A N SPORI

IN

After intravenous administration to experimental animals, bacterial endotoxins persist in the blood for some time before they are cleared mainly into the liver (Braude, 1964). Since they are not found on circulating blood cells, the very early main targets must be constituents of the plasma. One early target of endotoxin after its intravenous administration is the complement system. The property of lipopolysaccharides to interact with the complement system in vitro has been known for a long time (Pillemer et a / . , 1955). Recently, using lipopolysaccharides in physicochemically defined forms, it could be shown that the property to interact with complement in Litro is expressed only by lipopolysaccharides that are present in a high degree of aggregation (Galanos and Luderitz, 1976). In a more recent investigation carried out in rats, it was demonstrated that a high aggregation is also required for the interaction of lipopolysaccharide with C’ iri viva (Freudenberg and Galanos, 1978). The in vivu anticomplementary activity of lipopolysaccharide, however, is not related to its toxic properties. Thus the Iipopolysaccharide of Rhps. virirfis, which was shown to be nontoxic (Galanos r t a / . , 1 9 7 7 ~ )in mice, was highly anticomplementary iri v i w following its adminstration in rats, however in the absence of toxic effects (Freudenberg and Galanos, 1978). Skarnes (1968) was the first to identify lipopolysaccharide complexes formed with lipoprotein from the plasma. Recently lipopolysaccharide-HDL complexes in circulating plasma (and serum) of rats have been identified by crossed immunoelectrophoresis (Freudenberg et a l . , 1980a). These complexes were formed within 3 minutes of the administration of lipopolysaccharide. The binding of lipopolysaccharide to HDL leads to a reduction in the rate of lipopolysaccharide clearance from the blood (Mathison and Ulevitch, 1979). Bound lipopolysaccharide circulates in low-density form (Ulevitch et a / . , 1979), and it has been reported that the endotoxic activity of the complexed lipopolysaccharide in this form is reduced (Skarnes, 1968; Ulevitch and Jonston, 1978). I t is the interaction with HDL, however, that probably determines the distribution of lipopolysaccharide in vivo. The affinity of lipopolysaccharide for HDL is very high, and coating of red blood cells with lipopolysaccharide is completely inhibited in the presence of HDL. The active principle in the binding reaction is lipid A, and incubation of

124

OTTO LUDERITZ ET AL.

free lipid A with HDL immediately leads to complex formation (Freudenberg et a / . , 1980c). It should be noted that in other animal species lipoproteins other than HDL might also be involved in lipopolysaccharide transport. The clearance time for lipopolysaccharide from the blood depends on the immune status of the animal (host), the lipopolysaccharide structure (S, R form), and the degree of lipopolysaccharide aggregation as shown by investigating plasma clearance of [14C]lipopolysaccharidepresent in different salt forms. Under the conditions used, the half-time of clearance was less than 2 minutes and 30 minutes for Re lipopolysaccharide Na and triethylamine forms, respectively, and 6 hours and 7.5 hours for S lipopolysaccharide Na and triethylamine froms, respectively (M. A . Freudenberg and C. Galanos, unpublished data).

2. DISTRIBUTION OF LPS

IN

EXPERIMENTAL ANIMALS

Earlier studies had shown (see Braude, 1964) that endotoxin is cleared mainly into the liver, and to some extent into the spleen. This has since been confirmed in many laboratories. There is general agreement concerning the uptake of lipopolysaccharide by hepatic macrophages, and only a few authors have detected lipopolysaccharide in hepatocytes (Willerson et al., 1970; Zlydaszyk and Moon, 1976; G. Ramadori, C. Galanos, V. Hopf, and K . Meyer zum Buschenfelde, unpublished data). Recently immunohistochemical methods have been applied to study the kinetics of the distribution of Salrnorzellu S- and Re-form lipopolysaccharides in the rat. It was found (Freudenberg et nl., 1980b) that the first cellular targets for the S-form lipopolysaccharide were phagocytic cells, mainly Kupffer cells of the liver, macrophages of the spleen and some granulocytes in agreement with previous studies. Two to three days after injection, however, a redistribution of the lipopolysaccharide in the liver was observed, by this time the hepatocytes becoming strongly endotoxin positive. When Re-form lipopolysaccharide was used, it was from the beginning in Kupffer cells, granulocytes, and hepatocytes, indicating that hepatocytes may in principle also clear lipopolysaccharide from the blood. Both S- and R-form lipopolysaccharide preparations showed a decrease with time in the staining activity in the liver. Nine days after injection only very weak diffuse lipopolysaccharide staining was seen, indicating that part of the lipopolysaccharide was no longer present in the liver or that its antigenic specificity had been modified, or that both effects were taking place. Similar differences in the primary uptake of S- and Re-form lipopolysaccharides were detected in vivo and in witro in mice (Ramadori et ul., 1980). However, in a recent study in rabbits, association of the Re lipopolysaccharide with hepatocytes was not observed (Mathison and Ulevitch, 1979). There are considerable indications that the liver is an important organ of lipopolysaccharide excretion. In

125

LIPOPOLYSACCHARIDES OF GRAM-NEGATIVE BACTERIA

rabbits high concentrations of [ I Z 5 I llipopolysaccharide were observed in the gallbladders (Mathison and Ulevitch, 1979); in rats injected with [14C/ :~H]lipopolysaccharide,about 60% of the label was found in the feces for u p to 3 weeks following lipopolysaccharide treatment (Kleine, 1981). An interesting observation was made concerning uptake of lipopolysaccharide by the lung (Freudenberg e / al., 1980b). During the first hours after injection this organ was free of tissue-bound endotoxin. However, a large number of endotoxin-positive cells appeared with a delay of several hours when compared to the presence of bound endotoxin in the liver. It is possible that lipopolysaccharide-carrying cells originate from the liver and (or) spleen and are excreted in the lung. The presence of a number of lipopolysaccharide-carrying alveolar and bronchiolar macrophages between 24 hours and 5 days after endotoxin treatment supports this assumption. PROSTAGLANDIN 3. LIPOPOLYSACCHARIDE-INDUCED

IN

SYNTHESIS

MACROPHAGES

It is known that a number of endotoxin effects such as fever, early hypotension, skin necrosis, and abortion can be suppressed by acetylsalicylic acid (aspirin) or indomethacin (i.e., drugs that are inhibitors of prostaglandin biosynthesis). Furthermore, certain of the prostaglandins induce typical endotoxic reactions. These observations have led to the hypothesis that prostaglandins may represent mediators of lipopolysaccharide effects. In fact, macrophages could be identified as the cell type that, on incubation with lipopolysaccharide, would be stimulated to synthesis and excretion of prostaglandins of the E, and F2a type (Rietschel e/ al., 1980a). Three lines of evidence exist indicating that macrophages are indeed the source of prostaglandins mediating the lipopolysaccharide effects in vivo. It has been demonstrated in several laboratories that the mouse strain C3H/HeJ, which is genetically resistant to a number of endotoxin effects including lethality (lipopolysaccharide nonresponder), produces macrophages that cannot be stimulated by lipopolysaccharide to secrete PGE, and F,a. As expected, cells from genetically related lipopolysaccharide-responder strains were stimulated to prostaglandin synthesis and excretion (Wahl et a / . , 1979; Rietschel e/ al., 1980b). Second, mice can be rendered nonresponsive (tolerant) to lipopolysaccharide by repeated administration of sublethal doses of lipopolysaccharide. Macrophages from tolerant mice are completely refractory to the action of lipopolysaccharide and do not secrete prostaglandins E, and F,a (Schade and Rietschel, 1980). Third, rats fed with a diet devoid of essential fatty acids, which are precursors of prostaglandin biosynthesis, are highly resistant to lipopolysaccharide lethality (Cook er a / . , 1979). Although these results are consistent with the hypothesis that macrophage-

126

Om0 LUDERITZ ET AL.

derived prostaglandins play a role i n the mediation of lipopolysaccharide effects, further studies are needed to elucidate possible etiology or regulatory functions of prostaglandins. Most data in the past have been obtained in in vitro experiments with mouse macrophages obtained from the peritoneum or from the bone marrow. Preliminary results show that Kupffer cells (rat) and alveolar macrophages (rabbit) are also stimulated by lipopolysaccharide to release prostaglandins E, and F p (B. Bhatnagar, K. Decker, K . Tanamoto, U . Schade, and E. T. Rietschel, unpublished data). 4 . GALACTOSAMINE-INDUCED SENSITIZATION TO T H E LE I’HAI. EFFECTSOF LIPOPOLYSACCHARIDES Several experimental models exist by which the natural sensitivity of animals toward lipopolysaccharide (endotoxin) may be increased. Examples are adrenalectomy, and treatment with Bacillus Calmette-Guerin, actinomycin D, lead tetraacetate, a-amanitin (Schlievert et al., 1980),galactosamine (Galanos et ul., 1979b), or antigen-antibody complexes (Galanos, 1979). Increased susceptibility to endotoxin may have a role to play under natural conditions. It is known that outer membrane proteins of gram-negative bacteria are immunogenic, and it has been shown that bacterial protein-antibody complexes will enhance the lethal toxicity of lipopolysaccharide. Since a wide serological cross-reactivity exists among proteins of different bacterial genera, and since animals and humans are permanently in contact with gram-negative bacteria, preexistent bacterial protein antibodies could play a role during infection by increasing susceptibility of the host to lipopolysaccharide (Galanos, 1979). The susceptibility-increasing models mentioned previously have multiple effects on the organism as a whole, but several are also known to be poisonous to the liver. Galactosamine, however, represents a highly specific hepatotoxic agent, and mechanisms underlying “galactosamine” hepatitis have been extensively elucidated (Decker and Keppler, 1974). Administration of galactosamine and lipopolysaccharide (either together or with the lipopolysaccharide within 1 hour later) will lead to a dramatic decrease (Galanos e f al., of the LD,, in mice (by factors lo-“ to lop5),or rabbits 1979b). It is the lipid A part of lipopolysaccharide to which the animals are sensitized. If uridine is applied within 3 hours after the galactosamine treatment, the effect of galactosarnine is negated. In this model the early metabolic effects induced by galactosamine are sufficient for sensitization to endotoxin. At a time when the animals are highly susceptible, typical liver enzymes have not yet been released into the blood, indicating the absence of cell injury. This shows that temporary metabolic changes without clinical symptoms may render an organism highly susceptible to very small amounts of endotoxin, and this may be of clinical importance.

LIPOPOLYSACCHARIDES OF GRAM-NEGATIVE BACTERIA

127

It has been observed over many years that different animal species show large variations in their susceptibility to lipopolysaccharide. Mice and rats are relatively resistant, whereas rabbits and humans generally show a high susceptibility. Rabbits also exhibit a high degree of variation, however. On average, the LDIo0 is about 50 p g of endotoxin for a given strain, but in a single group of rabbits individuals are found that are very susceptible, whereas others can survive milligram amounts of lipopolysaccharide. It has now been shown that in the small percentage of rabbits that die with 0.1 to 1 p g of lipopolysaccharide, the kinetics of liver-enzyme release into the blood are similar to that observed in rabbits made sensitive to lipopolysaccharide by galactosamine. It might be concluded that in natural sensitization, biochemical alterations i n the liver, similar to those induced by galactosamine, are involved. The liver, apart from being the main organ of endotoxin clearance, could thus play a role in the initiation of endotoxic effects (C. Galanos and M . A . Freudenberg, unpublished data).

5. ROLE O F T H E POLYSACCHARIDE POKTION OF LIPOPOLYSACCHAKIDES I N CELLULAR RECOGNIT ION The fundamental importance of polysaccharides in recognition phenomena rests on their property of exhibiting compositional and structural diversity. Lipopolysaccharides are composed of a variety of different sugar residues that are linked in a number of different ways. They are thus extremely complex and diverse molecules with a high value of structural information. Inserted asymmetrically into the outer membrane with their carbohydrate determinants facing and extending outward, lipopolysaccharides are ideal recognition structures for phages, antibodies, or eukaryotic cells. It is this complexity and diversity of the polysaccharide moiety of lipopolysaccharides that make gram-negative bacteria useful probes for the detection and identification of carbohydrate-binding structures such as lectins o r lectin-like structures on eukaryotic cells. Evidence for the existence of lectin-like receptors on phagocytes comes from work on mouse peritoneal macrophages. Bacteria are bound to the macrophage membrane by a mechanism that appears to involve recognition of sugars derived from lipopolysaccharides by membrane-bound lectin receptors of the macrophage (Weir, 1980). The binding of bacteria by macrophages in monolayers can be inhibited by preexposure of the monolayer to a variety of monosaccharides. Glucose and galactose inhibit the binding of numerous bacterial species such as E . coli, Ps. ueruginosa, or S . typhimiirium. If one or both sugars are not present in mutant lipopolysaccharides, the missing sugars become noninhibitory, despite their ability to inhibit the binding of the wild-type organism. Thus S . f ~ ~ ~ ; core ~ mutants ~ z f of ~ the ; chemotype ~ ~ ~ Re, with no outer core or 0 chain, appear to bind via their inner-core components, and binding is not inhibited by any sugar that inhibits binding of the wild type. A similar picture

128

OTTO LUDERITZ ET AL.

emerges with Klebsiellu uerogenes, in which galactose fails to inhibit binding of the galactose-deficient core mutant MlOB. in a more recent approach bacterial mutants from Safmone/lu strains were selected that are able to bind to lectin-like structures expressed on activated T lymphocytes and absent on resting T lymphocytes (Lehmann et af., 1980). The selection procedure included two consecutive steps: the mutagenesis of nonadhering smooth strains from Sulmonellu and the subsequent enrichment of mutants adhering to T lymphocytes that had been activated in a mixedlymphocyte culture. Enrichment of the desired mutants was achieved by I g-velocity sedimentation, a procedure that separates cells on the basis of size differences. Accordingly, T-cell blasts and adhering mutants were separated from nonresponsive small lymphocytes as well as from nonadhering bacteria. Adhering mutants were shown to belong to the class of rough mutants with the chemotype Ra or Rb (Fig. 4).These mutants exhibit specific binding properties. They bind to a T-cell subpopulation, tentatively identified as T helper cells, whose proliferation and differentiation is required for the generation of killer or suppressor cells. Binding of the mutants is mediated by lectin-like receptor sites on the T-cell subset, which recognizes a sugar portion of Ra or Rb lipopolysaccharides on the bacterial surface. The involvement of polysaccharides in the binding process rests on the observation that adherence is specifically inhibited by lipid A-free Rb polysaccharides. Marginal or no inhibition was observed with polysaccharides derived from the wild-type strain or from Sulmonellu Rc, Rd, or Re mutants. There is evidence that the Rb polysaccharide-recognizing structures on activated T lymphocytes have a functional role for the differentiation of T-cell subsets. This conclusion is based on the observation that polysaccharides of the chemotype Rb that inhibited adherence of the mutants to activated T cells suppress also the generation of killer T cells. It is believed, therefore, that the interaction between the bacterial polysaccharides and the lectin-like structures on T helper cells prevents differentiation of these cells, or alternatively, mediates the induction of suppressor cells that inhibit the generation of killer cells. So far only a limited number of lectin or lectin-like structures have been identified on phagocytic and nonphagocytic cells. Selected bacterial polysaccharides may reveal more of these structures on lymphoid and nonlymphoid cells and may eventually show that there are in nature similar numbers of carbohydrate and complementary structures, the lectin or lectin-like proteins, that interact and mediate fundamental functions in biology (see also Mayer and Teodorescu, 1980). 6. LIPOPOLYSACCHARIDE DEGRADATION IN NATURE

Several biological systems have been identified that will degrade lipopolysac~ R8, , and others), whose recharide. A number of bacteriophages (el5,E ~ P22,

LIPOPOLYSACCHARIDES OF GRAM-NEGATIVE BACTERIA

129

ceptor site is lipopolysaccharide (from Salmonella, Escherichia, Shigella$exneri. Proreus), have been identified and shown to exhibit (end0)glycosidase activity (Iwashita and Kanegasaki, 1973; Reske et d.,1973; reviewed by Lindberg, 1977; Braun and Hantke, 1981). This activity resides in the spikes or tail parts, which consist of a single protein species. These enzymes recognize, absorb to, and cleave the 0 antigen, which is the specific phage receptor of the host. Interaction of phage or isolated enzyme with the respective lipopolysaccharide leads to the cleavage of specific linkages in the 0 chain and to the liberation of mono- and oligomeric repeating units (which may differ in their sugar sequence from the biological or chemical repeat unit). During this interaction and cleavage, core and lipid A remain intact. Slime molds constitute another system of biodegradation of lipopolysaccharides. These organisms, which are ubiquitous in soil, utilize bacteria for food. Dictyostelium discoideum has available acylesterases and amidases that degrade lipopolysaccharide by cleaving off the long-chain fatty acids from lipid A. The remaining polysaccharide-lipid A backbone is then secreted by the cells (Malchow et ul., 1969). Recently, Rosner ei d., ( I 979c) have succeeded in purifying two acyl amidases from D . discoideum. Reaction of amidase I with 0deacylated lipid A leads to the removal of the glucosamine I-linked 3-OH-14:O fatty acid. The reaction product formed (but not the original lipid A) then reacts as a substrate for amidase 11, which cleaves the glucosamine 11-linked 3-OH14:O fatty acid. This reaction sequence represents an elegant and specific way for preparing defined degradation products of lipid A , which may be useful in studies of structure-function relationships. Saddler et al. (1979a) have shown that the slime mold Physarium po/ycepha/um also reacts with lipopolysaccharide. Again, the lipid A part is degraded. In this case, however, only the nonhydroxylated fatty acids are removed. A similar degradation pathway for lipopolysaccharides was observed with the gut juice of the snail Helix pomatiu (Saddler et al., 1979b). A microorganism, Bacillus macerans, has been selectively grown from soil samples by culture on mineral medium supplemented by lipopolysaccharide. This organism is assumed to cleave the polysaccharide-lipid A linkage and to remove long-chain fatty acids from lipid A (Voets e t a / . , 1973; see also Saddler and Wardlaw, 1980). Whether the higher organism is capable of degrading lipopolysaccharide has hitherto not been established definitively. In connection with endotoxin inactivation by serum factors, it has been suggested that serum esterases might degrade lipopolysaccharide (Skarnes, 1968), but no further evidence has been presented for such reactions (see Johnson et a/.,1977). Current investigations (Kleine, 1981) on excretion products of rats and vervets injected with radiolabeled endotoxin indicated that more than 60% of the label is found in the feces and urine, mainly in the form of degradation products.

o n 0 LUDERITZ ET AL.

130

IV.

FINAL REMARKS

Lipopolysaccharides are still in fashion. It is tempting to believe, as many did 50, 20, or 10 years ago, that research during the next decade will solve all the remaining problems. Experience indicates, however, that with new answers new questions will arise. The past decade has furnished us with detailed chemical formulas of many lipopolysaccharides, and the structural principles of their architecture are now known for gram-negative bacteria of various families. Of equal importance has been the elucidation of their immunological principles, their pathways of biosynthesis, and their genetics. Using methods that have proved successful in the past, structures. biosynthesis, and genetics of lipopolysaccharides different from those hitherto studied will be investigated. Efforts in the research on lipopolysaccharides are now concentrated on their interaction with other outer membrane components. and their role in the molecular organization and biogenesis of the outer membrane. The function of the outer membrane and the communication of the cell surface with the environment are further topics of investigation. Readers interested in present highlights of research and perspectives in outer membrane biochemistry and physiology are advised to study the excellent reviews of Nikaido and Nakae (1979), Inouye (1979), Makela and Stocker (1981), Braun and Hantke (1981), and the recent papers of Osborn and her group (1980). Their discussions point to future research topics. Another central aspect in this field concerns the role of lipopolysaccharides in pathogenicity and protection against infection, as well as their physiological role in normal animal life. The reviews of Melchers (1980), and the other contributions at the Dahlem conference 1979 on “The Molecular Basis of Microbial Pathogenicity” provide facts and hypotheses in this field (Smith et ul., 1980). As a conseqeunce of the identification of lipopolysaccharide structures, a number of research groups have succeeded in synthesizing the immunodeterminant structures of the 0 chains of clinically interesting enterobacteria. Coupled to protein carriers, they provide potent, nontoxic immunogens that lead to specific high-titer antisera valuable for diagnostic purposes, and with the potential to protect against experimental infection (Svenungsson and Lindberg, 1978b; Josephson and Bundle, 1979). As immunization with R-mutant bacteria has been shown to induce cross protection against infection by a variety of bacterial pathogens based on the presence of a common (inner) core structure (Ziegler ef ul., 1979, 1981; McCabe erul., 1977; Diena et u l . , 1978), it certainly will be of importance that R-type-specific nontoxic immunogens (containing core-lipid A backbone coupled to proteins) and specific pure R antibodies (obtained by immunoadsorbants containing the antigen coupled to solid carriers) are now available. The same is true for studies on the role of anti-lipid A antibodies in

131

LIPOPOLYSACCHARIDES OF GRAM-NEGATIVE BACTERIA

endotoxin pathogenesis; these antibodies can now be produced with nontoxic immunogens and highly purified by immunoadsorbants (Section III,A,2). In the vast field of endotoxin research, the old idea of Menkin (1953) that most endotoxin effects are mediated by secondary, endogenous factors, has been widely accepted and substantiated. A number of lipopolysaccharide-inducible mediators have been discovered (Schlessinger, 1980). Lipopolysaccharide nonresponder mouse strains have provided an important model in this line of research. Standardized pure lipopolysaccharide has been made available for comparative studies. But we are still only beginning to understand the molecular basis of endotoxin action (Berry, 1977). The evaluation of the lipid A structure has provided the possibility of its chemical synthesis. Carbohy,drate chemists experienced in the synthesis of amino sugar-containing natural antigens ( e . g . , bacterial 0 antigen or blood-group determinants) agree that by using available techniques, lipid A synthesis from the chemical point of view presents n o special problems. Just before completion of this article, Prof. Shiba from Osaka University, Japan, gave us a presentation on his approach to synthesize the “fundamental structure” of lipid A (Fig. 14; lnage rt al., 1980a,b) and his ideas to prepare the diphosphorylated derivatives. Also Kiso et al. ( 1 980, 1981a.b) and Nashed and Anderson ( I 98 1) have published recently the chemical synthesis of intermediates suitable for lipid A synthesis. Therefore, we can assume that a “lipid A ” synthesis will soon be achieved. Unfortunately, however, some reservations have to be made as to the lipid A structure. Even in the case of Salmondla, only the main structural features are proven; a number of minor details remain to be evaluated (Section lI,D,I). In spite of a lack of complete knowledge of the lipid A structure, we feel able to provide some predictions on substructures necessary for the activity of lipid A . This would make chemical synthesis more rational, as simpler molecules may be synthesized. From studies of structure-activity relationships (Section III,B) i t has become evident that the polar head groups of lipid A are not essential for the nHO HO

H,OH I

YoR1

R

I

R = R l = 1L.O

11

R =

14.0

to R1

R1 = 3 - O H - l L : O

FIG. 14. Synthetic partial lipid A

htructures

(Inage et d., 1980a,b)

132

OTTO LUDERITZ ET AL.

biological activities; the phosphate group linked to glucosamine 1 may not even be necessary. It would follow that the acylated lipid A backbone represents the biologically effective structure of lipid A. The length of the fatty acids is not decisive and may vary within certain limits. That acylated hydroxy acyl esters are not a prerequisite for endotoxicity, follows from the finding that they are absent from certain active lipid A's (e.g., from Chr. violaceutn lipid A). Nothing is presently known about the role of the following factors for activity: Are the 3-hydroxy groups of amide- or ester-linked fatty acids essential'? Is their D-configuration important? Are 0-3-acylated hydroxy acyl amides essential? Is the (presently unknown) distribution of the ester-bound fatty acids on the backbone critical? An atomic model of lipid A, where the nonhydroxylated acyl units are attached directly to the backbone (not to the amide-bound hydroxy fatty acids as in Fig. 6), allows the acyl residues to be brought into parallel positions (like in Fig. 6). This may indicate that the fatty acid distribution on the lipid A backbone is not decisive for interaction of the molecules with cell membranes, which probably is a prerequisite for activity (Kabir et a / . , 1978). From our own investigations it would follow that the smallest lipid A substructure identified so far and endowed with some biological activities is represented by precursor I (Fig. IOa), containing the lipid A backbone (it seems that the presence of the glucosamine I-bound phosphate is not obligatory) and carrying two amide-bound and two ester-bound 3-hydroxy fatty acids (the latter in two unknown positions). This product exhibits antigenicity, mitogenicity, lethal toxicity, (weak) pyrogenicity, and (weak) complement reactivity, but strong Liinulus lysate activity. The two ester-bound acyl residues are important, since the completely 0-deacylated product has lost most activities. Substituents on the backbone other than acyl groups, such as glucosamine, and even core and 0 chains i n some of the cases (Rhsp. tenue), seem to reduce some endotoxic activities (Table VI). The chemical synthesis of the lipid A backbone substituted with two amidelinked 3-hydroxy fatty acids can be achieved according to the opinion of experts. A limited esterification with a suitable mixture of fatty acids should also be possible. Such synthetic products should be available in the near future and it will be of great interest to see whether they act as endotoxins. Alternatively, it may turn out that gram-negative bacteria have developed the synthesis of the one endotoxin structure, where the fatty acids are linked to defined positions. The synthetic product might then contain by chance the structurally correct active molecular species in a mixture with inactive ones. We must, however, also consider the prospect that the proposed structure of lipid A is still incorrect in one or more important details, or even incomplete. The de novo chemical synthesis of lipid A analogues or their resynthesis from lipid A degradation products represents a further promising approach. Table IV gives examples of such model substances all with lipid A-like activities, some

133

LIPOPOLYSACCHARIDESOF GRAM-NEGATIVE BACTERIA

Lipopolysocchoride (Endotoxin) l P e i j ICOO'lj I

6

Polysoccharide 0 Chain

Lipoprotein

I

r '

(Pel2 (FA16-7 I 1

I '

Lipid A

Core

(8-cell mitogen) IFAlj

L

l

-

Polypept d e

4

'

1

Lipid

Lipoteichoic Acid (Shwortzmon + I

Polyglycerol- P D

Peptidoglycon

1ipid

(Endotoxin-like1

Polysorcharide - Peptide

FIG. 15. acids.

Schematic structures and biological activities of bacterial cell-wall antigens. FA, fatty

structures being quite remote from lipid A . Korhana and his group are studying the effect of the nature of the amide-linked fatty acids on lipid A activity. With the aid of specific amidases from ameba, they are able to remove sequentially the amide-bound acyl residues from the lipid A backbone with the intent of replacing them by other acyl residues (Rosner el al., 1 9 7 9 ~ ) . In this context it should be emphasized that in gram-positive and gramnegative bacteria, cell wall components other than lipopolysaccharide may also be endowed with endotoxin-like activities (Wicken and Knox, 1980; Fig. 15). Like lipopolysaccharide, these constituents are amphipathic in nature (probably with the exception of murein, though it may contain lipoprotein). These findings indicate that at least some lipid A activities are not restricted to one specific structure, but are rather connected with general physicochemical properties. ACKNOWLEDGMENTS We would like to express our thanks to Dr. H. Formanek, University of Munchen, for his help in constructing the lipid A model (Fig. 6 ) . We thank our colleagues K. and B . Jann, H. Mayer, S. Schlecht, and G . Schmidt for critiques and suggestions. We also thank Miss Helga Kochanowski for preparing the drawings, Mrs. Ingrid Himmelspach and Mrs. Lore Lay for the photographs, and Mrs. Rosemary Schneider for typing the manuscript.

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Yu, F.. and Mizushima. S. (1977). Stimulation by lipopolysaccharide of the binding of outer membrane proteins 0-8 and 0-9 tci the peptidoglycan layer of Esc.hericlriri c.o/i K- 12. R i o c ~ h ~ n i . Riophys. Rrs. Conimun. 74, 1397- 1402. Ziegler, E. J . , McCutchan, J . A , . and Brdude, A . I . (1979). Treatment of gram-negative bacteremia with antiserum to core glycolipid. I . The experimental basis of immunity to endotoxin. Eur. 1. Cuncer 15, 71-76. Ziegler. E. J . , McCutchan. J . A , , and Braude. A . I . (1981). Successful treatment of human gramnegative bacteremia with antiserum against endotoxin core. Clirz. Res. 29, A576. Zlydaszyk, J . C.. and Moon. R . J . (1976). Fate of "Cr-labeled lipopolysaccharide in tissue culture cells and livers of normal mice. I n f i ~ t .Immun. 14, 100-105. Zurkowski, W . , and Lorkievicz. 2. ( 1979). Plasmid-mediated control of nodulation in Rlrizohiutn trrfidii. A w h . Mic.rohio/. 123, 195-201.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 17

Prokaryotic Polyter penes: Phy lo ge net ic Precursors of Ste ro Is1 GUY 0URISSON Lahoratoire dr Chimie Orgi~tiiyrtetles Subattmce.~Nutrrrrlles Crntre de Neuroc,hitnir-(inivrr.~it(~ Louis Pusteur Strashourg, France

AND MICHEL ROHMER Ecole Natiotiule Superirure de Chiniie de Mirlhoirse Mulhouse Frutrce

I. Introduction . . . . , . . 11. The Sterols of Prokaryotes

. , . . . . . . The “Absence” of Sterols . . . . The Apparent Exceptions . . . .

. . . . . . . . A. . . . . B. . . . . C. The Case of Unicellular Eukaryotes . . . . The Polyterpenoids of Prokaryotes . . . . . . . A. Distribution of Polyterpenoids in Prokaryotes

, , . . . , . . . . . . . . . . . . , , . . . . . , . . . . . . . . . . , , , . . . Structural Regularities in Membrane Polyterpenoids . . .

, . . . . . . . . . . . . . . , , . , , . . . 111. , . . . . . , . . . B. . . . . . C. The Functional Equivalence of Sterols and Prokaryotic Polyterpenoids . IV. The Prokaryotic Polyterpenoids as Phylogenetic Precursors of Sterols . . . A. General Features of the Coninion Polyterpene Biogenetic Pathway , . B. Hopanoids as Precursors o f Sterols . . . . . . . . . . , , . . . C. Carotenoids as Precursors of Cyclic Polyterpenoids . . . . , . . . . D. The Archaebacterial Polyterpenes. Biogenetic Considerations . . . . . V . Addendum . . . . . . . . . . . . . . . . . . . . . . . . , .

.

,

,

Tricyclohexaprenol as a Putative Prokaryoric Triterpene and a Putative Phylogenetic Precursor of Hopanoida . . . , , . . . . . . , , References . . . . . . . . . . . . . . . . . . , . . . . . , ,

. . . . . . . . . . . . . , . . . . . . . . . . . . . ‘

. .

. . . . . . . ,

. . ,

. . .

.

154 155 155

156 157 158 159

162 166 167 167 170 175 176 177 177 178

‘This article honors the memory of academician Frantitek Sorm, who has contributed so much to the chemistry and biochemistry of polyterpenes and steroids.

153

Copyrighr 0 19RZ by Academic Press. Inc All rights of reproduction in any form rewrved. ISBN 0-12-1.53317-4

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GUY OURISSON AND MICHEL ROHMER

1.

INTRODUCTION

In eukaryotes, sterols (Fig. 1) are ubiquitous components of the membranes. They are known to improve the mechanical properties of the phospholipid bilayers, in which they are oriented by the combined interactions of the hydrophilic polar “heads” with the aqueous medium, and the cooperative van der Waals interactions of the hydrophobic chains and of the steroid skeleton (Fig. 2). Phospholipid bilayers deprived of sterols show a sharper transition between the closely packed ordered arrangement at low temperatures, and the more fluid, liquid-crystal-like arrangement at higher temperatures. Furthermore, above this transition temperature, they display a lower stability. It is generally accepted that sterols act like impurities in a crystal, to replace the transition point by a transition range, but also-and this is essential-as rigid, oriented impurities, akin to the rigid inserts of a composite material, to stabilize the self-organized structure of the membrane in its fluid state (Demel e f a f . , 1967, 1972; Demel and de Kruyff, 1976). By contrast, prokaryotes normally contain no sterols (Section II,A), and exceptions to this statement (Section II,B) are in fact compatible with the generalization that this vast group of organisms is able to live with sterol-free membranes. The present article is devoted to a study of this apparent contradiction between the essential role assigned to sterols in eukaryotes, and their absence in prokaryotes. We shall see (Section 111) that, in many cases, prokaryotes contain polyterpenes (tri-, tetraterpenes, or larger molecules) of structures compatible with their being structural equivalents of sterols. We shall furthermore see that these substances are membrane constituents and can, in living microorganisms, replace sterols: They are also functional equivalents of sterols. Finally, we shall

R

FIG.I . Eukaryotic sterols. R = H , Cholesterol; R = CH:,, campesterol; R = C, H,, sitosterol.

155

STEROL EQUIVALENTS AND PRECURSORS

Ph FIG. 2 .

S

P

Eukaryotic membrane model. Ph, PhospholiGd; S , sterol; P, protein.

consider these polyterpenes as phylrtic unc~~smrs of sterols (Section IV): They are produced from similar precursors by similar biosynthetic reactions, and the enzymatic systems operating on these substrates appear to form an evolutionary line. Many of the ideas developed in Sections 1-111 (and partly in Section IV) have first been put forward by Nes (1974), in a review paper where, in our opinion, much that is reasonable is diluted by as much that is improbable or impossible; this paper has certainly contributed to kindle our interest in these questions. We have made no attempt here to provide a complete bibliography. Other key references can be found in Nes’s paper (1974), and in Ourisson et al. (1979a,b) and Rohmer et a l . (1979).

II. THE STEROLS OF PROKARYOTES A. The “Absence” of Sterols For nearly a century (Hammerschlag, 1889; von Behring, 1930), it has been known that sterols are “absent” from bacteria. Such an absolute statement is incorrect: it should be restricted by indications on the level of sensitivity of the analytical methods used, on their indifference to the actual nature of sterols, and on the range of strains studied. Even then, one could never safely exclude that, in a new isolate, some known or unusual sterols will be found, by a sensitive enough method. However, it remains remarkable that: 1 . In no case has it been possible thus far to demonstrate an active in viiw biosynthesis of sterols in prokaryotes (see, however, de Souza and Nes, 1968; Reitz and Hamilton, 1968, Weeks and Francesconi, 1978).

156

GUY OURISSON AND MICHEL ROHMER

0 A

QQ B

FIG.3 . Cholesterol-phospholipid interactions. ( I ) Closely packed hexagonal regular arrangement (crystal), cholesteroliphospholipid = 1 . (2) Disordered quasi-closely packed arrangement (liquid), cholesteroliphospholipid < I . A, Cross section of cholesterol molecule. 9, Cross section of phospholipid n-acyl chains.

2 . In every case where sterols have been found in prokaryotes, their concentration was at least one or several orders of magnitude lower than in eukaryotes. Contamination is furthermore impossible to exclude: in many cases, the better the control, the smaller the amounts of sterols detected (Bouvier, 1978; for a review leading to more optimistic conclusions, see Weeks and Francesconi, 1978; Hayami et ul., 1979).

Even if this residual level were in some cases genuine, no structural significance can be attributed to such small amounts. I n eukaryotic cells, the sterol concentration in membranes approaches a ratio of one sterol molecule for each molecule of phospholipid: this is the ratio that allows (in the limiting case of the fully ordered “crystalline” system) a closely packed hexagonal array of the lipidic partners, with each of the aliphatic chains of the fatty acids in van der Waals contact with two sterol molecules, imparting rigidity to the system (Fig. 3). (For a model of cholesterol-phosphatidylcholine complexes in membranes, see Huang, 1977a,b.) Certainly 10 times fewer rigid partners (e.g., sterols) could have no meaningful influence on the mechanical properties of the membrane.

6. The Apparent Exceptions 1 . T H EMYCOPLASMAS

These parasitic, wall-less bacteria normally contain sterols. However, these are obtained from their host. Few of them, like Acholeplasma laidfawii, can be grown on a sterol-free diet; this mycoplasma synthesizes polar carotenoids (Huang and Haug, 1974), a fact we shall discuss later (Sections III,A,2 and III,B,2; see also Razin, this volume).

STEROL EQUIVALENTS AND PRECURSORS

157

I

FIG.4.

4a-Methylsterols of Methyloc,occ,u.scapsulatfts (Bouvier et nl., 1976)

2 . Methylococcus Methylotrophs are aerobic bacteria characterized by extensive intracytoplasmic membranes. Their metabolism is exceptional as they grow on one-carbon substrates, such as methane. In the case of Methylococcus, triterpenes of the hopane family have been isolated (see Section III,A), but also 4a-methylsterols (Fig. 4) (Bird er a / . , 1971, Bouvier er al., 1976). Sterols proper are absent. This will warrant a later discussion (Section 1V,B,2). Thus these exceptions are not really contradictory with the generalization that prokaryotes do not normally contain sterols.

C. The Case of Unicellular Eukaryotes Unicellular eukaryotes fall outside the scope of this article; however, it is necessary to mention them in the present context. Unicellular fungi (e.g., yeast) or algae (Rhodo-, Phaeo-, Chlorophyceae) usually contain sterols quite similar to those of their pluricellular relatives (see, for instance, Goad, 1976, 1978). A particularly interesting case is that of the unicellular Euglenophyceae Euglena gracilis, which has been shown not only to contain sterols (Brandt et a / ., 1970),

A B FIG.5 . Triterpene precursors of cholesterol. (A) Cycloartenol (in plants). (B) Lanosterol (in yeast and mammals).

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GUY OURISSON AND MICHEL ROHMER

FIG. 6.

Triterpenes of T O ~ ~ ~ J ~ WpW y rV i j bI U m i ~ s (A) . Tetrahymanol; ( B ) diplopterol,

but also to synthesize them by the “green-plant route,” via cycloartenol (Fig. 5) (Anding et al., 1971; see Section IV,B). Furthermore, the chlorophyll-free euglenoid, Asrusia longu, also utilizes this pathway (Rohmer and Brandt, 1973), and is therefore, from this point of view, a normal etiolated or albino (unicellular) green plant. In contrast to these cases, it has been reported that the saprophytic fungi Phytophtoru and Pythium, which normally utilize the sterols of their host, can grow on sterol-free media, and do not then produce sterols (Hendrix, 1970; Wood and Gottlieb, 1978a,b). These fungi would warrant a further study of their other constituents and of their metabolism. The other singular case is that of Tetrahynirnu pyrtfiwmis, a ciliate protozoon. This organism is predatory “in the wild,” when it acquires its sterols from its food, and only transforms them slightly. In the laboratory, grown in a medium devoid of sterols, it lacks the ability to synthesize them, but instead produces triterpenes: diplopterol and mostly tetrahymanol (Fig. 6) (Conner et ul., 1968). These substances are typical prokaryotic constituents (Section III,A, I ) , and their production by an eukaryote is therefore remarkable. What is really needed is an investigation of the sterols or equivalents of a variety of protozoa growing in sterol-free media.

Ill.

THE POLYTERPENOIDS OF PROKARYOTES

We have seen in the preceding section that in one case (Acholeplasrnu), sterols and polar carotenoids may be interchangeable, and that in another one (the eukaryote Tetrahjrnena), sterols and triterpene alcohols are similarly related. In the present section, we shall compare the distribution in prokaryotes of polyterpenes of various families, their molecular structure, and their localization in the cells. This will lead to the conclusion that they are structurul and functional equivalents of the sterols of eukaryotes.

STEROL EQUIVALENTS AND PRECURSORS

159

A. Distribution of Polyterpenoids in Prokaryotes 1 . Tt1E HOIJANOIDS

TetrahymanoI and diplopterol, the triterpenoids of 7.~ ? ~ r ~(Section ~ ~ ~ II,C) / ~ i ~ . ~ are members of the family of hopanoids, which is widely distributed in prokaryotes (Rohmer et ul., 1979). The known prokaryotic hopanoids (Fig. 7) comprise these C:,,, substances, and more complex C,, (or CZti)C-pentosyl derivatives (Forster et a l . , 1973; Rohmer and Ourisson, 1976a,b,c; Langworthy and Mayberry, 1976; Langworthy et al. , 1976). These pentacyclic 3-deoxy triterpenes are found, i n amounts comparable to those of sterols in eukaryotes, in many of the prokaryotes investigated (Rohmer at ul., 1979). No clear taxonomic clue can be discerned, as they are found in some cyanobacteria as well as in eubacteria, in some gram-positive as well as in gram-negative strains, in some anaerobes as well as in aerobes. Furthermore, the existence of some of them as esters (Haigh et d., 1973) or as complex conjugates (Langworthy et ul., 1976) calls for caution in the interpretation of negative results: the “absence” of hopanoids in some strains may well be apparent, and only reflect the inadequacy of the extraction procedure at this early stage of our awareness of the importance of this family of substances. Hopanoids are also known in eukaryotes (Fig. 8): a few 3-deoxy members of the family in some lichens or ferns, and 3-oxygenated ones in secretions of some scattered taxa of higher plants (see Ourisson et a / . , 1979a). The significance of

FIG. 7.

Prokaryotic hopanaids. (A) Diploptene; (B) diplopterol; (C) bacteriohopane derivatives

160

GUY OURISSON AND MICHEL ROHMER

n

FIG.8 .

Eukaryotic hopanoids. (A) Diploptene; (B) zeorin; (C) hydroxyhopanone; (D) spergulagenin.

the first group is not obvious, the case of 3-oxygenated hopanoids is in fact irrelevant to the present study, as they are only some of the many variants of cyclization products of squalene oxide found in secretions of higher plants, of no precise structural significance (Ourisson et a / ., 1979b).

2. THEBACTERIAL CAROTENOIDS Prokaryotes contain an extraordinary variety of carotenoids (for a recent review, see Liaaen-Jensen, 1979). These incorporate in particular some structural features rarely or never found in higher organisms, some of which are directly relevant to the present discussion: acyclic C,,-carotenoids bearing terminally polar oxygen functions, carotenoid glycosides, or other polar carotenoids (the “carotenols”); C,,-carotenoids, often distally hydroxylated or glycosylated; and even C,,-carotenoids (Fig. 9). These may or may not coexist with hopanoids (for instance, they do coexist in nonsulfur purple bacteria). The published data provide little taxonomic significance for their distribution.

DI- A N D TETRATERPENOIDS 3. T H EARCHAEBACTERIAL Among prokaryotes, archaebacteria (Woese and Fox, 1977; Fox et ul., 1980) have quite unique lipids. Their phospholipids are alkyl erhers (not esters) of phosphorylated glycerol or of other polyols (Kates, 1972). The alkyl groups

STEROL EQUIVALENTS AND PRECURSORS

161

involved are also unusual, as they include the highly branched C,,-terpenoid phytanyl radical, its dehydroderivatives (De Rosa et al., 1976; Tornabene et al., 1978), and the corresponding C4,,20,20'-bisphytanyl dimeric radical engaged in macrocyclic tetraether assemblies (De Rosa ct al., 1977a,b; Tornabene et ul., 1979) (Fig. 10). These basic units can be further modified by partial cyclizations to form five-membered rings. Furthermore, from the study of molecular fossils isolated from sediments, it has been suggested that methyl-1 3-tetradecyl ethers, as well as the corresponding dimeric diethers present in sediments, most probably derive from archaebacteria (Chappe et a l . , 1979) (Fig. 1 I ) . Archaebacteria contain other polyterpenic constituents-squalene derivatives (Tornabene et u l . , 1979, Tornabene and Langworthy, 1979, Kates and Kuswaha, 1978) and carotenoids (Kates and Kuswaha, 1978) in particular-probably not engaged in the basic architecture of their membranes like the alkyl ethers, but of importance for the evolutionary considerations that follow (Section IV,D).

bH

7\

HO

Fic;. 9 . Some prokaryotic carotenoids. (A) 4-o-Glucopyranosyl-4,4'-diaponeurosporene (C.%,J; (€3) aphanizaphyll C4,,; (C) sdrcinaxanthin (C5(,);(D) bacterioruberin (C5,,).

162

GUY OURISSON AND MICHEL ROHMER

OR

RO

FIG. 10. Archaebacterial lipid ethers. C,, (phytanyl) and C,,, (bis-phytanyl) ethers.

B. Structural Regularities in Membrane Polyterpenoids We shall now show that some of the molecular features of the apparently hodgepodge variety of prokaryotic polyterpenoids just mentioned are uniquely compatible with their oriented incorporation into lipid bilayers. I . SELF-ORGANIZAIION

OF

BILAYERS-THE ROLE OF STEROLS

The self-organization of phospholipids into a bilayer can be attained only within a rather narrow range of chain lengths. If the chains are too short, homogeneous solutions can be obtained, bur too long chains cannot be ordered and lead to the formation of micelles (see, e.g., Tanford, 1978). A straight chain of about 15-20 carbon atoms for the hydrophobic portion of the molecule is needed. This leads to rather strict criteria for the inclusion, in the partially ordered layers of the membrane, of inserts able to impart some rigidity to the whole system. These inserts must be self-oriented, i.e., they must possess a

RO OR

FIG. I I .

Monomeric and dimeric lipid ethers from sedirncnts

STEROL EQUIVALENTS AND PRECURSORS

163

hydrophilic “head” and a lipophilic rigid “body. ” They must have a length of about 15 A. They must have a cross section enabling a close packing with the phospholipid chains, i.e., they must be approximately 6 A thick and 6 A wide. Finally, they must be, like the extended conformations of the lipid chains, grossly cylindrical. Thib is precisely the case for sterols, and the good fit of their molecular dimensions with those of n-acyl phospholipids is taken to be responsible for their efficiency in optimizing the mechanical properties of the membrane, whatever technique is used to evaluate it (Yeagle et NI., 1977).

2 . COMPARISON O F T H E MOLKUI.ARPROPERTIES O F PROKAKYOTIC POI-YTERPENOIDS-THE ROLE OF B R A N C H E D - C H ALIPIDS IN Among the prokaryotic polyterpenoids just described, many have structures perfectly compatible with their being incorporated in a sterol-like manner in

OH FIG. 12

Dimensions of cholesterol (top) and prokaryotic triterpenoids

164

GUY OURISSON AND MICHEL ROHMER

phospholipid bilayers (Fig. 12). This is the case for the hydroxylated hopanoids (i .e., excluding the hydrocarbon diploptene). For the polar carotenoids of prokaryotes, another arrangement has been proposed, with the molecule, rendered straight and rigid by the conjugation of the unsaturated chain, oriented like a rivet across both halves of the phospholipid bilayer. In this case again, the dimensions and amphipathy of the molecules (Fig. 13) are compatible with the dimensions of the lipid bilayer required for its self-organization (Fig. 13) (Ourisson et ai., 1979a). Finally, the archaebacterial di- and tetraterpenoid ethers themselves also have comparable dimensions and amphipathic character. However, in this case, the dimeric molecules are not intrinsically rigid, and it is only if they were incorporated in a bilayer that the inclusion of their head groups in the aqueous compartments could rigidify them, keeping them taut (Fig. 14). We have not yet discussed the cross sections, but only the lengths of these molecules. In fact, they are all comparable, and very slightly larger than those of n-acyl chains or sterols. They would probably be rather more efficient as rigidifying partners of flexible lipids of slightly larger cross sections than the n-acyl phospholipids. The ideal case is that of the archaebacterial ethers, comprising mixtures of monomers and dirners, as these have identical branched chains, and therefore can be perfectly fitted together. But it is also, in the other cases, quite HO

v

h

OH

36

FIG. 13. Dimensions of some bacterial carotenoids.

165

STEROL EQUIVALENTS AND PRECURSORS

A

B

C

Piti. 14. Model o f inclusion of rigid and nonrigid inserts in the bilayer. ( A ) Incorporation of a hopanoid into a phospholipid bilayer. ( R )Inclusion of a long rigid insert (e.g., carotenoid) across the bilayer. (C) Inclusion of a long nonrigid insert (e.g., archaebacterial tetraether) across the bilayer.

possibly linked with the known fact that the bacterial lipids are frequently inordinately rich in branched-chain or o-cycloalkyl acids (Kaneda, 1977), i.e., precisely in chains of slightly larger cross section than those of n-acyl phospholipids. In one case, Bacillus ucidocaldarius, it is known that glycosylated bacteriohopane triterpenoids (Langworthy et a / . , 1976) are found together with essentially branched and o-cyclized phospholipids (De Rosa et al., 1971). It is not known whether this is general, and coordinated analyses of both classes of lipids would be in order now.

3. PHYSICOCHEMICAL E V I D E N CFOR E T H E STRUCTURAL A N D PROKARYOTIC POLYTERPENOIDS E U L I I V A I . E ONFCSTEROI.S E There is limited but clear evidence for the in vitro equivalence of prokaryotic polyterpenoids and sterols, in their ability to strengthen the structure of artificial membranes. Thus the hopanoids of 3. ucirlocalduriiis were shown to have a condensing effect similar to that of cholesterol on dipalmitoylphosphatidylcholine, as measured by isobars and isotherms of monolayers of these phospholipids (Poralla et a / . , 1980, Kannenberg et al., 1980). For carotenoids, the evidence is more indirect. In Acholeplasma, the carotenoid content can be decreased drastically (by a factor larger than 50) by growing the organism in a medium containing propionate instead of acetate. As judged by several converging physical methods (spin-labeling, buoyant density, osmotic resistance, and glycerol permeability), the carotenoid pigments seem to display a condensing effect on the membrane (Huang and Haug, 1974; Rottem and Markowitz, 1979). However, the physiological significance of the presence or absence of carotenoids for the regulation of the membrane fluidity is not clear: a change in the ratio of unsaturated to saturated fatty acids is not observed by

166

GUY OURISSON AND MICHEL ROHMER

Rottem and Markowitz (1979), a conclusion that does not agree with that of Huang and Haug (1974). Furthermore, Acholeplastna can be grown deprived of sterols, and without production of carotenoids (Razin and Rottem, 1967). It is possible that an interpretation of these apparent contradictions will require the precise knowledge of the molecular structure of the polar carotenoids localized in the membranes, and other lipid partners (e.g., colorless carotenoid precursors?). The ether phospholipids of archaebacteria, and in particular the dimeric tetraterpene diethers forming bridges across membranes, will require adequate physicochemical studies. However, the fact that these lipids can of course only form “bilayers” excludes for their evaluation the use of the direct methods (isobars and isotherms) available for monolayers. It seems that the very large diethers do self-organize into artificial membranes, but it is not known whether the postulated riveting effect can be demonstrated quantitatively in vitro.

C. The Functional Equivalence of Sterols and Prokaryotic Polyterpenoids I t remains to show that the prokaryotic polyterpenoids just described do in fact play a role in vivo that is similar to that of sterols in eukaryotes. Two lines of evidence are available: the polyterpenoids, like the sterols, have been shown to be localized in membranes, and in a few cases changes in growth conditions bring about compensatory changes in the sterol-polyterpenoid biosynthesis by the organisms. 1 . LOCALIZATION OF POLYTERPENOIDS I N MEMBRANES

Tetrahymanol. the eukaryotic hopanoid of sterol-deprived Tetrahytnerza pyriformis, is localized in membranes (Ferguson et al.. 1975; Thompson et ul., 1971). The same is true of some bacterial carotenoids (Kushwaha et al., 1975; Anvar et a / . , 1977), and of the di- and tetraterpenoid ethers of archaebacteria. These statements should not be construed as implying that all the polyterpenoids of the groups mentioned are localized exclusively, or even predominantly in the membranes, and even when this is the case, they do not necessarily play the role postulated in this chapter. For instance, in a complex organism the membrane proteins themselves can interact preferentially with some of these substances, in particular with carotenoids (Cheesman er al., 1967; Zagalsky et a / . , 1970; Ji et a!., 1968). Furthermore, carotenoids are also partly localized in membranes of the endoplasmic reticulum of a eukaryote, the fungus Neurosporu crussa (Mitzka-Schnabel and Ran, 1980).

2. In Vivo SUBSTITUTION O F STEROLS BY POLYTERPENOIDS We have already mentioned the two most convincing cases of in vivo replacement of sterols by polyterpenoids: in Terrahymena, where sterol deprivation

STEROL EQUIVALENTS AND PRECURSORS

167

leads to hopanoid biosynthesis, in amounts comparable to the sterols that disappear (Conner et d . , 1968); and in Acho/c./7/usrnu,where sterol deprivation may be accompanied by the production of polar carotenoids (Smith, 1971), a conclusion that does not agree with that of Razin and Cleverdon (1965), however. It has also been possible to test the physiological equivalence of sterols and analogues with a sterol-dependent mycoplasma, Mycqdasrnu cupricolutn. This microorganism grows well in a medium supplemented with sterols (with an efficiency depending on the particular sterol used), but also with various triterpenoids (Odriozola et a / ., 1978). Unfortunately, neither hopanoids nor carotenoids have been tested; furthermore, the results obtained with some of the analogues tested [e.g., cycloartenol, which can replace sterols whereas lanosterol does not (Dahl et a / . , 1980: Buttke and Bloch, 1980)] are difficult to rationalize (Fig. 5 ; see also Razin, this volume).

IV. THE PROKARYOTIC POLYTERPENOIDS AS PHYLOGENETIC PRECURSORS OF STEROLS The prokaryotic polyterpenoids also share with the eukaryotic sterols a large part of their biosynthetic pathway. We shall first summarize this basic common feature, and then show that a limited number of mutations luto sensu, operating on a limited set of enzymes, can offer a plausible mechanism for a biochemical evolution leading eventually to the sterols. These considerations are quite hypothetical, but one can expect to obtain, in at least some of the cases, experimental evidence, confirmatory or otherwise.

A. General Features of the Common Polyterpene Biogenetic Pathway All the substances just mentioned are produced by branchings of one common biosynthetic pathway, leading from acetate to all polyterpenes. This pathway, limited to the steps directly pertinent to the present discussion, is summarized in Fig. 15. For sterols and all their polyterpene equivalents, all the first steps are identical in that the substrates are the same, and the nature of their chemical transfomiations, including fine stereochemical detail, is the same. It is therefore an inescapable assumption that for every one of the first steps the enzymes involved are probably very similar, or even identical in all living organisms. The same holds true for some of the later specific stages. For instance, the dimerization of farnesyl pyrophosphate to squalene, leading to sterols and triterpenoids, and that of its homologue geranyl-geranyl pyrophosphate to the precursors of carotenoids, appear to entail very similar enzymatic systems: (1) Both involve unusual inter-

CH,COOH

HO

-

xopp pp-+

&-

1

s

o

p

p

-

Monoterpenss

O2

Squalene epoxide

Qyopp-

Sterols

Diterpener

/

I Carotenoids

Archaebacterial bisphytanyl ethers

OR

RO

FIG. 15.

Polyterpene biogenetic pathway.

169

STEROL EQUIVALENTS AND PRECURSORS

mediates, cyclopropylcarbinols, of identical structure and stereochemistry (Fig. 16), (Altman et ul., 1972, Crombie et ul., 1972; Popjak et a / . , 1973). (2) Purified squalene synthetase can also dimerize geranyl-geranyl pyrophosphate, though with a lower efficiency (Qureshi et d., 1973). However, the fate of the cyclopropyl intermediate is usually different: it is reduced to provide squalene, and dehydrated, in the homologous series, to provide phytoene (though in some cases lycopersene is obtained in a manner similar to squalene, e.g., Davies and Taylor, 1 976). Clearly, we are dealing here with analogous enzymatic systems. In such cases, we shall postulate that they may both derive from the same common ancestor enzyme, by slightly divergent evolution. We shall assume that the divergence can be direct. For example, a one-amino acid change in a protein sequence can have drastic effects on the conformation, and therefore, if far from the active site, on the substrate specificity, while retaining the reaction selectivity. It could as well be indirect when the same enzyme is placed in different media; for example, the same enzyme in membranes of different lipid composition could adopt different conformations, with the same consequences as above. As to the mechanisms involved, they can be postulated to follow the pattern summarized by Ohno (1970; cf., e.g., Cohen et ul., 1977): a given gene, coding for a certain enzyme, would first be duplicated. Once two or more copies of the same gene coexist in the genome, one of them can undergo mutations. Minor mutations lead then to isoenzymes, and more important ones could alter sufficiently the substrate specificity to lead to biochemical functions other than the original one. An important consequence of this postulated mechanism is that the original gene can remain operative, and therefore the enzyme it codes for can

+ p

p

o

y

R

p OPP

-R

\

FIG. 16. Cyclopropane alcohols as intermediates in the biosynthesis of squalene (upper figure, R , geranyl) and precarotenoids (lower figure, R , larne\yl).

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GUY OURISSON AND MICHEL ROHMER

remain active and functional, while other routes are explored without harm by evolutionary drift. Replacement of the original gene by a new one (i.e., a change in a biosynthetic pathway) can then occur once the new functional unit has been achieved and the old one has become obsolete. Of course, such a description is extremely sketchy. It will nevertheless be sufficient to describe in outline the postulated phylogeny of the membrane constituents discussed here. Finally, it must be emphasized that, in a biosynthetic scheme such as that of Fig. 15, a sharp difference exists between those steps (the first ones) that are independent of molecular oxygen and those that require the intervention of atmospheric oxygen, such as the formation of squalene epoxide, the oxidative degradation of sterol precursors, and the oxidative transformations of carotenoids into epoxides, ketones, etc. Thus it has often been remarked that sterols can only have been formed in living organisms after the onset of photosynthesis, the major source of molecular oxygen (e.g., Nes, 1974; Bloch, 1976).

B. Hopanoids as Precursors of Sterols 1. BIOSYNTHESIS OF STEROLS

Sterols are produced by oxidative degradation of their C,,-tetracyclic precursors: lanosterol for fungi and vertebrates, and its isomer cycloartenol for green plants (Fig. 5) (see, e.g., Goad, 1971). The oxidative degradation will not be discussed here, except to mention that it goes by way of 4a-methylsterols. The tetracyclic precursors are themselves produced by an enzyme-mediated, acidcatalyzed cyclization of squalene (3S)-2,3-epoxide, and the stereoelectronic requirements of this reaction imply a particular folding of the acyclic precursor, as shown in Fig. 17 (Eschenmoser et al., 1955). This is a pre-chair, boat, chair, chair conformation that is a strained one, and it can only be achieved by the substrate-enzyme interactions. A plausible mechanism for the reaction implies, as shown in Fig. 17, the presence on the enzyme of a proton-donating site -A-H, of a first nucleophile N, at the other terminus of the hydrophobic enzyme cleft, and of a second nucleophile N, located in the middle of the cleft, and stabilizing the positive charge once this has migrated back toward C-9 (by a series of H and CH :I shifts) (Rees et c z l . , 1968). From this point on, there is a dichotomy between the lanosterol and the cycloartenol routes. In vertehrutes and fungi, the intermediate with NJC-9 bond eliminates H-8, to form the 8,9 double bond of lanosterol, which can then undergo oxidative demethylation at C-14 and C-4, to sterols. This must imply a basic site B. In higherplcrnts, the basic site B abstracts a proton not from C-8, but from the methyl group at C-10, to form the cyclopropane ring of cycloartenol. This difference may only result from a small difference in the relative position of the groups N, and B, i.e., it is compatible with a very minor change in the enzyme

STEROL EQUIVALENTS AND PRECURSORS

HO

171

& FIG. 17

Cyclization of cqualene epoxide into lanmtcrol ( A ) and cycloartenol (B)

systems implied in the two routes. This is made plausible by the fact that in the absence of a methyl group at C-4 of a squalene epoxide analogue, a cyclase from a higher plant normally producing cycloartenol gives directly the corresponding norlanosterol (Cattel et m l . , 1976). From this sketchy summary, we shall retain the conclusion that a major dichotomy in biosynthetic pathways can probably result from a displacement of one group in the enzyme, by as little maybe as I W---entirely compatible with a one amino acid change in a sequence. We can now turn to hopanoids. Their biosynthesis is much simpler. I t implies squalene itself, folded in the enzyme cleft in an all-pre-chair conformation, and attacked by the same two groups as above: an acidic site -A-H at one end, and a nucleophilic site N,at the other end. To shift the cyclization from tetrahymanol to diplopterol, all that is required is to move N , by about 1 A. In this case, the sites N, and B, which may well be retained in the enzyme, play no role. It is also to be assumed that the general shape of the cleft is very slightly changed, to favor the all-pre-chair conformation (Fig. 18). The enzyme system responsible for the biosynthesis of the precursors of sterols

172

GUY OURISSON AND MICHEL ROHMER

H /A

I

+

/ &c)“ FIG. 18.

Cyclization of squalcnc into a hopanoid (e.g., diplopterol)

(lanosterol or cycloartenol) can therefore by essenfidly fhe scitne as that implied for hopanoids, with limited changes compatible with limited mutations (one or a few one-amino acid mutations) in the protein sequence. The biosynthesis of hopanoids is in fact “more primitive”: ( 1 ) the precursor is squalene, not squalene epoxide, and therefore the biosynthesis is entirely anaerobic (the OH group of the amphilic molecule produced derives from wurer, whereas in sterols it derives from mo/ecii/ar o g g e n ) ; (2) the squalene-hopanoid transformation is only a cyclization, and does not imply extensive rearrangements and degradations: (3) the hopane cyclization implies an all-pre-chair conformation of the substrate, which is more stable than any other one; (4) finally, primitive characters of the squalene-hopanoid cyclase are also revealed by enzymatic studies, as we shall see.

2.

BIOSYNTHETIC

EXPERIMENTS

The eukaryotic cyclase of squalene epoxide is a very specific enzyme, even though it does convert a few analogues of squalene epoxide (Van Tamelen and James, 1977, Van Tamelen et a l . , 1977; Cattel ct al., 1976). However, it does not act on squalene itself, nor on the (3R)-epoxide, but only on the (3s)enantiomer. Squalene cyclases of microorganisms are much less specific. Three of them have been studied (Anding el a / . , 1976; Rohmer et a / . , 1980b,c; Bouvier et ul., 1980). An enzyme preparation of Acetohacter pasteurianum converts squalene into diploptene and diplopterol, both previously isolated from the organism as minor Companions of the C,,,-bacteriohopane polyols, which are, however, not biosynthesized by this in vitro system. Racemic squalene epoxide, which is not a normal constituent of Acetobacter, is itself cyclized, to give a mixture of 3u- and 36-hydroxy derivatives of diplopterol and diploptene. The same is true of the squalene cyclase of 7’. pyr[formis, which converts

173

STEROL EQUIVALENTS AND PRECURSORS

squalene to tetrahymanol (Caspi et d., 1968; Aberhart and Caspi, 1979), and (R,S)-squalene epoxide to the corresponding pair of 3a- and 3P-alcohols (Fig. 19) (Bouvier et u / . , 1980). Stereochemical details of the cyclization have been elucidated; they agree with the assumption that for the three substrates (squalene and its two epoxides) the active sites -A-H and N, of the enzyme maintain their relative positions (Bouvier ~t u l . , 1980). Finally, an enzymatic system obtained from M . cupsulufris cyclizes squalene to diplopterol and diploptene, and (R,S)-squalene epoxide to four alcoholsconsisting of the 3 a - and 3P-hydroxy derivatives of diploptene, just as in the previous two cases, and two tetracyclic alchohols, lanosterol and its 3a-hydroxy isomer (Fig. 19) (Rohmer er u / . , 1 9 8 0 ~ )This . experiment shows that the enzyme preparation in this case contains TWO distinct cyclases, one with a “all-pre-chair squalene” cleft, indifferent to the presence or absence of the epoxide group, and producing hopanoids, the other one with a “pre-chair-boat-chair-chairsqualene” cleft, unable to cyclize squalene, but able to act on the more reactive squalene

\

&

no

0

44

1

4

Methylococcus

Acetobacter

. I I )Tetrahymena I

FIG. 19. Cyclizations of squalene and (3/?.S)-squalene epoxide by cell-free systems from Acetohucter pusteuriunrtm. Tetrrrhymetiu pyrjformis, and Methdococcus capsidatus.

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GUY OURISSON AND MICHEL ROHMER

epoxides. Furthermore, in Methyfococtus the first cyclase must be present in ii tight compartment distinct from the one where squalene epoxide is produced, or else the squalene oxidase and the squalene epoxide cyclase must be efficiently coupled in order to avoid the in vivo formation of 3-hydroxy-hopanoids. The microorganism itself contains hopanoids, small amounts of lanosterol, and the 4a-methyl sterols derived from lanosterol by oxidative degradation. It is not known whether hopanoids and 4a-methyl sterols are both contained in membranes, and, if this is the case, whether they co-occur in the same membranes, or are segregated in the cell. Clearly, the lanosterol-producing cyclase of M . cupsulatus is intermediate in its selectivity between the hopanoid-producing one, and the lanosterol-producing cyclase of eukaryotes.

3. A POSSIBLE EVOLUTIONARY MECHANISM One can postulate a mechanism for an evolution leading from hopanoids to sterols as follows: 1 . One cyclase is present and is able to act on squalene, the only available substrate in an anaerobic environment, to produce hopanoids. 2 . Gene duplication, and mutations on one of the duplicate genes, produce isoenzymes still capable of cyclizing squalene. 3. The conditions becoming aerobic, a monooxygenase can produce squalene epoxide, which is cyclized first into 3-hydroxy-hopanoids. 4. Lanosterol, produced by one of the isoenzymes, can be further oxidatively degraded to 4a-methyl sterols or even sterols, potential membrane constituents. This would be the stage of Methylococcus, with a squalene epoxide cyclase “not yet” specific enough to distinguish between the R - and S-epoxides, but with a squalene oxidase specifically producing the S-epoxide. A later stage would be the appearance of specificity in the squalene epoxide cyclase. 5. Final degradation of the 4a-methyl group yields sterols, which are now able to replace the hopanoids fully.

In such a scheme, many additional facts lend credence to one or another of the stages. For instance, the case of Tetrahymena can, in this perspective, be explained by an inhibition of hopanoid biosynthesis by the sterols added, or by some of their metabolites, in a kind of feedback control. We can also note that there is still place for a missing link between hopanoids and lanosterol: the evolution of the hopanoid-producing cyclase could proceed first by the necessary change in conformation, to produce a pentacyclic analogue of lanosterol, and only later by the second necessary change in placement of the nucleophile N,, to produce the tetracyclic lanosterol itself. The pentacyclic intermediate would be an arborenol. In fact, isoarborinol (Fig. 20) is a known natural product, which has been found

STEROL EQUIVALENTS AND PRECURSORS

Fic. 20.

175

A putative constituent of an unknown aerobic microorganism, isoarborinol.

intact in several recent sediments where it can reasonably only have been formed by microorganisms (Dastillung ct ml., 1980a,b, Rohmer Pt ul., 1980a). We postulate it to be present in aerobic bacteria akin to methylotrophs.

C. Carotenoids as Precursors of Cyclic Polyterpenoids I.

C O M P A R I S O N OF T H E B l O S Y N T H E S l S O F CAKOIENOIDS

AND

SQUALENOIDS

A very large part of the biosynthetic pathway of carotenoids follows the steps valid for sqiialenoids. We have already illustrated this for the dimerization of the C,, geranyl-geranyl pyrophosphate, compared to that of its lower isoprenologue (Section IV,A). This is further shown by the existence, in some microorganisms, of C:,o squalene-derived carotenoids (Liaaen-Jensen, 1979). For the acyclic carotenoids and for squalene, all the enzymes required are essentially the same, except for those leading (anaerobically) to dehydrogenations in carotenoids. “The same” is to be understood here, as for the preceding section, as implying a very close structural analogy; the substrate specificity must, however, be different (favoring 2 x C,,, or 2 x C,,), and, in the case of carotenoids, the nonreductive pathway of evolution of the intermediate cyclopropane alcohol to phytoene appears to be predominant over the reductive one leading to lycopersene, the analogue of squalene. A close analogy can also be found, though without experimental support so far, in the cyclization step. The formation of cyclic carotenes, like the u - , /?-,or y-carotenes, implies an enzyme-mediated, acid-catalyzed reaction entirely analogous to the cyclization of squalene, except that in this case N 2 (or another nucleophilic site) can block the ion after one ring only is formed. The stereocheniistry involved is similar (Eugster, 1979).

2. A POSSIBLE EVOLUTIONARY MECHANISM

In exactly the same way as before, one can postulate a mechanism for the hypothetical evolution from carotenoids to hopanoids. The primitive stage would

176

GUY OURISSON AND MICHEL ROHMER

be one in which optimization of membrane rigidity and fluidity is achieved with acyclic carotenoids, hydroxylated or glycosylated at each end. Their biosynthesis implies, en route to geranyl-geraniol, the formation of famesol; therefore, due either to the limited specificity of the dimerization step, or to a mutation in a duplicated gene, squalene could be formed. Again, the most primitive type of cyclase may well be that leading to a-,jl-, and y-carotenes. It is easy to see how it can be modified in two ways: 1 . If the acidic site in the cyclase can carry an isoprenyl group instead of a proton, the same cyclization will produce isoprenologues, the C,,-carotenoids typical of several groups of bacteria. 2 . If the nucleophile N, is moved slighly away, and N, becomes operative, one can obtain the hopanoids as shown earlier (Section IV,B,I) from squalene.

In this hypothesis, the carotenoids, even if replaced by the hopanoids as membrane stabilizers (wholly or partly), are nevertheless not made thereby obsolete. They have acquired many other functions, in particular in relation with photosynthesis and protection against photooxidation.

D. The Archaebacterial Polyterpenes. Biogenetic Considerations The basic building block is, here again, geranyl-geraniol. This diterpene can in archaebacteria be partly or wholly hydrogenated, to produce the phytenyl and phytanyl residues found as ethers in their lipids (Kates et al., 1966; Langworthy et ul., 1974, Langworthy, 1977a,b; Tomabene et id., 1978, 1979; Tornabene and Langworthy, 1979). An archaebacterium, Halohacteriuin cutirubrum, can also produce carotenoids, and even C,,,and C,5, skeletons (Liaaen-Jensen, 1979). It is therefore no surprise that archaebacteria contain also squalene, and this is accompanied by its partially hydrogenated derivatives (Tornabene er al., 1978). The enzymes required for their formation are all present, possibly in a slightly modified form, for the biosynthesis of phytanyl ethers and of carotenoids. The active metabolism of molecular hydrogen, which appears to be characteristic of at least some of the archaebacteria, is even more striking in the steps of dimerization of the phytenyl units, or of internal cyclization of the bis-phytanyl ethers (De Rosa rt a / . , 1977a,b, 1980). Nothing is known of the biochemistry of these dehydrogenation steps (for a similar dehydrogenation in another bacterium, see Hanser ei mi., 1979). I n this phylum, the only enzyme that is apparently missing is a squalene hopanoid-cyclase. Too little is known yet, only a few years after the recognition of the concept of archaebacteria, to draw any conclusion as to the relative primitiveness of headto-head or tail-to-tail dimers of geranyl-geraniol derivatives, i.e., of carotenoids or of his-phytanyl ethers. It i s nevertheless remarkable that these opposite dimers

177

STEROL EQUIVALENTS AND PRECURSORS

are able to make use of the same building blocks to fulfill the samephysicochemical function.

V.

ADDENDUM

Tricyclohexaprenol as a Putative Prokaryotic Triterpene and a Putative Phylogenetic Precursor of Hopanoids Recent work on the structures of frequent components of the polycyclic hydrocarbons of sediments, the so-called extended diterpenes, has led to the structures shown in Fig. 21. These are still only incompletely proved ( A and B, by synthesis, but the CSl-C3()homologues only from mass spectra). This family of triterpenes, in part degraded in the sediment, cannot derive from squalene, but from h n u p r e m l D. Tricyclization of this would give tric~clohexuprenoIE, the structure of which resembles that of the sesterterpene cheilanthatriol F. Tricyclohexaprenol is not yet a known natural substance; from the wide distri-

A

B

R:H Me

C

GUY OURISSON AND MICHEL ROHMER

178

bution of its molecular fossils in sediments, it is clear that it must be of bacterial origin. Furthermore, its molecular dimensions and polarity would make it an ideal sterol surrogate. We therefore predict that it will be found in the membranes of some, still unknown, bacterium. Its biosynthesis would require the same enzymatic system as that depicted in Fig. 18, with the basic site B acting to stop the cyclization, and the additional “primitive” feature of an all-pre-chair, “all-Markownikoff” reaction. We therefore predict that tricyclohexaprenol will be found in bacteria more primitive than those containing hopanoids (P. Alrecht, F. de A. N . Radler, M . Rohmer, and G. Ourisson, unpublished observations). REFERENCES Aberhart, 1). J . , and Caspi, E. (1979). Nonoxidative cyclization of squalene by Te/rtrhwieriu pyr[formih. Incorporation of a 3P-hydrogen (deuterium) atom into tetrahymanol. J . A m . Clierri. S i r . 101, 1013-1019. Altman, L. J . , Ash, L., Koerski, R. C., Epstein, W . W., Larsen. B. R. Rilling, H. C., Muscio. F.. and Gregonis, D. E. (1972). Prephytoene pyrophosphate. A new intermediate in the biosynthesis of carotenoids J . A m . Cheni. Soc. 94, 3257-3259. Anding, C . , Brandt, R. D., and Ourisson, G. (1971). Sterol biosynthesis in Eiiglrmr grticilis Z . Sterol precursors in light-grown and dark-grown Eiigleriu grucilis Z . Eirr. J . Biorhern. 24, 259-263. Anding. C.. Rohmer. M . . and Ourisson, G . (1976). Nonspecific biosynthesis of hopane triterpenes in a cell-free syslern from Acelobucrrr rurwrm. J . A M . Chrwr. Soc. 98, 1274-1275. Anvar. M., Hasan Khan, T . . Prebble, J . , andzagalsky, P. F. (1977). Membrane boundcarotenoid in Micrococcus Iirteifs protects naphtoquinone from photodynamic action. Nariire (Londorij 270, 53 8 -540. Bird, C. W . . Lynch. C . M., Pirt, F. J . , Reind, W. W . , Brooks, C. J . W . , and Middleditch, B. S. (1971 ) . Steroid!, and squalene in Merliylococcm cuprulurus grown on methane. Nature (Lotidon) 230, 473. Bloch, K . (1976). On the evolution of a biosynthetic pathway. I n “Reflections on Biochemistry” ( A . Komberg, B. L. Horecker, L. Cornudella, and J . Orb, eda.), pp. 143-150. Pergamon, Oxford. Bouvier, P. (1978). Dr. Sc. Thcsis, Universitt Louis Pasteur. Strasbourg. Bouvier, P., Rohmer, M., Benveniste, P., and Ourisson, G . (1976). A”“”-steroids in the bacterium Mrrhylococcus cup.siilurus. Diochem. J . 159, 267-27 I . Bouvier. P . , Berger, Y . , Rohrner, M . , and Ourisson, G. (1980). Non-specific biosynthesis of gammacerane derivatives hy a cell-free system from the protozoon Terruh~meri~ pyriforinis. E w . J . Biochrin. 112, 549-556. Brandt, R . D.. Pryce, R. J . , Anding, C . , and Ourisson, G . (1970). Sterol biosynthesis in Eirgletiu gruc’ilis Z. Comparative study of free and bound sterols in light and dark grown Euglencr grurilis Z. Eirr. J . Biwhern. 17, 344-349. Buttke, 7. M., and Bloch, K . (1980). Comparative responses of the yeast mutant strain GL 7 to lanosterol, cycloartenol and cyclolaudenol. Biocltern. Biophy.s. Res. Cortimiin. 92, 229-236. Caspi, E., Zander, J . M., Greig, J . B., Mallory. F. B., Comer, R. L., and Landrey, J . R. (1968). Evidence for a nonoxidative cyclization of squalene in the biosynthesis of tetrahymanol. J . A m . Cherii. S o c 90, 3563-3565.

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Cattel, L., Anding, C . , and Benveniste, P. (1976). Cyclisation of I-fruns- l’-norsqualene-2,3-epoxide and I-cis-I ‘-norsqualen-2,3-epoxideby a cell-free system from corn embryo. Phytochernistp 15,931-935. Chappe, B . , Michaelis. W., Albrecht, P., and Ourisson, G. (1979). Fossil evidence for a novel series of archaebacterial lipids. Natiirrr,i.F.ror.st.h~~c,n 66,522-523. Cheesman, D. F., Lee, W . L., and Zagalsky. P. F. (1967). Carotenoproteins in invertebrates. B i d . Rei,. Cwnhrirfge Philo&. SOC. 42, 13 1 I60 Cohen, G . N., Saint Girons. I . , and Truffa-Bachi, P. (1977). The evolution of biosynthetic pathways. Trrnds Biochem. S i . 2, 97-99. Conner, R . L., Landrey. J . R . , Burns, C. H.. and Mallory, F. B. (1968). Cholesterol inhibition of pentacyclic triterpene hiosynthehis i n 72truhyenci p?r[fimni.s. J . Profoxmi. 15, 600-605. Crombie, L., Findley, D. A. R . , and Whiting, D. A. (1972). Lanthanide induced chemical shifts i n natural cyclopropanes; stereochemistry of chrysanthemyl and presqualene alcohols and esters. Tetrahedron Lerr. pp. 4027-4028. Dahl. C . E . , Dahl. J . S . , and Bloch, K . (1980). Effects of cycloartenol and lanoaterol on artificial and natural membranes. Biochetn. Bioplrr.~.R ~ ACornnirrri. . 92, 221 -22X. Dastillung, M., Albrecht, P . , and Ourisson, G . (1980a). Aliphatic and polycyclic alcohols in sediments: Hydroxylated derivatives of hopane and 3-methylhopane. J . Chem. R r s . , (S) pp. 168169: (M)pp. 2353-2374. Davies, B. H . , and Taylor, R. F. (1976). Carotenoid biosynthesis. The early steps. Pure A p p l . Chem. 47, 211-221. Dernel. R . A . , and de Kruyff, B. (1976). The function o f sterols in membranes Biocltirn. Bioph,ys. Acru 457, 109-132. Demel, R . A,, van Deenen, L. L. M.. and Pethica. B . A. (1967). Monolayer interactions of phospholipids and cholesterol. Eiochinr, Llroph?..~Acfcr 135, 1 1-19, Demel, K. A,. Bruckdorfer, K . R . , and van Deenen, L. L. M. (1972). The effect of sterol structure on the membrane permeability o f liposomes to glucose, glycerol and Rb+. Biochini. EiophJ,.c. Actu 255, 32 1-330. De Rosa, M . , Gambacorta, A , , Minale, L.. and Bu’lock, J . D. (1971). Cyclohexane fatty acids from a thermophilic bacterium. Clion. C o n n n r i ~pp. 1334-1 335 De Rosa, M.,De Rosa, S . , Gambacorta, A., and Bu’lock, J. D. (1976). lsoprenoid triethcr lipids from Culduricllu. Ph!.foc,/rerni.srr:r. IS, 1995- 1996. De Rosa, M.,De Rosa, S . , and Gambacorta. A. (19772). Lipid structures in the Cult/urie//ri groupof extreme thermoacidophile bacteria. ChPnr. Conrvrrtn, pp. 514-515. De Rosa, M., De Rosa, S . , Gambacorta. A.. Minale, L., and Bu’Lock, J . D. (1977b). Chemical ctructure of the ether lipids of therrnophilic acidophiiic bacteria of the Cnlcirrirlh group. PkUochr~nisfr:~ 16, 1961-1965. De Rosa, M . , Gambacorta, A . . Nicolaus. B . . Sodano, S . , and Bu’lock. J . D. (1-80). Structural regularities in tetraether lipids of Cnldurirlltr and their bioaynthetic and phyletic implications. Pl7ytocherni.str-~19, 833-836. De Souza, N . J . . and Nes, W. R . (196X). Sterols: Isolation from a bluegreen alga. Sc,irrrc~,162, 363. Eschenmoser. A.. Ruricka, L., Jeger. 0..and Arigoni, D. (1955). Eine stereochemische Interpretation der biogenetischen lsoprenregel bci den Triterpenen. Melv. Chini. Arrrr 38, 1890-1904. Eugsrer, C. H. ( I 979). Characterisation. cheniistry and stereochemistry o f carotenoids. P / , w Appl, Cheni. 51, 463-506. Ferguson, K . A . , Davis, F . M . . Conner, R . L , Landrey, J . R.. and Mallory, F. B. (1975). Effect of sterol replacement iit viro on the fatty acid composition of T r f r ~ r h ~ t n r m J . ~B. i d . Clictn. 250, 6998-7005. Forster, H. J . , Biernann. K . , Haigh. W . G . , Tattrie, N . H . , and Colvin. J . R . (1973). The structure of novel C,, pentacyclic terpenes from Acrrohucrer ~ryr‘imm.Bioc~hrni.J . 135, 133- 143. -

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Fox, G. E . . Stackebrandt, E., Hespell. R . B., Gihson. J . . Maniloff. J . . Dyer, T. A , , Wolfe. R . S., Belch. W . E., Tanner, R . S . Magrum, L. J . , Zablen. I<. B.. Blakemore, R . . Gupta, R . , Ronen, L.. Lewis. B . J . , Stahl, D. A . , Luehrscn, K. R., Chen, K . N . , and Woesc, C . R. (1980). The phylogeny of prokaryotes. Seierice 209, 457-463. Goad, L. J . (1971 ) . Sterol biosynthesis. Irr “Natural Substances Formed Biologically from Mevalonic Acid” (T. W. Goodwin. ed.), pp. 45-77. Pergainon, Oxford. Goad. L. J . ( 1976). The steroids of marine algae and invertebrate animals Biocherrr. Biophv.s. Perspec,t. Mtrr. Biol. 3, 213-318. Goad, L. J . ( I 978). The sterols of marine invertebrates: Composition, biosynthesis, metabolism. Irr “Marine Natural Products” (P. J . Scheuer, ed.), Vol. 2. pp. 75-172. Academic Press, New York.

Haigh. N. G . , Fdrster, H. J . . Biemann, K.. Tattrie. N. H., and Colvin, J . R . (1973). Induction of orientation of bacterial cellulose microfibrils by a novel terpenoid from Acetobucter .r?liriitm. Biorherri. J . 135, 145-149. Harnmerschlag, A. (1889). Bacteriologisch-chemische Untersuchung der Tuberkelbacillen. Moriutsh. Chrrrr. 10, 9-18. Hanser, H., Hazlewood, G . P., and Dawson, R . M . C. (1979). Membrane fluidity of a fatty acid auxotroph grown with palmitic acid. Ntrritre (Lortdon) 279, 536-538. Hayami, M., Okabe. A . , Sasai. K., Hayashi, H . , and Kanemasa. Y . (1979). Presence and synthesis of cholesterol in stable staphylococcal L-forms. J . Bncreriol. 140, 859-863. Hendrix, J . W. (1970). Sterols in growth and reproduction of fungi. A N I I I I . Rev. Phyropurhol. 8, 1 1 1-130, and references cited therein. Huang, C . H. (1077a). A structural model for the cholesterol-phosphatidylcholinecomplexes in bilayer membranes. Lipids 12, 348-356. Huang, C. H. ( I 977b). Configurations of fatty acyl chains in egg phosphatidycholine-cholesterol mixed bilayers. Cherri. P ~ ? . ALipids . 19, 150-158. Huang, L., and Haug, A . (1974). Regulation of‘ membrane lipid fluidity in A ~ l i o l e ~ ~ itiirilaw~iit i~~ttr~ Effect of carotenoid pigment content. Biochirri. Biophys. Actti 352, 36 1-370. Ji, T . E., Hess, J . L.. and Benson, A. A . (1968). Nature of p-carotene association in chloroplast lamellas. Iri “Comparative Biochemistry and Biophysics of Photosynthesis” (K. Shibata, A . Takamiya, A. I . Jagendorff, and R . C. Fuller, eds.), pp. 36-49. Untv. ofTokyo Press, Tokyo. Kaneda. T. (1977). Fatty acids of the genus Bacillii.~:An example of branched chain preference. Boi./eriol. Re\.. 41, 391-418. Kannenkrg, E., Poralla, K., and Blume. A. (1980). A hopanoid from the thermoacidophilic Bacill i i s cii.irlor.trltlaritt.s condenses membranes. Nururwissrnschufen 67, 458-459. Kates. M. (1972).Ether-linked lipids in extremely halophilic bacteria. I n “Ether Lipids. Chemihtry and Biology” ( F . L. Snyder, ed.), pp. 351-398. Academic Press, New York. Kates, M., and Kuswaha, S . C. (1978). Biochemistry of the lipids of extremely halophilic bacteria. f n “Energetics and Structure of Halophilic Microorganisms” (S. R. Caplan and M. Ginzburg, eds. ) * pp. 461 -480. ElsevieriNorth-Holland Biomedical Press, Amsterdam. Kates, M.. Palameta, B., Joo, C. N., Kushner, D. J . , and Gibbons, N . E. (1966). Aliphatic diether analogs of glyceridc-derived lipids. 1V. The occurrence of di-0-dihydrophytylglycerol ether containing lipids in extremely halophilic bacteria. Biochrtnisrrs 5, 4092-4099. Kuswaha.S. C . , Kates, M . , and Martin, W. G . (1975). Characterization and composition of the purple and red membranes from Hulohucterirrrti crtririrhrurn. Cirri. J . Biochem. 53, 284-292. Langworthy, T. A. (1977a). Comparative lipid composition of heterotrophically and autotrophically grown Sidfblohus ucidocaldarius. J . Bucteriol. 130, 1326-1332. Langworthy, T. A. (1977b). Long-chain diglycerol tetraethers from Therrrioplasmu acidophihtm. Biochirn. Binph?s. Actu 487, 37-50. Langworthy, T. A . . and Maybeny, W . R . (1976). A 1.2.3.4-tetrahydroxypentane substituted pentacyclic triterpene from Bircillrra uc-idocaldcrriirs. Biochim. Riophvs. Actir 431, 570-577.

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Langworthy, T . A., Mayberry, W. R . , arid Smith, P. F. (1974). Long-chain glycerol dietherc and polyol dialkyl glycerol triether lipids of S u / / i h / i u . c [rc.irloc.tr/l[rricts. J . Hucrrriol. 119, 106116.

Langworthy, T. A , , Mayberry, W. R . . and Smith, P. F. (1976). A sulfonolipid and novel glycosainidylglycolipids from the extreme thermoacidophile Buci//u.\ ~ r c ~ i t / o c ~ t r / t / ~ ~ r i r , . ~ . Bioc,/iiin. Biophys A r t u 431, 550-569. Liaaen-Jensen, S . (1979). Carotenoids, a chemosysteinatic approach. Porc, App/. Chern. 51, 661 675. Mitzka-Schnabel, V . . and Ran, W. (1980).The subcellular distribution ofcarotenoids in Nrrrrosportr C ~ U S S N P/iy/~r/renristry . 19, 1409- I4 I3 Nes, W. R . (1974). Role of sterols in membranes. Lipid.\ 9, 596-612. Odriozola. J . M., Waitzkin, E . , Smith, T . L., and Bloch. K. (1978). Sterol requirement of Myc’oplusmu c~rpricdum.Pro(.. N u t / . AM^. &.i. (J.S.A. 75, 4107-4109. Ohno. S . (1970). “Evolution by Gene Duplication. “Springer-Verlag, Berlin and New York. Ourisson, G . , Alhrecht, P., and Rohnier. M. (1979a). The hopanoids: Palaeocheinistry and biochemistry of a group of natural products. Pure App1. Chern. 51, 709-729. Ourisson, G.. Rohnier, M . , and Anton, R . (197Yb). From terpenes t o sterols: Macroevolution and microevolution. Hecotit Adi,. Phvrot~hrin.13, 131 -162 Popjak, G.. Edmond, J . , and Wong, S (1973). Absolute configuration of preaqualene alcohol. J . A t n Chertt. So(,. 95, 2713-2714. Poralla, K., Kannenberg, E . , and Blume, A . (1980). A glycolipid-containing hopane isolated from the acidophilic therniophilic Rtrc~i//u.\crcrt/occi/t/nrirrs haa a cholcatei-ol-like function in ineiiibranes. FEBS Lett 113, 107-1 10. Qureshi, A. A , , Barnes, F. J . , and Porter. J . W. (1973). Biosynthesis of prelycopersene pyrophosphate and lycopersene by squalene synthetase. J . B i d . Chern. 248, 2755-2767. Razin, S . , and Cleverdon. R. C. (1965). Carotenoida and cholesterol in membranes of M w o p / u . w i u loidluwii. J . Geri. Microhiol. 41, 409-415 Razin, S . , and Rotteni. S (1967). Role of carotenoids and cholesterol in the growth of M\cop/tisniu /uit//awii. J . Buc/er.io/. 93, I 1 X I - I 1 X2 Rees. H. H., Goad. L. J.. and Goodwin. T . W. (1908). Studies in phytosterol biosynthesis. Mechanism of biosynthesis of cycloaitenol. Rioc.hrvtr. J . 107, 417-426. Reitz. R . C . , and Hamilton. J . G. (1968). The isolation and identification of two sterols from two species of blue-green algae. Cornp. Bior,hmt. Physiol. 25, 401 -416. Rohmer, M., and Brandt. R . D. (1973). Les stkrols ct leurs precurseurs chez Astusiu longu Pringsheim. Eitr. J . Riochrin. 36, 446-454. Rohmer, M . , and Ourisson, G. ( 1976a). Structure deb bacteriohopanetetrol5 d’Acetohucter .ry/rnirni, Tetrtrh~~dron L m . pp, 3633-3636. Rohmer, M., and Ourisson. G. (1976b). Derives du bactkriohopane: Variatiuns structurales et repartition. Tctra/ic,dron Lert. pp. 3637-3640. Rohnier, M . , and Ourisson. G . ( 1 9 7 6 ~ )Methylhopanes . d’Acetohuc,rrr xylitiuin et d’Acetohuc~rer rutzcens: Une nouvelle famille de composes triterpeniques. Trtruhedrt~nLett, pp. 3641 -3644. Rohmer, M . . Bouvier, P., and Ourisson. G. (1979). Molecular evolution of biomembranes: Structural equivalents and phylogenetic precursor\ of sterols. P r o c . N u t / . Acad. Sci. U.S.A. 76, 847--851. Rohnier, M., Dastillung, M . , and Ourisson, G. (1980a). Hopanoids from C:,,, to C:%5in recent muds. Chemical markcrs for bacterial activity. Nururiri.tsr.nschujiL.n 67, 456-458. Rohmer, M., Anding, C., and Ourisson, G. (1980b). Nonspecific hiosynthesis of hopane triterpenes by a cell-free system from Acrtohrrcter [Ju.\tc,iiriuriiiiti. E w . J . Biocheni. 112, 541 -547. Rohmer, M . , Bouvier, P., and Ourisson. G. ( 1 9 8 0 ~ )Non-specific . lanosterol and hopanoid biosynthesis by a cell-free system from the bacterium M e / h y / o c o m r s cupsir/utu.c. Eitr. J . Biocheni. 112, 557-560. ~

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Rottem, S . . and Markowitz, 0. (1979). Carotenoids act as reinforcers of the Acholepltrsrna h i d luwii lipid bilayer. J . Bucteriol. 140, 944-948. Smith, P. F., (1971). 111 “The Biology of Mycoplasmas.” Academic Press, New York. Tanford, C. (1978). The hydrophobic effect and the organization of living matter. Science 200, 1012-1018. Thompson, A , . Jr., Bamherry, J . R., and Nozawa, Y. (1971). Further studies of the lipid composition and biochemical properties of Tetruhynerra pyrifornris membrane system, Biochenristry 10, 4441-4447. Tornabene, T . G . , and Langworthy. T. A. (1979). Diphytanyl and dibiphytanyl glycerol ether lipids of methanogenic bacteria. Scierrcr 203, 51 -53. Tornabene, T . G . , Wolfe, R . S . , Balch. W. E . , Holzer, G . . Fox, G. E . , and Oro, J . (1978). Phytanyl-glycerol ethers and squalenes in the archaebacterium Methatiobacterium therinouutotrophicuin. J . Mol. EL^. 11, 259-266. Tornabene, T. G . , Langworthy, T. A , , Holzer, G . , and Oro, J . (1979). Squalenes, phytanes and other isoprenoids as major neutral lipids of methanogenic and thermoacidophilic Archaebacteria. J . M o l . Evul. 13, 73-83. Van Tamelen, E. E . , and James, D. R. (1977). Overall mechanism of terpenoid terminal epoxide polycyclizations. J . A m . Clrern. Soc. 99, 950-95 I . Van Tamelen, E. E., Pedlar, A. D., Li, E., and James, D. R . (1977). Cyclization studies with norand homosqualene 2,3-oxide. J . Arn. Chein. Soc. 99, 6778-6780. yon Rehring, H. (1930). Enthalten Bakterien Sterine? Hoppe-Seyler’.\ Z. Physiol. Chew. 192, 112-1 13. Weeks, 0. B., and Francesconi, M. D. (1978). Occurrence of squalene and sterols in Celluloniofias deliydrogertam (Arnandi 1942) comb. nov. Hester 1971. J . Bucteriol. 136, 614-624. Woese, C. R., and Fox, G. E. (1977). Phylogenetic structure of the prokaryotic domain: The primary kingdoms. Proc. Nutl. Acutf. Sci. U.S.A. 74, 5088-5090. Wood. S. G . , and Gottlieb, D. (197th). Evidence from mycelial studies for differences in the sterol biosynthetic pathway of Rhyzoctoniu soluiii and Phptophtoro cinnurnuni. Biochern. J . 170, 343-354. Wood, S. G., and Gottlieb, D. (1978b). Evidence from cell-free systems for differences in the sterol biosynthetic pathway of Rhizocronru s o l m i and P h y t o p h r u cinnumoni. Biochern. J . 170, 355-363. Yeagle, P. L., Martin, R . B., Lala, A. K . , Lin, H. K . , and Bloch, K . (1977). Differential effects of cholesterol and lanosterol on artificial membranes. Pruc. Nutl. Acad. Sci. U.S.A. 74, 49244926. Lagalsky, P. F., Ceccaldi, G . J . . and Daumas, R. (1970). Comparative studies on some decapod crustacean carotenoproteins. Conip. Biochern. Phpsiol. 34, 579-607.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 17

Sterols in Mycoplasma Membranes S H M U E L RAZlN Depurttnerir of Meitihrutic~orid Liltru.srrrrc~irreRrseurdi The Hebrew Uniwrsif ~ - H u d m ~ oMrriiw fi I School Jerirsulem, lsruel

I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . Cholesterol Uptake . . . . . . . . . . . . . . . . . . . . . A . Cholesterol Donors . . . . . . . . . . . . . . . . . . . B . Membrane Components Involved in Cholesterol Uptake . . . C. Location of Sterols in the Membrane . . . . . . . . . . . Ill. Role of Sterols . . . . . . . . . . . . . . . . . . . . . . . A . Regulation of Membrane Fluidity . . . . . . . . . . . . B . Structural Features of Sterol Molecules Required for Function C. New Concepts of Sterol Function . . . . . . . . . . . . D. Why Do Mycoplasmas Require Sterols'! . . . . . . . . . 1V. Conclusions . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . 11.

1.

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183

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185

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185 186 190 191 191

193 197 198 200 201

INTRODUCTION

Mycoplasmas are easily distinguishable from all other prokaryotes by their lack of a cell wall and intracytoplasmic membranes. Hence, mycoplasma cells have a single membrane-the plasma membrane. The genome size of mycoplasmas varies between 5 x lox and 1 X 10:' daltons, the lowest recorded values for self-replicating prokaryotes. As a consequence of their limited genetic information, the mycoplasmas depend on a supply of many nutrients from their animal, plant, or insect hosts. Most distinctive among these nutritional requirements are those for fatty acids and sterols, components that are essential for membrane biosynthesis. The requirement of mycoplasmas for fatty acids will be discussed extensively by Melchior and by McElhaney (this volume). For a general review on the cell biology of mycoplasmas the reader is referred to Razin (1978a). 183

Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-1533 17-4

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SHMUEL RAZIN

The requirement of mycoplasmas for sterols is unique among prokaryotes, though very recently Treponerna hyodysenteriue has been shown to share this property (Lemcke and Burrows, 1980). Since none of the mycoplasmas is capable of cholesterol synthesis, they depend on its exogenous supply (Razin, 1975; Razin and Rottem, 1978). The sterol-requiring mycoplasmas incorporate large quantities of cholesterol into their plasma membrane from their host or from the serum component of artificial growth media. The levels of cholesterol incorporated into the membrane of the sterol-requiring mycoplasmas included in the genera M y o p l u s t n u , Ureuplusma, and Spiroplustnu are comparable to those found in plasma membranes of eukaryotes, that is about 25-30% by weight of total membrane lipids (Argaman and Razin, 1965, Mudd et ul., 1977; Patel et u l . , 1978, Razin, 1978a; Razin et al., 1980). Sterol requirement is not ubiquitous among mycoplasmas. The group of mycoplasmas capable of growing without sterols is presently classified as a family named Acholeplasmataceae, having thus far a single genus: Acholeplusma. The trivial term achoieplasmas is also used for describing the sterolnonrequiring mycoplasmas (Razin, 1978a). As can be seen in Table I, the Acholeplustna species are more restricted in their ability to incorporate free and esterified cholesterol as well as exogenous phospholipids than the sterolrequiring mycoplasmas. In addition to the mycoplasmas that show a strict requirement for cholesterol or closely related sterols, and to the acholeplasmas which do not require sterols at all, several goat mycoplasmas exhibit a less stringent requirement for sterols, but TABLE I CHOLESTEROL A N D PHOSPHOLIPID UPTAKEFROM SERUMB Y GROWING Acholeplusma A N D Mq'coplosma SPECIES" ~

Lipids in cells (pmol/gm cell protein)

Organism" A , iuidluwii

A . arunthum A . gmrru/trrum M . ~iilliseprirrrtn M. humini,t

M. trrginini M . pnrumoniw M . rwpricolrtm

Free cholesterol

Esterified cholesterol

Sphingomyelin

Phosphatidylcholine

10.2 3.7 28.5 76.0 76.4 58.2 85.9 67.2

0 0 0 4.7 30.6 27.5 58.0 67.7

0 0 0 15.5 32.7 37.8 40.3 20.8

0 0 0 37.8 20.3 31.9 16.6 40. I

Data from Razin rr tzl. (1980) The organisms were grown with 5% (viv) horse serum for 20-24 hours at 37°C. M. pneumonitrr was grown with 10% ( v i v ) horse serum for 4 days at 37°C. "

"

STEROLS IN MYCOPLASMA MEMBRANES

185

cannot grow in their total absence (Rottem ef al., 1973a; Odriozola er al., 1978). This wide spectrum of sterol requirements has made the mycoplasmas most useful tools for studying basic problems concerning mechanisms of sterol transfer and role in biomembranes, topics that will be discussed in detail in the following sections. Moreover, the facility by which the type of membrane sterol and its content can be manipulated in mycoplasmas (Razin and Rottem, 1976) has been exploited extensively in studies on the mechanism of action of agents, such as polyene antibiotics and thiol-activated bacterial toxins, which damage cell membranes by specifically complexing with membrane sterols (reviewed in Razin, 1981).

II. CHOLESTEROL UPTAKE

A. Cholesterol Donors Serum lipoproteins are the natural donors for animal mycoplasmas in vivo and in conventional mycoplasma media iri vitro. They can be replaced by artificial donors including phoqpholipid-cholesterol vesicles (Kahane and Razin, 1977, Razin t t (I/. , 1980; Efrati t t ul., I98 I a), Tween 80-cholesterol micelles (Razin and Tully, 1970; Razin pt (11.. 1974). or simply by an ethanolic solution of cholesterol (Razin and Tully, 1970). The use of ethanolic solutions of sterols suffers from the deficiency that when added to the aqueous growth medium some of the sterol may come out of solution, crystallize, and cosediment with the cells or membranes on centrifugation (Razin and Rottem, 1976). The use o f Tween 80-cholesterol solutions is not recommended either, as the detergent Tween 80 may damage the membrane and inhibit mycoplasma growth (Razin and Tully, 1970). Moreover, to achieve high cholesterol concentrations in aqueous media, the concentration of Tween 80 has to be raised to relatively high levels (up to 0.1%). At this concentration the detergent may perturb the membrane lipid bilayer and influence cholesterol uptake. The finding that cholesterol uptake by resting Achol~plusrnuImidlarvii cells and isolated membranes in the presence of 0.1% Tween 80 reached high values with no sign of saturation (Razin et ul., 14174) supports the above suggestion. Similar experiments carried out with phosphatidylcholine-cholesterol vesicles or with serum showed cholesterol uptake by mycoplasma cells and membranes to reach an equilibrium (Razin ct ul., 1980). The major factor that determines the effectiveness of serum lipoproteins and lipid vesicles as cholesterol donors is the molar ratio of free cholesterol to phospholipid in the donor. Cooper (1978) has postulated that the higher the free cholesterol :phospholipid ratio of the donor is relative to that of the membrane, the more effective it is in donating cholesterol. This postulate is supported by our

186

SHMUEL RAZlN

0.5

E i) 0.4-

c 4

0

0.27

057

1.10

1.72

CHOCESTEROC-WSPHATIDYLCHOUNE MOLAR RATIO

FIG. 1 . Growth response of Mycopltrsmcr horninis to cholesterol (15 pgiml), provided in the growth medium as cholesterol-phosphatidylcholine vesicles of different molar ratins. The improved growth-promoting activity o f vesicles with higher cholesterol: phospholipid ratio can be scen. (From Kahane and Razin. 1977.)

observation (Kahane and Razin, 1977) that cholesterol-phosphatidylcholine vesicles with a molar ratio of 0.3:l serve as inefficient cholesterol donors and only permit poor growth of the sterol-requiring Mycoplasmu horninis, whereas cholesterol-phosphatidylcholine vesicles at a molar ratio of I :1 and above are very effective cholesterol donors and support excellent growth (Fig. I ) . Likewise, the significantly higher molar ratio of free cholesterol to phospholipid in human low-density lipoproteins (about 0.8:1) as compared to that of highdensity lipoproteins (about 0.15:l) appears to be responsible, at least in part, for the better performance of the low-density lipoproteins as cholesterol donors to mycoplasmas (Slutzky et ul., 1976, 1977, 1981). B. Membrane Components Involved in Cholesterol Uptake I . LIPIDS The mechanism by which eukaryotic cells control the amount of exogenous cholesterol incorporated into their plasma membrane from serum lipoproteins, and the reasons for the great variations i n the cholesterol content among the various membranes of mammalian cells have not been clarified (McCabe and Green, 1977). Elucidation of the factors controlling the amount of cholesterol taken up by mycoplasma membranes may contribute to better understanding of this basic biological problem. The wall-less mycoplasmas offer several unique

STEROLS IN MYCOPLASMA MEMBRANES

187

advantages for investigating this issue as their plasma membrane interacts directly with exogenous lipid donors, and the cholesterol taken up is not esterified or modified in any other way (Razin, 1975, 1978a; Razin and Rottem, 1978). Moreover, mycoplasma lipid composition and membrane fluidity can be manipulated in a controlled manner, facilitatirg the study of the physical factors influencing cholesterol uptake by biological membranes (Razin et al., 1974; Razin, 1978b). As mentioned above, the sterol-nonrequiring Acholeplasrna species incorporate much less cholesterol than the sterol-requiring Mycoplasmu species (Argaman and Razin, 1965; Razin, 1974; Razin et ul., 1980; Table I). Hence, elucidation of the factors responsible for restricting cholesterol uptake in Acholeplasma may provide some clues as to the solution of the general problem of control of cholesterol uptake i n biomembranes. Cholesterol uptake by isolated mycoplasma membranes (Gershfeld et al., 1974) and intact cells (Le Grimellec and Leblanc, 1978; Rigaud and Leblanc, 1980) can be defined as a physical adsorption process, independent of growth and metabolic activity. Rigaud and Leblanc (1980) go even further and conclude that cholesterol incorporated by resting cells has an identical disposition and function in the membrane as cholesterol incorporated during growth. Our recent finding that the marked difference in cholesterol uptake between Acholeplasma and Mycoplastnu species is retained in their isolated membranes (Razin et al., 1980) indicated that the mechanism restricting cholesterol uptake in Acholeplasma is a physicochemical one, being associated with the membrane per se. This mechanism probably depends on a specific composition and/or organization of membrane components. Nevertheless, cholesterol uptake can be influenced by cell integrity and growth indirectly, as growing M . capricolurn cells take up exogenous phospholipids, whereas isolated membranes do not. The phospholipids taken up are incorporated into the membrane and serve as additional binding sites for cholesterol (Razin et al., 1980). The dependence of cholesterol uptake on membrane phospholipid content has been clearly demonstrated in mycoplasmas. The marked decrease in membrane phospholipid content relative to that of the protein on aging of mycoplasma cultures was accompanied by a parallel decrease in membrane cholesterol, whereas an increase in membrane phospholipid content resulting from chloramphenicol treatment of cultures was accompanied by an increase in membrane cholesterol (Razin, 1974). Hydrolysis of membrane phospholipids by phospholipase A2 decreased the cholesterol-binding capacity of isolated A . laidlawii and M . capric:olurn membranes roughly in proportion to the amount of phospholipid hydrolyzed (Efrati et ul., 1981a). On the other hand, an increase in the phospholipid content of M .capricolum membranes following the uptake of phosphatidylcholine and sphingomyelin from serum was accompanied by an increase in cholesterol uptake (Razin et ul., 1980). These results support the principle expressed by Cooper ( I 978) that the amount of cholesterol incorporated into

188

SHMUEL RAZlN

biomembranes depends on the amount of phospholipid available for interaction with it.

It is conceivable that phospholipids that interact with membrane protein, the so-called boundary, annular, or halo lipids, are less available for interaction and binding of cholesterol (Cooper, 1978). The possibility that Acholeplmma membranes contain a higher percentage of boundary lipids than membranes of Mycoplasmu species was tested by the dry-ether extraction procedure. Dry ether supposedly extracts the unbound fraction of membrane phospholipids representing the protein-free lipid bilayer. The results failed to show any significant difference in the quantity of ether-extractable phospholipids between the Acholeplasma and Mycoplusina membranes tested (Kutner, 1979). Moreover, digestion and removal of over 50% of A . luidluwii membrane proteins, a procedure that might be expected to release at least some of the boundary lipids from interaction with protein, did not increase cholesterol binding (Efrati ei a / ., 198 1 a). Glycolipids are major components in Acholeplasma but not in Mycoplasma membranes. In A . laidluwii, glycolipids and phosphoglycolipids constitute over 60% of the polar lipid component (Wieslander and Rilfors, 1977; Efrati et a / . , 198 la). Hence, another possible explanation for the differences in cholesterol uptake between Acholeplusma and Mycopb.sinu species can be based on the notion that glycolipids have lower affinity for cholesterol than phospholipids. Several recent reports claim that sphingomyelin has a higher affinity for cholesterol than phosphatidylethanolamine or glycolipids (van Dijck et al., 1976; Demel rt ill,, 1977, McCabe and Green, 1977). If glycolipids have indeed a lower affinity for cholesterol, it could be argued that a bilayer rich in glycolipids, like that of Acholeplasmu, will incorporate less cholesterol. Phospholipase A, effectively hydrolyzes the “pure” phospholipids of A . laidluwii membranes, leaving the glycolipids and phosphoglycolipids intact (Bevers et ul., 1977). We could recently show that the complete hydrolysis of “pure” phospholipid (phosphatidylglycerol and diphosphatidylglycerol), which make only 30% of the polar lipids of A . luidluwii membranes, caused a decrease of about 55% in cholesterol uptake. This suggests that the glycolipids have a lower binding capacity for cholesterol. Yet it is not clear whether the residual cholesterol uptake is due to the “pure ” glycolipids, phosphoglycolipids, or both. Monoglucosyldiglyceride of A . laidlawii shares some properties with phosphatidylethanolamine. These include low hydration capacity, relatively high transition temperature, and the presence of a reversed hexagonal-phase structure (Wieslander ot a / . , 1978). If the claim that phosphatidylethanolamine has lower affinity for cholesterol is correct (van Dijck et a / . , 1976; Demel P t a / . , 1977), then it can be argued that the A . laidlawii glucolipid shares this property. However, direct proof for this suggestion is still lacking (Razin et ul., 1981). It should be mentioned in this context that the membrane lipid bilayer may also show different affinities for various sterols and sterol derivatives. Thus when

189

STEROLS IN MYCOPLASMA MEMBRANES

F"I

OLEATE-ENRICHED CELL

0

-

AIDATE E N R l C m D CELL: 0

I 1

1 2

I

3

INCUBATION TIME (hour.)

Effect of temperature on cholcsierol upiake by oleate- (0)and elaidate-enriched (0) A. Itritlltnl~iicells at 4°C. Cholesterol uptake is minimal by the elaidate-enriched cells in which nieinbrane lipids are in the gel state. but significant in the oleate-enriched cells in which membrane lipids are still in the liquid-crystalline \late. (From Razin. 1978b.3

FK.2 .

cholesterol is added to the growth medium of M . capricolum with lanosterol, there is a preferential uptake of cholesterol by the growing cells (J. S. Dahl et ul., 1980). When free and esterified cholesterol are present in the medium, the free cholesterol will be preferentially incorporated (Slutzky et a l ., 1976). The dependence of cholesterol uptake on membrane fluidity has been convincingly demonstrated with A . laidluwii cells enriched with elaidate or oleate (Razin, 1978b). The transfer of elaidate-enriched cells in culture from 37 to 4°C virtually arrested cholesterol incorporation into the cell membrane. Cholesterol uptake continued, though at a lower rate, in the oleate-enriched cells undergoing a similar temperature shift-down. (Fig. 2 ) It can be concluded that the incorporation of exogenous cholesterol into the cell membrane of living mycoplasmas is rapid when the lipid bilayer is in the liquid-crystalline state, and very slow when the lipid bilayer is in the gel state. Although it is possible that some of the membrane lipid is in the gel state in growing mycoplasmas (McElhaney, 1974, see also McElhaney, this volume), it is doubtful that this factor plays a role in determining the final cholesterol content of the membrane, though it certainly may affect the rate of cholesterol uptake (Razin ef a / . , 1974).

2 . PROTEINS Protease-sensitive receptors with high affinity for lipid vesicles (Pagan0 et a / ., 1978) or for low-density lipoproteins (Basu et a / . , 1978) have been detected on

190

SHMUEL RAZIN

the surface of some eukaryotic cells. Recent findings in our laboratory appear to suggest that the sterol-rich Mycoplusmu species differ from the sterol-poor Ac~holeplasrnaspecies i n possessing cell surface proteins with high affinity for exogenous lipid donors. Thus the addition of trypsin to logarithmic cultures supplemented with cholesterol-phosphatidylcholine vesicles had no effect on cholesterol uptake by A . laicllawii cells, but decreased cholesterol uptake by M . cupricolutn cells by about 50% (Efrati et a / . , 1981a). Moreover, growing Mycoplusmu cells take up significant quantities of exogenous phospholipids from serum or lipid vesicles, in addition to free and esterified cholesterol, whereas Acholeplusmci cells take up only low amounts of free cholesterol (Razin et ul., 1980). This finding can also be taken in support of the hypothesis that Mycoplusmu cells have receptors on their surface for exogenous lipid donors. These receptors facilitate a closer or more prolonged contact with the donor, needed for phospholipid transfer (Bloj and Zilversmit, 1977). The possibility that serum lipoproteins actually adhere and remain attached to the mycoplasma cell surface has been investigated with lipoproteins in which the protein moieties were selectively labeled with 1251. Experiments with growing cells and isolated A . luidlawii membranes speak against the prolonged adherence of lipoprotein particles to the membrane, as up to 45% of the free cholesterol of the lipoprotein particle could be taken up by the membranes with little or no concomitant uptake of labeled protein, esterified cholesterol, and phospholipids (Slutzky el ul., 1976, 1977). The finding that growing cells and isolated membranes of A . laicllawii take up only the cholesterol component of phosphatidylcholine-cholesterol vesicles indicates that lipid vesicles do not adhere to Acholeplusma membranes either (Kahane and Razin 1977, Razin et ul., 1980). On the other hand, our finding that growing cells of a variety of Mycoplasmu species take up, in addition to free cholesterol, significant quantities of esterified cholesterol and phospholipids from serum lipoproteins (Razin er al., 1980) may suggest adherence of the lipoprotein particles to the sterol-requiring Mwoplrtsma. Recent experiments in our laboratory (H. Efrati, S . Eisenberg, and S . Razin, unpublished data) show that M . capricolum binds about six to eight times more low-density lipoproteins than A . luidlawii, according to I n s I measurements, supporting the receptor hypothesis. However, the amount of labeled lipoprotein that is protein bound is much less than expected from the amounts of esterified cholesterol and lipoprotein phospholipids taken up by M . capricolurn. Hence, it appears that almost all of the free cholesterol and a significant part of the esterified cholesterol, phosphatidylcholine, and sphingomyelin found in the M . capricdurti membrane do not constitute part of lipoprotein particles attached to the cell surface.

C. Location of Sterols in the Membrane The above conclusion, that a significant part of the esterified cholesterol found in M . cupricolum membranes is not part of attached lipoprotein particles, is

STEROLS IN MYCOPLASMA MEMBRANES

191

supported by the finding that extensive proteolytic digestion of M . c ~ ~ p r i c d t r r n membranes, a treatment that is expected to remove any adherent lipoprotein particles, did not affect the cholesteryl ester content of the membrane (Efrati et u / . , 198 l a ) . Furthermore, [I4Clcholesteryl oleate taken up by growing M . ctrpricolurn could not be removed by extensive washing of the isolated cell membranes in deionized water and 0.5 M NaCl, or by intensive ultrasonic irradiation of the membranes (Melchior and Rottem, 1981). Hence, it is clear that the association of’ cholesteryl esters with M . c u p ~ i c d u ~membranes n is very tight. The above findings pose an enigma, as cholesteryl esters do not commonly form a significant component of biological membranes. Cholesteryl esters have a low solubility (about 5 molt%) in lipid bilayers, and when present in excess of this they form a separate phase (Small and Shipley, 1974). In M . cupricolum grown with 4% serum (Razin (’1 al, 1980; Melchior and Rottem, 1981) or with human low-density lipoproteins (H. Efrati, unpublished data), the molar ratio of esterified cholesterol to free cholesterol approaches 1 ,and the absolute amounts of cholesteryl esters associated with the membrane far exceed the maximal values of cholesteryl esters that can be incorporated into phospholipid bilayers. Recent calorimetric studies by Melchior and Rottem (1951) suggest that the large amounts of cholesteryl esters form large droplets or pockets in the M . cupricolum membrane, with little or no association with other membrane components. The disposition and transbilayer movement of free cholesterol and related sterols in mycoplasnia membranes has been extensively studied by Rottem and Bittman, and will be discussed in detail by Rottem (this volume).

111.

ROLE OF STEROLS

A. Regulation of Membrane Fluidity The presence of sterols in membranes of all but the most primitive cells suggests that they perform an essential function in higher forms of life. However, the exact mode of action of sterols in biomembranes is still unclear. The total dependence of the sterol-requiring mycoplasmas on exogenous sterols for growth, and their inability to modify chemically the sterols taken up (Razin, 1975; Freeman et NI., 1976; Odriozola et a / . . 1978; Lala et a l . , 1979; Rottem and Markowitz, 1979a; C . E. Dahl et ( I / , , 1980a) make these organisms especially well suited for studying the role of sterols in membranes. The widely accepted notion that cholesterol and related sterols function as regulators of membrane fluidity, first proposed on the basis of studies on artificial lipid bilayers (Chapman, 1966), has gained strong experimental support from studies on mycoplasma membranes. The idea as summarized by Rothman and Engelman (1972) is that cholesterol produces an “intermediate fluid condition” in the membrane lipid bilayer by virtue of its peculiar molecular shape. Cholesterol o r

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SHMUEL RAZIN

related sterols (containing a planar ring system, a free hydroxyl group at the 3p position, and a hydrocarbon side-chain) are oriented in the membrane in such a way that the ring system is aligned parallel to the acyl chains of membrane phospholipids, while the hydroxyl group anchors the sterol molecule to the polar surface of the bilayer. The rigid ring system of the cholesterol molecule, which is about twice as large as its flexible hydrocarbon side-chain, interacts hydrophobically with the upper portions (C-2 to c-10) of the phospholipid acyl chains, exerting a condensing effect on this region. On the other hand, the bulky ring system separates the lower portions of the phospholipid acyl chains from each other, increasing the freedom of motion of the chains in this region. Hence, the insertion of cholesterol into a phospholipid bilayer allows a liquid-like configuration for the phospholipid acyl chains at the cholesterol tail region and a more condensed configuration at the ring region. The net result of cholesterol action is to exert a condensing effect on phospholipids at temperatures above their thermal phase transition. and to prevent the cooperative crystallization of the hydrocarbon chains at temperatures below that of the phospholipid phase transition. Complete elimination of the phase transition takes place only if the molar ratio of cholesterol to phospholipids exceeds 1.2 (Demel and de Kruyff, 1976). The condensing effect of cholesterol, as expressed by decreased membrane fluidity and permeability, could be observed in A . laidlawii (McElhaney et al.. 1970; de Kruyff et ul., 1972, 1973; Stockton et a / . , 1977; Butler et ul., 1978; Davis et ul., 1980). However, the relatively low amounts of cholesterol that can be incorporated into the A . luidluwii membrane (Table I ) suffice only to reduce the energy content of the phase transition of membrane lipids, but not to eliminate it. Hence, the sterol-nonrequiring A . laidlawii is certainly not the ideal model for studying cholesterol effects and its role in membranes. A much better model for this purpose is that proposed by Rottem et a / . , (1973a). The goat mycoplasmas, M . tnqcoidrs subsp. capri and the closely related M . cnprirolum, can be cultivated with little cholesterol, though growth is less than optimal under these conditions (Razin, 1967; Rottem et a / . , 1973a,b; Archer, 1975; Clejan et ( I / . , 1978; Le Grimellec and Leblanc, 1978; C. E . Dahl et d., 1980a; J . S . Dahl e f a / . , 1980). In this way the cholesterol content of the membranes can be reduced to less than 3% of the total membrane lipid, as compared with about 25% in membranes of the same organisms grown with optimal amounts of cholesterol (Rottem et d. 1973a; Clejan et d., 1978). The most remarkable difference between the cholesterol-poor and the cholesterol-rich membranes is that a thermotrophic phase transition can be demonstrated only in the former (Rottem et ul., 1973b; Melchior and Rottem, 198 I ) . Differential-scanning calorimetry revealed an endothermic phase transition centered at about 25°C in the cholesterol-poor membranes, whereas no transition was observed in the cholesterol-rich ones. Other techniques, such as fluorescence polarization with diphenylhexatriene as a probe, and freeze-fracturing, further confirmed these

STEROLS IN MYCOPLASMA MEMBRANES

193

findings. Chilling of the cholesterol-poor membranes to 4°C prior to the quick freezing caused the aggregation of the intramembranous particles, leaving over two-thirds of the fracture faces particle-free (Rottem et u l . , 1973a). Aggregation of the intramembranous particles, believed to contain integral membrane proteins, is a manifestation of the gelation of the lipid domain (Verkleij et ul., 1972; James and Branton, 1973). No aggregation of particles was discernible in the cholesterol-rich membranes, even when quenched from 4°C (Rottem et ul., 1973a). The experiments carried out with the cholesterol-poor M . rnycoiiles (Rottem er ul., 1973a,b) provided the first clear-cut evidence with membranes of growing cells that cholesterol regulates membrane fluidity during changes in growth temperature, or following alterations in fatty acid composition of membrane lipids. In accordance with this supposition, growth of the cholesterol-poor organisms was almost completely arrested at 25"C, the temperature at which most of the membrane lipids crystallized, whereas the cholesterol-rich cells of the same organism grew well, though at a much slower rate than at 37°C (Rottem et al., 1973a). There can be little doubt that the near-arrest of growth of the cholesterol-poor organisms at 25°C was associated with the steep decline in the membrane-bound ATPase and transport activities that have been shown to occur at this temperature (Rottem c't d., 1973b; Le Grimellec and Leblanc, 1980).

6. Structural Features of Sterol Molecules Required for Function Numerous studies with artificial membranes have indicated that the structural features of the sterol molecule essential for modulating membrane fluidity include a planar (trans-fused) tetracyclic ring system, an equatorial hydroxyl group at position C-3, and a branched aliphatic side-chain at least eight carbon atoms long (Demel and de Kruyff, 1976). These are exactly the structural features of sterols capable of promoting mycoplasnia growth (Razin, 1975). Accordingly, cholesterol, p-sitosterol, stigmasterol, and ergosterol enable mycoplasma growth, whereas coprostanol and epicholesterol cannot substitute for cholesterol and may even inhibit growth (Rottem et id., 1971; Freeman et al., 1976). The report of Odriozola ~t a / . (1978) that M . iupricoium can grow on a variety of sterols that do not fulfill some of the structural requirements stated above, therefore, came as somewhat of a surprise. Sterols allowing M . cupricolum growth included cholesteryl methyl ether and cholesteryl acetate, in which the 3 p hydroxyl group of the sterol is blocked; p-amyrin, which lacks the flexible aliphatic side-chain; and lanosterol, which contains three extra methyls on the sterol a-face, known to weaken hydrophobic interactions between the otherwise planar sterol a-face and phospholipid acyl chains (Lala et id.,1979; C . E. Dahl

194

SHMUEL RAZIN

CHOCESTEROL

-

’.

LANOSTEROL

2

~cI-&N-cn,-C-O

CHOLESTERYL BETAINATE

CYCL09RTENOL

FIG. 3 .

CYCLOLAUDENOL

Structural formulas of sterols tested for growth promotion of M . cupricol~on.

et ul., 1980a,b; J . S. Dahl e t a / . , 1980; Fig. 3). However, as will be shown, the broad sterol specificity of M. crrpricolum can be considered as an advantage, and has been utilized by Bloch and his associates in a most elegant way to prove the validity of the old generalizations concerning the structural requirements for optimal sterol function in membranes. Nevertheless, the data obtained with M. c q w i c d u m indicate that sterols may fulfill functions additional to regulating membrane fluidity. 1 . PLANARIT OY F T H E STEKOI. NUCLEUS

Bloch (1976) has speculated that the compulsory removal during the late stages of cholesterol biosynthesis of methyl substituents attached to the a-face of lanosterol is linked to its membrane function. Thus demethylation at C-14 and C-4 results in a planar a-face, facilitating the hydrophobic interaction of the sterol with contiguous phosphoIipid acyl chains in the lipid bilayer. The data obtained with M . cripricolirm provide strong support for this hypothesis (C. E. Dahl et al., 1980a,b; J . S. Dahl et u l . , 1980). Sterol effectiveness in growth promotion and in increasing cell membrane viscosity progressed in the order lanosterol < 4,4-dimethylcholestanol 5 4P-methylcholestanol < 4 a methylcholestanol < cholestanol < cholesterol (Table 11). Since the corresponding steps in cholesterol biosynthesis occur in the same order, C. E. Diihl el 01. (1980a,b; J . S. Dahl et al., 1980) conclude that the nuclear modifications of the lanosterol structure by oxidative demethylation serve to improve the membrane function o f the sterol molecule. Cycloartenol, a 9,I9-cyclopropane sterol

195

STEROLS IN MYCOPUSMA MEMBRANES

isomeric with lanosterol (Fig. 3) showed an ability intermediate between that of lanosterol and cholesterol in promoting M . ccipricolum growth and increasing membrane microviscosity (C. E . Dahl et a / . , 1980b). The reason for the enhanced effectiveness of cycloartenol over lanosterol apparently lies in the fact that in cycloartenol the 14a-methyl gIoup is not protruding on the a-face of the molecule as i n lanosterol (Fig. 3), promoting more effective van der Waals contacts between the phospholipid fatty acyl chains and the sterol a-face. 2. T H E 3P-HYDROXYL

GROUP

A free hydroxyl group is a universal feature of all cholestane derivatives associated with biological membranes. This fact and the inability of steroid hydrocarbons, steroid ketones, or sterol esters to modulate certain properties of artificial membranes-including fluidity-constitute the principal support for the current view that an unsubstituted sterol hydroxyl is essential for sterolphospholipid interactions. There is also wide agreement that the perpendicular orientation of cholesterol in the bilayer depends on the free hydroxyl group that anchors the sterol to the polar region of the membrane. This generalization appears to hold also for mycoplasmas, since cholesteryl esters, in which the 3P-OH group is blocked, failed to support growth of sterol-requiring mycoplasmas (Edward and Fitzgerald, 1951). Moreover, the steric configuration of the 3-OH group appears of importance as the 3a-OH isomers of cholesterol and cholestanol, epicholesterol and epicholestanol respectivley , were much less effecTABLE I 1 E F F E ~OTF VARIOUSMETHYLATED CHOLESTANE DERIVATIVES ON Mycopkrsmu C(lp'i('O/Um GROWTH AND ON T H E MI(.ROVIS(.OSIIY OF ITS M F M B R A NAEN D OF PHOSPHATIDYLCHOLINE VESICI.ES" Microviscosity (q)"

Sterol added to growth medium (10 pgiml)

Growth rate (mass doubling time in hours)

Absorbance of late log culture (at 640 nm)

Mycoplasma membranes

Phosphatidylcholinesterol vesicles

Cholesterol Cholestanol 401-Methylcholesterol 4,4-Dimethylcholesterol 4,4-Dimethylcholestanol 4P-Methylcholestanol Lanosterol

1.8 2.5 4.0 4.6 5.6 5.4 6.0

0.45 0.40 0.32 0.31 0.30 0.23 0.18

5.0.5 4.72 4.53 4.31 4.09 3.69 3 . I7

4.00 3.7s 3.20 3.00 2.60 2.60 I .20

Data of C. E. Dahl c f ( I / . (1980a). Determined according to fluorescence polarization of diphenylhexatriene incorporated into the mycoplasma membrane and measured at 37°C. or into phosphatidylcholine vesicles containing 50 mol% sterol. and measured at 25°C. "

"

196

SHMUEL RAZlN

tive than the natural 3P-OH isomers in modulating fluidity and permeability of artificial as well as of A . Iaidlarvii membranes (de Kruyff et a / . , 1972), and SP-cholestan-3a-ol failed to support growth of M . mycoides subsp. cnpri (Archer, 1975). Again, as with the requirement for planarity of the sterol nucleus, M . capricolu1n has also been shown to be more relaxed with regard to the requirement for an unblocked 3P-OH group. Cholesteryl acetate and cholesterol methyl ether promoted growth of this mycoplasma though the growth rate was lower,and yield of organisms smaller than with cholesterol (Lala et d l . , 1979). It should be pointed out that the two cholesterol derivatives were recovered unchanged from the mycoplasma cells. Nevertheless, the finding that cholesteryl methyl ether resembled cholesterol in its effects on membrane microviscosity led Lala et ul. (1979) to propose that the polarity of the OCH, moiety suffices for keeping this sterol perpendicularly oriented in the membrane. Similarly, data by Luken et (11. ( 1980) obtained with dipalrnitoyl phosphatidylcholine-cholestan-3-onevesicles suggest that the polar nature of the 3-keto group of the sterol keeps the molecule in a perpendicular position in the bilayer, as reflected by the effects of the sterol on the freedom of motion of the phospholipid chains. Hence, it appears that sterols lacking a 3P-OH group are still capable of modulating membrane fluidity, provided that they possess another polar group and a planar ring structure. Recent studies in o u r laboratory (Efrati et ul., 1981b) show that cholesteryl betainate, an hydrophilic synthetic cholesteryl ester (FIg. 3), can substitute for cholesterol in M . mpricoluin growth. However, it is extremely important to point out here that cholesteryl betainate under the same conditions failed to support growth of several other sterol-requiring mycoplasmas, including M . gallisep~ic~iitn, M . urgitiirii, and M . hoviinis. Thus conclusions drawn from studies with M . capricdum may not be valid for other sterol-requiring mycoplasmas. 3 . T H EAI.IPHA,IIC SIDE-CHAIN A branched and flexible aliphatic side-chain at least eight carbon atoms long is considered as one of the structural features essential for sterol function (Demel and de Kruyff, 1976), and, in fact, sterols devoid of a side-chain failed to support growth of sterol-requiring mycoplasmas (Edward and Fitzgerald, 195 I ; Rodwell, 1963; Rottem et d.,1971). Again, the more recent data from Bloch’s group show that M . capricolum forms an exception to this rule in being able to grow with 0-amyrin, a pentacyclic triterpene with no side-chain. However, growth on P-amyrin was much slower and poorer in yield than with cholesterol. Nevertheless, the same group of workers has also shown that the addition of a methyl group at C-24 of the isooctenyl side-chain, as in cyclolaudenol (Fig. 3), de-

STEROLS IN MYCOPLASMA MEMBRANES

197

creased markedly the growth-promoting and bilayer-condensing activities of the sterol, indicating that the presence of an alkyl substitution at the side-chain weakens considerably fatty acyl chain-sterol interactions (C. E. Dahl et ul., 1980b). This conclusion is strengthened by the findings of Clejan et ( I / . (1981) that the plant and fungal sterols, p-sitosterol, stigmasterol, and ergosterol, which differ from cholcsterol in having an alkyl substituent at C-24, are less effective than cholesterol i n growth promotion of M . cupricolun~.

C. New Concepts of Sterol Function As can be seen in Table 11, sterol structure appears to determine growth rates of M . capricolirrn in proportion to its ability to raise the microviscosity of the receptor membrane. The superior position of cholesterol in this series reflects the fact that this sterol possesses all the structural features optimal for modulating fluidity, including the planarity of the a-face in the region of C-14, absence of a methyl substituent at C-4, insertion of a 5,6-double bond, an unsubstituted 3P-OH, and a flexible and unsubstituted aliphatic side-chain. Thus the data obtained by Bloch’s group support strongly the role of cholesterol as a regulator of membrane fluidity during changes in growth temperature or after alterations i n the fatty acid composition of membrane lipids (Razin, 1975). The ineffectiveness of lanosterol as a regulator of membrane fluidity (Table 11) is retlected well in the findings of J . S . Dahl cJt a / . (1980) showing that the fatty acid requirements of M . cupricolrrrii grown with lanosterol are much stricter than when growth takes place with cholesterol. Thus elaidate alone, or mixtures of myristate-elaidate, niyristate-palmitate, or palmitate-linoleate, failed to support growth in the presence of lanosterol, in contrast to the results obtained with cholesterol. On the other hand, the finding that lanosterol supports suboptimal growth of M . cupricdi.rrn, despite its total incompetence in altering fluidity of artificial and biological membranes, indicates that membrane sterols can play a role other than to regulate bulk lipid fluidity. It has been suggested (Yeagle et u l . , 1977; Odriozola P / ul., 1978) that a more primitive function of sterols in membranes may be to separate phospholipid head groups and thus act as spacer molecules. For this purpose the bulky and rigid polycyclic ring structure that lanosterol shares with cholesterol may suffice (see Section II1,D). Early efforts by Rodwell e f ul. (1972) and by us (Rottem et ul., I973a; Clejan et al., 1978) to obtain cholesterol-free M . niycoides subsp. cupri and M . capricolum membranes have failed, indicating that small amounts of cholesterol in the membrane (less than 3% of total membrane lipids) are indispensable for growth of the goat mycoplasmas. As these minute amounts of cholesterol cannot possibly exert a fluidizing effect on the bulk lipid bilayer, I have proposed (Razin, 1981) that cholesterol fulfills a more specific role, such as activation of

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SHMUEL RAZlN

membrane-bound enzyme systems, in addition to its role as a regulator of membrane fluidity. Support for this hypothesis has recently been obtained by Dahl e/ 01. (1981). Their study was prompted by the finding that combination of a low level of cholesterol (0.5 pg/ml), unable to support M . cupricolurn growth by itself, with a 20-fold greater quantity of lanosterol, which supports slow growth, stimulates the bacterial growth rate by approximately 2-fold. Under these synergistic conditions, the bulk physical state of the membrane was the same as in membranes from cells grown with lanosterol alone (1. S. Dahl et u l . , 1980). These observations suggested that cholesterol may serve more than one function in membranes. Further study of this phenomenon (Dahl et ul., 1981) revealed that the minute amounts of cholesterol enhanced considerably the uptake of unsaturated fatty acids in the lanosterol-grown M . cup,vicolu/n. Dahl et ul. (1 981) concluded, therefore, that cholesterol exerts in this way some control on phospholipid biosynthesis. This apparent control is not dictated by the bulk physical state of the lipid bilayer, but may be dictated by more specific interactions of the sterol with other membrane components. It can be argued that the small amounts of cholesterol concentrate in specific areas in the membrane, increasing viscosity in these microdomains. However, the finding that 3amethylcholesterol, a sterol unable to raise microviscosity of mycoplasma membranes, exerts the same synergistic response as cholesterol (Dahl et ul., 1981) speaks against this possibility. The most plausible explanation for the interesting observations of Dahl et ul. (1981) is that cholesterol interacts specifically and enhances the activity of an enzyme(s) involved in fatty acid activation, transfer, or incorporation into membrane phospholipids, but experimental data to support this explanation are still missing.

D. Why Do Mycoplasmas Require Sterols? Mycoplasmas appear to be unique among prokaryotes in their sterol requirement. Obviously, it is tempting to associate this requirement with the lack of a cell wall-the single most important property distinguishing the mycoplasmas from all other prokaryotes. The notion that cholesterol increases the tensile strength of the mycoplasma membrane, facilitating survival and growth without the protection of a rigid cell wall, has been proposed long ago (Razin, 1967). Cells of the cholesterol-poor M . mycoides subsp. cupri were in fact found to be quite fragile, frequently undergoing lysis even in the growth medium (Razin, 1967; Rottem er ul., 1973a). Furthermore, the K+ level was found to be much lower in these cells than in the sterol-rich organisms, reflecting a higher permeability of the sterol-poor membrane to K + and H+ (Le Grimellec and Leblanc, 1978). The cholesterol-poor organisms were also susceptible to attack by pancreatic phospholipase A,, whereas the cholesterol-rich organisms resisted this

STEROLS IN MYCOPLASMA MEMBRANES

199

enzyme, probably due to tighter packing of the phospholipids in the membrane by cholesterol (Rigaud and Leblanc, 1980). Nevertheless, cholesterol requirement by mycoplasmas cannot be attributed solely to the lack of a cell wall, as Acholeplasmci species and the wall-less bacterial L forms do not require sterols for growth (Razin, 1973). Hence, any explanation for the sterol requirement by mycoplasma should take into account and explain also the sterol independence of acholeplasmas. A plausible explanation of the need of mycoplasmas for sterols can be based on the proven ability of cholesterol to act as a regulator of membrane fluidity. Present evidence suggests that cells employ two devices for adjusting membrane fluidity in response to environmental changes: raising or lowering the ratio of saturated to unsaturated fatty acids by preferential synthesis or incorporation of exogenous fatty acids (Marr and Ingraham, 1962; Rock and Cronan, this volume), or by changing the levels of membrane-associated sterol (Sinensky, 1978). The sterol-requiring mycoplasmas appear to lack the mechanism of regulating membrane fluidity by altering their fatty acid composition, as they are incapable of fatty acid synthesis, and their ability to select exogenous fatty acids for modulating membrane fluidity is rather poor (Razin, 1978a). This deficiency is illustrated well by the very strict fatty acid requirement of M . c q w i c v l i r r n cultivated o n lanosterol, a sterol unable to modulate membrane fluidity (see Section 111,C). Acholciplusmi species, on the other hand, are capable of regulating their ) acid biosynthesis, incorporamembrane fluidity at various levels of (10 w i ~fatty

tion and elongation of exogenous fatty acids, and their utilization for synthesis of complex membrane lipids (Razin, 1978a, 1981; see also Melchior, this volume). In addition, acholeplasmas synthesize carotenoids, which may also function in regulation of membrane fluidity (Huang and Haug, 1974; Rottem and Markowitz, 1979b). and A , laidlu,c,ii was shown to be capable of modifying the ratio of monoglucosyldiglyceride to diglucosyldiglyceride in response to factors modulating membrane fluidity (Christianson and Wieslander, 1978). As was already mentioned (Section IIl,C), sterols may also act in membranes in the capacity of spacer molecules, separating the phospholipid head groups. In this way the sterols decrease the charge density of the membrane, lower its electrostatic free energy, and stabilize the bilayer structure. This property appears particularly of importance in mycoplasmas, where essentially all the phospholipids synthesized are of the acidic type-mostly phosphatidylglycerol and diphosphatidylglycerol. Neutral lipids, such as phosphatidylcholine, sphingomyelin, and phosphatidylethanolamine, which may also act as spacers (Smith, 1980), are not synthesized by mycoplasmas (Razin, 1975). Gram-negative bacteria synthesize large quantities of phosphatidylethanolamine, and gram-positive bacteria synthesize glycolipids. which can also act as spacer molecules (Smith, 1980). The sterol-nonrequiring acholeplasnias, unlike the sterol-requiring

200

SHMUEL RAZlN

mycoplasmas, synthesize large quantities of glycolipids (Section II,B,l). Hence, it can be argued that the spacer function in acholeplasmas is fulfilled by their glycolipids, whereas in the sterol-requiring mycoplasmas it is fulfilled by sterols. Wieslander tlr ( I / . (1980) develop this idea further by postulating that cholesterol by virtue of its wedge-shaped molecule increases the distances between the polar head groups of membrane phospholipids, diluting the surface charge density. In addition, cholesterol induces local regions in the membrane having a nonlamellar or reversed hexagonal phase, a property shared by the acholeplasmal glycolipid, monoglucosyldiglyceride. According to this hypothesis, a membrane will act optimally when its bilayer consists of a lamellar and other mesophase structures; this necessitates a mixture of lipids rather than one lipid species. The balance between the various lipid species must be kept within certain limits so as to create local regions forming transitory nonlamellar phases, which may be advantageous to some membrane features such as movement of membrane components between bilayer halves, fusion, exo- and endocytosis, and facilitated transport (Wieslander et a / . , 1980).

IV.

CONCLUSIONS

The requirement of mycoplasmas for sterols, unique among prokaryotes, has been utilized to show that sterols function as regulators of membrane. fluidity during changes in growth temperature or alterations in the fatty acid composition of membrane lipids. T o accomplish this role the sterol should have a planar ring system, a free 3P-hydroxyl group and an aliphatic side-chain. Cholesterol fulfills all three structural requirements and is usually superior to other sterols in growth promotion of mycoplasmas. The recent finding that M . cupricolum can grow with sterols that do not fulfill all three structural requirements indicates that sterols may have additional functions to that of regulating bulk lipid fluidity. Thus the finding that lanosterol, a sterol not competent in modulating membrane fluidity, supports suboptimal growth of M . c ~ i p r i c ~ i l r r /has n , been taken to suggest that sterols may act also as spacer molecules. In this capacity, the bulky ring structure of the sterol separates the acidic phospholipid head groups, in this way decreasing the charge density of the membrane and stabilizing its structure. The recent work by Dahl et al. (1981) indicates that cholesterol may have in addition a more specific role in control of membrane phospholipid synthesis. species do not require sterols for growth can be The fact that Ac~holeplus~~ru explained by the ability of these mycoplasmas to regulate membrane fluidity by altering the fatty acid composition of membrane lipids, a property not shared by the sterol-requiring mycoplasmas. Moreover, Arholrplus/no membranes contain large quantities of glycolipids, which share with sterols the capacity of acting as spacer molecules.

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201

Some progress has recently been made in understanding the factors controlling the transfer of sterols to mycoplasma membranes. Among serum lipoproteins, those having the highest cholesterol :phospholipid ratio, such as human lowdensity lipoproteins, are the best cholesterol donors. Transfer of free cholesterol to Acholeplasrrici occurs during a transient contact of the lipoprotein particle with the membrane. In sterol-requiring mycoplasmas the contact appears to be tighter, or more prolonged, as it suffices for transfer of some of the lipoprotein phospholipid and esterified cholesterol to the membrane. It is proposed that the sterol-requiring mycoplasmas possess on their surface receptors, probably of a protein nature, responsible for better contact of the lipoprotein particle with the membrane. Another, or an additional explanation for the limited capacity of Acholeplusrna membranes to take up cholesterol, can be based o n the large quantities of glycolipids found in these membranes. Preliminary evidence suggests that the affinity of these glycolipids for cholesterol is lower than that of phospholipids, but more experimental data are needed to support or refute this suggestion. ACKNOWLEDGMENTS Our recent studies on cholesterol in mycoplasma nieinbranes were supported by grants from the United States-lmel Binational Science Foundation (BSF) and the Israel Commission for Basic Research. REFERENCES Archer, D. B. (197.5). Modification of the membrane composition of Mwopla.sniu ny-oitie.s subsp. rtrpri by thc growth medium. J . Grri. Mic,rohio/. 88, 329-338. Argaman, M.. and Razin, S. (1965). Cholesterol and cholesterol ebters in mycoplasma. J . G r t ~ . Microhiol. 38, 153-168.

Basu. S. K . , Goldstein, J . L.. and Brown. M . S. (1978). Characterization of the low-density lipoprotein receptor in mcmbranes prepared lrom human fibroblasts. J . B i d . Chrrn. 253, 3853-3856. Bevers, E. M.. Singal. S . A , . Op den Kamp. J . A. F., and van Deenen, L. L. M . (1977). Recognition of different pools of phosphatidyl glycerol in intact cells and isolated membranes of Ar.ho/rp/tr.\~riciItritllt~itvi by phospholipase A,. Biochrrrri.clr:\. 16, 3943-3948. Bloch, K . (1976). On the evolution of a biosynthetic pathway. In “Reflections on Biochemistry” ( A . Kornberg, B. L. Horecker, L. Cornudella, and J . Or6, eds.), pp. 143-150. Pergamon, Oxford. Bloj, B . , and Zilversniit. D. B. (1977). Complete exchangeability of cholesterol in phosphatidyicholine/cholesrerol vcsicles of different degrees of‘unsaturation. Rirdrrrrristr? 16, 3943-394x.

Butler, K. W . , Johnson, K C . . and Smith, I . C. P. (1978). Achn/rpltr.\rncr ktiidlrwii membranes: An electron spin resonance \tudy o f the influence on niolecular order of fatty acid composition and cholesterol. Arc./i. Bioc.htw7. Biophys. 191, 289-297. Chapman, D. (196h). Liquid crystals and cell membranes. A m N . Y . A c u d Sci. 137, 745-7.54. /dChristiansson. A . , and Wieslander. A. (1978). Memhrane lipid metabolism in Athoic~~iitrsnicr ltrwri A EF 22. Influence o f cholesterol and temperature shift-down on incorporation of fatty acids and synthesis of membrane lipid species. Eur. ./. Riorhem. 85, 65-76.

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Clejan, S., Bittman. R., and Rotteni. S . (1978). Uptake, transbilayer distribution, and movement of cholesterol in growing Mycoplusmcr c c i p r i c ~ d ~cells. m Biochemistry 17, 4579-4583. Clejan, S., Bittman, R . . and Rottem, S . (1981). Effects of sterol structure and exogenous lipids on the transbilayer distribution of sterols in the membrane of Mycoplusmu cqwicolfim.Bi(JChemi.ttn: 20, 2200-2208. Cooper, R. A . (1978). Influence of increased membrane cholesterol on meinbrane fluidity and cell function in human red blood cells. J . S i p ~ i t r f dSfriic't. . 8, 413-430. Dahl, C. E.. Dahl, J . S . . and Bloch, K. (1980a). Effect of alkyl-substituted precursors ofcholesterol on artificial and natural membranes and on the viability of M,~r,oplasmu cupricottrm. B l o C h P m i S f f ? 19, 1462-1467. Dahl. C. E.. Dahl, J . S., and Bloch, K . (1980b). Effects ofcycloartenol and lanosterol on artificial and natural membranes. Biocfiem. Biuphw. Res. Commun. 92. 221 -228. Dahl, J . S . , Dahl, C . E., and Bloch, K. (1980). Sterols i n membranes: Growth characteristics and membrane properties of M W T J ~ ~ Wc~upricolum TIU cultured on cholesterol and lanosterol. B~orhemisrry19, 1467-1471. Dahl, J . S . , Dahl, C. E . , and Bloch, K . (1981). Effect of cholesterol on macromolecular synthesis and fatty acid uptake by Mvcqhsmu tupric.o/um. J . B i o l . Chrm. 256, 87-91. Davis, J . H . , Bloom, M . . Butler, K. W., and Smith. 1. C. P. (1980). The temperature dependence of molecular order and the influence of:cholesterol on Acholeplasmu luidlawii membraneb. B i ( ~ ~ / f i f JBlOp/I,l',S. ?. A C f U 597, 477-491 . de Kruytf, B.. Demel, R. A , , and van Deenen, L. I*. M . (1972). The effect of cholesterol and epicholesterol incorporation on the permeability and on the phase transition of intact Acholepicismti / [ ~ i d / m , cell i i membranes and derived liposomes. Biochim. Biophys. Acru 255, 33 1 341. de Kruyff, B., van Dijck. P. W. M.. Goldbach, R. W.. Demel. R. A . , and van Deenen, Is. L. M. (1973). lnlluence of fatty acid and sterol composition on the lipid phase transition and activity of membrane-bound enzymes in Acholep/u.sr~rcrIuidluwii. Biochim. Biciphy,~.Acru 330, 2642x2.

Demel, R . A , , and dc Kruyff, B. (1976). The function of sterols i n membranes. Biochim. Riophys. Acrci 457, 109- 132. Demel, R . A , , Janaen, J . W. C. M., van Dijck, P. W. M . , and van Deenen, L. L. M . (1977). The preferential interaction of cholesterol with different classes of phospholipids. Biochim. B ~ o ~ ~ Acru J u . 465, 1-10,

Edward, D. G. If.. and Fitzgerald. W. A . (1951). Cholesterol in the growth of organisms of the pleuropneumonia group. J . GLW.Microhiol. 5, 576-586. Efrati. H., Rottern, S . , and Razin. S . (1981a). Lipid and protein membrane components associated with cholesterol uptake by mycoplasmas. B i ( ~ c . l ~ Biophys. i~?. Acru 641, 386-394. Efrati, H . , Shinitzky. M., and Razin, S. (1981 b). Effect of charged cholcsteryl esterson mycoplasma growth. FLUS L P I I .122, 59-63, Freeman. B. A , , Sisscnstein. R . . McManus, T . T . , Woodward, J . E . , Lee, I . M . , and Mudd, J . B. ( I 976). Lipid composition and lipid metabolism of Spiroplmmti cirri. J . Bnc~rerial. 125, 946-954. Gershfeld. N . L.. Wormscr, M . . and Razin, S . (1974). Cholesterol in inycoplasma membranes. I . Kinetics and equilibrium studies of cholesterol uptakc by the cell membrane of Acholeplusmi / d / t w i i , R i o d i I ~ ?Biop/ry.\. ~. A[,ftr 352, 37 1-384. Huang, L., and Haug, A . (1974). Regulation of membrane lipid fluidity in Ach/ep/tic.rrru /uid/uwii: Effect of carotenoid pigment content. Biochir,i. Biophys. Acvo 352, 361 -370. James, R., and Branton, D. (1973). Lipid- and temperature-dependent structural changes in Acholeplotmti laidltrwii cell membranes. Riothim. R i ( ~ p h y .Acttr ~ . 323, 338-390.

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Kahane, I., and Razin, S . (1977). Cholesterol-phosphatidylcholine dispersions as donors of chalesterol to mycoplasma membranes. BlcJchi/?J.E i u p l ? A ~ c m 471, 32-38. Kutner, S. (1979). Uptake of exogenous phospholipids by mycoplasmas. M . Sc. Thesis, Hebrew University, Jerusalem. Lala, A. K . , Buttke, T . M., and Bluch, K. (1979). On the role of the sterol hydroxyl group in membranes. J . B i d . Clwrri. 254, 10582-10585. Le Grimellec, C . , and Leblanc, G . (1978). Effect o f membrane cholesterol on potassium transport in Myc,op/mrnu ntycwirles var. copri (PG 3). Biochirn. B i o p h y ~Acru 514, 152-163. Le Grimellec, C . , and Leblanc. C. (1980). Temperature dependent relationship between K’ influx, Mg ‘--ATPase activity, .transmembrane potential and membrane lipid composition in mycoplasma. Eiochim. Bioplzys. Ae,trr 599, 639-65 I . Lemcke. R. M.. and Burrows. M. R. (IY80). Sterol requirement for the growth of ~ ~ ~ ~ o w ~ ~ /iyodysrn/rriut,. J . G P J IM . i r w h i o / . 11 6, 539-543 Luken. D. W . , Esfahani, M . , and Devlin, T . M. (1980). Effect of sterols on diphenylhexatriene fluorescence in lecithin vesicles. FEES Lrlr. 114, 48-50. McCabe, P. J . . and Green. C . (1977). The dispersion of cholesterol with phospholipids and glycolipids. Chem. Phys. Lipids 20, 319-330. McElhaney, R. N . (1974). The effect of alterations in the physical state ofthe membrane lipids on the ability of A ~ I O / P ~ / U W/ uJi Ud / c i ~ ? iB to grow at various temperatures. J . M d . Eiol. 84, 145157.

McElhaney, R. N . , de Gier, J . . and van Deenen, L . L. M . (1970). The effect of alterations in fatty acid composition and cholesterol cnntent on the permeability of M~cop/ti.srriu/ [ ~ i d / u w iBi cells and derived liposomes Bloc,/iim. Eiopli?.~.Acrtr 219, 245-247. Marr, A . G . . and Ingraham. J . L. (1962) Effect of temperature on the composition of fatty acids in E . c , o / i . J . Buc.lcrio/. 84, 1260-1267 Melchior, D. L.. and Rotteni, S. (1981). The organization of cholesterol esters in M J ~ ~ o ~ / u . s u J ~ ~ cwprico/irm membranes. Eur. J . Hiu~.hein.117, 147-153. Mudd, J . B.. Ittig, M.. Roy, B . , Latrille, J.. and Bove, J . M . (1977). Composition and enzyme activities of Sptrop/usmu c,i//-i membranes. J . E t i c ~ r i n l .129, 1250- 1256. Odnozola, J . M . , Waitzkin. E . , Smith, T. L . , and Bloch, K. (1978). Sterol requirement of M.wopltismu ccrprir.o/um. Pro<..N u / / . A m d . .%.I. U.S.A. 75, 4107-4109. Pagano. R. E . , Sandra. A . . and Takeichi. M . (1978). Interactions of phospholipid vesicles with mammalian cell\. Artrr. N . Y . Accid. k i . 308, 185-199. Patel, K . R., Smith, P. F.. and Mayberry, W. R . (I97X). Comparison of lipids froin S p i ~ ~ p / t i . ~ ~ ~ i ~ cirri and corn stunt spiroplasma. J . Buc.~rrio/.136, 829-831. Razin, S . (1967). The cell membrane of mycoplasma. A I I ~ JN. . Y . A u ~ c i .Sci. 143, 11.5-129. Razin, S. (1973). Physiology of mycoplasmas. A h . M i ~ r ~ Ph\,.\io/. h. 10, 1-80. Razin, S. (1974). Correlation of chole\terol to phospholipid content in membranes 01‘ grnwing mycoplasmas. FEES Let t . 47, 81 85. Razin, S. (1975). The mycoplasma membrane. Prosy. Sio-/. Ma/trbr. S r i . 9, 257-312 Razin, S. ( l 9 7 8 a ) . The mycoplasmas. Microhiol R r i , . 42, 414-470. Razin, S . (l978b). Cholesterol uptake is dependent on membrane fluidity in mycoplasmas. Eiochirtt. Eiophxs. Acru 513, 401 -404. Razin, S . (1981 ). The mycoplasma membrane. Irr “Organization of Procaryntic Cell Membranes” ( B . K . Ghosh. c d . ) , Vol. I. pp. 165-250. CRC Press. Boca Raton, Florida. Razin, S., and Rotteni. S. (1976). Technique\ for the manipulation of mycoplasrna membranes. Irr “Biochemical Analysis of Membranes” ( A . H. Maddy. ed.). pp. 3 -26. Chapman CC. Hall, London Razin, S., and Rotteni, S. (1978). Cholehterol in membrane\: Studies with mycoplasnias. Trerti/.\ RiOC/tf’/lJ. SC’;. 3, 51 - 5 5 .

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Razin. S . , and Tully, J . G . (1970). Cholesterol requirement of mycoplasmas. J . Boctrriol. 102, 306-3 10. Razin, S . , Wormser. M., and Gershfeld, N . 1. (lY74). Cholesterol in mycoplasma membranes. 11. Components of Acho/ep/u.tmu kricllrrwii cell membranes rebponsible for cholesterol binding. Biochim. Biophys. Actu 352, 385-396. Rarin, S . , Kutner, S , Efrati, H.. and Rottem, S . (1980). Phospholipid and cholesterol uptake by mycoplasma cells and membranes. Bioc.hirn. 5ioplry.s. Actcr 598, 628-640. Razin, S.. Efrati. H.. Kutner. S . , and Rotteni, S . (1981). Cholesterol and phospholipid uptake by mycoplasmas. Re\,. /nfr(,/.Dis. (in press). Rigaud, J . L.. and Lehlanc, G . (1980). Effect of membrane cholesterol on action ofphospholipase A2 in M,scup/u.\mo rrrvcoitles var. ctrpri. Evidence for lysophospholipase activity. Eict.. J . BiOc'ht'tJl. 110, 77-84. Rodwell, A . W . (1963). The steroid growth requirement of Mycoplasmu mycuidrs. J . G m . Microhiot. 32, 91-101. R~tlwell,A. W . , Peterson. 1 . E.. and Rodwell, E. S . (1972). Macromolecular synthesis and growth of mycoplasmas. Pothog. M w ~ p l u s m u s ,Cihu Found. S y p . I972 pp. 123- 139. Rothman. J . E . , and Engelman, D. M. (1972). Molecular mechanism for the interaction of phospholipid with cholesterol. Nafrtre ( L o d o t r ) 237, 42-44. Rottem. S . . and Markowitz, 0 . ( 197Ya). Membrane lipids of Mwqdusma gu//iseptic.irm: A disaturated phosphatidylcholine and a phosphatidylglycerol with an unusual positional distribution o f fatty acids. 5ioc/remi.r/ry 18, 2090-293s. Rottem, S . , and Markowitz, 0. (197%). Carotenoids act as reinforcers of the .4cho/rp/crsmu laidlerwii lipid bilayer J . Bacrrrid. 140, 944-948. Rottem, S., Pfendt, E. A , , and Hayflick, L. (1971). Sterol requirements ofT-strain mycoplasmas. J . Bacterial. 105, 323-330. Rotreni, S . . Yashouv, Y . . Ne'eman, Z . , and Razin. S . (1973a). Cholesterol in mycoplasma membranes. Composition. ultraatructure and biological properties of membranes from Mwy/u.sritu ntycoicles var. c y r r cells adapted to grow with low cholesterol concentrations. Riochim. Biophvs. Acftr 323, 495-508. Rortem. S . . Cirillo, V . P.. de Kruyfi. B., Shinitzky, M., and Razin. S . (1973b). Cholesterol i n mycoplasma membranes. Correlation of enz,ymic and transport activities with physical state of lipids in membranes of MwopIamitci rnjcoides var. capri adapted to grow with low cholesterol concentrations. Biochim. Biophys. Actu 323, 509-5 19. Sinensky, M. (1978). Defective regulation of cholesterol biosynthesis and plasma membrane fluidity in a Chinese hamster ovary cell mutant. Proc. Ntrfl. Acad. Sci. U . S . A . 75, 1247-1249. Slutrky, G. M., Razin, S., Kahane. I . , and Eisenberg, S . (1976). Serum lipoproteins a5 cholesterol donors to mycoplasma membranes. Biocham. Biophys. Res. Cummuti. 68, 529-535. Slutzky, G . M . , Raiin, S . , Kahane, I . . and Eisenberg, S . (1977). Cholesterol transfer from serum lipoproteins to mycoplasma membranes. Biochemist)? 16, 5 158-5163. Slutzky, G . M . , Razin, S . , Kahane, I . , and Eisenberg. S . (1981). Serum lipoproteins as cholesterol donors to mycoplasma cells and membranes. /n "High-Density Lipoproteins" (C. E. Day, ed.), pp. 307-318. Dekker. New York. Small, D. M., and Shipley. G . G . (1974). Physical-chemical basis of lipid deposition in atherosclerosis. Science 185, 222-229. Smith, M. W . (1980. Physical studies of bacterial lipids and model systems. Ph.D. Thesis, Brown Univeraity, Providence, Rhode Island. Stockton, G. W . , Johnson, K . G., Butler, K . W . . Tulloch, A . P.. Boulanger, Y . , Smith, I . C. P., Davis, J. H., and Bloom, M . (1977). Deuterium NMR study of lipid organization in Acholrpiasmo Itritlfawii membranes. Narirrc. (London) 269, 267-268. van Dijck, P. W. M.. dc Kruyff, B., van Deenen, L. L. M . , de Gicr, J . , and Demel, R. A. (1976).

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The preference of cholesterol for phosphatidylcholine in mixed phosphatidylcholine-phosphatidylethanolamine bilayers. Bioc.hirn. Bioph?s. Actu 455, 576-587. Vcrkleij, A . J . , Ververgaert, P. H . J . . van Deenen, I.. L. M . , and Elbers, P. F. (1972). Phase transitions of phospholipid bilayers and membranes of A d w l ~ p l t i s r r i c i hiidltiuii B visualized , J . 288, 326-332. by freeze fracturing electron microscopy. Rioc~hirn.R ~ O [ J ~ \Acfu Wieslander, A , , and Rilfors, L. (1977). Qualitative and quantitative variations of membrane lipid species in A~~holep/a,sr7rti luid/riiz.ii A . B i o c h i i ~ B . i o p h ~ sActu . 466, 336-346. Wieslander, A , , Ulmius. J . . Lindblom, G.. and Fonkll, K . (1978). Water binding and phase structures for different A ~ , h o / e p / ~ i . \ , ~I ~i t i r l l t i i r ~ imembrane i lipids studied by deuteron nuclear magnetic resonance and X-ray diffraction. Riot,/iit?i.Biophys. Actti 512, 241 -253. Wieslander, A , , Christiansson, A . , Rilfors. L., and Lindblom, G . (1980). Lipid bilayer stability in membranes. Regulation of lipid composition in Acliolepkmr~ia l ~ i d l u w i ias governed by molecular shape. Bioc.hrmist,? 19, 3650-3655. Yeagle, P. L . , Martin, R . B . , Lala, A . K.. Lin. H . K., and Bloch, K . (1977). Differential effects of cholesterol and lanosterol o n artificial membranes. Proc. Nufl. Acud. Sci. U . S . A . 74, 49244926.

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CURRENT TOPICS I N MEMBRANES AND TRANSPORT, VOLUME 17

Regu lation of Bacterial Membrane Lipid Synthesis CHARLES 0 . ROCK

I. 11.

111.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation o f Membrane Lipid Synthesis . . . . . . . . . . . . . . . A . Control of Membrane Phospholipid Acyl-Group Composition . . . . . B. Regulation of the Rate o f Phospholipid Synthesis . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1.

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207 208 208 222 226 227

INTRODUCTION

Early investigations into microbial lipids focused on their structure and biosynthesis. At present, most of the microbial-lipid structures are known and although many molecular details have yet to be resolved, the metabolic pathways giving rise to these structures have been defined. Due to the large amount of information available on this topic and the space limitations of this chapter, as well as to avoid overlap with other recent reviews, we have limited our discussion to those areas of current research activity. Most notably, this includes investigations into the biosynthesis and regulation of lipid metabolism in Eschrrichim coli. Therefore, we wish to alert the reader to several reviews that emphasize topics not covered in detail by us. These include the structure of microbial lipids (Goldfine, 207

Copyright 0 1YR2 by Academic Press. Inc All rights of reproduction in any fonn reserved. ISBN 0~12-153317.4

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1972; Goldfine, this volume), function of microbial membrane lipids (Cronan and Gelman, 1975; Melchior and McElhaney, this volume), biosynthesis of fatty acids (Bloch, 1971; Bloch and Vance, 1977; Volpe and Vagelos, 1976; Silbert, 1975; Rock and Cronan, 1981), phospholipid synthesis, and its regulation and topology (Cronan, 1979; Raetz, 1978, Finnerty, 1978; Clark and Cronan, 1981).

II. REGULATION OF MEMBRANE LIPID SYNTHESIS

A. Control of Membrane Phospholipid Acyl-Group Composition I . REGULATION O F 1-HEPOSITIONAL DISTRIBUTION O F ACYLMOIETIES Naturally occurring phospholipids are generally characterized as having a saturated fatty acid at position 1 and an unsaturated fatty acid at position 2 (see van Deenan, 1965). The a?-glycerol-3-phosphate acyltransferase (acyltransferase) is the first committed step in phospholipid biosynthesis and is responsible for esterifying one (or both) fatty acids to glycerol-P to form the intermediate, phosphatidic acid. Since post-de m v o biosynthetic redistribution of acyl moieties does not occur in E . roli (Cronan and Prestegard, 1977), the most likely origin of acyl-group asymmetry is at the level of the acyltransferase. The acyltransferase activity is located on the inner membrane and is thought to proceed by two consecutive acylation reactions (Cronan and Gelmann, 1975). The first reaction catalyzes the transfer of a saturated acid to the one position forming 1-acyl-srz-glycerol 3-phosphate ( I-acylglycerol-P), and the second reaction catalyzes the transfer of an unsaturated acid to the 2 position of the first intermediate to form phosphatidic acid. The question of the specificity of these membrane-bound enzymes has been the subject of a large and confusing literature (for review, see Cronan, 1978). Several workers have reported that saturated acyl-CoA substrates yield I-acylglycerol-P, whereas unsaturated acyl-CoA substrates yield 2-acylglycerol-P (Ray e f ul., 1970; Sinensky, 1971). Other investigators have reported that both unsaturated and saturated fatty acids are capable of forming I-acylglycerol-P and find no specificity in the acyl transfer reaction (Okuyama and Wakil, 1973). Acylation specificity of the acyl transfer system has also been studied in the presence of a mixture of saturated and unsaturated acyl-CoAs (Ray ef al., 1970; Nishihara et a l . , 1976). These results have shown that the positional distribution of fatty acids in phosphatidic acid (Ray et ul., 1970; Kito er u l . , 1978) or phosphatidylglycerol (Nishihara et a / . , 1976) is similar to the distribution in vivo. However, these studies do not delineate the number or specificity of the intermediate reactions responsible for

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209

conferring the specificity. Several groups have attempted to purify the acyltransferase in the hope that these properties could be assayed in purer preparations (Kito et a / . , 1978; Snider and Kennedy, 1977; Ishinaga et al., 1976). Unfortunately, the acyltransferase is inactivated by most detergents used in membrane solubilization (Kito et a l . , 1978; Snider and Kennedy, 1977; Ishinaga et al., 1976), and the Michaelis constant for glycerol-P has been found to increase 20-fold following solubilization (Kito et al., 1978). These results show that the solubilized acyltransferase preparations have kinetic properties that do not reflect the activity of the protein in its native environment. The resolution of this contradictory information seems to reside in the examination of the kinetic mechanism of the acyltransferase(s). This conclusion is supported by experiments using unsaturated fatty acid auxotrophs. When E . coli mutants that are unable to synthesize unsaturated fatty acids are deprived of an exogenous unsaturated fatty acid supplement, a significant accumulation of disaturated molecular species is observed (Silber, 1970). Restoration of unsaturated fatty acids to the medium results in t h e synthesis of molecular species having the typical fatty acid positional asymmetry. These data demonstrate that acylation specificity is not absolute in viiv, and is controlled in part by the supply of fatty acids. Most investigations into the kinetics and specificity of acyltransferase in vitro have used acyl-CoAs as acyl donors, but recent data suggest that acyl-ACP and not acyl-CoA is the acyl donor in vivo (Rock et al., 1981~).Acyl-CoA synthetase is the only known enzyme for the synthesis of CoA thioesters in E . coli (Overath et a l . , 1969). This enzyme is a component of the P-oxidation regulon (Overath et a l . , 1969) and is repressed by growth on glucose (Pauli et al., 1974). Mutants defective in acyl-CoA synthetase activity are unable to transport and subsequently P-oxidize fatty acids, but they show no growth abnormalities on other carbon sources (Overath et al., 1969). Acyl-CoAs have not been detected in vivo, whereas some evidence, albeit indirect, has been presented for the presence of acyl-ACP iri vivo (Elovson and Vagelos, 1975). E . c d i also contains two thioesterases (Bonner and Bloch, 1972; Barnes and Wakil, 1968; Barnes et u / . , 1970) that are very active on acyl-CoA substrates, but have only marginally detectable activity toward acyl-ACP substrates (Spencer et ul., 1978). An additional complication with acyl-CoA substrates is that they are potent inhibitors of acyltransferase by virtue of their detergent properties (Kito and Pizer, 1969; van den Bosch and Vagelos, 1970). This property necessitates the inclusion of serum albumin in the assay in order to ameliorate the detergent effects of the substrate. Thus the actual free acyl-CoA concentration is unknown, making rigorous interpretation of kinetic results impossible. In light of these considerations, it is doubtful that the arguments presented to explain the specificity of the acyltransferase system on the basis of in vitro assays using acyl-CoA substrates are relevant to the in vivo situation (see also Cronan, 1978). In in vitro studies utilizing acyl-CoAs as substrates, Okuyama and co-workers 1976) have reported that acyl(Okuyama and Yamada, 1979; Okuyama et d.,

21 0

CHARLES 0. ROCK AND JOHN E. CRONAN. JR.

transferase specificity is dependent on the glycerol-P concentration. As the glycerol-P concentration is increased, the specificity decreases. From these data, they suggested that abnormally high intracellular concentrations of glycerol-P would result in the synthesis of phospholipids with unusual positional distributions (Okuyama rt ( i f . , 1976). Kito er u l . (1978) have disputed these results but these in vim experiments seem to have no physiological significance, since the levels of glycerol-P are tightly regulated in E . coli (Bell and Cronan, 1975; Clark et al., 1980). Recently, Goelz and Cronan (1980) have determined the positional distribution of acyl moieties i n phospholipids derived from strains that possess intracellular glycerol-P concentrations ranging from 0.18 to 2.8 mM. They find that these changes in the intracellular concentration of glycerol-P have no effect on the acyl-chain distribution and conclude that phospholipid structure is not modulated by the supply of glycerol-P in v i w . Van den Bosch and Vagelos (1970) were the first workers to examine the specificity of acyltransferase using acyl-ACP substrates. Unfortunately, these results are suspect because the chemical procedure employed to synthesize the acyl-ACP substrates results in a dramatic loss of the secondary structure and biological activity of ACP (Schultz, 1975). Ray and Cronan (1975) have used a cell-free fatty acid synthase system derived from spinach chloroplasts (Jaworski and Stumpf, 1974) to prepare labeled palmitoyl-ACP for acyltransferase studies. This procedure yielded native unmodified acyl- ACP, but unsaturated acyl-ACPs cannot be prepared. Ray and Cronan (1975) found that the p l s B acyltransferase possessed similarly altered activities for both acyl-ACP and acyl-CoA substrates, indicating that the two acyltransfer reactions have at least some components in common. However, they also found that Mg2+ stimulates acyltransfer from CoA donors 1.5-fold, but has no effect on acyl transfer from acyl-ACP; and that g~anosine-5'-diphosphate-3~-diphosphate (ppGpp) is a potent inhibitor of acyl transfer from acyl-CoA but does not affect the rate when acyl-ACP is used as an acyl donor. An identical result had been previously reported by Lueking and Goldfine ( 1975), who utilized chemically prepared acyl-ACP. The discovery (Ray and Cronan, 1976a) and purification (Rock and Cronan, 1979a) of the inner-membrane enzyme acyl-ACP synthetase has provided a method for the preparation of native acyl-ACP species. This enzyme ligates both saturated and unsaturated fatty acids to ACP and the enzyme is most active on chain lengths found in E . coli phospholipids. Rock and Garwin (1979) have outlined the technology required to prepare milligram quantities of homogeneous acyl-ACP of defined chain length (see also Rock P r ul., 198 1b). Physical studies on these thioesters have shown that the protein moieties of ACP and acyl-ACP possess the same Stokes radius and sedimentation constant (Rock and Cronan, 1979b). AcyI-ACPs have been found to be more stable to pH-induced denaturation than ACP, and unlike acyl-CoAs, acyl-ACPs do not exhibit properties typical of detergents (Rock and Cronan, 1979b; Rock c'f a / . , 1981a). Rock er a / . ( 198 1 c) have used these acyl-ACP substrates to study acyltransferase reactions

BACTERIAL MEMBRANE LIPID SYNTHESIS REGULATION

21 1

present in E . c ~ d inner i membranes. Two kinetically distinguishable glycerol-P sites were found on the inner membranes. The first system was characterized as having a K , for glycerol-P of 90 p M . This system is the same as described by previous investigators. Palmitoyl-CoA, palmitoyl-ACP, and cis-vaccenoyl-ACP were found to be substrates for this system. Although the acyl-ACP and acylCoA were found to have kinetically indistinguishable glycerol-P sites, distinct acyl-donor binding sites were inferred from experiments using ACP as an acyltransferase inhibitor. A second enzyme system possessing a K , for glycerol-P of 700 p M was observed when palmitoleoyl-ACP was used as a substrate. Two degradative reactions were also identified. When palmitoyl-ACP was the substrate, a significant accumulation of diacylglycerol was observed, whereas other substrates did not yield this product. Second, glycerol-P was found to be converted to glycerol during the course of the incubation. Glycerol formation was dependent on the addition of acyl donor and was most pronounced when palmitoleoyl-ACP was the substrate. Taken together, these data support the role of acyl-ACP as the in vivn acyl donor and provide evidence for a multiplicity of sites (if not proteins) involved in acyltransferase function. Although considerable effort has been devoted to this topic, the biochemical details of acyltransferase function and its role in regulation of membrane-lipid structure remain obscure. It is our view that acyl-ACP is the physiological acyl donor, but more definitive experimental support is needed. In particular, measurements of the ACP-and CoA-pool composition would be helpful in determining the contributions of these substrates to acy] transfer reactions in vivo. More work is also required to define the role of acyl-ACP substrates in acyl transfer reactions in viiro. Studies of Rock et ril. ( 1 9 8 1 ~are ) a first step in this direction, but these data have raised more questions than they have answered. Finally, the application of DNA-cloning techniques for the amplification and subsequent purification of acyltransferase should greatly assist the study of this enzyme. Bell and co-workers (Larson et d.,1980; Lightener e f al., 1980) have recently succeeded in cloning a structural gene for the MI-glycerol-3-phosphate acyltransferase by selecting for a cloned fragment of DNA that complements strains carrying the pl!sB mutation. These workers have revised the genetic map position of the p l s B locus and have identified a protein having an apparent molecular weight of83.000 in strains overproducing acyltransferase activity (10-to 15-fold) as a subunit of the acyltransferase. Although it appears that other proteins may be involved in acyl transfer in v i w , this work should greatly increase the sophistication of future acyltransferase experiments. OF 2. REGULATION

I HE

SA I U K A T E U : U N S A l U K A I E D RATIO

i i . Resporise to Altcred Grobtdi Trinperutirre. Marr and Ingraham ( 1 962) first noted that E . coli adjusts the fatty acid composition of its phospholipids i n response to the growth temperature. As the temperature of growth is lowered, the

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CHARLES 0 . ROCK AND JOHN E. CRONAN, JR.

proportion of cis-vaccenic acid i n the membrane phospholipids increases. As described elsewhere (Melchior, this volume), similar behavior has been observed in virtually all organisms examined. It seems clear that this regulatory mechanism functions to lower the temperature at which the order-disorder lipidphase transition occurs, and thus optimizes membrane function at the lower temperature (Cronan and Gelmann, 1975; Melchior and McElhaney, this volume). The molecular mechanism of temperature regulation has been studied in a number of organisms, but only in E . c d i is the proposed mechanism firmly based on genetic and biochemical evidence. The temperature control mechanism of E . c d i is not an inducible process, as shown by two lines of evidence (Garwin and Cronan, 1980). First, temperature regulation is evident and virtually complete within 30 seconds following shift of an E . roli culture from 42 to 24°C. This time course is much too rapid for the transcription and translation needed for enzyme induction. In the same experiments, 5 minutes were required for induction of P-galactosidase. A second line of evidence is that temperature control is not affected by inhibitors of RNA and protein synthesis under conditions where macromolecular synthesis is almost completely (> 95%) inhibited (Garwin and Cronan, 1980). Since temperature control does not require new macromolecular synthesis, it follows that the temperature control mechanism is the property of an enzyme(s) present at all temperatures. In principle, temperature regulation could be exerted at the level of fatty acid synthesis, or the level of fatty acid incorporation into phospholipid, or both. A control mechanism acting solely at the level of fatty acid synthesis would function by temperature control of the level of cis-vaccenic acid synthesis. Although E . roli has two unsaturated fatty acids, palmitoleic acid and cis-vaccenic acid, only the level of the latter acid fluctuates with growth temperature. The amount of palmitoleic acid is invariant. Pulse-labeling experiments from several laboratories have demonstrated that decreased temperature results in an increased synthesis of cis-vaccenic acid synthesis rather than a decrease in the synthesis of saturated fatty acids (Cronan, 1975; Nishihara trt u l . , 1976; Okuyama et a l . , 1977). Temperature control of an enzyme specifically involved in cis-vaccenic acid synthesis could therefore result in the regulation of phospholipid fatty acid composition. The other distinct possibility is that temperature control is only exerted at the level of incorporation of fatty acids into phospholipid. In this hypothesis the same ratio of unsaturated to saturated fatty acids would be synthesized at all temperatures, and temperature control would be exerted by acyltransferase through transfer of the mixture of fatty acid species appropriate to the temperature. To decide between control exerted at the level of fatty acid synthesis and/or at that of phospholipid synthesis, Cronan (1975) examined the ratio of saturated to

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213

unsaturated free fatty acids accumulated in cultures of E . coli in which acyl transfer was blocked. Acyl transfer was blocked by starvation of glycerol 3-phosphate auxotrophs (either g p 4 or p/sB) for glycerol 3-phosphate (Cronan er al., 1975). Starvation of these auxotrophs results in the accumulation of free fatty acids dueto the inhibition of acyl transfer caused by the lack of acyl acceptor. These free acids are the product of rle n o w synthesis (Cronan et u / . , 1975), and thus if fatty acid synthesis per se is under temperature control, the ratio of unsaturated to saturated fatty acids should be increased as the temperature of incubation is decreased. The results of these experiments are clear; the relative amount of unsaturated free fatty acid increases 3-fold upon a decrease in incubation temperature of 42 to 15°C. These results therefore argue strongly that a major site of temperature control is at the level of fatty acid synthesis. It is possible that a second site of control is also present at the level of incorporation of fatty acids into phospholipid. This question has been approached by t w o types of experiment: ( I ) supplementation of cultures of E . c o l i with mixtures of radioactive saturated and unsaturated fatty acids (Sinensky, 197 I ; Cronan, 1975); and ( 2 ) in vitro studies on the acyltransferases (Nishihara et a / . , 1976; Kit0 et d., 1978; Okuyama et al., 1977). The former type of experiment is the stronger. Sinensky (1971) reported that cultures of E . coli supplemented with an equimolar mixture of palmitic and oleic acids incorporated these acids into the cellular phospholipids in a temperature-dependent manner. For a number of technical reasons, the interpretation of these results was not straightforward, but a later experiment (Cronan, 1975) eliminated the possibility that differences in fatty acid uptake and/or activation of the CoA esters were differentially temperature sensitive. However, the interpretation of both experiments suffers from lack of knowledge as to how exogenous fatty acids are incorporated into phospholipids (see earlier discussion). It is not clear that the incorporation of intracellularly transported exogenous acids proceeds by the same mechanism as that of endogenously synthesized acids (Ray and Cronan, 1976b), and thus interpretations based on this assumption are weak. The in vitro experiments (Nishihara et ul., 1976; Kit0 et al., 1978; Okuyama et al., 1977). in which the specificity of the acyltransferase for saturated versus unsaturated acyl donors were assayed at various temperatures, are also equivocal. These studies have been done with acyl-CoA substrates that are probably not the physiological donors (Rock et d., 1981a). Also, the possible effect of temperature on the rnicellar behavior of the CoA substrates was not measured. A third problem is that at least two acyltransferase activities are present in the membrane fractions assayed and this could greatly complicate interpretation. For these reasons, we do not believe there is any compelling evidence for temperature control at the level of acyl transfer of endogenously synthesized fatty acids. However, such a mechanism cannot be rigorously excluded. It seems that if control at the level of acyl transfer of endogenous acids does

21 4

CHARLES 0 . ROCK AND JOHN E. CRONAN, JR.

exist, it is secondary both temporally and quantitatively to the regulatory mechanism acting at the level of fatty acid synthesis. This conclusion is based on the finding that the altered ratios observed in the free fatty acid experiments described previously are quantitatively similar to those observed for normal cells at a given temperature (Cronan, 1975). Temperature control of fatty acid synthesis in E . coli can be simply restated as the problem of temperature control of the elongation of palmitoleic acid to cis-vaccenic acid. This statement comes from the following findings: (1) cisvaccenic acid (but not palmitoleic acid) increases with decreased temperature; (2) temperature control is exerted by increasing the relative rate of cis-vaccenic acid synthesis rather than by decreasing palmitic acid synthesis; and (3) a mutant of E . coli (formerly called Cvc, now calledJabF) has been isolated that is defective in temperature control and is deficient in the elongation of palmitoleic and to cis-vaccenic acid in vivo (Gelmann and Cronan, 1972). The ,fLbF mutant was isolated as a putative suppressor of a mutant VubA) deficient in unsaturated fatty acid synthesis (Gelmann and Cronan, 1972). This strain is severely deficient in cis-vaccenic acid synthesis both in vivo and in a cell-free fatty acid synthetase system. The deficiency in elongation of palmitoleic acid to cis-vaccenic acid it1 vivo results in a somewhat increased amount of palmitoleic acid in the membrane phospholipids (Gelmann and Cronan, 1972). When isolated, candidates for the deficient enzyme in this mutant were nil, since the same enzymes were thought to synthesize both cis-vaccenic acid and palmitoleic acid (Cronan and Vagelos, 1972). However, D'Agnolo et d.,(1975) subsequently showed that one of the enzyme activities of fatty acid synthesis exists in two forms. The enzymatic activity is that of P-ketoacyl-ACP synthase, the activity that catalyzes the condensation of malonyl-ACP with the growing fatty acyl chain. The reaction is as foIlows: 0

11 R-C-S-ACP

0

I1

0

/I

+ IIOC--CH,--C-S-ACP

0

0

!I I1 RC-CH2-C-SACP

-k

C 0 2 + ACP-SH

D'Agnolo et ul., (1975) showed that two forms of this enzyme exist in E . coli. P-ketoacyl-ACP synthases I and 11 differ in their elution from hydroxylapatite, pH optima, heat stability, and substrate specificity. These workers had also shown that the fubB class of E . coli unsaturated fatty acid auxotrophs is deficient in synthase I. Since the fuhF mutants are also altered in unsaturated fatty acid synthesis, it seemed likely that these mutants might lack /3-ketoacyl-ACP synthase 11. Garwin and co-workers ( 1 980a) have recently demonstrated that fubF mutants do lack synthase 11. They have further demonstrated that the fabB locus is the structural gene for synthase I and that fahB fubF double mutants lack all fatty

BACTERIAL MEMBRANE LIPID SYNTHESIS REGULATION

21 5

acid synthetic ability. This latter finding argues strongly that synthases I and 11 are the only E . coli enzymes able to catalyze the elongation reaction of fatty acid synthesis (Garwin er u l . , 1980a). This finding has permitted a detailed genetic analysis of the fuhF gene that indicates that the same lesion is responsible for P-ketoacyl-ACP synthase I1 deficiency, the cis-vaccenic acid deficiency, and the loss of temperature control. It therefore follows that synthase I1 plays a major role in the mechanism of temperature regulation. How does synthase 11 regulate the fatty acid composition of the membrane phospholipids? The finding (Garwin and Cronan, 1980) that adjustment of the phospholipid composition to a new temperature does not require transcription or translation indicates that the activity (rather than the amount) of synthase I1 is altered by temperature. The simplest hypothesis is that P-ketoacyl-ACP synthase I1 is an enzyme that functions better than the other fatty acid-biosynthetic enzymes at low temperature, but functions relatively poorly at elevated temperatures. This thermolability of synthase 11 could be an inherent property of the enzyme or a property bestowed by a temperature-sensitive modification (e.g., phosphorylation, adenylation, methylation) enzyme. The possibility of enzyme modification raises the question of the relationship between 6-ketoacyl-ACP synthases I and 11. All of the differing properties originally reported by D’Agnolo and co-workers (1975) are equally compatible with synthases I and I1 being coded by two unrelated genes or with synthase I1 being a modified form of synthase 1. This latter hypothesis was tenable because the differing propertics of synthases I and I1 reported by D’Agnolo c’t ul. ( 1 975) (pH optima, heat stability, elution from hydroxylapatite, substrate specificity) are those often found to occur between the modified and unmodified forms of enzymes. The molecular weight difference reported was small (< 15%) and was based only on gel filtration, a technique sensitive to the conformational changes that often accompany modification. A definitive study of the relationship between P-ketoacyl-ACP synthases I and I1 of E . roli has recently been reported by Garwin er ul. (1980b). These workers demonstrated that synthases I and I1 have distinctly different molecular weights, antigenic sites, peptide maps, amino acid compositions, and substrate specificities. Synthase 1 has a molecular weight by sedimentation equilibrium of 80,000 and is composed of two similarly sized subunits. The subunits of synthase I are identical (Prescott and Vagelos, 1970). Synthase I1 is a protein of molecular weight 85,000 and is likewise composed of two similarly sized, probably identical subunits. Peptide maps of the two proteins (generated by three proteases of differing specificity and CNBr) demonstrated that synthases I and I1 share few, if any, common peptides. Synthases 1 and I1 are also unrelated by immunological criteria. The characterization of these proteins demonstrates that synthase I1 is not a modified form of synthase I, and thus the properties of synthase I1 should reflect its proposed role in temperature regulation.

21 6

CHARLES 0. ROCK AND JOHN E. CRONAN. JR.

The physiological and genetic evidence (Gelmann and Cronan, 1972; Garwin ct al., 1980a) dictates that ( I ) P-ketoacyl-ACP synthase I1 should catalyze the elongation of palmitoleoyl-ACP much more effectively than does synthase I; and (2) this difference should be accentuated at lower assay temperatures. Both of these predictions have been borne out (Garwin et ul., 1980b). At 37”C, synthase I1 elongates palmitoleoyl-ACP at a rate 12-fold greater than that given by synthase I. This differential is increased to over 30-fold at 27°C and at this temperature, the Michaelis constant of synthase I1 for palmitoleoyl-ACP is 2-fold lower than at 37°C. Palmitoleoyl-ACP was the only substrate tested for which synthases I and I1 have different activities (Garwin et ul., 1980b). In conclusion, the primary regulatory site in temperature control of membrane-phospholipid synthesis in E . coli is P-ketoacyl-ACP synthase 11. Synthase I1 functions much better than the other condensing enzyme, synthase I, in the elongation of palmitoleic acid to cis-vaccenic acid. The cis-vaccenic acid is then incorporated into both positions 1 and 2 of the phospholipid sn-glycerol 3-phosphate backbone (Cronan, 1978), resulting in diunsaturated phospholipid molecules (Kito et al., 1978) that lower the lipid-phase transition (Cronan and Gelmann, 1975). It should be noted that the amount of palmitoleic acid cannot be greatly increased in E . t d i , since this acid can be incorporated only into position 2 of the phospholipids (Cronan and Vagelos, 1972; Baldassare et al., 1976), and thus only saturated:unsaturated phospholipid species can be synthesized. h. Control (it Constant Growth Temprratures (Isothermal Control). Our knowledge of the mechanisms(s) whereby E . coli regulates its phospholipid acyl-group composition at a given growth temperature, “isothermal control, ” is incomplete. As discussed above (Section 11, A, l ) , some control is exerted at the level of the acyltransferase, but acylation specificity is not absolute in vivo, and thus a major site of control must be at the level of fatty acid synthesis. The mechanism of isothermal control has not yet been determined because it proved necessary that the mechanism of temperature control must first be understood. Since temperature control can now be eliminated by the &bF mutation, some recent progress on the isothermal-control mechanism has been made. The enzyme, P-hydroxydecanoyl thioester dehydrase, is responsible for the introduction of the double bond into the unsaturated fatty acids of E. coli (Bloch, 1971). The enzyme converts P-hydroxydecanoyl thioester to a mixture of cis-3decanoyl and trans-2-decanoyl thioesters. The properties of this enzyme led Broch and co-workers (Brock et al., 1967) to propose that the ratio of the cis-3 to the tr-uns-2-decenoates produced by the enzyme directly determined the ratio of unsaturated to saturated acyl groups synthesized by E . coli. After reduction to decanoyl-ACP, the rruns-2-decenoyl-ACP would be elongated to a saturated fatty acid, whereas several cycles of elongation of the cis-3-decenoyl-ACP would result in the unsaturated fatty acids of E . coli. This proposal was weakened by the isolation of mutants CfabA ) lacking the P-hydroxydecanoyl

BACTERIAL MEMBRANE LIPID SYNTHESIS REGULATION

21 7

thioester dehydrase (Silbert and Vagelos, 1967). These mutants had genetic lesions in the structural gene for the dehydrase (Cronan and Gelmann, 1973), but required only an unsaturated fatty acid for growth; saturated fatty acid synthesis was not defective. Similar observations were made using an irreversible specific inhibitor analogous to the cis-3-decenoate substrate (Kass, 1968; Kass and Bloch, 1967), and a second E . c d i enzyme that dehydrated P-hydroxydecanoyl-ACP to tr~~tz.s-2-decanoyl-ACPwas discovered (Birge c’t a / ., 1967; Birge and Vagelos, 1972). These results, coupled with mechanistic studies on the dehydrase (Rando and Bloch, 1968) indicating that trans-2-decanoate is an intermediate in the formation of ci.s-3-decenoate, suggested that the dehydrase did not produce free frutiJ -2-decenoyl-ACP i r i vivo. P-Hydroxydecanoyl thioester dehydrase was therefore supposed to play no role in saturated fatty acid biosynthesis. With this background, the results of Clark, Polacco, and Cronan (unpublished results) were unexpected. These workers have produced strains of E . cwli having greatly elevated levels of the dehydrase. These strains are (1) a putative promoter mutant that produces 10- to 12-fold more dehydrase than normal, and (2) an in vitro cloned fabA gene that overproduces the dehydrase about 5-fold. Surprisingly, both of the strains overproduce saturated rather than unsaturated fatty acids. From these results, it seems clear that overproduction of the dehydrase leads to greater levels of trans-2-decanoyl-ACP. and that the dehydrase probably produces free trans-2-decenoyl-ACP in wild-type strains. These findings indicate that the dehydrase reversibly interconverts the ACP thioesters of P-hydroxydecenoate, cis-3-decenoate, and trans-2-decenoate in vivo, and thus the levels of the other enzymes that react with these intermediates must be important in isothermal control. P-hydroxydecanoyl-ACP can be converted to the trans-2-derivative by the “other” dehydrase (Birge et a / . , 1967; Birge and Vagelos, 1972), whereas the rruns-2-decanoyl-ACP can be reduced by enoyl-ACP reductase (Prescott and Vagelos, I972), and the cis-3-decenoyl-ACP elongated by P-ketoacyl-ACP synthase I (D’Agnolo et a / . , 1975). The ratios of the P-hydroxydecanoyl thioester dehydrase to these other enzymes thus may play a key role in the regulation of saturated versus unsaturated fatty acid synthesis. Clark, Polacco, and Cronan (unpublished observations) have strengthened this suggestion by varying the levels of bothP-ketoacyl-ACP synthase I andp-hydroxydecanoyl thioester dehydrase in the same bacteria. Preliminary results indicate that a decrease in synthase I activity coupled with an increase in the dehydrase activity has a greater effect on the saturated:unsaturated ratio than the sum of the two manipulations each done in a separate bacterial strain. This synergistic effect coupled with the previous results of Cronan ( 1 974), who showed that a 2-fold overproduction of the dehydrase produced slightly more unsaturated fatty acids, indicate that it is not the dehydrase level per se that determines isothermal control, but rather the ratios of the dehydrase to other enzymes acting on the dehydrase substrates (Bloch,

21 8

CHARLES 0.ROCK AND JOHN E. CRONAN, JR.

197 1; Kass and Bloch, 1967). It is tempting to propose physical interactions between the dehydrase and the other enzymes, but no data to support this notion are available. It should be noted that the presence or absence of P-ketoacyl-ACP synthase I1 has no effect on isothermal control (Clark er al., 1981), except that the unsaturates consist only of palmitoleate (Gelmann and Cronan, 1972). 3 . REGULATIONO F F A I IY ACIDC H A I NLENGTH Palmitate, palmitoleate, and cis-vaccenate comprise the bulk of the fatty acids found in E . coli membrane phospholipids (Cronan and Vagelos, 1972). One likely candidate for the regulation of fatty acid chain length is at the level of the P-ketoacyl-ACP synthase reaction that catalyzes the condensation of malonylACP with acyl-ACP, thus initiating a new round of elongation (see previous discussion). Garwin et u1. (1980a) have used native acyl-ACP substrates to assay the activity of purified P-ketoacyl-ACP synthases I and I1 in v i m . These workers found that palmitoleoyl-ACP is a much better substrate for synthase I1 than for synthase I. The conclusion from these data that P-ketoacyl synthase 11 is primarily responsible for the elongation of palmitoleate to cis-vaccenate in vivo is strongly supported by the finding that cells deficient in the synthesis of cisvaccenate lacked measurable levels of P-ketoacyl-ACP synthase I1 (Garwin et u / , , 1980b). Garwin er a l . (1980a) have also reported that palmitoyl-ACP and cis-vaccenoyl-ACP are extremely poor substrates for both synthase I and I1 in keeping with the role of these enzymes in regulating fatty acid chain length. Although the P-ketoacyl-ACP synthases may play a significant role in the regulation of fatty acid chain length, Cronan cr a / . (1975) have provided evidence that other factors are involved in vivo. These workers report that when phospholipid synthesis is arrested at the acyltransferase level (glycerol starvation of a plsB strain) in strains unable to degrade fatty acids, free fatty acids accumulate in the cell. Most significantly, these fatty acids were found to be abnormally long as compared to the normal chain lengths found in E . coli (Cronan et a / . , 1975). Therefore, these workers concluded that fatty acid synthesis continues in the absence of phospholipid synthesis and that acyl transfer is an important determinant in the regulation of fatty acid chain length. These conclusions were questioned by N u n n and co-workers (1977), who found in similar experiments that the accumulation of free fatty acids observed by Cronan P r u / . (1975) was due to the increased specific activity of the intracellular acetate pool i n glycerolstarved cultures. Taking this effect into account, Nunn et ( I / . (1977) conclude that fatty acid synthesis is indeed arrested in the absence of acyltransferase activity. However, this result does not explain why the small amount of free fatty acids appearing in glycerol-starved cultures have abnormally long chain lengths. Additional in v i v o experiments are needed to define more precisely the role of the two P-ketoacyl-ACP synthases, acyltransferase, and perhaps other factors in the

BACTERIAL MEMBRANE LIPID SYNTHESIS REGULATION

21 9

regulation of fatty acid chain length. The most helpful experiments in this regard would seem to be the examination of the ACP-pool composition in the absence of acyl transfer activity, and the application of DNA cloning procedures to determine the effect of increased acyltransferase gene dosage on fatty acid composition.

4. CONVERSION OF UNSATURATED ACIDS'TO T H E I R CYCLOPROPAN E DERIVATIVES Most bacteria, including E . coli, convert a major fraction of the unsaturated moieties of their membrane phospholipids to cyclopropane derivatives (Christie, 1970; Goldfine, 1972; Goldfine, this volume). This conversion occurs predominantly as bacterial cultures enter stationary phase, and it can be considered an in situ modification of the phospholipid bilayer. Only one group of bacteria, the Rhodopseudomonadacae, that possesses a suitable unsaturated fatty acid content fails to cyclopropanate their lipids. The most interesting questions concerning the synthesis of cyclopropane fatty acids (CFA) are ( I ) why are these acids formed, and ( 2 ) how are the double bonds of the phospholipids of the membrane hilayer made accessible to the enzyme-catalyzing methylenation?

a . Function qf Cyclopropane Fatty Acids. The function of CFA is unknown. Despite their prevalence in bacteria, no definitive reason for the conversion of phospholipid unsaturated moieties to cyclopropane derivatives is apparent. The cyclopropane rings formed are cis-cyclopropanes (Cronan ef al., 1974), and the acyl-chain bond angles and physical properties of phospholipids containing these acids are very similar to those of phospholipids containing the homologous unsaturated fatty acid (Cronan ef al., 1974; Silvius and McElhaney, 1979; McElhaney , this volume). Law and co-workers ( 1963) proposed that conversion of double bond to cyclopropane rings would protect the lipids from oxidation. However, monounsaturated acids are difficult to oxidize, cyclopropane rings can be oxidized about as readily, and CFA are formed only under reducing rather than oxidizing conditions. Cronan (1 968) proposed that cyclopropane formation might protect phospholipids from hydrolytic turnover in vivo, but later direct experiments (Cronan et al., 1974) preclude this explanation. The most definite demonstration of the enigmatic nature of CFA is the isolation of mutants of E . roli containing < 0.5% of the normal CFA content by Taylor and Cronan ( 1976). These workers sought temperature-sensitive mutants by an [3 H-methyl lmethionine suicide procedure, but the mutants isolated grow and survive normally under all conditions tested-including stationary phase under extremely aerobic and completely anaerobic conditions in a wide variety of media. This lack of a phenotype for the CFA mutants is puzzling. CFA synthesis

220

CHARLES 0 . ROCK AND JOHN E. CRONAN, JR.

is metabolically expensive, occurs at a time when energy is limited, and has been maintained during bacterial evolution. These circumstantial arguments indicate that CFA should be essential, unfortunately the mutanfs lack a phenotype. The regulation of CFA synthesis in E . coli is as obscure as the function of these acids. Although the bulk of these acids are synthesized only upon entry of the cultures into stationary phase (Law et a l . , 1963; Cronan, 1968), the responsible enzyme-CFA synthase-is present in exponentially growing cultures in levels as high as those found in stationary-phase cells (Cronan, 1968; Taylor and Cronan, 1979). The amount of CFA synthesis is not controlled by either the level of S-adenosyl methionine (SAM), the methyl donor, or the level of unsaturated phospholipids, the methyl acceptor (Cronan et al., 1974). Low 0, tension seems to be the major but not the only factor that triggers CFA synthesis in vivo (Ohlrogge et ul., 1976). A recent finding is that strains that overproduce CFA synthase (due to an in vim-constructed plasmid carrying the structural gene) synthesize CFA throughout log phase (Grogan and Cronan, unpublished observations). The regulatory mechanism that shuts down CFA synthesis in log phase can therefore be overcome by overproduction of CFA synthase. CFA synthase was discovered by Law and co-workers (Chung and Law, 1964; Zalkin et al., 1963), who partially purified the enzyme from Clostridiurn hutryicwin (Chung and Law, 1964) and also studied extracts of Serrutia marcescens (Zalkin er d., 1963). These workers showed that this enzyme used SAM as the methylene donor to form CFA from the unsaturated fatty acid moieties of phospholipids dispersed in a bilayer. Only phospholipid substrates were functional; fatty acids, diglycerides, CoA esters, and so on, were inactive (Thomas and Law, 1966). The clostridial enzyme was active on ether- as well as ester-linked unsaturated chains (Chung and Goldfine, 1965), thus precluding a mechanism requiring hydrolysis and reformation of the ester bond. It should be noted that the mechanism proposed by Law and co-workers (Law, 1971) from in vitro experiments has been fully supported by it7 vilw experiments. Phospholipids synthesized during early log phase are fully converted in stationary phase (Cronan et a l . , 1974). Since redistribution of phospholipid acyl chains does not occur in E . c d i (Cronan and Prestegard, 1977). and CFA synthesis does not require concomitant unsaturated fatty acid synthesis (Cronan et a l . , 1974), the intact phospholipids of the membrane lipid bilayer must be the methyl acceptor in vivo. The it? vivo methylene donor is SAM, as the SAMase of phage T3 immediately halts CFA synthesis in vivm (Cronan er a l . , 1979). h. Cyclopropune FNttj Acid Synthase. Law and co-workers expended several years of work attempting to purify the CFA synthase of C. butyricum (Chung and Law, 1964). However, the enzyme was very labile and this lability precluded purification. Similar stability problems plagued attempts to purify the enzyme from E . coli until Taylor and Cronan (1979) realized that this enzyme is unstable in the absence of phospholipid. This stabilizing property of phos-

BACTERIAL MEMBRANE LIPID SYNTHESIS REGULATION

221

pholipid explained the difficulties encountered in recovering activity after protein fractionation, in that protejn-fractionation schemes readily separate protein and lipids. In the absence of phospholipid, the CFA synthase of E . coli has a half-life of < 12 hours at 4°C. The requirement for lipid precluded the use of conventional protein fractionation methods, but the enzyme could be purified over 500-fold by flotation with phospholipid vesicles (Taylor and Cronan, 1979; Taylor et ul., I98 I ) . Recently. Grogan and Cronan (unpublished observations) have succeeded in cloning CFA synthase by in vitro techniques and have obtained a 12- to 15-fold overproduction of this enzyme. CFA synthase seems to be a protein having a molecular weight of about 90,000 that is loosely bound to the inner membrane of E . cnli (Taylor and Cronan, 1979). It is one of few (and the only discrete) enzymes known to act on the nonpolar portion of phospholipids dispersed in a vesicle. The double bond of the substrate phospholipid molecule must be 9 to 1 1 carbon atoms removed from the glycerol backbone of the phospholipid molecule (Marinari et u l . , 1974; Ohlrogge et ul., 1976). Therefore, the site of action of the enzyme is well within the hydrophobic region of the phospholipid bilayer. CFA synthase binds only to vesicles made of phospholipids containing either unsaturated or cyclopropane fatty acid moieties (Taylor and Cronan, 1979). Vesicles made of saturated phospholipids neither bind to nor inhibit the reaction of CFA synthase with vesicles of unsaturated phospholipids. CFA synthase is equally active on each of the phospholipids of E . c d i . phosphatidylglycerol, phosphatidylethanolamine, and cardiolipin. The enzyme also has some activity on phosphatidylcholine (Taylor and Cronan, 1979). I n vivo, CFA synthase seems able to react with all the phospholipid molecules of both monolayer leaflets of both (inner and outer) membranes of E . coli, since virtually all the cellular phospholipids become methylenated (Ishinaga et nl., 1979; Nakayama et ul.. 1980). The consideration suggests that CFA synthase may react with both the inner and outer monolayers of synthetic lipid bilayers. Indeed, Taylor and Cronan (1979) have reported that the partially purified enzyme reacts with phospholipids (defined by selective-labeling techniques) to reside on the inner as well as the outer phospholipid monolayer of unilamellar vesicles. Furthermore, the vesicles remained intact, the contents not mixing with the external medium (Taylor and Cronan, 1979). This finding is surprising and suggests that CFA synthase might penetrate the lipid bilayer and/or cause transbilayer movement ( ‘ ‘flip-flop”) of phospholipid molecules. Arguments have been put forth that membrane biogenesis might require specific proteins to facilitate the transbilayer movement of phospholipid molecules (for review, see Rothman and Lenard, 1977). Although these arguments are equivocal (Cronan, 1979), CFA synthase could be such a protein. When homogeneous enzyme is available, further work is indicated to test this hypothesis. If CFA synthase penetrates the bilayer, it might be expected that penetration would be inhibited by

222

CHARLES 0. ROCK AND JOHN

E. CRONAN, JR.

an ordered lamellar phase in the vesicles. However, the enzyme shows no dependence on the amount of ordered lipid i n the substrate vesicle (Taylor and Cronan, 1979). In conclusion, further study of CFA synthase should result in a detailed model of how this novel protein reacts with groups within the hydrophobic region of the lipid bilayer. This interaction should give general information on the mechanism whereby proteins and lipids interact, and may also give information on the topology of lipid synthesis it7 v i v n .

B. Regulation of the Rate of Phospholipid Synthesis 1 . CANDIDATE REGULATORY SITES

The classical rate-limiting step in fatty acid and, hence, phospholipid synthesis, is acetyl-CoA carboxylase, the enzyme catalyzing the first committed step in fatty acid synthesis (Volpe and Vagelos, 1976). Acetyl-CoA carboxylase, the sole biotin-containing protein in E. coli, converts acetyl-CoA to malonyl-CoA. Although, the enzyme from E . coli is well studied (Alberts and Vagelos, 1972), there is no in vivo evidence as to how (or if, this enzyme is regulated. Recent cloning experiments on severaI of the enzymes of phospholipid biosynthesis indicate that these enzymes do not catalyze rate-limiting steps in phospholipid synthesis. For example, cells containing > 10-fold overproduction of the sn-glycerol-3-phosphate acyltransferase (Larson et al., 1980) and the enzymes of phosphatidylethanolamine synthesis (Raetz, 1978) have been constructed, and all these strains fail to increase their rate of phospholipid synthesis (Snider, 1979). Another attractive candidate for a rate-limiting step is the conversion of apoACP to its active form, holoACP, via incorporation of a 4'phosphopantetheine group from CoA. Discrete enzymes catalyze the transfer from CoA to upoACP or the hydrolysis of holoACP to apoACP, and the turnover of the prosthetic group is very rapid (Prescott and Vagelos, 1972). Since holoACP is required for fatty acid synthesis (Prescott and Vagelos, 1972), and acyl-ACP is probably the in vivo substrate for the sn-glycerol-3-phosphate acyltransferase (Section 11, A, I ) , a deficiency in holoACP should slow membrane-phospholipid synthesis. Recently, Polacco and Cronan (198 1) have isolated a mutant having a defective hofoACP synthase. Under nonpermissive conditions, this strain is unable to transfer 4'-phosphopantetheine from CoA to upoACP, whereas the turnover of holoACP to upoACP occurs normally. This strain should allow the testing of the possibility that holoACP synthesis is the rate-limiting step in membrane-phospholipid synthesis.

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2. COORDINATION O F MACROMOLECULAR A N D PHOSPHOLIPID S Y N IHESIS

The exponential phase of bacterial growth is typically referred to as “balanced,” since the myriad cellular events are well coordinated. The regulatory mechanisms responsible for coordination have proved difficult to isolate experimentally during balanced growth, but are somewhat easier to analyze when the system is perturbed. Nutritional stress is one such perturbed metabolic system, and considerable data on the regulation of phospholipid synthesis during this physiological response is available. The stringent response results when the availability of any amino acyl-tRNA species becomes limiting, and is characterized by (1) a dramatic reduction in stable RNA accumulation; (2) an increased rdte of protein turnover; (3) heterogeneous effects on the transcription of mRNA species; (4) reduced rate of translation; and ( 5 ) a 3- to 4-fold decrease in the lipid biosynthetic rate (Gallant, 1979). A single-site mutation, r r l A , abolishes this entire set of adjustments, conferring a phenotype that is termed relaxed. These regulatory effects are mediated by a family of novel nucleotides, most notably guanosine 5’-diphosphate-3’-diphosphate(ppcpp), that accumulate in r e / + but not r r l A strains (Gallant, 1979). Several laboratories have reported that the rate of phospholipid synthesis decreases following amino acid starvation in w / + but not relA strains (Sokawa ef ul., 1968; Golden and Powell, 1972; Merlie and Pizer, 1973; Polakis el al., 1973; Nunn and Cronan, 1974, 1976a; Spencer et u / . , 1977; Snider, 1979). The major nucleotide that accumulates during the stringent response is ppGpp (Gallant, 1979), and it seems likely to be the effector of rrlA gene control of lipid synthesis. Strains harboring the genetic lesion, .~pnT(Laffler and Gallant, 1974), are defective in ppGpp turnover and possess higher basal levels of ppGpp during balanced growth. In ~ p o Tstrains, ppGpp reaches a higher level during the stringent response and persists for a longer period of time following the readdition of amino acids. Nunn and Cronan (1976b) have utilized various spoT strains and growth conditions to vary the intracellular content of ppGpp, and found a quantitative correlation between the decreased rate of phospholipid synthesis and ppGpp concentration. Carbon-source shiftdown and temperature upshift also result in an intracellular increase in ppGpp, but not pppGpp (Gallant er a / ., 1977; Chaloner-Larsson and Yamazaki, 1977; Kainuma-Kuroda et a / ., 1980). The accumulation of ppGpp during these metabolic adjustments occurs independently from the rclA gene (Friesen et a / ., 1978) and appears to be under the control of the genetic locus r e l X , whose gene product has not yet been identified (Pao and Gallant, 1978). Kainuma-Kuroda el ul. (1980) have recently shown that phospholipid synthesis is arrested concurrent with the appearance of ppGpp in both r e / + and relA strains subjected to temperature upshift and carbon-source

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shiftdown. Phospholipid synthesis also is relieved of relA gene control in relB mutants, strains that are defective in ppGpp accumulation after amino acid starvation due to an alteration in the protein synthesis machinery (Mosteller, 1978). These results are important in that they demonstrate that phospholipid synthesis is affected by the presence of ppGpp, rather than another effect due to the relA locus. The recent objections to the proposed role of ppGpp in lipid metabolism put forth by Raetz ( 1 978) are laid to rest by these experiments. These data provide compelling, albeit circumstantial, evidence that ppGpp is the effector of lipid synthesis it1 vivo, and have led several laboratories to examine the effects of ppGpp in vitro. Inhibition of phosphatidylglycerol phosphate synthetase (Merlie and Pizer, 1973) and P-hydroxydecanoyl thioester dehydrase (Stein and Bloch, 1976) by ppGpp has been observed, but these results clearly do not bear on the in vivo situation, since no preferential decrease in the formation of the product of either of these enzymes is observed during the stringent response (Merlie and Pizer, 1973; Spencer et d., 1977; Snider, 1979). Acyltransferase is also inhibited by ppGpp (Ray and Cronan, 1975; Lueking and Goldfine, 1975; Merlie and Pizer, 1973). In contrast to the in vivo situation, the inhibition of acy ltransferase is irreversible in vitro, and acyl transfer from acylCoA is affected whereas acyl transfer from the in v i i ~acyl ~ donor, acyl-ACP, is unaffected (Ray and Cronan, 1975; Lueking and Goldfine, 1975). Polakis et u l . ( I 973) reported ppGpp to be a reversible inhibitor of the carboxyltransferase component of acyl-CoA carboxylase. Reversibility of the effect was not tested, and the maximal inhibition of this enzyme was 50% in contrast to the i n vivo results that show inhibition of the target enzyme should be almost complete at high ppGpp concentrations (Nunn and Cronan, 1976b). Cronan (1978) has suggested that ppGpp may inhibit CoA-utilizing enzymes by occupation of their CoA-binding sites similar to the inhibition of these enzymes by pAp. To date, pGp and other analogous nucleotides have not been tested. Although in vitro experiments will be needed to explore the mechanisms of stringent control, ambiguities in the data dictate that the available results should be interpreted with caution and point to the need for more discerning in ~iivoexperiments to decide the issue. The problem of "artifacts" has also plagued the interpretation of in i'itro results on the effect of ppGpp on stable RNA synthesis (Gallant, 1979). The relevance of a new nucleotide ppGp (Pa0 and Gallant, 1979) also remains to be ascertained. What then can be concluded from available iri vivo experiments about the target enzyme? Most investigators agree that the target site is at or before the acyltransferase step (Cronan, 1978), since an effect is seen on 32Pincorporation, and the accumulation of membrane-bound intermediates was not found (Snider, 1979). Stringent control of fatty acid synthesis has been directly observed (Nunn and Cronan, 1976a; Spencer cJta l . , 1977). and the syntheses of saturated, unsaturated, and P-hydroxy fatty acids were affected to the same extent. Nunn and

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Cronan (1974) have examined stringent control in strains that require exogenous fatty acids for phospholipid synthesis and growth. Incorporation of exogenous fatty acids into phospholipid was inhibited by amino acid starvation of r e / + but not relA strains, and they conclude that regulation may also occur at the acyltransferase step. However, these experiments suffer from the caveat that the pathway for incorporation of exogenous fatty acid is unknown and may or may not proceed via an ACP thioester intermediate. These results serve to narrow the choices somewhat, but several candidate enzymes still exist. I n summary, the role of ppGpp in the regulation of lipid synthesis would appear to be well established; however, the identity of the enzyme(s) affected is still unproven. To resolve the issue, additional iri iiiw experiments are required to identify the target enzyme, followed by the in iitro analysis of ppGpp effects on the kinetics of the target enzyme(s). 1s macromolecular synthesis arrested when phospholipid synthesis is inhibited? Strains harboring the p l s B or gpsA require exogenous glycerol or glycerol-P for growth, and when they are deprived of the supplement, phospholipid synthesis and accumulation cease immediately (Bell, 1974; McIntyre and Bell, 1975; McIntyre et d., 1977). Macromolecular synthesis does not cease immediately but continues for about one generation, and the membranes of these starved cells have been found to contain considerably more protein than normal (Mclntyre and Bell, 1975; Mclntyre et a / . , 1977). Intracellular nucleotide triphosphate levels.reniain high during the period of starvation (McIntyre er a / . , 1977). Thus these investigators have concluded that macromolecular synthesis is not dependent on phospholipid synthesis. However, the strains utilized for these experiments possessed both re/A and spoT alleles, two genes that dramatically effect macromolecular synthesis under other starvation conditions. If re/ is required for cessation of macromolecular synthesis under these conditions as it has been shown to be in amino acid starvation (see previous discussion), these conclusions could be in error. +

3.

I N T E R R E l A l IONSHIPS W I T H

CELLULAK ENERGY METABOLISM

Another mechanism for finely tuning the biosynthetic rate is suggested by a relationship between the acyltransferase and adenylate kinase. A class of mutants, p / s A , were isolated as temperature-sensitive mutants defective in phospholipid synthesis (Cronan c’t ( { I . , 1970). They have been characterized as possessing an abnormally thermolabile acyltransferase compared to their parent strains, both irr viiw and iir vim) (Cronan er N / . , 1970; Cronan and Godson, 1972; Glaser P r “ I . , 1973, 1975). At the nonpermissive temperature, the rate of phospholipid, DNA, R N A , and protein synthesis decreased, as did the ATP concentration (Glaser cr d., 1975). This last effect was found to be due to the inactivation of adenylate kinase (Glaser ef a / . , 1975). Specific inhibition of

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phospholipid synthesis occurred at the lower end of the nonpermissive temperature range, whereas at higher temperatures, ATP synthesis and thus macromolecular synthesis was decreased (Glaser et nl., 1973, 1975; Ray et a / . , 1976). Temperature-sensitive mutants in adenylate kinase (ndk) have been independently isolated (Cousin and Buttin, 1969). A comparison of the properties of p l s A and adk mutants have shown they have the same phenotype (Glaser ~t al., 1975) and fall into the same complementation group (Esmon et al., 1980). These results suggest that the locus adk ( p l s A ) is the structural gene for adenylate kinase. The question remains as to whether adenylate kinase is a regulator of acyltransferase i r i vivo. Glaser et a / . ( 1 975) and Cronan ( 1 978) have proposed that adenylate kinase may be a subunit of the acyltransferase. To test this hypothesis, Goelz and Cronan (1982a) have recently purified the adenylate kinase of E . roli to homogeneity. This protein exists in soluble form as a monomer having a molecular weight of 27,500, although antigens that crossreact with anti-adenylate kinase antibody are also found in purified E . coli inner membranes (Goelz and Cronan, 1982a). These workers (Goelz and Cronan, 1982b) have also shown that antibodies raised against homogeneous adenylate kinase can inactivate the membrane-bound acyltransferase. Furthermore, incubation of E . co/i inner membranes having a temperature-sensitive acyltransferase with homogeneous adenylate kinase from a normal strain relieves the thermolability of the acyltransferase. These experiments are strong (albeit less than conclusive) evidence for a functional interaction between the acyltransferase and adenylate kinase. It is also clear that the abnormal temperature sensitivity does not require that the adenylate kinase also be temperature sensitive. Goelz and Cronan (1 982b) have shown that one of the original plsA strains has an adenylate kinase that is temperature stable both iri vitro and iri rsivo (as reflected in the ATP pools). The adenylate kinase of this mutant is altered, as shown by its aberrant behavior upon purification, and thus an altered but not temperature-sensitive adenylate kinase is sufficient to cause the plsA phenotype. The acyltransferase-adenylate kinase interaction model has attractive features other than providing an explanation for the plsA phenotype. The model also provides (1) a possible mechanism to attune the rate of phospholipid synthesis to the cellular energy charge and (2) an explanation for the stimulation of acyltransferase activity by ATP (Merlie and Pizer, 1973; Rock et u l . , 1981c). The recent advance in acyltransferase purification provided by the cloning experiments discussed above should soon provide the means for a definitive test of the model.

111.

CONCLUSIONS

Although our knowledge of the mechanisms regulating bacterial membranelipid biosynthesis is fragmentary, the pace of recent progress is heartening.

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Recent advances in genetic cloning procedures have begun to have a profound effect. This approach, coupled with other genetic procedures and newly developed biochemical technology, should enable the field to move forward rapidly. A good illustration of this promise is the major progress on the snglycerol-3-phosphate acyltransferase (Section 11, A, 1) permitted by genetic cloning and by synthesis of native acyl-ACP substrates. A similar combination of genetic and biochemical approaches has led to a detailed knowledge of the mechanism of temperature control (Section 11, A, 2 , a ) . P-Ketoacyl-ACP synthase I1 is required for temperature control. This enzyme is not involved in isothermal control. P-Ketoacyl-ACP synthase I1 therefore seems tailored for its role in temperature regulation, and temperature control seems to be superimposed on the normal (isothermal) regulatory mechanism. Teleologically, it appears that E . coli developed temperature control by simply evolving a temperature-sensitive isozyme to catalyze the synthesis of the required unsaturated fatty acid. Although simple regulatory mechanisms such as that of temperature control may probably explain much of the phenomenology of the regulation of bacterial lipid synthesis, the regulation of phospholipid synthesis by the relA gene may be an exception. The unreliable nature of results obtained in v i m means that more sophisticated approaches must be developed to ascertain the physiological relevance of ppGpp inhibition of a given enzyme. It seems clear that mutants in which lipid synthesis has become refractory to elevated ppGpp concentrations must be isolated. The target enzymes in these mutants should be resistant to ppGpp inhibition in virro, thereby providing a criterion for in vivo relevance. Since relA gene control seems to be exerted at least at two sites, selection of these mutants will not be straightforward but should be possible. ACKNOWLEDGMENTS Unpublished observations from our laboratories were supported by the National Institutes of Health (grant GM 28035 and GM 29053 to C.O.R., grants A1 1.56.50and GM 26156 to J.E.C.). the National Science Foundation (grant 79-25689 to J.E.C.). National Cancer Institute Cancer Center [(CORE) grant CA 217651. and ALSAC to C.O.R. REFERENCES Alberts, A , . and Vagelos, P. R . (1972). Acyl-CoA carboxylases. /ti “The Enzymes” (P. D. Boyer, ed.), 3rd ed., Vol 6 , pp. 37-82, Academic Press, New York. Baldassare, J . J . , Rhinehan, K . B., and Silbert, D. F. (1976) Modification of membrane lipid: Physical properties in relation to fatty acid structure. Biochemistry 15, 2976-2994. Barnes, E. M., Jr., and Wakil. S . J. (1968). Studies of the mechanism of fatty acid synthesis. XIX. Preparation and general properties of palmitoyl thioesterase. J . B i d . Chrm. 243, 2955-2962. Barnes, E. M., J r . , Swindell, A. C . , and Wakil, S. J . (1970). Purification and properties of a palmitoyl lhioesterdse I1 from Escharichiu coli. J . Biol. Chem. 245, 3122-3128. Bell, R. M. (1974). Mutants of Escherichiu coli defective in membrane phospholipid synthesis: Macromolecular synthesis in an sri-glycerol-3-phosphateacyltransferase K, mutant. J . B u teriol. 117, 1065-1076.

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Bell, R. M., and Cronan, J . E . , Jr. (1975). Mutants of E.rch,richirr c d i defective in membrane phospholipid synthesis: Phenotypic supression of s~i-glycerol-3-phosphate acyltransferase K, mutants by loss of feedback inhibition of the biosynthetic sri-glycerol-3-phosphate dehydrogenase. ./. Biol. C/wrn. 250, 7153-7158. Birge. C. H . . and Vagelos. P. R . (1972). Acyl carrier protein. XVII. Purification and properties of P-hydroxyacyl-acyl carrier protein dehydrase. J . B i d . C h c ~247, . 4930-4938. Birge, C. H . , Silbert, D. F.. and Vagelos, P. R. (1967). A P-hydroxydecanoyl-ACP dehydrase specific for saturated fatty acid biosynthesis in E . coli. B i o c h ~ t t iBiophys. . Res. Comrnroi. 29, 808-8 14. Bloch, K . (1971). P-hydroxythioester dehydrase. / t i “The Enzymes” (P. D. Boyer, ed.). 3rd ed., Vol. 5, pp. 441-464. Academic Press, New York. Bloch, K . , and Vance, D. (1977). Control mechanisms in the synthesis of saturated fatty acids. A i r t ~ i ( RCI.. . B i o c h n . 46, 263-298. Bonner, W . M., and Bloch, K. (1972). Purification and properties of fatty acid thioesterase I from EscAaridiro 1.nli. ./. Biol. C/reni. 242, 3123-3133. Brock. 0. J . H.. Kass, L. R . , and Bloch, K . (1967). P-hydroxydecanoyl thioester dehydrase. 11. Mode of action. J . Biol. Chern. 242, 4432-4440. Chaloncr-Larsson, G.. and Yarnazaki, H. (1977). Adjustment of RNA content during temperature up-shift in E.sf/ferrc.hiuc d i . Biochetn. Ricyh.\”. RES.C‘orrittiuii. 77, 503-508. Christie, W . W . (1970). Cyclopropane and cyclopropane fatty acids. 7 o p . Lipid C’hem. 1, 1-89. Chung, A. E . , and Goldfine, H. (1965) Enzymatic synthesis of cyclopropane fatty aldehydes. Nutrrrc (Loritlorr) 206, 1253-1254. Chung, A . E., and Law, J . H. (1964) Cyclopropane fatty acid synthetase: Partial purification and properties. Bioc.heitii.c/p 3, 967-974. Clark. D.. and Cronan, J . E., Jr. (1981). Bacterial mutants for the study of lipid metabolism. Meth. E r i ~ y t ~ r o72, l . 293-307. Clark, D. P., Ltghtner, V.. Edgar, R . , Modrich, P., Cronan, 1. E., Jr., and Bell, R, M. (1980). Regulation of phospholipid hiosynthesis in /k~Ac~ricliiacolic Cloning of the structural gene for the biosynthetic s,i-glycerol-3-phosphatedehydrogenase. J . Biol. Cherii. 255, 714-717. Cousin, D., and Buttin, G . (1969). Mutants thermoaensibles d’tscheridiiu coli K12. 111. Une mutation letale d’E. coli affectant I’activite de I’adenylate kinase. A i i r i . Insr. Pu.steur, Ptrris 117, 612-630. Cronan, J . E . . Jr. ( 1968). Phospholipid alterations during growth of Esc~herichitrc d i . J . Bw/c,riol. 95, 2054-2061 Cronan, J . E . , J r . (1974). Regulation of the fatty acid composition of the membrane phospholipids of Lschrrichirr colr. Pro(,. Nu//. Acad. Sci. U.S.A. 71, 373-3762, Cronan, J . E., Jr. (1975). Thermal regulation ofthe membrane lipid composition of Eschprichiu coli: Evidence for the direct control of fatty acid synthesis. J . R i d . Chem. 250, 7074-7077. Cronan, J . E . , J r . (1978). Molccular biology of bacterial membrane lipids. Aiirrrr. Rev. Bioc.hem. 47, 163- I 89. Cronan, J . E . , Jr. (1979). Phospholipid synthesis and assembly. / / I “The Bacterial Outer Membrane” (M. Inouye, ed.). pp. 35-65. Wiley, New York. Cronan, J . E . , J r . , and Gelmann, E. P. (1973). An estimate ofthe minimum amount uf unsaturated fatty acid required for growth of Escherrchirr coli. J . B i d C’hcwi. 248, 118-1 195. Cronan, J . E . , J r . , and Gelniann. E. P. (1975). Physical properties of membrane lipids: Biological relevance and regulation. B u c t e r d . Rev. 39, 232-256. Cronan, J . 6 . . Jr.. and Godson, G . N . (1972). Mutants of Escherichiu c d i with temperaturescnsitive lesions in membrane phospholipid synthesis: Genetic analysis of glycerol 3-phosphate acyltransferase mutants. Mol. Gen. Genet. 116, 199-210. Cronan, J . E . , J r . , and Prestegard, J . H . (1977). Difference decoupling nuclear magnetic resonance:

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Goelz. S. E.. and Cronan. J . E.. Jr. (1982b). Adenylate kinase of Eschcric,hiu c,olic Evidence for a functional interaction i n phospholipid synthesis. Biochemistry (in press). Golden, N . G . . and Powell, G. L. (1972). Stringent and relaxed control of phospholipid metdbolism in Escherichiu coli. J . B i d . Chern. 247, 6651-6658. Goldfine. H . (lY72). Comparative aspects of bacterial lipids. A h . Micro. Phy.siol. 8, 1-58. Ishinaga. M., Nishihara, M., and Kito, M. (1976). Purification and positional specificity of .sn-glycerol-3-phosphate acyltransferase from E.wherichiu coli. Biochirn. B i o p h . ~ . Ar,ru 450, 269-272. [shinaga, M.. Kanainoto, R., and Kito, M. (1979). Distribution of phospholipid molecular species in outer and cytoplasmic membranes of Esc.huric.hin d i . J . Biochem. (Tok.yo) 86, 161- 165. Jaworski, J . G . . and Stumpf, P. K. (1974). Fat metabolism in higher plants. Enzymatic preparation of E . coli stearyl-acyl carrier protein. Arch. Biochem. Biophys. 162, 166-173. Kainuma-Kuroda, R.. Goelz, S . , and Cronan, J . E., Jr. (1980). Regulation of membrane phospholipid synthesis in Escherichiu coli during temperature upshift. J . Bacterial. 142, 362-365. Kass. L. R. (1968). The antibacterial activity of 3-decynoyl-N-acetylcysteamine, inhibition in vivo of P-hydroxydecanciyl thioester dehydrase. J . B i d . Chem. 243, 3223-3228. Kass, L. R., and Bloch, K . (1967). On the enzymatic syfithesis of unsaturated fatty acids in Escherichiu c d i . Pro<..Nnfl. Acad. Sci. U.S.A. 58, 1168-1 173. Kito, M., and Pizer, L. I. (1969). Phosphatidic acid synthesis in Escherichia coli. J . Bucferiol. 97, 1321-1327. Kito, M., Ishinaga, M.. and Nishihara, M. (1978). Function of phospholipids on the regulatory properties of solubilized and membrane-bound sn-glycerol-3-phosphate acyltransferase of Escherichiu coli. Biochitn. Biuphys. Actn 529, 237-249. Laffler, T . , and Gallant, J . (lY74). spoT, a new genetic locus involved in the stringent response in E . coli. Cell 1, 27-30. Larson, T. J . , Lightner, V . A., Green, P. R., Modrich, P., and Bell, R. M. (1980). Membrane phospholipid synthesis in Escherichia coli. Identification of the sn-glycerol 3-phosphate acyltransferase as the p l s B gene product. J . B i d . Chern. 255, 9421-9426. Law, J . , Zalkin, H . , and Kaneshiro, T. (1963). Transmethylation reactions in bacterial lipids. Biochitn. Biophjs. Actn 70, 143-151. Law, J . H . (1971). Biosynthesis of cyclopropane rings. Ace. Chem. Res. 4, 199-203. Lightener, V . A., Larson, T. J . . Taileus, P., Kontor, G. D., Raetz, C. R. H., Bell, R. M . . and Modrich, P. (1980). Membrane phospholipid synthesis in Escherichia coli. Cloning of a structural gene ( p l s B ) of the sn-glycerol 3-phosphate acyltransferase. J . B i d . Chem. 285, 94 13-9420. Lueking, D. R., and Goldfine, H. (1975). The involvement of guanosine 3'-diphosphate in the regulation of phospholipid biosynthesis in Escherichia coli: Lack of ppGpp inhibition of acyl transfer from acyl-ACP to sn-glycerol 3-phosphate. J . Biol. Chern. 250, 491 1-4917. McIntyre, T. M.. and Bell, R . M. (197.5). Mutants of Escherichia coli defective in membrane phospholipid biosynthesis. Effect of cessation of net phospholipid synthesis on cytoplasmic and outer membranes. 1.Biol. Chcm. 250, 9053-9059. McIntyre, T. M., Chamberlain. 8. K.. Webster, R. E., and Bell. R . M. (1977). Mutants of Escherichia coli defective in membrane phospholipid biosynthesis. Effects of cessation and reinitiation of phospholipid synthesis, macromolecular synthesis and phospholipid turnover. J . Biol. Chem. 252, 4487-4493. Marinari. L. A., Goldfine. H . , and Panos, C. (1974). Specificity of cyclopropane fatty acid synthesis in E . colic Utilization of isomers of monounsaturated fatty acids. Biochrmisfry 13, 1978- 1983. Marr. A. G . , and Ingraham, J . L. (1962). Effect of temperature on the composition of fatty acids in Escherichiu coli. J . Bacreriol. 84, 1260-1267.

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Merlie. J. P.. and Pizer, L. I . (1973). Regulation of phospholipid synthesis in E d w r i c ~ h i t rc d i by guanosine tetraphosphate. J . B n m r i o l 116, 355- 366. Mosteller, R . D. (1978). Evidence that glucosc starvation mutants are altered in the w l B locus. J . Rucwriol. 133, 1034-1037. Nakayama, H . , Mitsu, T . , Nishihara. M.. and Kito, M. (1980). Relationship between growth temperature of E . c,oli and phase transition temperatures of its cytoplasmic and outer membranes. B i o d i i m . B i o p l i ~ A~ . m 601, 1-10. Nishihara, M . , Iahinaga. M . . Kato, M . , and Kito. M. (1976). Temperature-sensitive forniation of the phospholipid molecular species i n k-sc herichici c,oli membranes. Bioc~hin7.Bioph.v.\. A c m 431, 54-61 Nunn. W . D.. and Cronan, J . E . , Jr. (1974). I-rl gene control of lipid synthesis in Il.vd7eric.hiu c d i . Evidence for eliminating fatty acid syntheais as the sole regulatory site. J . Biol. L‘hrwi. 249, 3994-394rb. Nunn. W . D . , and Cronan, J . E., Jr. (1976a). Evidence lor a direct effect o n fatty acid synthesis in r e l A gene control of membrane phospholipid synthesis. J . M o l . B i d . 102, 167- 172. Nunn. W . D . , and Cronan, J . E . , Jr. (1976b). Regulation of membrane phospholipid synthesis by the r e l A gene: Dependerce on ppGpp levels. B i o c h m i s t r y 15, 2546-2550. Nunn, W . D., Kelly, D. L . , and Stumfall, M. Y. (1977). Regulation of fatty acid synthesis during the cessation of phospholipid synthesis in Eschcvkhiu coli. J . Bac,/Wio/. 132, 526-53 I . Ohlorogge, J . B., Guistone, F. D., Ismail, I . A.. and Lands, W . E. M. (1976). Positional specificity of cyclopropane ring formation from cis-octadecenoic acid isomers in Escherirhirt c d i . Biorhiin. Biophys. Acttr 431, 257-267. Okuyama, H . , and Wakil, S . J . (1973). Positional specificitiea of acyl coenzyme A: Glycerophosphate and acyl coenzyme A: Aminoacylglycerophosphate acyltransferases in Escheric+?ict coli. 1. Biol. Cherii. 248, 5 197-5205 Okuyama, H . , and Yamada, K. (1979). Specificity and selectivity of diacylglyarophosphate synthesis i n Esc.hcv+hiu u d i . Biochiin. Biophys. Actu 573, 207-21 1 . Okuyama, H . , Yamada, K . . Ikezawa, H . , and Wakil, S. J . (1976). Factors affecting the acyl selectivity of acyltransferases in Escherichia coli. J . Biol. Cheni. 251, 2487-2492. Okuyama, H., Yamada, K . , Kameyama, Y . , Ikezawa, H., Akarnatsu, Y., and Nojima, S . (1977). Regulation of membrane lipid synthesis in E.wherkhirr coli after shifts in temperature. Biorhernistn 16, 2668-2673. Overath. P., Pauli, G . , and Schairer, H . U . (1969). Fatty acid degradation in Eschrrichio coli. An inducible acyl-CoA synthethase, the mapping of old mutations and the isolation of regulatory mutanta. Eitr. J . Biochcw 7, 559-574. Pao, C. C . , and Gallant, J . (1978). A gene involved in the metabolic control of ppGpp synthesis. M i d . G e n . Genet.158, 271-277. Pao, C. C., and Gallant. J . (1979). A new nucleotide involved in the stringent response in €.scherichitr c.oli: Guaiiosine-S’-diphosphate-3’-monophosphate. J . B i d . Chem. 254. 688-692. Pauli. G., Ehring, R . , and Overath, P. (1974). Fatty acid degradation in Escherichiu c.oli: Requirement of cyclic adenosine monophosphate receptor protein for enzyme synthesis. J . Bocrrriol. 117, 1178-1183. Polacco, M. L., and Cronan, J . E . , Jr. (1981). A mutant of E.\chrric+~iuc d i conditionally defective in the synthesis of /iolo-acyl carrier protein. J . Biol. C ‘ l i c m . 256, 5750-5754. Polakia, S . E., Guchhait, R. B . , and Lane. M. D. (1973). Stringent control of fatty acid synthesis in E . cnli: Possible regulation of acetyl-CoA carboxylaae by ppGpp. J . B i d . C h w . 248, 79577966. Prescott, D. J . , and Vagelos, P. R. (1970). Acyl carrier protein. XIV. Further studies on P-ketoacyl acyl carrier protein synthatase from E ~ h e r i c h i c ic.oli. J . Biol. Chrm. 245, 5484-5490. Prescott, D. J.. and Vagelos, P. R. (1972). Acyl carrier protein. A d v . Enzymol. 36, 269-31 I .

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Raetz, C. R. H. (1978). Enzymology, genetics. and regulation of membrane phospholipid synthesis in Eschrrichici coli. Mirrohial. Rev. 42, 614-659. Rando, R. R., and Bloch, K . (1968). Mechanism of action of P-hydroxydecanoyl thioester dehydrase. J . B i d . Chem. 243, 5627-5634. Ray, T. K . , and Cronan, J . E.. Jr. (1975). Acylation of sn-glycerol3-phosphate in Escherichia coli: Study of reaction with native palmitoyl-acyl carrier protein. J . Biol. Chern. 250, 8422-8427. Ray, T . K., and Cronan, J . E., Jr. (1976a). Activation of long chain fatty acids with acyl carrier protein: Demonstration of a new enzyme, acyl-acyl carrier protein synthetase in Escherichiu coli. Pror. Nurl. Acad. Sci. U . S . A . 73, 4374-4378. Ray, T. K . , and Cronan, J . E., Jr. (1976b). Mechanism of phospholipid biosynthesis in Escherichia coli: Acyl-CoA synthetase is not required for the incorporation of intracellular free fatty acids into phospholipid. Biuchem. Biuphys. Res. Commuri. 69, 506-513. Ray, T. K . , Cronan, J . E., J r . . Mavis, R. D., and Vagelos, P. R. (1970). The specific acylation of glycerol-3-phosphate to monoacylglycerol-3-phosphate in Escherichia coli. Evidence for a single enzyme conferring this specificity. J . B i d . Chrm. 245, 6442-6448. Ray, T. K . , Cronan, J . E., Jr., and Godson, G. N. (1976). The specific inhibition of phospholipid synthesis in p l s A mutants of Escherichia coli. J . Bacteriol. 125, 136-141. Rock, C. 0.. and Cronan, J . E., Jr. (1979a). Solubilization, purification, and salt activation of acyl-acyl carrier protein synthetase from Escherichiu coli. J . B i d . Chem. 254, 71 16-7122. Rock, C. O., and Cronan, J . E., Jr. (1979b). Reevaluation of the solution structure of acyl carrier protein. J . B i d . Chem. 254, 9778-9785. Rock, C. O., and Cronan, J . E., Jr. (1982). Solution structure of acyl carrier protein. In “Membranes and Transport: A Critical Review” (A. Martonosi, ed.). Plenum, New York (in press). Rock, C. 0.. and Garwin, J . L. (1979). Preparative enzymatic synthesis and hydrophobic chromatography of acyl-acyl carrier protein. J . B i d . Chem. 254, 7123-7128. Rock, C. O., Cronan. J . E., J r . , and Armitage, I . M. (1981a). Molecular properties of acyl carrier protein derivatives. J . Biol. Chrm. 256, 2669-2674. Rock, C. O., Garwin, J . L., and Cronan, J . E., Jr. (1981b). Preparative enzymatic synthesis of acyl-acyl carrier protein. Meth. EnZ.?mO/. 72, 397-403. . synthesis in Escherichia Rock, C. O . , Goelz, S. E., and Cronan, J . E., Jr. ( 1 9 8 1 ~ )Phospholipid colic Characteristics of fatty acid transfer from acyl-acyl carrier protein to sn-glycerol 3-phosphate. J . B i d . Chem. 256, 737-742. Rock, C. O., Goelz, S. E . , and Cronan, J. E . , Jr. (1981d). ATP stimulation of the sn-glycerol 3-phosphate acyltransferase of Escherichiu coli. Arch. Biochem. Biophys. 211, 113-1 18. Rothman, J . E., and Lenard, 1. (1977). Membrane asymmetry. Scienc.u 195, 743-753. Schultz, H . (1975). On the structure-function relationship of acyl carrier protein of Escherichia coli. J . Biol. Chem. 250, 2299-2304. Silbert, D. F. (1970). Arrangement of fatty acyl groups in phosphatidylethanolamine from a fatty acid auxotroph of Escherichiu coli. Biochemistry 9, 363 1-3640. Silbert, D. F. (1975). Genetic modification of membrane lipid. Annu. Rev. Biochern. 44, 315-339. Silbert, D. F., and Vagelos, P. R. (1967). Fatty acid mutant of E . coli lacking a p-hydroxydecanoyl thioester dehydrase. Proc. Narl. Acad. Sci. U . S . A . 58, 1579-1586. Silvius, J . R., and McElhaney, R. N . (1979). Effects of phospholipid acyl chain structure on thermotrophic phase properties. 2. Phosphatidylcholines with unsaturated or cyclopropane acyl chains. Chem. Phys. Lipids 25, 125-134. Sinensky, M. (1971). Temperature control of phospholipid biosynthesis in Escherichia coli. J . Bacreriol. 106, 449-455. Sinensky, M. (1974). Homeoviscous adaption-a homeostatic process that regulates the viscosity of the membrane lipids in Escherichia coli. Proc. Null. Acud. Sci. U . S . A . 71, 522-525.

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Snider, M. D. (1979). Control of membrane lipid synthesis in Ewherichiu coli during growth and during the stringent response. J . Biol. Chem. 254, 7197-7202. Snider, M. D., and Kennedy, E. P. (1977). Partial purification of glycerophosphate acyltransferase from Escherichiu coli. J . Bacteriol. 130, 1072- 1083. Sokawa, Y . , Nakoa, E . , and Kaziro, Y . (1968). On the nature of the control by the RC gene in E . coli: Amino acid-dependent control of lipid synthesis. Biochem. Biophjs. Rrs. Cornniuti. 33, 108-1 12. Spencer, A., Muller, E . , Cronan, J . E., J r . , and Gross, T. A . (1977). r e l A gene control of the synthesis of lipid A fatty acyl moieties. .I. Ructeriol. 130, 114-1 17. Spencer, A . K., Greenspan, A . D., and Cronan, J . E . , Jr. (1978). Thioesterases I and I1 of Escherichiu coli: Hydrolysis of native acyl-acyl carrier protein thioesters. J . B i d . Chrm. 253, 5922 -5926. Stein, J. P., J r . , and Bloch, K . E. (1976). Inhibition of E . r d i P-hydroxydecanoyl thioester dehydrase by ppGpp. Biochem. Biophys. Res. Cornrnim. 73, 881-884. Taylor, F. R., and Cronan, J . E . , Jr. (1976). Selection and properties of mutants of Escherichia coli defective in the synthesis of cyclopropane fatty acids. J . Bacreriol. 125, 518-523. Taylor, F. R., and Cronan, J . E . , Jr. (1979). Cyclopropane fatty acid synthease of Eschericbiu wli: Stabilization, purification, and interaction with phospholipid vesicles. Biochemist? 18, 3292-3300. Taylor, F. R . , Grogan, D., and Cronan, J . E.. Jr. (1981). Cyclopropane fatty acid synthase from Escherichia rot;. Merh. Enz.vrno/. 71, 133-139. Thomas, J . , and Law, J . (1966). Biosynthesis of cyclopropane compounds. 9. Structural and stereochemical requirements for the cyclopropane synthetase substrate. J . B i d . Chern. 241, 5013-501 8 . van Deenan, L. L. M. (1965). Phospholipids and biotnembranes. Prog. Chem. Fats Orher L i p i d ~ 8 , Part 1, 1 - 1 15. van den Bosch, H . , and Vagelos, P. R. (1970). Fatty acyl-CoA and fatty acyl carrier protein as acyl donors in the synthesis of lysophosphatidate and phosphatidate in Escherichia coli. Biochim. Bii~pphys.Acta 218, 233-248. Volpe, J. J . , and Vagelos, P. R. (1976). Mechanisms and regulation of biosynthesis of saturated fatty acids. Physiol. Rev. 56, 339-417. Zalkin, H . , Law, J . , and Goldfine, H. (1963). Enzymatic synthesis of cyclopropane fatty acids catalysed by bacterial extracts. J . B i d . Chern. 238, 1242-1248.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 17

Transbilayer Distribution of Lipids in Microbial Membranes

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . 11. Assessment of Transbilayer Distribution of Membrane Lipids . . . 111. Transbilayer Distribution of Outer Membrane Lipids . . . . . . A. Lipopolyaaccharides . . . . . . . . . . . . . . . . . . B . Phospholipids . . . . . . . . . . . . . . . . . . . . . IV.

V.

. . . . . . . . . . . . .

. . . . . . .

. . . . . . . . . . . . C. Translocation of Lipids between the Outer and Cytoplasmic Membranes , . . Tran5bilayer Distribution of Cytoplasmic Membrane Lipids . . . . . . . . . . A . Glycolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Phospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . C . Cholesterol . . . . . . . . . . . . . . . . . . . . . . . . . . . . How Lipid Asymmetry Is Maintained . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1.

235 236 239 239 240 243 244 244 246 253 256 256

INTRODUCTION

Asymmetry of membrane lipids, meaning that lipids in the outer leaflet are different from those in the inner leaflet of the bilayer, is a new dimension added to the concept of membrane organization during the last decade (Bretscher, 1972; Rothman and Lenard, 1977; Op den Kamp, 1979). Compared with protein asymmetry, lipid asymmetry seems not to be absolute. Thus the various lipid species are present in both leaflets but in different proportions (Rothman and Lenard, 1977). Lipid asymmetry, first described in the red cell membrane by Bretscher (1972), has been demonstrated in other animal cells, bacteria, and viruses-establishing lipid asymmetry as a general property of biological membranes (Rothman and Lenard, 1977; Op den Kamp, 1979). In this chapter I will 235

c)

Copyright 1982 hy Academic Prer,. Inc All rights of reproduction in any form r c w v r d . ISBN 0- 12- 1533 17-4

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outline the available evidence for lipid asymmetry in microbial membranes and discuss possible mechanisms for the maintenance of this asymmetry. The membranes dealt with include the outer membrane of gram-negative bacteria; the cytoplasmic membrane of gram-positive bacteria; the mycoplasma membrane; intracytoplasmic membranes, and the lipid-containing envelope of PM2 bacteriophage. The membranes of enveloped animal viruses, which incorporate preformed host-cell lipids by budding through the cytoplasmic or nuclear membrane of the host cells (Lenard and Compans, 1974; Lenard, 1978), represent the cell membrane of the animal host cells and will not be discussed here.

II. ASSESSMENT

OF TRANSBILAYER DISTRIBUTION OF MEMBRANE LIPIDS

A variety of techniques have been developed to provide specific information on the transbilayer distribution of membrane lipids (Op den Kamp, 1979). The most common are those based on the degradation of phospholipids by lipolytic enzymes, on nonenzymatic chemical modification of membrane phospholipids, and on immunochemical techniques. The basic assumption underlying all these procedures is that the enzyme. chemical reagent, or antibody will have access in intact cells only to lipids localized in the outer leaflet of the lipid bilayer. Hence, by determining the percentage of a specific lipid hydrolyzed or chemically modified on treatment of intact cells, one can estimate its localization in both leaflets. Complementary experiments will be those utilizing isolated unsealed membrane fragments where the enzyme, chemical agent, or antibody has access to lipids exposed on both membrane leaflets, provided that the isolated membrane fragments do not reseal and that lipid localization does not differ significantly from that in membranes of intact cells. Three major conditions have to be fulfilled prior to the adoption of any enzymatic, chemical, or immunochemical procedure for lipid localization: 1 . The lipids in the outer leaflet are accessible to the agent. 2. The agent does not lyse the cells, penetrate them, or induce alterations in lipid distribution. 3. The time required for the reaction is faster than the rate of the transbilayer movement of the lipid from one leaflet to the other. Under certain conditions lipids in the outer leaflet are inaccessible to the agent. These include (1) presence of a penetration barrier; (2) masking of the lipid by another integral membrane component; (3) a membrane with a lipid bilayer in the crystalline phase; and (4)close association of membrane lipids with intrinsic membrane proteins. The cell wall may constitute a penetration barrier in all of the wall-containing

LIPID DISTRIBUTION IN MICROBIAL MEMBRANES

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bacteria. The selective permeability of the lipid-containing outer membrane of gram-negative bacteria will protect the cytoplasmic membrane lipids from hydrolytic enzymes and chemicals of a molecular weight larger than 600 (Nakae and Nikaido, 1975; Nikaido and Nakae, 1979). Thus phospholipase C cannot attack the lipids of intact E . coli cells unless the cells are pretreated with Tris and EDTA (Duckworth et u l . , 1974). Since after Tris-EDTA treatment it is difficult to separate quantitatively the cytoplasmic membrane from the outer membrane, the localization of membrane lipids in the cytoplasmic membrane of gramnegative bacteria has not been studied. Gram-positive organisms offer a more promising system for study, mainly due to the ease by which their cell wall can be digested and stable protoplasts isolated (Duckworth et at., 1974; Bishop et ul., 1977; Barsukov et ul., 1976), and to the high permeability of the cell wall of intact organisms to labeling agents (Rothman and Kennedy, 1977a). Masking of membrane lipids by other membrane components might also influence the rate of phospholipid hydrolysis or even completely prevent it. For example, phosphatidylglycerol, the major phospholipid of M . hominis membranes, resisted hydrolysis by phospholipase C (Rottem et al., 1973) and failed to interact with specific anti,phosphatidylglycerol antiserum (Schiefer et al., 1975a), or to bind polycationic ferritin (Schiefer et al., 1976) unless surface membrane proteins were first removed by proteolytic digestion. A masking effect of the lipopolysaccharide, protein, or divalent cations was suggested as a possible explanation for the inaccessibility of the outer membrane phospholipids of wild-type smooth variants of gram-negative bacteria to phospholipase (Hasin et al., 1976; van Alphen et al., 1977). The accessibility of membrane phospholipids to enzymes is governed also by the physical state of membrane lipids, primarily by lipid phase transition and phase separation processes. At temperatures above the phase transition, phosphatidylglycerol of Acholeplastiza laidluwii membranes is susceptible to phospholipase A2 treatments, whereas below the phase transition none of the phosphatidylglycerol is hydrolyzed. At intermediate temperatures only that part of phosphatidylglycerol that is in a fluid state will be hydrolyzed (Bevers et al., 1977, 1978a). The accessibility of membrane lipids is affected also by interaction of the phospholipids with intrinsic membrane proteins. Thus about one-third of the phosphatidylglycerol of A . laidluwii that is not hydrolyzed by phospholipase A2, though present in a fluid state, becomes accessible to the enzyme after disrupting protein-lipid interactions by chemical modifications of membrane protein (Bevers et al., 1979). The effects of compactness of the membraneous lipid layer, surface charge density of the membranes, local pH, steric hindrance, and energy source on the accessibility of membrane phospholipids to phospho1ipases.- have been discussed in detail elsewhere (Zwaal and Roelofsen, 1976; Op den Kamp, 1979).

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It is necessary in localization studies to demonstrate the intactness of the cells throughout the reaction by showing that the cells do not lose cytoplasmic components; thus the agent has no access to the lipids in the inner leaflet of the membrane. The possibility that the labeling agent has access to the inner leaflet due to its permeability through the cell membrane should be considered. Permeability of the label is unlikely when protein labels such as phospholipases or immunoglobulins are used, but is likely with the low-molecular-weight chemical agents. The permeability of low-molecular-weight chemical labels may dramatically differ among different membranes. Thus trinitrobenzenesulfonic acid (TNBS), used to label external amino groups of membrane lipids, does not penetrate artificial phospholipid bilayers (Roseman et d . , 1975), as would be expected from its polarity, size, and net negative charge. However, it was found to cross the erythrocyte membranes (Arroti and Garvin, 1972; Haest et a l . , 1981), apparently via the anion channel, whose specificity appears to be quite broad (Rothstein et al., 1976). The permeability of Bacillus megaterium to TNBS was found to be temperature dependent (Rothman and Kennedy, 1977a). TNBS was impermeable at 3"C, partially permeable at 15"C, and fully permeable at 37°C (Rothman and Kennedy, 1977a), allowing its use for localization studies at low temperatures (Paton et al., 1978). Nevertheless, using another strain of B . megaterium, Demant et ul. (1979) showed that TNBS hardly penetrated the protoplast independent of temperature and reagent concentration, so the reagent could be used for localization studies even at 37°C. Although phospholipases are not likely to penetrate through the cytoplasmic membrane, and cells may remain intact even after extensive degradation of the phospholipids (Demant et al., 1979; Paton et a!., 1978; Bishop et al., 1977), these enzymes may perturb membrane structure. Such perturbations may be due to the loss of polar head groups at the membrane surface, the accumulation of diglycerides in the lipid bilayer, and after phospholipase A2 treatment to the accumulation of lysophospholipids. Lysophospholipids in particular tend to disrupt the lipid bilayer. Thus the use of phospholipase A2 was limited to systems containing an active membrane-bound lysophospholipase A2 (van Golde et ul., 1971; Gatt et a / . , 1982). As the rate of transbilayer movement of phospholipids in several bacterial membranes seems to be very fast (see Section IV,B,2), the time required for the reaction of many of the agents-particularly the phospholipases-will always be slower. This does not allow their use in localization studies unless the experiments are performed under conditions where transbilayer movement is very slow or nonexistent. The finding that the transbilayer movement is temperature dependent (Bevers et al.. 1977; Bishop et al., 1977; Demant et d.,1979) may therefore enable the use of even the slow-reacting phospholipases in localization studies carried out at low temperatures.

LIPID DISTRIBUTION IN MICROBIAL MEMBRANES

111.

239

TRANSBILAYER DISTRIBUTION OF OUTER MEMBRANE LIPIDS

Gram-negative bacteria are bounded by two membranes, the cytoplasmic membrane and the outer membrane. The cytoplasmic membrane is responsible for the major metabolic functions, including respiratory pathways, biosynthetic activities, and transport. The major function of the outer membrane is to serve as a barrier protecting the underlying peptidoglycan and cytoplasmic membrane by controlling access of nutrients and agents such as detergents, enzymes, and antibodies (Nikaido and Nakae, 1979). The outer membrane also differs from the cytoplasmic membrane in its composition. Although their phospholipid content is similar, only the outer membrane contains lipopolysaccharides. The hydrocarbon chains of both the phospholipids and lipopolysaccharide molecules are arranged in the outer membrane at right angles to the plane of the membrane to form its hydrophobic interior, which seems to correspond to the bilayer structure of biological membranes (Nikaido and Nakae, 1979). This section will try to answer the following questions: How are the lipopolysaccharide and the phospholipid molecules distributed between the outer and inner leaflets of the membrane? What is their diffusion rate within the plane of the membrane; their rate of transbilayer movement from one leaflet of the membrane to the other leaflet; and the rate of translocation between the two membranes? Of the various outer membranes studied, the outer membranes of Sulmonellu hphirnurium and E . cofi are probably the most thoroughly investigated.

A. Lipopolysaccharides Although Shands demonstrated in 1966 by immunoferritin labeling that the lipopolysaccharide in spheroplasts of S. hphirnuriurn is located on both leaflets of the outer membrane (Shands, 1966), it is now generally accepted that the lipopolysaccharide is located exclusively on the outer leaflet of the outer membrane. This conclusion is based primarily on the elegant work of Muhlradt (Muhlradt rt al., 1973, 1974; Miihlradt and Golecki, 1975) using ferritin-labeled antibodies, and by degradation experiments using galactose oxidase (Nikaido and Nakae, 1979). Both investigations were performed on wild-type strains of S . Qphirnurium. However. when cell envelopes are treated with lysozyme, disturbing the peptidoglycan layer, a rapid rearrangement of the lipopolysaccharide occurred, leading to a symmetrical distribution of the lipopolysaccharide on the outer membrane surfaces (Muhlradt and Golecki, 1975). This finding may explain the results obtained by Shands ( 1 966) with spheroplasts prepared by penicillin treatment. Using the immunoferritin technique, Muhlradt et u l . (1974) also measured the lateral-diffusion coefficients of lipopolysaccaride in the outer leaflet

240

SHLOMO ROTTEM

of the outer membrane. Their values of D = 3 X cm2 sec-’ are much lower than recent values of D = 2.0 ? 0 . 9 x cm2 sec-’ obtained by fluorescence redistribution after photobleaching (Schindler et al., 1980). A direct comparison of the results obtained in the two studies is difficult, however, because the faster rates were obtained with a lipopolysaccharide mutant (R strain), whereas the slower rates of Muhlradt et al. (1974) were obtained with wild-type (S strain) bacteria that have a lipopolysaccharide with a much longer polysaccharide chain as well as a much higher protein content in their outer membrane. The condensing effects of membrane proteins and lipopolysaccharides on membrane fluidity are well established (Rottem and Leive, 1977; Rottem and Samuni, 1973). The lipopolysaccharide is synthesized in the cytoplasmic membrane, and is then translocated to the outer membrane. In this respect it resembles the phospholipids, but unlike membrane phospholipids, which were found to move back from the outer membrane to the cytoplasmic membrane at a fast rate (Jones and Osborn, 1977a,b), the lipopolysaccharide, once translocated to the outer membrane, remains in it. It is not known whether this unidirectional movement is imposed by the mechanism of the transmembrane movement per se or by subsequent lipopolysaccharide-protein or lipopolysaccharide-phospholipid interactions within the outer leaflet of the outer membrane (Bayer, 1975; Schindler er ul., 1980). The latter possibility gains support from the findings of Miihliadt and Golecki (1975) that trypsinization of S . typhimurium cells or digesting the peptidoglycan with lysozyme will induce transmembrane movement of the lipopolysaccharide from the outer to the inner leaflet of the outer membrane.

8. Phospholipids Evidence for an asymmetric distribution of phospholipids between the outer and inner leaflets of the outer membrane of gram-negative bacteria has been presented by the group of Nikaido (reviewed by Nikaido and Nakae, 1979). According to Nikaido and co-workers (Smit et ul., 1975; Nikaido, 1976; Kamio and Nikaido, 1976; Nikaido et al., 1977), the outer membrane of wild-type strains of S . typhimurium is highly asymmetric with the lipopolysaccharides located exclusively in the outer leaflet and the phospholipids (primarily phosphatidylethanolamine, which constitutes the major phospholipid of the outer membrane) in the inner leaflet. The transbilayer distribution is different in “deep rough” mutants of S . typhimurium (i.e., Rdl, Rd2, and Re strains). These laboratory strains produce lipopolysaccharides lacking 85-95% of the saccharide residues found in the lipopolysaccharide of the wild type. In these rough strains lipopolysaccharide molecules are still present exclusively in the outer leaflet,

LIPID DISTRIBUTION IN MICROBIAL MEMBRANES

241

whereas the phospholipids are present in both the outer and inner leaflets of the outer membrane (Nikaido, 1976). These conclusions on phospholipid distribution are based on the following observations:

1 . The phospholipids of intact cells of a wild-type S. typhimurium were found to be inaccessible to phospholipase C treatment or to CNBr-activated dextran that covalently labels the amino groups of phosphatidylethanolamine (Kamio and Nikaido, 1976). In deep rough mutants, however, the outer membrane phospholipids were partially hydrolyzed by phospholipase C and bound the CNBractivated dextran. 2. Calculations show that the phospholipid content of the outer membrane of wild-type S. typhimurium cells is not sufficient to cover even one half of a bilayer (Smit et ul., 1975), whereas deep rough mutants of either E . coli or S . typhirnurium contain in their outer membrane the same number of lipopolysaccharide molecules per unit area, but a lower protein content and a higher phospholipid content (Koplow and Goldfine, 1974; Smit er a/., 1975), far beyond what could be accommodated into the inner half of the membrane (Smit et al., 1975). 3. Freeze-fracturing of the outer membrane of deep rough mutants is easier than that of the wild-type strain. This is consistent with the assumption that in the deep rough mutants the structure of the outer membrane is close to a phospholipid bilayer that can be easily fractured, whereas in the wild-type strains bilayer regions are absent, rendering the membrane much more resistant to fracturing (Smit er ul., 1975). Furthermore, the outer fracture face of outermembrane preparations is studded with a dense array of intramembraneous particles, 4-10 nm in diameter (see review by Osborn and Wu, 1980), that were shown to represent protein-lipopolysaccharide complexes (Verkleij et ul., 1976, 1977). Smooth areas corresponding to phospholipid regions were not found on the outer fracture face but were abundant on the inner fracture face of outer-membrane preparations of S. typhimurium. 4 . The model of Nikaido and co-workers, emphasizing the absence of phospholipids in the outer leaflet of the outer membrane of wild-type S. typhimurium cells, gains further support from the observation that small hydrophobic molecules that are freely permeable through most biological membranes and through the outer membrane of deep rough mutants do not penetrate the outer membrane of wild-type S. typhimurium cells (Nikaido, 1976). Since the hydrophobic molecules have first to diffuse through the hydrophobic hydrocarbon region of the membrane, it is assumed that the hydrophobic molecules can diffuse through regions of ‘‘phospholipid bilayer” but not through an envelope with an outer leaflet that is primarily composed of lipopolysaccharide molecules whose highly saturated hydrocarbon chains are tightly clustered together (Nikaido et ul., 1977).

242

SHLOMO ROTTEM

According to Nikaido’s concept of an asymmetric outer membrane structure, with lipopolysaccharide exclusively in the external leaflet and phospholipids predominantly in the inner leaflet (Nikaido and Nakae, 1979), the hydrocarbon chains of the phospholipids do not intermix with those of the lipopolysaccharide; thus the physical behavior of the outer membrane phospholipids should be similar to that of the phospholipids of the cytoplasmic membrane and should not be affected by the presence of lipopolysaccharide molecules. Indeed, Nikaido et a / . ( 1 977), using electron-paramagnetic-resonance spectrometry of nitroxide-labeled fatty acids, showed that phospholipid hydrocarbon chains in the outer membrane show a degree of fluidity very similar to that in the cytoplasmic membrane. However, there is no general agreement on this issue. It was shown (Rottem et a / . , 1975; Rottem and Leive, 1977) that phospholipid hydrocarbon chains of Proreus rnirahilis and E . coli J5 moved less freely in the outer membrane than in the cytoplasmic membrane, and that the restricted mobility in the outer membrane is determined by the amount as well as by the length of the saccharide chain of the lipopolysaccharide molecules (Rottem and Leive, 1977). This conclusion was supported recently by deuterium-magnetic-resonance quadrupolar echo spectroscopy (Davis et a / . , 1979; Nichol et d., 1980), showing that in E . d i the orientational order in the fluid state was greater in the outer membrane than in the cytoplasmic membrane, and that the liquid crystalline-gel transition in the outer membrane was upshifted by about 7°C compared to cytoplasmic membranes. Similar results suggesting the cooperative melting of phospholipid and lipopolysaccharide were obtained by X-ray diffraction and fluorescent polarization techniques (Emmerling er u / . , 1977; Nakayama er a/., 1980). Other X-ray and fluorescence studies of Overath e t a / . (1975) showed that the outer and cytoplasmic membranes had phase transitions occurring over the same temperature range. However, the findings that only 25-40% of the phospholipids of the outer membrane-compared with 60-80% of the cytoplasmic membraneparticipated in such phase transition (Overath et ul., 1975), and that part of the phospholipids of the outer membrane resisted extraction with aqueous acetone-conditions that suffice to extract essentially all the phospholipids from the cytoplasmic membrane (Rottem et a / . , 1975)-indicate the existence of two lipid environments. It may be suggested that the part of outer membrane phospholipids that resists extraction and does not participate in the phase transition is more tightly bound to the outer membrane, probably due to its interaction with another membrane component such as protein or lipopolysaccharide. If the hydrocarbon chains of outer membrane phospholipids do intermix with hydrocarbon chains of the lipopolysaccharide molecules, phospholipids must be present also in the outer leatlet of the outer membrane. In this case the inaccessibility of outer membrane phospholipids to phospholipase or to CNBr-activated dextran might be due to a shielding effect by the long carbohydrate chains of the lipopolysaccharide molecules (Hasin e f a / ., 1976)-or by a combination of the

LIPID DISTRIBUTION IN MICROBIAL MEMBRANES

243

carbohydrate chains, some outer membrane protein, and divalent cations (van Alphen et a / ., 1977). The difficulty in obtaining fracture faces of wild-type outer membranes might be due to the large protein content in this membrane, and to the fact that most outer membrane proteins transverse the outer membrane (Osborn and Wu, 1980). Smooth areas in the outer fracture, though not found in S. ephirnuriurn (Smit et a / . , 1975), were found in E . co/i K-12 (Verkieij er u / . , 1976, 1977), suggesting that in E . coli phospholipids are present also in the outer leaflet. The low permeability of small hydrophobic molecules through the outer membrane of wild-type strains may result from a smaller amount of phospholipids in the outer leaflet as well as from a high surface pressure of the phospholipid in the outer leaflet that prevents the partition into the membrane of the hydrophobic compounds. One cannot exclude, however, the possibility that the controversy concerning the intermixing of the hydrocarbon chains of outer membrane phospholipids and lipopolysaccharide is a result of a possible reorganization of outer membrane components upon isolation of the outer membrane.

C. Translocation of Lipids between the Outer and Cytoplasmic Membranes Bidirectional translocation of phospholipids between the cytoplasmic and outer membranes of gram-negative bacteria occurs at a rapid rate (Jones and Osbom, I977b; Donohue-Rolfe and Schaechter, 1980). The translocation process appears to be nonspecific with respect to the phospholipid head group. Yet, the outer membrane of S . typhirnurium is enriched with phosphatidylethanolamine as compared with the acidic phospholipids. This is apparently due to secondary lipid-protein or lipid-lipopolysaccharide interactions within the outer membrane (Jones and Osborn, 1977b). The mechanism of translocation was studied in E . d i by Donohue-Rolfe and Schaechter (1980), who found that transiocation of phosphatidylethanolamine is not coupled with membrane protein synthesis and is not driven by ATP or by newly synthesized phospholipids. However, the dissipation of the proton gradient across the membrane inhibited translocation, suggesting that translocation is driven by the proton-motive force. Two major steps are involved in lipid translocation. The lipids synthesized in the inner leaflet of the cytoplasmic membrane move first to the outer leaflet, and then are transferred across the periplasmic space to the outer membrane. The proton-motive force can thus affect the first, the second, or both steps. The mechanism that allows a rapid transbilayer movement of lipid may certainly require the proton-motive force. However, very few systems have been investigated to date to allow generalizations. Thus the transbilayer movement of phosphatidylethanolamine was found to be independent of proton-motive force (Langley and Kennedy, 1979), whereas in Mycop/usnrcr

244

SHLOMO ROTTEM

cupricolutn the transbilayer movement of cholesterol was inhibited by ionophores that dissipate the electrochemical gradient across the membrane (Clejan et ul., 1978). The transfer step of the lipids from the cytoplasmic to the outer membrane may also be driven by the energy derived from a gradient. This transfer is believed to proceed via zones of adhesion between the cytoplasmic and outer membranes (Muhlradt et al., 1973; Bayer, 1975), perhaps representing fused areas of the outer leaflet of the cytoplasmic membrane with the inner leaflet of the outer membrane. Thus fused areas may allow lipids of the inner membrane to move by lateral diffusion to the outer membrane and vice versa. It is possible that the stability of these zones of adhesion is dependent on the proton-motive force, so that dissipation of the proton gradient would result in loss of the adhesion sites, preventing the flow of lipids between the two membranes (Donohue-Rolfe and Schaechter, 1980).

IV. TRANSBILAYER DISTRIBUTION OF CYTOPLASMIC MEMBRANE LIPIDS

A. Glycolipids Studies on the localization of glycolipids in the bacterial cell membrane are not abundant. Information about the transbilayer distribution of carbohydrate moieties of mycoplasmas was obtained by using specific antibodies or lectins (plant proteins that interact with carbohydrates). Immunological studies (Schiefer et al., 1977), agglutination experiments with lectins (Schiefer et al., 1974), and electron microscopic visualization of concanavalin A-surface carbohydrate complexes (Schiefer et al., 1975b) indicated that the carbohydrate moieties, presumably of glycolipids, are exposed on the external surface of mycoplasmas. The fact that in all the Acholeplasma and Mycoplasma species tested, the amount of labeled lectins bound to intact cells was almost the same as that bound to isolated membranes (Kahane and Tully, 1976), brought these authors to the conclusion that mycoplasmal glycolipids are completely external. The concept of the asymmetric distribution of glycolipids in A . laidlawii was further promoted by Gross and Rottem ( 1979), who applied lactoperoxidase-mediated radioiodination (Hubbard and Cohn, 1972) to study lipid asymmetry. The glycolipids diglucosyldiglyceride and monoglucosyldiglyceride, which account for about 30% of the total membrane lipids, were found by this method to be located in the outer half of the bilayer (see Fig. 1). Yet the phospholipids and phosphoglycolipids were found almost equally distributed between the inner and outer leaflets of the bilayer. The ferritin-avidin-biotin labeling procedure of Heitzmann and Richards (1974), used to label membrane protein, was also applied to study asymmetry in the purple membrane of Hulobacterium halohium (Henderson et al., 1978). The

245

LIPID DISTRIBUTION IN MICROBIAL MEMBRANES

--

100-

OUTSIDE

80 60

-

TL

DPG

40-

E . Y

0

w

20-

z W

’

W a t -

aua

0-

W

20 -

40

60

~

INSIDE

80 FIG. 1 . Asymmetric distribution of phospholipids, glycolipids, and phosphoglycolipids in membranes of A . /uid/awii expressed as mole percent. Abbreviations: TL, total lipids: MGDG, monoglucosyldiglycende; DGDG, diglucosyldiglyceride; PG, phosphatidylglycerol; DPG, diphosphatidylglycerol; GPMGDG, glycerophosphorylmonoglucosyldiglyceride; GPDGDG, glycerophosphoryldiglucosyldiglyceride. (From Gross and Rottem, 1979; Razin, 198 l .)

biotin hydrazide reacts with free aldehydes, formed after periodination of the sugar residues to hydrazones. Since glycosylation of proteins is rare in bacteria (Shaw , 1970), the biotin hydrazide reacts apparently with sugar-containing lipids. However, this reagent may also react with phosphatidylglycerol, the predominant lipid of many bacteria (see Goldfine, this volume), since periodate oxidizes cis-diols in general. The transbilayer distribution of triglycosyldiether and glycolipid sulfate, which together comprise about 30% of the total purple membrane lipids, appears to be highly asymmetric with most of the label present on the outer half of the membrane (Henderson et al., 1978). The finding of the external location of glycolipids in the cell membrane of bacteria corresponds nicely with the results obtained with eukaryotic cells and enveloped viruses (for reviews, see Rothman and Lenard, 1977; Op den Kamp, 1979).

246

SHLOMO ROTTEM

B. Phospholipids 1 . T R A N S B ~ L ADISTRIBUTION YER

Phospholipid distribution in the cytoplasmic membrane of bacteria was mainly studied with gram-positive bacteria whose cell wall can be easily digested and stable protoplasts isolated, or with the wall-less mycoplasmas. The major approaches employed were (1 ) chemical modification of phospholipids, mainly of those containing primary amino groups, and (2) enzymatic degradation of membrane phospholipids using a highly purified phospholipase. Most of the information available relates to phosphatidylethanolamine and phosphatidylglycerol (Tables I and 11). Chemical modification of phosphatidylethanolamine, the major phospholipid in many bacterial cells, using TNBS and isethionyl acetimidate (IAI), was applied to a variety of prokaryotic cells (Table I). The mild labels appear to leave the membrane intact and it is relatively easy to establish conditions under which the cells are impermeable to the label (Op den Kamp, 1979). Yet the results obtained vary to a large extent. Thus by using TNBS more than 90% of the phosphatidylethanolamine was found in the outer leaflet of the plasma membrane of Bacillus amyloliquejuciens (Paton et al., 1978), whereas only a slight preference of phosphatidylethanolamine for the outer leaflet was found in Bacillus subrilis (Bishop rt al., 1977), a symmetrical distribution of phosphatidylethanolamine was found in Bacillus tnegaterium (Demant et ul., 1979), and a preference for the inner leaflet was found in another strain of B . megureriurn (Rothman and Kennedy, 1977a). Are these variations due only to the different species and strains used? Do these variations reflect variations in culture age, and a different physiological state of the cells? Are these variations due to artifactual distribution obtained due to a perturbation in the membrane structure? These questions cannot yet be easily answered, but a study on the interaction of TNBS with monolayers of aminophospholipids (Bishop et al., 1979) indicated that the bulky label may cause spatial restrictions at the monolayer surface, decreasing the rate and preventing the reaction between TNBS and phosphatidylethanolamine from going to completion. Furthermore, fatty acid composition of the lipid and the presence of negatively charged phospholipids such as phosphatidylglycerol or diphosphatidylglycerol markedly affected the trinitrophenylation of phosphatidylethanolamine. It seems, therefore, that the interpretation of studies with TNBS must be approached with great caution. TNBS was also used to determine the localization of phosphatidylethanolamine in intracytoplasmic membrane vesicles (Shimada and Murata, 1976). These membraneous structures present in photosynthetic bacteria (chromatophores) or in the nitrogen-fixing Azotobacter vinelandi (Oppenheim and Marcus, 1970) appear to form spherical vesicles that can be isolated by disruption of the cells followed by sucrose-density-gradient centrifugation

TABLE I TRAN5BILAYER

DISTRIBUTION OF PHObPH4TlDYl

E l H4NOI A V I \ E I W MICROBlAl

MEMHK\YES"

Transbilayer distnbution' Organism

PE contenth

Bacillus rnrgoteriurn K M B . tnegureriurn MK 10 B . subtilis B . an2\lo/iqitefacierrs

69 35 28-45 30

Chrornutiuni ritiosurn" AiorobacrcJr bjinrlundi" Envelope of PM2 phage

55 64

21

Outer leaflet 33 50 3 60 90 30 45

Inner leaflet 66 50 ND 70 55 395

Method TNBS, IAl TNBS, Pl'ase C TNBS. Pl'ase C Pl'ase A,, Pl'ase C. TNBS TNBS TNBS Diazonium salt

Reference Rothman and Kennedy (1977a) Dernant rt d . ( I 979) Bishop ~f a / . (1979) Paton C I ti/. ( 1978) Shimada and hlurata (1976) Shirnada and Murata (1976) Schifer P I a/.( 1974)

" Abbreviations used: PE,phosphatidylethanolamine: PL, phospholipids; TNBS, trinitrobenzenesulfonic acid; IAI, isethionyl acetimidate; Pl'ase, phospholipase; ND, not determined. ' Percentage of total membrane PL. Percentage of total PE. " Intracytoplasmic membrane.

TABLE I1 TRANSBILAYER DISTRIBUTION OF PHOSPHATIDYLGLYCEROL I N MICROBIAL MEMBRANES" Transbilayer distribution" Organism

PG content*

Outer leaflet

Inner leaflet

Method

Reference

Bacillus megaterium Micrococcus lysodeikticus Acholeplasma /aidlawii B A . laidlawii OR Mycoplasma gallisepticum PM2 phage

6 35 30 13

>40

ND 33 >20 42

Pl'ase C Exchange protein Pl'ase A. Radioicdination Pl'ase A, Diazonium NaB3H,

Demant et al. (1979) Barsukov et al. (1976) Bevers et a [ . (1977) Gross and Rottem (1979) Markowitz et al. (1981) Schafer et a / . ( I 974)

"

45 64

66

>so 58 >60 85

>25 ND

+

Abbreviations used: PG, phosphatidylglycerol; PL, phospholipids; TNBS, trinitrobenzenesulfonic acid; ND, not determined; Pl'ase, phospholipase

* Percentage of total membrane PL. Percentage of total PG.

LIPID DISTRIBUTION IN MICROBIAL MEMBRANES

249

(Cusanovich and Kamen, 1968). In both systems phosphatidylethanolamine has a preference for the inner leaflet of the membrane (Shimada and Murata, 1976). A method to modify not only the amino group of phosphatidylethanolamine, but also the hydroxyl group of the free glycerol of phosphatidylglycerol, was developed by Schafer et al. (1974) for the study of the localization of lipids in the envelope of a lipid containing bacteriophage PM2. In this envelope phosphatidylethanolamine and phosphatidylglycerol represent more than 95% of the total lipids. By treating PM2 envelope with diazonium salt of sulfanilic acid, an azoamino compound was formed from phosphatidylethanolamine, whereas the hydroxyl group of the free glycerol of phosphatidylglycerol was oxidized to a monoaldehyde. Thus the phosphatidylethanolamine could be labeled by using a 35S-labeled diazonium salt, whereas the phosphatidylglycerol could be labeled by reducing the monoaldehyde formed with NaB3H,. Since the diazonium salt does not penetrate the membrane, only lipids in the outer leaflet will be labeled, but at high LiCl concentrations the probe becomes permeable, labeling the lipids in both leaflets. The results clearly showed an asymmetric distribution of the lipids in the envelope of PM2 virus, with most of the phosphatidylglycerol occupying the outer leaflet, interacting with the positively charged protein that covers the surface of the phage, whereas phosphatidylethanolamine resides in the inner leaflet of the viral envelope (Schafer et al., 1974). Inferences about transbilayer distribution of phospholipids in the host Pseudomnnas BAL-3 1 cells could not be drawn, since the lipid composition of the phage markedly differed from that of its host (Braunstein and Franklin, 1971). Although many complicating factors are involved in the use of phospholipases in lipid distribution studies (Op den Kamp, 1979; Section 11), phospholipase C was used to substantiate the data obtained with TNBS in B . subtilis, B . megaterium, and B . atnyloliquefaciens (Bishop et a/., 1977; Demant et al., 1979; Paton et al., 1978), and to extend the study to phospholipids that do not contain the active amino group that interacts with TNBS. The temperaturedependent hydrolysis of phospholipids in intact cells was taken to indicate a temperature-dependent transbilayer movement triggered by degrading phospholipid at the outer leaflet of the membrane (Demant et al., 1979). This phenomenon will be further discussed in Section IV,B,3. Phospholipases were intensively used also to study transbilayer localization of phosphatidylglycerol, a major phospholipid in mycoplasmas. The studies were carried out with A . luidlawii and M . gallisepticurn using phospholipase A2 (Bevers et al., 1977, 1978b; Markowitz e f al., 1981). These organisms possess an active lysophospholipase (van Golde et al., 1971; Gatt et a l . , 1982) capable of degrading the lysophospholipids formed by phospholipase A2. The results showed that at least 50% of phosphatidylglycerol in A . laidlawii and 60% in M . gallisepticum are in the outer leaflet of the membrane (see Table 11). It is interesting to note that in A . luidlawii a fraction of phosphatidylglycerol(20-30% of the

250

SHLOMO ROTTEM

total ) was not accessible to the enzyme even in unsealed membrane fragments. It was later shown that the phosphatidylglycerol in the pool not accessible to the enzyme consists of lipid molecules in a crystalline-gel state, whereas the lipids vulnerable to attack are those in a fluid state(Bevers et ul., 1978a). A somewhat more general procedure for labeling membrane phospho- and glycolipids of A . luidlawii was reported by Gross and Rottem (1979) (Fig. I ) . This procedure is based on the lactoperoxidase-mediated radioiodination technique widely used to determine protein asymmetry (Hubbard and Cohn, 1972). The iodine preferentially labeled the fatty acid esterified at the p position of the glycerol, by what seems to be an a-substitution process (Benenson ef uf., 1980). The enzymatic iodination may be the method of choice for determining lipid asymmetry, especially when taking into consideration that many of the other methods are based on a modification of only a specific moiety. This method labels most phospho- and glycolipids in biological membranes. The radioiodination of intact A . luidlawii cells and isolated membranes clearly suggests that glycolipids are preferentially located in the outer leaflet, whereas phosphatidylglycerol, diphosphatidylglycerol, and the phosphoglycolipids are present in both leaflets with a slight preference for the outer one (Fig. 1). Gram-positive cocci resemble mycoplasmas in lacking aminophospholipids. Like mycoplasmas, the membranes of these cells contain primarily phosphatidylglycerol and diphosphatidylglycerol (see Goldfine, this volume). Because of the difficulties involved in obtaining protoplasts, very little is known about transbilayer distribution and movement of lipids in these organisms. A study with M i c r o c w c w s lysodeikticeus (Barsukov et ul. 1976) revealed that of the three major phospholipids of this organism, phosphatidylglycerol was located preferentially in the ouer leaflet, phosphatidylinositol in the inner leaflet, and diphosphatidylglycerol was equally distributed between the inner and outer leaflets. This study was performed with a water-soluble nonspecific lipid-exchange protein (Wirtz, 1974). Exchange is presumably possible with phospholipids in the outer leaflet of intact cells, which are exposed to the aqueous medium. Moreover, the exchange process is unlikely to perturb the equilibrium state of the membrane (Rothman ct a / . , 1976; Rousselet et al., 1976; Bloj and Zilversmith, 1976.) ~

2 . TRANSBILAY MOVEMEN ER r An indication that transbilayer movement of phospholipids in model membranes is very slow was first brought up by Kornberg and McConnell (1971). They prepared dispersions of phosphatidylcholine containing a spin-labeled group attached to the choline moiety. When the impermeable reducing agent ascorbic acid was added to the vesicles, the signal arising from molecules exposed on the outer half was abolished and the rate of decay of the residual

251

LIPID DISTRIBUTION IN MICROBIAL MEMBRANES

signal provided a measure of the rate at which the labeled phosphatidylcholine moved from the inner to the outer leaflet of the bilayer. The measured rate of transbilayer movement was very slow, with a half time of days. The rates of transbilayer movement were considerably faster when the same technique was applied to excitable membrane vesicles (McNamee and McConnell, 19731, or when a variety of other techniques were applied to study transbilayer movement of lipids in red blood cells (Bloj and Zilversrnith, 1976; Renoij et a l . , 1976; van Meer et al., 1980), mitochondria (Rousselet et al., 1976), or enveloped viruses (Shaw et ul., 1979). For example, the half time for transbilayer movement of phosphatidylcholine in the red blood cell membrane is on the order of a few hours (Bloj and Zilversmith, 1976; van Meer et al., 1980). In bacteria it seems that the rates are even much higher. Rothman and Kennedy (1 977b) showed that in B . megaterium newly synthesized phosphatidylethanolamine molecules first incorporated into the inner leaflet of the bilayer are rapidly redistributed and appear in the outer leaflet. The half time for such redistribution was calculated to be about 3 minutes at 37"C, at least 30,000 times faster than the rate of transbilayer movement of phosphatidylethanolamine in an artificial bilayer (Roseman et a l . , 1975). Fast transbilayer movements of phosphatidylethanolamine in the cytoplasmic membrane of B . suhtilis (Bishop et ul., 1977) and B . niegaterium (Demant et ul., 1979), and of phosphatidylglycerol in membranes of A . luidluwii (Bevers et al., 1977), were deduced from studies with phospholipases (phospholipase C in the former cases and phospholipase A, in the latter case). In these studies the complete hydrolysis of the phospholipids at 37°C was taken to suggest a temperature-dependent transbilayer movement stimulated by the phospholipid hydrolysis at the outer leaflet (Fig. 2 ) . It was further suggested (Demant et al., 1979; Op den Kamp, 1979) that

increased

Equilibrium distribution in resting cells

decreased

FIG.2. A model showing the induction of transbilayer movement of phospholipids by disturbing the existing equilibrium distribution of the phospholipids in the two membrane leaflets. Left, cfr n o w phospholipid biosynthesis. Right, phospholipid degradation. (From Demant er d.. 1979; Op den Kamp, 1979.)

252

SHLOMO ROTTEM

when the equilibrium distribution of a specific phospholipid between the outer and inner leaflets is disturbed either by a selective removal of the phospholipid from the outer leaflet, or by introducing newly synthesized phospholipid molecules into the inner leaflet, transbilayer movement is enhanced (Fig. 2 ) . Thus even in an artificial membrane, a unilateral distortion of lipid structure may result in an increase in transbilayer movement (de Kruijff and Baken, 1978).

3. POSSIBLE MECHANISMS OF TRANSBILAYER MOVEMENTS The mechanism that allows for the rapid transbilayer movement of lipids in bacteria is as yet unknown. It is presumed that transbilayer movement does not involve the passage of polar head groups through the hydrocarbon core, as this will require high activation energy (Rothman and Lenard, 1977). It is also likely that the mechanism will include in bacteria special structures or processes not present in artificial membranes, red blood cells, mitochondria, or other biological membranes where the rate of transbilayer movement is found to be relatively slow. A possible mechanism, recently suggested by Cullis, de Kruijff, Verkleij, and co-workers (for review, see Cullis and de Kruijff, 1979; de Kruijff ef al., 1980), is that lipid transbilayer movement proceeds through the transitory formation of intrabilayer-inverted lipid micelles. The intrabilayer structures are in exchange with surrounding lipid bilayers on either side of the membrane. This mechanism is consistent with the findings by :IIP nuclear magnetic resonance (NMR), X-ray diffraction, and freeze-fracturing studies, indicating that several lipid components of biological membranes adopt on hydration nonbilayer structures [hexagonal (HI,)phase], whereas other lipids have a preferred bilayer structure. Mixed systems containing bilayer and hexagonal-phase lipids will exhibit a typical 31P-NMR spectra and upon freeze-fracturing, “lipid particle” structures that appear to arise from lipids in an inverted micellar configuration (van Dijck er al., 1976; Cullis and de Kruijff, 1978). The fast transbilayer movement observed in microbial systems (Rothman and Lenard, 1977; Op den Kamp, 1979) will then be due to a lipid composition consistent with the occurrence of such nonbilayer structures. In fact, typical hexagonal-phase lipids are phosphatidylethanolamine (Cullis and de Kruijff, 1979), monoglucosyldiglyceride (Wieslander ef al., 1980), and cardiolipin in the presence of Ca2+ ions (Cullis et al., 1978). These lipids are widely present in microbial membranes (see Goldfine, this volume). The fast rate of transbilayer movement in bacteria may be due to membrane proteins shown to increase considerably the transbilayer movement of phospholipids in artificial membranes (de Kruijff et a l . , 1978; van Zoelen et a [ . , 1978). But the association of a large amount of protein with the membrane is not in itself sufficient to explain the rapid rate of transbilayer movement in bacteria, since in other biological membranes having about the same protein content,

LIPID DISTRIBUTION IN MICROBIAL MEMBRANES

253

transbilayer movement is slow or nonexistent (Rothman et al., 1976; van Meer et al., 1980). It is therefore more plausible to base the explanation for the fast rate of transbilayer movement in bacteria on the presence of a catalytic protein in the membrane. Such protein might be analogous to the specific phospholipid exchange proteins found in animal cells known to facilitate phospholipid exchange between the various membranes within the cell (Wirtz, 1974). The catalytic protein may also be a transmembrane protein that forms channels through the membrane (Rothman and Kennedy, 1977b; Langley and Kennedy, 1979). Such channels could provide an intramembraneous hydrophilic surface with which the phospholipid head groups could interact during passage between the two hydrophobic leaflets, enabling intermixing of the two membrane leaflets by rapid lateral diffusion (Langley and Kennedy, 1979). Another possibility is that the fast transbilayer movement of phospholipids in bacteria is coupled to cell viability through processes such as membrane biosynthesis or ion and proton gradients across the membrane. In fact, enzymes participating in phospholipid biosynthesis were suggested to function in the distribution of the product between the two leaflets of the red blood cell membrane (Hirata and Axelrod, 1978). It is also reasonable to assume that by utilizing energy derived from an ion gradient, a carrier protein could transfer phospholipid molecules from one leaflet to the other (Donohue-Rolfe and Schaechter, 1980). The recent finding that in B . megaterium transbilayer movement is completely independent of phospholipid and protein biosynthesis and is not coupled to energy metabolism (Langley and Kennedy, 1979) makes these possibilities unlikely for B . megaterium, but these data do not yet allow general conclusions to be made.

C. Cholesterol Mycoplasmas are essentially the only prokaryotes requiring cholesterol for growth, thus making them useful tools for studying cholesterol distribution and movement in membranes. Cholesterol localization in mycoplasma cells was first attempted by an immunochemical procedure using cereolysin, a bacterial hemolysin that specifically binds to cholesterol, and then treating the cells with ferritin-anticereolysin (Pendleton el al., 1972). The high density of the label found on the surface of M . gallisepticum cells was taken as an indication that most of the mycoplasmal cholesterol is located at the surface of the membrane where it is readily accessible to the cereolysin. No attempts were made to apply this technique to cholesterol distribution studies, however. In several recent studies filipin was used to probe the transbilayer distribution of cholesterol in membranes of M . gallisrpticurn and M . capricolum (Bittman and Rottem, 1976; Clejan et al., 1978, 1981; Bittman et al., 1981). The filipin-cholesterol asso-

254

SHLOMO ROTTEM

ciation studies are based on the observations that the binding of polyene antibiotics to sterol-containing membranes is easily monitored by absorbance or tluorescence intensity measurements. Large changes in the fluorescence polarization and circular dichroism of filipin accompany its association with cholesterol. The major obstacle in utilizing filipin for membrane studies is the membrane perturbations caused by this probe. The extent of these perturbations depends on the experimental conditons, such as temperature, period of exposure, or antibiotic/sterol molar ratio. Stopped-flow kinetic measurements of filipincholesterol association represent a means by which filipin-induced membrane disruption can be minimized, especially if high cho1esterol:filipin molar ratios and low temperatures are used together with very short reaction times. The initial rates of filipin-cholesterol association were significantly lower with intact mycoplasma cells than with isolated membranes (Fig. 3 ) . Since filipincholesterol association process follows second-order kinetics (first order in filipin and first order in cholesterol), and the initial rate of interaction of filipin and cholesterol is sensitive to sterol accessibility and concentration, the ratio of the second-order rate constants in the unsealed isolated membrane relative to the intact cell is a measure of the cholesterol distribution. These rate constants

Cholesterol

in membranes ( g M )

FIG. 3 . Initial rates of filipin binding to unesterified cholesterol in intact M . gullisepricurn cells (0)and isolated membranes (0).Inset: A plot of the logarithm of the initial rate versus the logarithm of cholesterol concentration, showing that the binding reaction is first order with respect to cholesand membranes ( 0 ) .(From Bittman and Rottem, 1976.) terol in both cells (0)

LIPID DISTRIBUTION IN MICROBIAL MEMBRANES

255

indicate a symmetrical distribution of cholesterol in the two leaflets of the bilayer of M . gulfisrpticum membranes, whereas in M . cupricolum about two-thirds of the free cholesterol i s localized in the outer leaflet of the lipid bilayer. The nature of the alkyl side chain of the sterol affects the extent of sterol translocation and subsequently the transbilayer distribution in M . cupricolurn membranes (Clejan C I ul., 1981). Sterols with a side chain resembling that of cholesterol are distributed between the inner and outer leaflets to the same extent as cholesterol. However, sterols containing a 24a-ethyl group, (p-sitosterol and stigmasterol), a 24P-methyl group, and a A" bond (ergosterol and stigmasterol) remain localized predominantly i n the outer leaflet of the bilayer. Confirming the results obtained with filipin were the results obtained by exchange studies of ['4C]cholesterol between resting M . gallisepticurn cells and human high-density lipoproteins (HDL; Rottem rt a / . , 1978). This study indicates that cholesterol exists in M . gullisc,pticum cells in two different environments. One, representing about 50% of the total unesterified cholesterol, is readily exchanged with exogenous cholesterol, whereas cholesterol in the other environment exchanges at exceedingly slow rates. Since over 90% of the cholesterol in isolated membranes was exchanged rapidly, it is likely that these environments represent the inner and outer leaflets of the lipid bilayer. The aforementioned exchange studies suggested that in resting M . guflisepticurn cells the rate of transbilayer movement of cholesterol from the inner to the outer leaflets of the bilayer is exceedingly slow or nonexistent. One must assume, however, that in growing cells, the rate of transbilayer movement of cholesterol is much faster, since cholesterol taken up from the medium is first incorporated into the outer leaflet of the lipid bilayer, and then about 50% of it is translocated to the inner leaflet within the 16- to 20-hour growth period. Evidence for rapid transbilayer movement of cholesterol in growing M . cqv-icolum cells was recently obtained. Transfer of cholesterol-poor M . cupricolum cells to a cholesterol-rich medium resulted in an approximately 6-fold increase in the free cholesterol content of the membrane within 4 hours of incubation. The secondorder rate constants for filipin-cholesterol association indicated that the transbilayer distribution of cholesterol was essentially invariant throughout the growth period, with about 50% of the cholesterol located at the outer leaflet and 50% at the inner leaflet of the bilayer. However, when growth was inhibited translocation became much slower, and cholesterol accumulated in the outer leaflet of the bilayer. The conclusion that part of the free cholesterol in M . cupricolum is incorporated and transferred to the inner leaflet of the membrane in a growth-dependent process (Clejan et al., 1978) was further supported by showing that cross-linking of surface proteins of M . cupricolum by dimethylsuberimidate caused cholesterol to be localized predominantly in the outer leaflet of the bilayer (Bittman et ul., 1981). The mechanism of inhibition by dimethylsuberimidate differs from the

256

SHLOMO ROTTEM

action of ionophores and chloramphenicol (Clejan et d.,1978). The crosslinking reagent may inhibit translocation by modulating the packing of membrane components at or within the membrane surface or by denaturing a surface protein that may be involved in the movement of cholesterol in M . c u p r i d u r n membranes.

V. HOW LIPID ASYMMETRY IS MAINTAINED In principal, asymmetric distribution of lipids may be maintained by the lack of spontaneous transbilayer movement of the lipid molecule. This might be the case with the lipopolysaccharide of the outer membrane of gram-negative bacteria after being translocated from the site of synthesis in the inner leaflet of the cytoplasmic membrane to the outer leaflet of the outer membrane by a unidirectional and specific process (Muhlradt and Golecki, 1975; Jones and Osborn, 1977b). The asymmetry of the lipopolysaccharide molecules in the outer membrane is absolute, as these molecules reside only in the outer leaflet of the membrane (see Section 111,A). Lack of spontaneous transbilayer movement of phospholipids may also be the case of artificial lipid vesicles (Kornberg and McConnell, 1971). Yet, in bacterial membranes i t seems that phospholipids may move from one leaflet to the other at a relatively fast rate (Bevers e t a / . , 1977; Bishop e r a / . , 1977; Demant et a l . , 1979; Rothman and Kennedy, 1977b; Langley and Kennedy, 1979). Thus asymmetry may represent in this case a state of a delicate equilibrium, in which the asymmetric distribution of lipids reflects the differential interaction of the lipid in the two leaflets of the membrane with other membrane components such as membrane proteins, lipopolysaccharides, or ions. These components are known to be preferentially localized in either the outer or inner leaflets (Rothman and Lenard, 1977; Nikaido and Nakae, 1979). If asymmetry represents an equilibrium state where rapid transbilayer movement exists, whenever this equilibrium state is disturbed, the system may try to reestablish the equilibrium by rapid transbilayer movement (Demant et a l . , 1979; Op den Kamp, 1979). This may occur upon degrading the phospholipid molecule, affecting other membrane components or altering the ionic environment or membrane potential. The existing techniques for determining asymmetry therefore should be carefully controlled so that conditions are established to arrest transbilayer movement. REFERENCES Arroti, J . J . , and Garvin, J . E. (1972). Reaction of human serum albumin and human erythrocytes with tritiated 2,4,6-trinitrobenzenesulfonic acid and tritiated picryl chloride. Biochim. Biophys. A r m 255, 79-90. Barsukov, L. I., Kulilov, V . I . , and Bergelson, L. D. (1976). Lipid transfer protein as a tool in the study of membrane structure. Inside-out distribution of the phospholipids in the protoplasmic membrane of Micrococcus lysodeikricus. Biochem. Biophys. Res. Commun. 71, 704-71 1,

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Bayer, M. E. (1975). Role of adhesion zones in bacterial cell surface function and hiogenesis. ln "Membrane Biogenesis" (A. Tzagaloff, ed.), pp. 393-427. Plenum, New York. Benenson, A , , Mersel, M . . Pinson, A., and Heller, M. (1980). Enzymatic radioiodination of phospholipids catalyzed by lactoperoxidase. A M / . Biochem. 101, 507-5 12. Bevers, E . M., Singal, S . A., Op den Karnp. J . A. F., and van Deenen, L. L. M. (1977). Recognition of different pools of phosphatidylglycerol in intact cells and isolated membranes of Acholeplasmu luitllu~viiby phoapholipase A i . Biochemistry 16, 1290- 1295. Bevers, E . M., Op den Kamp, J . A. F., and van Deenen, L. L. M. (1978a). Physicochemical properties of phosphatidylglycerol in membranes of Acho/ep/usmu luidltiivii. Eur. J . Biochem 84, 35-42. Bevers, E. M., Leblanc, C . , Le Grimellec, C.. Op den Kamp, J . A. F., and van Deenen, L. L. M. (1978b). Disposition of phosphatidylglycerol in metabolizing cells of Acho/ep/usmu luitlltrwii. FEBS Lert. 87, 49-51 Bevers, E. M., Wang. H . H . , Op den Kamp, J . A. F., and van Deenen, L. L. M. (1979). On the interaction between intrinsic proteins and phosphatidylglycerol in the membrane of Ac,holc>plusmu luidluwii. Arch. Biochern. Biophys. 193, 502-508. Bishop, D. G . , Op den Kamp, J. A . F., and van Deenen, L. L. M . (1977). The distribution of lipids in the protoplast membranes of Buc.i/lus suhtilis. A study with phospholipase C and trinitrobenzenesulphonic acid. Eur. J . Bioc,hrm. 80, 38 1-391 . Bishop, D. G . , Bevers, E. M . . van Meer, G . , Op den Karnp, J . A. F., and van Deenen, L. L. M. (1979). A monolayer study of the reaction of trinitrobenzene sulphonic acid with amino phospholipids. Biochim. Biophys. A m 551, 122- 128 Bittman, R., and Rottem. S . (1976). Distribution of cholesterol between the outer and inner halves of the lipid bilayer of mycoplasma cell membranes. Biochem. Biophys. Res. Commrrn. 71, 3 18-324. Bittman, R . , Blau, L., Clejan, S . , and Rottem, S . (1981). Determination of cholesterol asymmetry by rapid kinetics of filipin-cholesterol association: Effect of modification in lipids and proteins. Biochemistty 20, 2425-2432. Bloj, B., and Zilversmith, D. B. (1976). Asymmetry and transposition rates of phosphatidylcholine in rate erythrocyte ghosts. Biochemistn 15, 1277-1283. Braunstein, S. N . , and Franklin, R. M. (1971). Structure and synthesis of a lipid containing bacteriophage. V . Phospholipids of the host BAL-31 and of the bacteriophage PM2. Virology 43, 685-695. Bretscher, M . S . (1972). Asymmetrical lipid bilayer structure for biological membranes. Nufure (London), New Biol. 236, 11-12. Clejan, S . , Bittman, R., and Rottem, S . (1978). Uptake, transbilayer distribution and movement of cholesterol in growing Myr.c~p/usmucupritnlum cells. Biochemistty 17, 4579-4583. Clejan, S . , Bittman, R., and Rottem, S . (1981). Effects of sterol structure and exogenous lipids on the transbilayer distribution of sterols in the membrane of Mycoplastnu capricolum. Biochemistry 20, 2200-2206. Cullis, P. R., and de Kruijff, B. (1978). Polymorphic phase behaviour of lipid mixtures as detected by 31PNMR. Evidence that cholesterol may destabilize bilayer structure in membrane systems containing phosphatidylethanolamine. Biochim. Biophys. Actu 507, 207-21 8. Cullis, P. R . , and de Kruijff, B. (1979). Lipid polymorphism and the functional roles of lipids in biological membranes. Biochim. Bicipphys. Actu 559, 399-420. Cullis, P. R . , Verkleij, A . J . , and Ververgaert, P. H . J . T. (1978). Polymorphic phase behavior of cardiolipin as detected by "P NMR and freeze-fracture techniques. Effect of calcium, dibucaine and chorpromazine. Biochim. Biophys. Acra 513, 11-20. Cusanovich, M. A . , and Kamen, M. D. (1968). Light induced electron transport in Chromutium strain D. I . Isolation and characterization of Chromurium cbromatophores. Biochim. Biophys. Acra 153, 376-396.

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Davis. J. H., Nichol, C. P . , Weeks, G., and Bloom, M. (1979). Study of the cytoplasmic and outer membranes of Escherichiu coli by deuterium magnetic resonance. Biochemisry 18, 2 10321 12.

de Kruijff, B., and Baken, P. (1978). Rapid transbilayer movement of phospholipids induced by an asymmetrical perturbation of the bilayer. Biochim. Biophys. Actu 507, 38-47. de Kruijff, B.. van Zoelen. E. J . J., and van Deenen, L. L. M. (1978). Glycophorin facilitates the transbilayer movement of phosphatidylcholine in vesicles. Biochim. Biophys. Actu 509, 537542. de Kruijff, B . , Cullis, P. R., and Verkleij, A. J. (1980). Nonbilayer lipid structures in model and biological membranes. Trends Bioc.hem. 5 i . 5, 79-8 1 . Deniant, E. J . F., Op den Kamp, J . A. F., and van Deenen, L. L. M. (1979). Localization of phospholipids in the membrane of Bacillus meguteriurn. Eur. J . Biochem. 95, 613-619. Donohue-Rolfe, A , , and Schaechter, M. (1980). Translocation of phospholipids from the inner to the outer membrane of Escherichiu coli. Proc. Nurl. A u ~ t l Sci. . U.S.A. 77, 186771871. Duckworth, D. H., Bevers, E. M., Verkleij, A . I., Op den Kamp, J . A. F., and van Deenen. L. L. M. (1974). Action of phospholipase A, and phospholipase C on Escherichiu coli. Arch. Biochem. Biophys. 165, 379-387. Emmerling, G . , Henning, U., and Gulik-Krzywicki, T. (1977). Order-disorder conformational transition of hydrocarbon chains in lipopolysaccharide from Eschrrichiu coli. Eur. J . Biochem. 78, 503-509. Gatt, S., Morag, B., and Rottem, S. (1982). Utilization of membranous lipid substrates by membranous enzymes. Hydrolysis of lysophospholipid by lysophospholipase in membranes of Mycopplusmu Kullisepticum. J . Bucreriol. (in press). Gross, Z . , and Rottem, S. (1979). Lipid distribution in Aclioleplusmu luidluwii membrane. A study using the lactoperoxidase-mediated iodination. Biochim. Biophys. Acru 555, 547-552. Haest, C. W. M., Kamp, D., and Deuticke, B . (1981). Penetration of 2,4,6-trinitrobenzenesulfonate into human erythrocytes. Consequences for studies on phospholipid asymmetry. Biochim. Biophys. ActU 640, 535-543. Hasin, M . , Razin, S . , and Rottem, S. (1976). The outer membrane of Proteus miruhilis. 111. Specific labeling and enzymic hydrolysis of the protein and phospholipid components of the outer and cytoplasmic membrane. Biochim. Biophys. Acru 433, 229-239. Heitzrnann, H.. and Richards. F. M. (1974). Use of the avidin-biotin complex for specific staining of biological membranes in electron microscopy. Proc Nurl. Acad. Sci. U . S . A . 71, 35373541. Henderson, R . , Jubb, J . S . . and Whytock. S. (1978). Specific labeling of the protein and lipid on the extracellular surface of purple membrane. J . Mol. B i d . 123, 259-274. Hirata, F.. and Axelrod. J . (1978). Enzymatic synthesis and rapid translocation of phosphatidylcholine by two methyltransferases in erythrocyte membrane, Proc. Nurl. A u l d . Sci. U 3 . A .75, 2348-2352. Hubbard, A . L.. and Cohn, Z.A . (1972). The enzymatic iodination of the red cell membrane. J . Cell Biol. 55, 390-405. Jones, N . C.,and Osborn, M . J . . (1977a). Interaction of Snlmonelltr ryphirnrrrium with phospholipid vesicles. J . B i d . Chem. 252, 7398-7404. Jones, N. C., and Osborn, M. J . (1977h). Translocation of phospholipids between the outer and inner membranes of Strlmonellu typhimurrrrm. J . Biol. Chrm. 252, 7405-7412. Kahane, I., and Tully, J. G. (1976). Binding of plant lectins to mycoplasma cells and membranes. J . Bucreriol. 128, 1-7. Kamio, Y ., and Nikaido, H. (1976). Outer membrane of Sulmonellu typhimurium: Accessibility of phospholipid head groups to phospholipase C and cyanogen bromide activated dextran in the external medium. Bioc~hemistry15, 2561 -2570.

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Koplow, J . , and Goldfine, H. (1974). Alterations in the outer membrane of the cell envelope of Heptose-deficient mutants of Escherichia coli. J . Bacteriul. 117, 527-543. Komberg, R. D., and McConnell, H . M. (1971). Inside-out transitions of phospholipids in vesicle membranes. Biochenrisrt? 10, 1 I 1 I 1 120. Langley, K. E., and Kennedy. E. P. (1979). Energetics of rapid transmembrane movement of compostional asymmetry of phosphatidylethanolarnine in membranes of Bacillus mrgurrrium, Proc. Narl. Arud. Sci. U.S.A. 76, 6245-6249. Lenard, J . (1978). Virus envelopes and plasma membranes. Annu. Rev. Biophys. Bioeng. 7, 139165. Lenard, J . , and Compans, R. W . (1974). The membrane structure of lipid-containing viruses. Biochim. Biophys. Acta 344, 51 -94. McNamee, M. G . , and McConnell, H. M. (1973). Transmembrane potentials and phospholipid flip-flop in excitable membrane vesicles. Bioc,heniisty 12, 2951 -2958. Markowitz, Z . , Gross, Z . , and Rottem. S . (19x1). Localization of phosphatidylglycerol in the membrane of Mywp1a.rmu gallisepricum. Isr. J . Med. Sci. 17, 22. Muhlradt, P. F., and Golecki. J . R. (1975). Asymmetrical distribution and artifactual reorientation of lipopolysaccharide in the outer membrane bilayer of' Salmonella ryphirnurium. Eur. J . Biochenr. 51, 343-352. Muhlradt, P. F., Menzel, J . , Golecki, J . R . , and Speth, V. (1973). Outer membrane of Salmonella. Sites of export of newly synthesized lipopolysaccharide on the bacterial surface. Eur. J . Biochem . 35, 47 I - 48 I . Muhlradt, P. F., Menzel, J . , Golecki, J . R . , and Speth, V. (1974). Lateral mobility and surface density of lipopolysaccharide in the outer membrane of Salmonella ryphirnurium. Eur. J . Biochem. 43, 533-539. Nakae, T . . and Nikaido, H. (1975). Outer membrane as a diffusion barrier in Salmonella fyphitnuriurn-penetration of oligosaccharides and polysaccharides into isolated outer membrane vesicles and cells with degraded peptidoglycan layer. J . B i d . Chem. 250, 7359-7365. Nakayama, H., Mitsui, T . , Nishihara, M., and Kito, M. (1980). Relation between growth temperatures of Escherichiu <,oliand phase transition temperatures of its cytoplasmic and outer membranes. Biochim. Biophys. Acra 601, I - 10. Nichol, C. P., Davis, J . H . , Weeks, G., and Bloom, M. (1980). Quantitative study of the fluidity of Escherichiu cnli membranes using deuterium magnetic resonance. Biochemisrry 19,45 1-457. Nikaido, H . ( I 976). Outer membrane of Sulmonella typhimurium. Transmembrane diffusion of some hydrophobic substances. Biochim. Biophys. Acta 433, 118-132. Nikaido, H . , and Nakae, T . (1979). The outer membrane of gram-negative bacteria. Adv. Micro. Physiol. 20, 163-250. Nikaido, H., Takeuchi, Y . , Ohnishi, S. I., and Nakae, T. (1977). Outer membrane of Salmonella typhimurium. Electron spin resonance studies. Biochim. Biophys. Arm 465, 152-164. Op den Kamp, J . A. F. (1979). Lipid asymmetry in membranes. Annu. Rev. Biochem. 48, 47-71. Oppenheim, J . , and Marcus, L. (1970). Correlation of ultrastructure in Azorobacrer vinelundi with nitrogen source for growth. J . Bacterial. 101, 286-291. Osbom, M. J . , and Wu, H. C . P. (1980). Protein of the outer membrane of gram-negative bacteria. Annu. Rev. Microbiol. 34, 369-422. Overath, P., Brenner, M., Gulik-Krzywicki, T . , Schechter, E . , and Letellier, L. (1975). Lipid phase transitions in cytoplasmic and outer membranes of Esrherichiu coli. Biochim. Biophys. Acra 389, 358-369. Paton, J . C., May, B. K . , and Elliott, W . H. (1978). Membrane phospholipid asymmetry in Bacillus umyloliquefaciens. J . Bacreriol. 135, 393-401. Pendleton, I . R., Kim, K . S . . and Bemheimer, A . W . (1972). Detection of cholesterol in cell membranes by use of bacterial toxins. J . Bacferiol. 110, 722-730. ~

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Razin, S . (1981). The mycoplasma membrane. In “Organization of Prokaryotic Cell Membranes” (B. K. Gosh, ed.), Vol. I. pp. 165-250. CRC Press, Inc., Boca Raton. Renooij, W., van Golde, L. M. G . , Zwaal, R. F. A,, and van Deenen, L. L. M. (1976). Topological asymmetry of phospholipid metabolism in rat erythrocyte membranes. Eur. J . Biochem. 61, 53-58. Roseman, M., Litman, B. J . , and Thompson, T. E. (1975). Asymmetric exchange of vesicle phospholipids catalyzed by the phosphatidylcholine exchange protein. Measurement of inside-outside transitions. Biochemistry 14, 2809-28 16. Rothman, J. E., and Kennedy, E. P. (1977a). Asymmetrical distribution of phospholipids in the membrane of Bacillus megaferium. J . Mol. B i d . 110, 603-618. Rothman, J . E., and Kennedy, E. P. (1977b). Rapid transmembrane movement of newly synthesized phospholipids during membrane assembly. Proc. Narl. Acad. Sci. U.S.A. 74, 1821-1825. Rothman, J . E., and Lenard, J . (1977). Membrane asymmetry. Science 195, 743-753. Rothman, J . E., Tsai, D. K., Dawidowicz, E. A , , and Lenard, J . (1976). Transbilayer phospholipid asymmetry and its maintenance in the membrane of influenza virus. Biochemistry 15, 23612370. Rothstein, A., Cabantchik, Z . I . , and Knauf, P. (1976). Mechanism of anion transport in red blood cells: Role of membrane proteins. Fed. Proc. Fed. A m . Soc. Exp. B i d . 35, 3-10. Rottem, S . , and Leive, L. (1977). The effect of variation in lipopolysaccharide on the fluidity of the outer membrane of Escherichia coii. 1. B i d . Chem. 252, 2077-2081. Rottem, S . , and Samuni, A . (1973). Effect of proteins on the motion of spinlabeled fatry acids in mycoplasma membranes. Biochim. Biophys. Acra 298, 32-38. Rottem, S . , Hasin, M., and Razin, S . (1973). Differences in susceptibility to phospholipase C of free and membrane-bound phospholipids of Mycoplasmu hominis. Biochim. Biophys. Acta 323, 520-53 I . Rottem, S . , Hasin, M., and Razin, S. (1975). The outer membrane of Proteus mirabilis. 11. The extractable lipid fraction and electron-paramagnetic resonance analysis of the outer and cytoplasmic membranes. Biochim. Biophys. Acta 375, 395-405. Rottem, S . , Slutzky, G. M . , and Bittman, R. (1978). Cholesterol distribution and movement in the Mycop/usmu gallisepticum cell membrane. Biochemisriy 17, 2123-2726. Rousselet, A , , Colbeau, A . , Vignaus, P. M . , and Devaux, P. F. (1976). Study of the transverse diffusion of spin labeled phospholipids in biological membranes. 11. Inner mitochondria1 membrane of rat liver: Use of phosphatidylcholine exchange protein. Biochim. Biophys. Acta 426, 372-384. Schafer, R., Hinnen, R., and Franklin, R. M. (1974). Structure and synthesis of a lipid-containing bacteriophage properties of the structural proteins and distribution of the phospholipids. Eur. J . Biochem. 50, 15-27. Schiefer, H. G . , Gerhardt, U., Brunner, H., and Krupe, M. (1974). Studies with lectins on the surface carbohydrate structures of mycoplasma membranes. J . Bacteriol. 120, 8 1-88. Schiefer, H. G . , Gerhardt, U . , and Brunner, H. (1975a). Immunological studies on the localization of phosphatidylglycerol in the membranes of Mvcoplusma horniriis. Hof~pr-,%yier’s Z . Physiol. Chem. 356, 559-565. Schiefer, H. G . , Krauss, H., Brunner, H., and Gerhardt, U. (1975b). Ultrastructural visualization of surface carbohydrate structures on mycoplasma membranes by concanavalin A. J . Bucreriol. 124, 1598-1600. Schiefer, H. G . , Krauss, H., Brunner, H., and Gerhardt, U. (1976). Ultrastructural visualization of anionic sites on mycoplasma membranes by polycationic ferritin. J . Bacteriol. 127, 461 -468. Schiefer, H. G., Gerhardt, U . , and Brunner, H. (1977). Localization of a phosphoglycolipid in mycoplasma membranes using specific antilipid antibodies. Zentrulhl. Bukteriol., Purusitenkd., Infektionskr. H y g . , Aht. I : Orig.. Reihe A 239, 262-269.

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Shands. J . W. (1966). Localization of somatic antigen on gram-negative bacteria using ferritin antibody conjugates. A n n . N . Y . Acad. Sci. 133, 292-298. Shaw, M. J . , Moore, N . F., Patzer, E. J . , Correu-Freire, M. C . , Wagner, R. R., andThompson, T . E. ( 1979). Compositional asymmetry and transmembrane movement of phosphatidylcholine in vesicular stomatitis virus membranes. Biochemistq 118, 538-543. Shaw, N . (1970). Bacterial glycolipids. Bucteriol. Rev. 34, 365-377. Shimada, K. and Murata, N . (1976). Chemical rrlodification by trinitrobenzenesulfonate of a lipid and proteins of intracytoplasmic membranes isolated from Chromatiurn vinosum and Azotobacter rainelandi. Biochiin. I3iophx.s. Actu 455, 605-620. Schindler, M., Osborn, M . J . , and Koppel, D. E. (1980). Lateral diffusion of lipopolysaccharide in the outer membrane of Salmonella ryphirnurirrrrr. Nature (London) 285, 261 -263. Smit. J . , Kamio, Y . , and Nikaido, H. (1975). Outcr membrane of Salrnorrellu ryphirnurirmc Chemical analysis and freeze-fracture studies with lipopolysaccharide mutants. J . Bacteriol. 124, 942-958. van Alphen, L., Lugtenberg, B., van Boxtel, R . , and Verhoef, K . (1977). Architecture of the outer membrane of Eschwichiu coli. I. Action of phospholipase A, and C on wild type strains and outer membrane mutants. Bioehirt7. Biophys. Actu 466, 257-268. van Dijck, P. W . M . . de Kruijff, B . , van Deenen. L. L. M . , de Gier, J . , and Demel. R . A . (1976). The preference of cholesterol for phosphatidylcholine in mixed phosphatidylcholinephosphatidylethanolamine bilayers. Biochirn. Biophys. Acta 455, 576-587. van Golde, L. M . G . , McElhaney, R. N . , and van Deenen, L. L. M. (1971). A membrane-bound lysophospholipase from M~coplusmaluicllawii strain B . Biochirn. Biophys. Acra 231, 245249. van Meer, G . , Poonhuis, J. H. M., Wirtz, K . W . A , , Op den Kamp, J . A. F., and van Deenen, L. L. M. (1980). Transbilayer distribution and motility of phosphatidylcholine in intact erythrocyte membranes. Eur. J. Biocheni. 103, 283-288. van Zoelen, E. J . J . . de Kruijff, B., and van Deenen, L. L. M. (1978). Protein-mediated transbilayer movement of lysophosphatidylcholine in glycophorin-containing vesicles. Eiochim. Bi~phy.>. Actu 508, 97-108. Verkleij, A . J . , Lugtenberg, E. J . J . , and Ververgaert, P. M. J . T . (1976). Freeze etch morphology 01 outer membrane mutants of Escherichia coli. Biochini. Biophys. Acta 426, 581 -586. Verkleij. A . J . , van Alphen, L., Bijvelt, J . , and Lugtenberg, B. (1977). Architecture of the outer membrane of t . ~ h e r i c h ci ~d ~i K12. 11. Freeze fracture morphology of wild type and mutant strains. Biochirn. Eiophvs. Actci 466, 269-282. Wieslander, A . , Christiansson, A , , Rilfors, L.. and Lindblom, G. (1980). Lipid bilayer stability in membranes. Regulation of lipid composition in Ac~holeplasniu laitfluwii as governed by molecular shape. Bioc9wmistry 19, 3 6 3 - 3 6 5 5 , Wirtz, K . W . A. (1974). Transfer of phospholipids between membranes. Biochim. Biophys. Acru 344, 95-1 17. Zwaal. R . F. A. and Roelofsen, B. (1976). Applications of pure phospholipases in membrane studies. It7 “Biochemical Analysis of Membranes” (A. H. Maddy, ed.), pp. 352-377. Chapman & Hall, London.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 17

Lipid Phase Transitions and Regulation of Membrane Fluidity in Prokaryotes DONALD L. MELCHIOR Department of B i o c h e t n i s t ~ Universie of Massuchuwtts Medicul Schoo/ Worcester, Mussachirwtt~

I. Introduction . . . . . . . . . . . . . . Lipid Phases . . . . . . . . . . . . . Membrane Bilayer Transitions . . . . . . A . General Properties . . . . . . . . . 3 . Lateral Phase Separations . . . . . . C. E x a m p l e s , , . . . . . . . . . . . IV. Fluidity-Modulating Lipids . . . . . . . . V . Patching . . . . . . . . . . . . . . . A . Fluid Bilayers . . . . . . . . . . . B. Membrane Proteins . . . . . . . . . VI. Biological Consequences of Membrane State VII. Biological Control . . . . . . . . . . . References . . . . . . . . . . . . . . 11. 111.

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INTRODUCTION

Normal cell function requires fluid membrane bilayers. Although some lipid species occurring in prokaryotic membranes do not form bilayers in solution, the total lipids comprising these membranes form stable bilayers. In addition to selecting mixtures that form stable bilayers for their membrane lipids, prokaryotes maintain their membranes in a fluid state under varying environmental conditions. This article will discuss why cells require fluid membranes as well as some of the strategies they use to maintain their membranes in a fluid state. The roles different lipid classes play i n membrane function is considered from the perspective of their physical behavior. To begin, we shall consider the bulk properties of membrane lipids in water. 263

Copynght 1982 by Academic Press, Inc All righrs ot reproduction in any form reserved. ISBN O-i2-I53317-4

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II. LIPID PHASES Extracted lipids in water spontaneously associate to produce a variety of mesophases or supramolecular structures whose conformations depend on temperature, concentration, the presence of various ions, as well as the nature of the lipid molecules themselves (Luzatti, 1968; Hauser and Phillips, 1979). At physiological temperature and in excess water (i.e., more added water does not alter the state of the lipid), the only case considered in this chapter, the preferred conformation for naturally occurring lipid mixtures and even for most purified phospholipids is the bilayer. Nonetheless, some isolated individual membrane lipid species do not form bilayers, or do so only under certain conditions. For example, phosphatidylcholine (PC) readily forms bilayers in aqueous suspension (Chapman et al., 1967; Small, 1967; Cullis et al., 1976; Ulmius, et al., 1977), whereas phosphatidylethanolamine (PE) exists in a bilayer conformation only below certain temperatures above which it assumes a hexagonal conformation (Fig. 1 ) (Cullis and deKruyff, 1978b). Sphingomyelin also forms a lamellar phase at lower temperatures (Shipley et al., 1974; Untracht and Shipley, 1977) and goes to a hexagonal phase at higher temperatures (Yeagle et al., 1978). Studies on the important prokaryotic polar lipids monoglucosyldiglyceride (MGDG) and diglucosyldiglyceride (DGDG) reveal that DGDG assumes the bilayer phase whereas MGDG forms a hexagonal phase (Wieslander et al., 1978). Similarly, monogalactosyldiglyceride extracted from Pelargonium leaves forms a hexagonal phase in water whereas digalactosyldiglyceride forms a bilayer phase (Shipley et al., 1973). Sterols such as cholesterol incorporate readily into phospholipid bilayers (Demel and de Kruyff, 1976), but alone exist in water in a crystalline state (Loomis et al., 1979). A useful theory has been developed to explain why different lipid classes self-assemble into different types of aggregates (Israelachvili et ui., 1977, 1981). This theory involves an interplay of thermodynamics, interaction forces, and molecular geometry. It has been applied with an emphasis on geometric considerations to specific situations by Cullis and de Kruyff (1979) and Wieslander et al. (1980). According to this approach, due to the hydrophobic effect (Tanford, 1973), lipid molecules aggregate in water so as to minimize the contact of nonpolar regions with water while allowing the contact of head groups with water. Packing is governed by repulsive forces arising from electrostatic headgroup repulsion as well as steric head-group and acyl-chain interactions. Lipid mblecules may be considered to be shaped as cylinders or cones or wedges of varying solid angles (Fig. 1). Bilayers are readily formed by cylinder-like molecules such as PC. Molecules with smaller head groups such as PE can form bilayers at lower temperatures. With increasing temperature, however, the increased motion of the fatty-acyl chains of PE molecules results in their becoming more squat in shape. Eventually they can no longer pack easily in a bilayer but

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___c_-

TEMPERATURE OC

FIG. 1 . The phase behavior of mixed-chain phosphatidylethanolamine in excess water. At low temperature this lipid exists as crystalline bilayers (the gel o r Lp state). With increasing temperature the bilayers undergo a reversible endothermic transition commonly called the bilayer phase transition. The resulting state (the liquid crystalline or L, state) is composed of fluid bilayers. The lipid hydrocarbon chains are more disordered in the fluid bilayers than in the crystalline bilayers. This results in more cone-shaped molecules as illustrated in the top of the figure (adapted from Wieslander e t a / . , 1980). With further increase in temperature the lipid molecules become still more cone-shaped and their preferred conformation is the inverted hexagonal HII state. The transition to this state requires less heat than the bilayer phase transition.

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DONALD L. MELCHIOR

assume a hexagonal phase of water cylinders in a hydrocarbon matrix (Fig. 1). Since increased unsaturation in the PE acyl chains gives rise to a more conelike molecule than PE with saturated chains, unsaturated PE forms a hexagonal phase at lower temperatures than saturated PE. Similarly, although DGDG and digalactosyldiglyceride form bilayers, the absence of one sugar residue from the head group of either of these classes results in the more conelike molecules MGDG and monogalactosyldiglyceride, which form hexagonal phases in water. Geometric considerations can also explain the stabilization and destabilization of bilayers by various lipid classes as well as their perturbation by environmental factors. An example is the stabilization of PE bilayers by PC (Cullis and de Kruyff, 1978a) or sphingomyelin (Cullis and Hope, 1980). A geometric approach is consistent with the stabilization by cholesterol of PE, soya PE/egg PC, and dioleoyl phosphatidylethanolamine (DOPE)/dioleoyl phosphatidylcholine (DOPC) bilayers, as well as the destabilization by cholesterol of soya PE/ dipalmitoyl phosphatidylcholine (DPPC) and DOPE/DPPC bilayers (Cullis and de Kruyff, 1978a; Cullis et u f . , 1978). Another application of a geometric approach to explain bilayer stability has been used on mixtures of egg PE and phosphatidylserine (PS). At 37”C, egg PE alone in water assumes a hexagonal conformation. If about 20 mol% PS is included with the PE, a bilayer conformation results. The addition of Ca2+ to the PE/PS suspension produces the hexagonal conformation. This behavior may result from an altered shape of the Ca”-PS complex or from Ca2+-inducedlateral segregation of the PS molecules and consequent formation of unstable regions of PS-rich bilayer (Cullis and Verkleij, 1979). The preceding examples, from studies on model membrane systems, illustrate that not all lipid classes or all mixtures of lipid classes form stable bilayers. Nonbilayer phases, however, have not been found in intact biomembranes (Cullis and de Kruyff, 1978a,b; Untracht and Shipley, 1977). No doubt the many varieties of lipids in biomembranes have evolved to fulfill various roles. Whereas some are preeminently bilayer formers, others may be designed to interact with membrane proteins, to provide membranes with a desired surface charge, or to act as receptor sites. Some lipids may be primarily metabolic intermediates. The structure of many of these molecules may not allow the formation of bilayers by the isolated lipids, but together with sufficient bilayer formers they can exist in bilayers. Despite any predilection for individual membrane lipid species to exist as nonbilayer phases, lipids have evolved to fulfill two basic requirements. They must form a stable bilayer together with membrane protein, and this bilayer must be under physiological conditions in a sufficiently fluid state. Both of these requirements are bulk properties of the ensemble of membrane lipids and are affected not only by membrane lipid composition but by such factors as temperature, pH, and ionic environment. Prokaryotes exist in environments subject to changes in these factors and their lipid composition in many instances reflects

MEMBRANE PHASE TRANSITIONS

267

this. In large part, this chapter is concerned with the influence of environment on the state of the prokaryotic membrane, how this influence is reflected in membrane-related cellular processes, and how prokaryotes modify their membrane lipids in response to environmental factors. To start, we will consider properties of the stable membrane bilayer.

111. MEMBRANE BILAYER TRANSITIONS A. General Properties The membrane bilayers of most organisms are entirely or mostly fluid at physiological temperatures, but at lower temperatures undergo a major reversible change of state (Melchior and Steim, 1976, 1979; Overath and Thilo, 1978). This phenomenon, which has been variously termed a phase change, an orderdisorder transition, a gel-liquid crystal transition, or simply a transition, was first found in Acholeplasnru l u i ~ l u w j membranes i by differential scanning calorimetry (DSC) (Steim, 1968; Steim et ul., 1969). The effect was soon shown calorimetrically in live A . laidlawii cells (Reinert and Steim, 1970; Melchior et al., 1970), and subsequently has been established by a variety of methods as a general property of any membrane containing little or no cholesterol. As first demonstrated by X-ray diffraction of A . laidfawii (Engelman, 1971), it consists of a change in the lipid hydrocarbon chains within the bilayer from a liquid-like state at physiological temperatures to an ordered array at low temperatures. The bilayer conformation is retained throughout. This transition is seen in Fig. I as the transition from state I1 to state I. Although the transition in membranes is, of course, not identical to crystallization and melting in ordinary liquids, extensive studies of model systems (for reviews, see Lee, 1977a,b; McConnell, 1978) show that the events occurring in the bilayer are surprisingly similar to those occurring during phase changes in many more common materials. Lowtemperature ordering of lipid bilayers can be considered to be analogous to crystallization in other multicomponent liquids. Membrane bilayers are composed of many different lipids and, like other mixtures, would be expected to melt over a wide range of temperatures. Melting of most materials, whether consisting of a single component or a mixture, is accompanied by an increase in volume and an absorption of heat. The thermodynamic features of the melting of A . luidluwii membranes exemplifies the behavior of most other membranes. The increase in membrane volume during melting, measured by the change in buoyant density (Melchior ef ul., 1977; Melchior and Steim, 1979), is about 0.71%. If the membrane is approximately one-third lipid (Razin, 1975; Melchior et al., 1970), this change is about 2% of the bilayer volume, an estimate in good agreement with volume increases during

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DONALD L. MELCHIOR

melting of extracted Escherichia coli lipids (Overath and Trauble, 1973). It is less than the 3.5% expansion accompanying the melting of pure dipalmitoyl lecithin (Nagel, 1973; Blazyk et nl., 1975; Macdonald, 1978; Melchior ef al., 1980), and almost an order of magnitude less than the expansion that takes place during the melting of n-eicosane, a 20-carbon n-alkane (Templin, 1956). The small membrane expansion relative to that of hydrocarbons is not surprising because the bilayer retains considerable organization after melting. X-Ray diffraction reveals that the A . laidlawii membrane thins by about 17% during melting (Engelman, 1971); this thinning translates to a change in membrane area of about 23% if the bulk expansion is about 2%. These features of the bilayer transition are illustrated in Fig. 1 . Unlike a synthetic phospholipid with uniform fatty acid chains, a membrane cannot be assigned a unique melting point. The melting range is broad, some 20-40°C in the membranes presented in Figs. 2 and 5, and the melting profile is asymmetric. Such asymmetry occurs commonly, but by no means universally, in other membranes as well, and depends upon the lipid composition of the membrane. The heat uptake in biomembranes, like the volume change, is less than that of pure synthetic phospholipids. Significantly, the heat uptake of the lipids within the membrane is also less, by about 10-20%, than that of the equivalent mass of lipid extracted from the same membranes. This difference between native and reconstituted bilayers presumably reflects lipids that are associated with membrane proteins and therefore in a conformation different than those in the bilayer (Steim et al., 1969; Melchior et ul., 1970). The calorimeter scans of A . laidlawii shown in Fig. 2 illustrate some additional attributes of membrane order-disorder transitions. The progression of membrane peak positions from those shown in curves b to d to f occurs as the membranes are progressively enriched in lipids containing fatty acids of decreasing melting points. This dependence of bilayer melting points on fatty acid composition has been extensively studied with synthetic phospholipids (Ladbrooke and Chapman, 1969), and roughly parallels the bulk melting points of the constituent fatty acids. Increasing the chain length of both fatty acids of saturated phospholipids by two carbons elevates the bilayer melting point by about 15-20°C; dimyristoyl lecithin bilayers melt at 23"C, dipalmitoyl at 42"C, and distearoyl at 54°C. Introduction of a single double bond in both of the chains greatly depresses the melting point: dioleoyl lecithin bilayers melt at -22°C. Synthetic phospholipids also demonstrate that melting points depend upon lipid class as well as fatty acid composition (for review, see Hauser and Phillips, 1979): dipalmitoyl lecithin melts at 42°C and dipalmitoyl phosphatidylethanolamine at 60°C. The narrow, nearly symmetrical peak seen in Fig. 2f is characteristic of membranes whose lipids are highly enriched in a single fatty acid; in the case illustrated they contain about 80% oleate. Similarly narrowed, low-temperature calorimeter peaks have also been reported in E . c d i membranes and whole cells enriched in cis-monoene

269

MEMBRANE PHASE TRANSITIONS

E

b

? $

O

;

LL

-

0

aJ

I

20

20

0

40

Temperature.

60

-180

"C

Flti. 2 . Characteristics of the membrane transition as illustrated by differential scanning calorimetry of A . /uidluwii. Membrane melting occurs at lower temperatures as membranes are enriched in lipids containing fatty acids of decreasing melting points. Curves a and b show respectively, extracted lipids and membranes of stearate-enriched membranes; curves c and d show extracted lipids and membranes of cells grown in unsupplemented growth medium; and curves e, f, and g show extracted lipids, membranes, and whole cells of oleate-enriched membranes. The higher temperature peak in curve d is the thermal denaturation of membrane protein. In curve b this denaturation overlaps the membrane lipid transition. The narrow, nearly symmetrical peak in curve f is characteristic of membranes whose lipids are highly enriched in a single fatty acid; here about 80% oleate. (From 1969.) Steim er d.,

fatty acids to about 90% (Baldassare ef al., 1976). The melting of the extracted membrane lipids in A . luidluwii, and in E . c d i as well (Schechter et a / . , IY74), is quite similar to that of the intact membranes.

B. Lateral Phase Separations Since the melting of membranes cannot be defined by one temperature, an alternative means is necessary. If calorimetry is used, the temperature of the beginning and end of the peak together with t h e temperature of the peak maximum are sometimes designated (Overath and Thilo, 1978). The complete description of phase changes in a multicomponent system is best displayed in a phase diagram (Findlay, 195 I ) , which is essentially a graph of the dependence of thermodynamic state upon such variables as temperature, pressure, and composition. In membranes and model bilayers, the most relevant phase diagrams are those in which the onset and completion o f melting are plotted against bilayer

270

DONALD

L. MELCHIOR

composition. A multicomponent phase diagram that would describe in detail the behavior of a real membrane composed of many lipid classes and fatty acids would be prohibitively difficult to determine experimentally or understand conceptually. Fortunately, the behavior of real systems can be reasonably inferred from simpler binary systems of synthetic lipids, and at the same time comparison of the properties of synthetic mixtures with those of real membranes gives considerable insight into Nature's choice of lipids. It appears that the phase diagrams of lipid bilayers may be more straightforward than those sometimes seen in other materials that can assume more complex geometries in three dimensions. However, depending upon the similarities in the physical properties of the constituent lipids, binary lipid phase diagrams can range from those indicating nearly ideal mixing both in the lipid and solid state to those that are quite nonideal and indicate immiscibility in the solid state (Mabrey and Sturtevant, 1976). This range of behavior was first described by calorimetric methods (Phillips et ul., 1970), but the concept was extended largely by spin-label partitioning of Tempo (McConnell, 1976). The type of diagram shown in Fig. 3 is the simplest, and shows three regions. At the highest temperatures the entire system is liquid; at the lowest temperatures it is solid; and within the temperature and composition range enclosed by the solidus and liquidus curves (the region of the calorimeter peak), both phases are present in equilibrium. The solid region is a solution of the two components, which cocrystallize in all proportions. The diagram is characteristic of mixtures of lipids with very similar physical properties, such as dimyristoyl iecithindipalmitoyl lecithin or dipalmitoyl lecithin-distearoyl lecithin (Shimshick and McConnell, 1973), which show single, broad, reasonably symmetrical peaks in

FIG. 3 . Idealized phase diagram of a two-component lipid hilayer whose components are completely miscible in the liquid and solid states. (For details see text.)

MEMBRANE PHASE TRANSITIONS

271

the scanning calorimeter. The compositions and amounts of the liquid and solid phases at any temperature can be obtained directly from the diagram. Imagine a binary bilayer containing mole fraction X of component B. At all temperatures above the liquidus line at temperature T1, the system is a single liquid phase of uniformly mixed lipids. When the temperature is lowered the first crystalline material appears at temperature T1, which is the high-temperature end of the calorimeter peak. The appearance of solid phase can also be seen as a change in partitioning of a spin-labeled probe, such as Tempo, between the fluidmembrane regions and the exterior aqueous phase (Kleeman et al., 1974). This initial solid phase has composition X , , determined by the intersection of a horizontal dashed line at TI with the solidus curve. It is greatly enriched in the higher melting component but is not pure B. If the temperature is then lowered a little from T1 to T 2 , additional solid material will join the solid areas that first appeared in the bilayer at T I . The solid now has composition Xtr whereas the remaining fluid area, at composition X 3 , is enriched in the lower melting component. The ratio of the liquid portion to the solid portion is given by the ratio of the horizontal line X X 2 to the line X X 3 . This process continues until the solidus line is intersected at temperature T,, where the bilayer is maximally ordered. This point corresponds to the lower end of the calorimeter peak or a second change in Tempo partitioning. Since the entire system is solid at temperature T s , the solid has composition X . The last of the bilayer to crystallize has composition X , , which is enriched in A but is not pure A . The solid regions appear as patches, which can usually be seen by freezefracture electron microscopy (Ververgaert et al., 1973). This separation, in the plane of the bilayer, of solid regions from fluid regions is usually termed lateral phase separation. The term is probably best reserved specifically for the crystallization process just described, since other phenomena, such as liquid-liquid separations (Wu and McConnell, 1975), can also occur in bilayers. Lateral phase separation is a reversible process that occurs continuously as temperature is lowered and the crystalline areas grow in size. Even this simplest of phase diagrams for a binary lipid mixture makes it clear that at no temperature does pure A or pure B separate; at any point in its growth the solid phase contains both components. This is also true for natural lipid mixtures and biomembranes, which, although they contain many components, are governed by the same thermodynamic considerations. Thus the broad peaks seen by calorimetry of biomembranes should not be viewed as the superimposition of many narrow peaks arising from the separate crystallization of each pure component in turn. Lateral phase separation into domains within bilayers implies lateral diffusion in the bilayer plane. Such diffusion measured by spin-labeled phospholipid in egg lecithin above its transition proves to be rapid (Devaux and McConnell, 1972). cm2 sec-' (Edidin, Diffusion coefficients are in the neighborhood of 2 X

272

DONALD L. MELCHIOR

1974) corresponding to an average displacement for a single molecule of about cm sec-l. Coefficients obtained from spin-labeled steroids in sonicated lipid vesicles (Trauble and Sackman, 1972) or inferred from nuclear magnetic resonance (NMR) in coarse lipid dispersions (Lee et d., 1973) have the same order of magnitude. The diffusion coefficient for the protein rhodopsin in the retinal rod membrane at 20°C was measured as 4 X lop9 cm2 sec-' (Po0 and Cone, 1974). Rates of diffusion in ordered bilayer regions have not been clearly established (McConnell, 1978). Some studies suggest, however, that it may be quite slow (Overath et al., 197 1 ; Tsukagoshi and Fox, 1973; Bevers et al., 1978). As the physical properties of the lipids in mixtures become more disparate, phase diagrams depart more and more from ideality. Some binary mixtures that are markedly nonideal but are probably still sufficiently similar to the case illustrated in Fig. 3 to produce solid solutions (i.e., cocrystallization) at all compositions (Mabrey and Sturtevant, 1976), are dimyristoyl lecithin-distearoyl lecithin and dipalmitoyl lecithin-dipalmitoyl phosphatidylethanolamine. Finally, where the melting points of the individual lipids are quite different from one another, immiscibility occurs in the solid at low temperature and the phase diagrams resemble Fig. 4.If such a lipid mixture, of composition X , is lowered in temperature, solid material first appears at temperature T I and has composition X I . If the temperature is lowered to T 2 . solid of composition X 2 will be in equilibrium with liquid of composition X s . The reservoir of liquid continuously changes in size and composition until TB, when a solid of composition X,, is in equilibrium with pure A . Suddenly, the remaining pool of pure A crystallizes at T , , the melting point of A . At temperatures below Tf, in contrast to the system described by Fig. 3, the two components do not form a solid solution. Instead,

-1 T Tl

FIG. 4. Idealized phase diagram of a two-component lipid bilayer whose components are not completely miscible in the solid state. (For details see text.)

T;

T.

I I

Solid A \ * S o l i d Soln I

J x3

SbliA ;Sol"

, , I

x,

I

XOX, Xl 1

MEMBRANE PHASE TRANSITIONS

273

two solids are present, with pure solid A in equilibrium with a saturated solution of A i n B. Two peaks are seen in the calorimeter. For descending temperatures, a higher temperature peak begins in the neighborhood of T , . It is skewed toward higher temperatures but covers the entire range from T , to TI. At T , a single sharp peak, characteristic of the freezing of pure A , appears. Examples of binary systems that show this behavior are dioleoyl lecithin-dipalmitoyl lecithin (Phillips et a/., 1972) and dilauroyl lecithin-destearoyl lecithin (Mabrey and Sturtevant, 1976). It is possible that the construction of a phase diagram and its interpretation in terms of the composition and amounts of each phase in the system at equilibrium might not be as straightforward for a bilayer as it is for other more conventional systems (Lee, 1977a,b). In principle, transitions in pure substances should be highly cooperative with discrete melting points, but in bilayers of a pure synthetic phospholipid such as dipalmitoyl lecithin, the order-disorder transition is slightly broader than in crystalline hydrocarbons. However, analysis of phase diagrams obtained by precise differential scanning calorimetry (Mabrey and Sturtevant, 1976) strongly supports a conventional interpretation. Calorimeter peaks calculated from phase diagrams agreed rather well with the experimental curves. Deviations from ideality can be explained simply by a van der Waals energy factor in a theoretical model that agrees well with experimental findings (Cheng, 1980). Studies by ' T - N M R of equimolar dipalmitoyl-dielaidoyl phosphatidylcholine bilayers also support the conventional interpretation of bilayer phase behavior (Brfilet and McConnell, 1976). A literal interpretation is further indicated by verifying the predictions of phase diagrams with freeze-fracture electron microscopy (Grant et ul., 1974; Verkleij and Ververgaert, 1975; Luna and McConnell, 1978). For bilayers of synthetic PC, solid regions of the bilayer produce striated patches whereas the liquid phase presents a smooth appearance (Verkleij rt ul., 1972). When mixtures of PCs, which show two peaks in DSC, are quenched at temperatures above the melting temperature of their highest melting PC component, a smooth fracture face is observed that is indicative of a fully fluid bilayer. When the same mixture is quenched from temperatures between the two peaks seen in DSC, patches of striated lipid (crystalline lipid regions) are interpersed in smooth regions (melted lipid). When the mixture is quenched from below the lower PC's transition temperature, the smooth regions are gone and striated regions of both PC species are observed (Ververgaert et ul., 1973). The general properties of the phase diagrams of synthetic binary mixtures are displayed by biomembranes, even though their lipid composition is usually quite complex. Broad, single transitions are very common and presumably indicate cocrystallization at low temperatures. Unless the fatty acid composition of the membranes is deranged by manipulating an exogenous supply, multiple

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calorimeter peaks are not ordinarily seen under natural conditions. Such multiple transitions are consistent with Fig. 4,and may indicate the formation of immiscible solid phases (Baldassare et al., 1976). Comparison of binary mixtures with most biological membranes emphasizes the importance of an almost universal attribute of naturally occurring lipids: positional specificity of fatty acid chains. The function of positional specificity is seen by contrasting the crystallization of bilayers containing equimolar mixtures of dioleoyl lecithin and dipalmitoyl lecithin (Phillips et al., 1970) with the crystallization of bilayers composed solely of 1-stearoyl-2-oleoyl lecithin. In the former case, two endotherms are obtained. The lower temperature transition is characteristic of the lower melting lecithin (dioleoyl), whereas the higher temperature transition, although broadened, resembles that of dipalmitoyl lecithin. In the latter case, however, one sharp transition is seen intermediate in temperature to that of the lecithins having either one or the other of the fatty acid chains (Phillips et a / . , 1972). The heat of transition is less for such intermolecular mixing of fatty acid chains than it is for the intramolecularly mixed system. Evidently the mixing of chains within molecules minimizes the spread of melting points of membrane lipids, and hence encourages uniform mixing of the lipids within the bilayer. A series of studies on A . laidawii membranes using phospholipase A2 from pig pancreas illustrates much of the membrane phase behavior just discussed (Bevers et a / . , 1977, 1978). Phospholipase Az hydrolyzes fatty acid esters on the 2-position of phospholipids. In A , luidluwii only phosphatidylglycerol (PG), which comprises about 30% of the total lipid, serves as a substrate for this enzyme. Accumulation of lytic degradation products does not occur due to the presence of a very active membrane-bound lysophospholipase. Membranes treated with phospholipase A2 below the onset of the bilayer transition show no PG hydrolysis. This is because the tight packing of membrane lipids in the crystalline state prevents the enzyme from penetrating the membrane surface and hydrolyzing its substrate (Demel et al., 1975). As the membranes melt with increasing temperature, the extent of PG hydrolysis reflects the progress of the membrane transition, with its concomitant increase in membrane surface area. In experiments in which membrane PG is enriched with specific fatty acids, it is found that as the bilayer melts with increasing temperature, PG molecules with higher melting points are progressively hydrolyzed. Utilizing this simple biochemical approach on membranes enriched in specific fatty acids, behavior indicative of both cocrystallization and multiple bilayer transitions could be demonstrated. These enzymatic studies nicely support conclusions from more indirect physical techniques. They demonstrate that as the bilayer melts, fluid regions grow at the expense of crystalline regions, with progressively higher melting point lipids segregating out of the crystalline regions into fluid regions. Thus, in the course of the membrane transition, there is a change in the relative proportions of fluid and crystalline regions rather than a gradual increase in overall fluidity.

MEMBRANE PHASE TRANSITIONS

275

C. Examples Membranes from a variety of prokaryotes have been shown to crystallize at lower than physiological temperature. In a few cases the membranes of these microorganisms are partially ordered at growth temperature, although there seems to be no physiological necessity for such ordering. The first demonstrations of the order-disorder transition in membranes were made with A . laidlawii by calorimetry (Steim, 1968; Steim et al., 1969), and subsequently other calorimetric investigations of this organism have been made (Melchior et al., 1970; de Kruyff et ul., 1973a; McElhaney, 1974). Transitions have been seen calorimetrically in many other prokaryotic membranes as well, including E . coli (Steim, 1970, 1972; Baldassare et al., 1976), Micrococcus lysodeikticus (Ashe and Steim, 1971), Bucillus subtilis and Staphylococcus aureus (Haest et al., 1972), Bacillus steurathermopliilus (McElhaney and Sousa, 1976; Reizer, 1978), Yersiniu enterocolitica (Abbas and Card, 1980), Veillonella parvula and Anaerovibrio lipolytic'a (Verlkeij et a / . , 1975), and the membranes of Thermophilus aquaticus, blue-green algae, yeast, Chromatiurn D, and Streptococcus faecalis (G. B. Ashe and J . F. Blazyk, unpublished). Cells have been shown to remain viable after calorimetry (Reinert and Steim, 1970; Melchior et a / . , 1970). The order-disorder transitions behave as would be expected from studies of model systems containing many components: they are broad, strongly dependent upon fatty acid composition, and of lower enthalpy than melts of homogeneous synthetic lipids. The shapes of calorimeter peaks vary, but generally the lowtemperature beginning tends to be so gradual that defining the onset is often difficult. The completion at high temperature is often more abrupt. Exceptions to this common shape occur, but generally cyrstallization in intact membranes is about the same as in extracted lipids, and their shape can be rationalized on the basis of fatty acid composition. Crystallization has been detected and studied by X-ray diffraction in both A . luidluwii (Engelman, 1971) and E . coli (Overath et ul., 1975; Schechter et al., 1974; Letellier et id., 1977; Nakayama et ul., 1980), and revealed by 13C- and 'H-NMR in A . laidlawii (Metcalfe et al., 1972; Stockton et a / . , 1975; Smith et d.,1979; Kang et ul., 1981) as well as in E . coli (Steim, 1970; Gally et a / . , 1979; Kang et ul., 1979, 1981; Davis et a / . , 1979). The most extensive physical studies and correlations with biological function have taken place with these latter two organisms, which we shall now consider in more detail. Razin (1975, 1978) has given a thorough account of the physical properties of A . luidlawii membranes, so that we shall consider only those aspects that are particularly relevant to the transition. Acholeplusma laidlawii is an attractive organism for membrane studies, since it readily accepts fatty acids from the growth medium and incorporates them into its membrane lipids (Tourtellotte, 1972). Since it has no cell wall, membranes can be prepared by simple osmotic

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DONALD L. MELCHIOR

lysis. As expected, transition temperatures depend upon the fatty acid supplement. They can be shifted by at least 70°C (Fig. 2). Two peaks are seen in the calorimeter: a lower temperature reversible one due to lipid and a higher temperature one arising from protein (Steim ef al., 1969; Reinert and Steim, 1970; Melchior et ul., 1970) (Fig. 2d). The lipid peak is nearly unaffected by thermal protein denaturation or by pronase digestion of most of the membrane protein, and is essentially the same in membranes, whole cells, and extracted lipids. The results imply that polar lipid-protein interaction is not extensive. A similar conclusion has been reached through careful 'H-NMR studies (Kang et ul., 1981). Judged by comparing heats of transition of membranes with extracted lipids, 90 10% of the lipids in the membranes are in the bilayer conformation, an estimate later verified by Tempo partitioning (Metcalfe et al., 1972) and X-ray diffraction (Engelman, 1971). The behavior of the broad lipid transition is roughly predictable from model systems containing many components. The similarity between the melt in intact membranes and in lipid model systems was emphasized by subsequent X-ray diffraction studies (Engleman, 1971). Nearly the same range of melting was found as in the calorimeter. The most striking feature of the diffraction pattern in A . luidlawii, as well as in pure lipids and in other membranes, is the change in reflections in the neighborhood of 4 A arising from fatty acid chains. As temperature is lowered through the region of the order-disorder transition, a diffuse band at about 4.6 A characteristic of fluid hydrocarbons is eventually entirely replaced by a sharp reflection at 4. I5 A characteristic of hexagonally packed chains. As discussed earlier, during the melting the thickness of the membranes decreases and lateral expansion occurs. Electron density profiles agree well with the bilayer model. An orderly arrangement of protein was excluded, but its localization by X-ray diffraction still remains unclear. It is interesting to note that the motion of spin-labeled free fatty acids (Rottem et al., 1970) and fatty acids esterified to lipids (Tourtellotte et al., 1970) is greater in extracted lipids than in intact membranes, and that pronase digestion of the membrane protein increases the mobility of spin labels (Rottem and Samuni, 1973). An increased packing density of membrane lipids around intrinsic membrane proteins is suggested by the ability of intrinsic membrane protein to protect a portion of membrane PG from hydrolysis by porcine phospholipase A2 at temperatures above the completion of the membrane phase transition (Bevers et ul., 1979). In addition to the phospholipase A2 studies described in Section 11, lateral heterogeneity in A . luidfuwii membranes has been directly demonstrated by Wieslander and colleagues (1979). Cells were grown in media supplemented with varying ratios of the high-melting-point fatty acid, palmitic acid, and the tow-melting-point fatty acid, oleic acid. Membranes were then prepared by osmotic lysis at 22"C, a temperature within or above the bilayer transition of the different membrane preparations. Each membrane preparation was submitted to

*

MEMBRANE PHASE TRANSITIONS

277

countercurrent distribution using a phase system that separates on the basis of membrane surface properties. The membrane fragments from each preparation of cells, with the exception of those supplemented solely with oleic acid, could be divided into at least two subpopulations. Both these “left-handed” and “righthanded” subpopulations had the same buoyant density. Analysis by gel electrophoresis of membrane protein in the different subpopulations revealed very small quantitative differences. However, significant differences existed in the activity of NADH dehydrogenase, NADH oxidase, and ATPase between the different membrane subpopulations. In contrast to their protein content, the lipid compositions of each of the subpopulations differed from one another. A quantitative comparison of the subpopulations showed that as cells were grown with decreasing ratios of palmitate/oleate, the “right-hand” population grew at the expense of the “left-hand” population. The membranes of cells supplemented solely with oleic acid showed only the “right-hand” population. Thus cells lysed at temperatures within their membrane transition appear to form two types of membrane fragments corresponding to crystalline and fluid bilayer regions. Since the membranes of cells supplemented solely with oleic acids were fully fluid at the temperature of cell lysis, only one population of membrane fragments was produced. The cell envelope of E . coli, like other gram-negative bacteria, is structurally more complex than that of A . luidlawii or gram-positive bacteria. The cell envelope consists of two membranes (Costerson er ul., 1975; Leive, 1973; DiRenzo et al., 1978; Nikaido and Nakae, 1979). The inner plasma membrane is in immediate contact with the cytoplasm. Exterior to the plasma membrane, and separated from it by the periplasmic space, is a thin peptidoglycan (murein) layer composed of polysaccharide chains cross-linked by short peptides to form a porous meshlike network encapsulating the entire cell. The final layer of the envelope is the outer membrane, which, in addition to phospholipid and protein, contains the lipopolysaccharide of the envelope (Inouye, 1979). The morphological Complexities, especially the presence of two lipidcontaining membranes, give rise to thermal complexities. The first published reports of a transition in E . coli, obtained by DSC and pulsed NMR in wild-type strain B650 grown at 37”C, revealed a broad melting range, beginning gradually in the neighborhood of 10°C and terminating a few degrees above growth temperature (Steim, 1970). These early results have proven to be deceptively straightforward, however, and more recent DSC reveals complications (Steim, 1972; Melchior and Steim, 1Y76). Subsequently, the order-disorder transition has been examined by X-ray diffraction (Dupont et u l . , 1972; Schechter et ul., 1972; Sackmann et ul., 1973; Trauble and Overath, 1973; Overath and Trauble, 1973; Overath et ul., 1975; Letellier et ul., 1977; Nakayama et ul., 1980), fluorescent probes (Overath and Trauble, 1973; Trauble and Overath, 1973; Sackmann et ul., 1973; Cheng et ul., 1974; Thilo and Overath, 1976; Thilo et

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DONALD L. MELCHIOR

ul., 1977; Tecoma et u l . , 1977), and spin probes (Overath and Trauble, 1973; Baldassare et al., 1973; Linden el ul., 1973a,b; Kleeman et al., 1974; Overath et al., 1975; Tecoma et d., 1977), NMR (Gally et ul., 1979; Kang et al., 1979, 1981; Davis et a l . , 1979; Nichol et a / . , 1980), and scanning calorimetry (Steim, 1970, 1972; Haest et al., 1972; Melchior and Steim, 1976; Overath and Thilo, 1978). The data are sometimes confusing and results from different laboratories are difficult to compare because of variations in bacterial strains, methods of growth and isolation of membranes, and methods used to detect transitions. In addition, a few obvious and unexplained discrepancies have appeared. One example is the detection by pyrene fluorescence of two transitions in outer membranes but none in the cytoplasmic membrane (Cheng et al., 1974). Nevertheless, although details differ, the majority of the results from different laboratories are in fundamental agreement. The inner (cytoplasmic) membrane of E . coli undergoes crystallization in essentially the same way as other bacterial membranes, and the transition temperature can be changed by manipulating fatty acid supplements in the growth medium or by changing growth temperature. Lateral phase segregation occurs, of course, in all cases. Generally, in both wild-type cells and unsaturated fatty acid auxotrophs a single transition occurs that corresponds to one peak in the calorimeter. Presumably the lipids are miscible in the solid membrane and cocrystallization occurs (Fig. 3). However, when the fatty acid composition of at least some fatty acid auxotrophs (fad E, fab E l , containing a temperature-sensitive mutation affecting total fatty acid synthesis and also defective in p oxidation) is manipulated, two transitions are seen (Baldassare et ul., 1976). In this case, the lipids are not miscible in all proportions in the solid, and fractional crystallization occurs into two solid phases (Fig. 4).In wild-type cells, Nature appears to have chosen fatty acid compositions that circumvent this eventuality. Esherichiu coli has played an especially important role in elucidating structural-functional relationships, largely because of the possibilities presented for genetic manipulation. Much of the information concerning crystallization in E . coli membranes comes from fluorescent probes, and especially from the use of N-phenyl- 1-naphthylamine in the laboratories of Overath, Traiible, and colleagues. The method is rapid, requires minimal equipment, and appears to report the order-disorder transition specifically and with reasonable fidelity. Agreement with X-ray diffraction and calorimetry is adequate (Overath and Thilo, 1978), although transitions characterized by fluorescence are frequently narrower than those seen by calorimetry and X-ray diffraction. Pronounced hysteresis, not seen by other methods, often occurs (Overath and Trauble, 1973), and care must be taken to avoid concentration-dependent artifacts. Tempo partitioning, which responds to the fraction of membrane in the fluid state, has proven useful to trace the order-disorder transition in model systems, as discussed earlier. It has also been used to define transitions in E . coli fatty acid auxotrophs (Linden et al., 1973b; Kleeman et al., 1974). Unlike intrinsic motional spin probes such as

MEMBRANE PHASE TRANSITIONS

279

spin-labeled stearic acid, the use of Tempo is based on a thermodynamic principle and might be expected to define dependably the entire course of the orderdisorder transition. Again, however, one gets the impression that transitions seen by partitioning are narrower than those seen by methods, such as X-ray and calorimetry, that do not introduce extraneous probes. Calorimetry, especially when combined with X-ray diffraction to provide structural information, appears to be the most straightforward method. Both calorimetry and X-ray diffraction respond specifically to order-disorder transitions, and although the calorimeter does not give structural information directly, it appears to report such transitions in more detail than can be obtained from X-ray diffraction. X-ray diffraction lacks the sensitivity of calorimetry, and unless great care is taken, minor changes are not detected. Thus the width of transitions reported by X-ray diffraction can be narrower than the true widths. Using inner cytoplasmic membrane vesicles from unsaturated fatty acid auxotrophs, Schechter o f a]. (1974) followed the course of transitions by observing the high-angle reflection at 4 . 2 A as a function of temperature. T h e intensity of the peak, which is characteristic of ordered fatty acid chains, is a measure of the amount of unmelted bilayer. Their X-ray investigation was accompanied by freeze-fracture electron microscopy and Arrhenius plots of transport of proline and glucose. The transition in elaidate membranes was narrow and well defined (about 25-42°C). In others it was broad, extending from about 7 to 28°C in oleate membranes, from below 2 to about 38°C in linoleate membranes, and from about 10 to 46°C in linolenate membranes. Since growth temperatures were 39°C for elaidate-supplemented cells and 37°C for the others, the membranes were entirely o r almost entirely fluid under physiological conditions. Unless total fatty acid synthesis is inhibited, the cis-unsaturated acids produce much higher transition temperatures in E . coli than in A . lriidluwii because of the pronounced tendency of the organism to compensate for the uptake of exogenous fatty acids by adjusting endogenous biosynthesis. Extracted lipids showed essentially the same transitions with minor shifts in temperature except for linoleate-enriched lipids, which were shifted down by about 15°C compared to membranes. On heating and cooling, linolenate cells and extracted lipids underwent hysteresis at the low-temperature end of the transition. Based o n a standard of egg lecithin mixed with 5 % water at o"C, in which all of the hydrocarbon chains are organized, about 55% of the chains were ordered in the elaidate membranes below the transition temperature and about 35 -40% in the cis-unsaturates. Subsequent diffraction of separated inner and outer membranes of fatty acid auxotrophs grown in trans-18:1, trans-16:1, and cis-16:l supported these observations (Overath er ul., 1975), and revealed transitions in both fractions. The temperature ranges of the inner and outer membrane transitions were similar. Below the transition, 60-80% of the hydrocarbon chains were ordere'd in the cytoplasmic and 24-40% were ordered in the outer membranes. Escherichiu coli has been the subject of calorimetric investigations for some

280

DONALD L. MELCHIOR

time (Steim, 1970, 1972; Melchior and Steim, 1976). The results for cytoplasmic membranes are in general agreement with fluorescence and X-ray measurements. However, in whole cells and in envelopes containing both inner and outer membranes, two reversible lipid transitions occur. The first is characteristic of live cells, and a second appears only after exposure to high temperature for short periods of time (seconds), storage for prolonged periods of time (weeks) at 4"C, treatment with trypsin, preparation of membranes by lysozyme-EDTA, or preparation of envelopes by sonication or the French press. This second, anomalous peak is not present in live cells in growth medium (Thilo and Overath, 1976). After appearing, however, it too is reversible. Comparison of phospholipasenegative mutants with parent strains, as well as the lack of correlation of lipid hydrolysis with the behavior of the peak in wild-type cells, indicates that the second transition is not an artifact due to phospholipase activity. The extent of expression of the second transition depends upon the severity of treatment that the preparations undergo. Rigorous physical or chemical operations, including heating to high temperature, treatment with trypsin, sonication, or the French press, usually result in the production of a major peak. No envelope preparations, no matter how prepared, were entirely free of the second melt. Scans of separated outer and inner membranes assign the first reversible transition, characteristic of live cells, to the inner cytoplasmic membrane. The second transition, characteristic only of perturbed cells, occurs in the outer membrane. In wild-type cells, the separate inner and outer order-disorder transitions are best resolved by growing cells at low temperatures. The effect is seen in Fig. 5, curves a and b, which are calorimeter scans of E . coli K12 W945 at 37°C in minimal salts with glucose. The lower temperature peak in Fig. 5a, extending from - 10 to 40"C, characterizes the living cells and occurs in the plasma membrane. This transition, characteristic of the melting of the native plasma membrane, is broad and ill-defined, and extends from low temperatures to the neighborhood of the growth temperature. Provided the calorimeter is not scanned above physiological temperatures, it is reversible. Multiple protein-denaturation peaks occur in the neighborhood of 50-80°C; a scan taken after heating to 100°C (curve b) reveals minor changes in the first transition and shows a new transition, arising from the outer membrane lipids, centered at about 40°C. Subsequent heatings do not change this pattern. The same pattern of behavior characterizes unsaturated fatty acid auxotrophs: a single reversible cytoplasmic membrane transition in whole cells, and a second reversible transition that appears in the outer membrane after heating or other perturbations. Resolution of the two is difficult even in mutants, although by judicious choice of fatty acid supplement, complete resolution is sometimes possible. In elaidate-forced mutants the two peaks are essentially superimposed, and cannot be resolved. The best resolution is obtained in wild-type cells grown in minimal medium at 20°C (Steim, 1972; Melchior and Steim, 1976). Resolu-

281

MEMBRANE PHASE TRANSITIONS

2 20

0

20

40

60

80

0

Temperature, C

FIG. 5 . Differential scanning calorimeter (DSC) scans of whole cells of wild-type E . coli K12 W945 grown at 37°C (indicated by arrows) in minimal medium, scanned through protein denaturation (curve a) and after protein denaturation (curve b). The cytoplasmic membrane transition extends from - 10 to 40°C. The higher temperature outer membrane transition appears in curve b after heating. Scans of the same cells grown in the same medium with an inhibitor of unsaturated fatty acid synthesis (DNAC) appear as curves c and d . If the cytoplasmic membrane transition is elevated beyond that shown in curve c , growth ceases. In curve d the outer membrane transition and the cytoplasmic membrane transition are superimposed. (From Steim, 1972.)

tion is further enhanced by adding ethylene glycol, which shifts the outer membrane transition to higher temperatures but, as in other organisms, has little effect on the cytoplasmic membrane melt. X-Ray studies on wild-type E . coli B (Nakayama er al., 1980) grown at various temperatures demonstrated that whereas the cytoplasmic membrane transition was always complete by growth temperature, that of the isolated outer membrane was not. This reflects the fact that although both the inner and outer E . coli membranes increase their unsaturated fatty acid content as growth temperature is lowered, the outer membrane always has a higher content of saturated fatty acids (Lugtenberg and Peters, 1976; Ishinaga et uf., 1979; Nakayama et al., 1980). It was also found that whereas the extracted inner membrane lipids show a transition similar to that of the inner membrane, extracted outer membrane lipids do not show a transition similar to the outer membrane (Nakayama er al., 1980). The calorimetric results are consistent with X-ray diffraction, if it is assumed that the transition seen by X-ray in outer membrane preparations arose because of preparative procedures. By X-ray, 25-40% of the hydrocarbon chains in the

282

DONALD L. MELCHIOR

outer membranes were found to participate in the outer membrane transition (Overath et a/., 1975). By calorimetry, in many preparations similar percentages are also found to participate, but the size of the transition, and hence the percentage of participation, can be increased by heating. Evidently the outer membrane is a very labile structure. Preparation of envelopes, and especially separating outer from inner membranes, results in at least a partial expression and sometimes a major expression of the higher temperature melt, which is really a property of denatured outer membranes.

IV. FLUIDITY-MODULATING LIPIDS Among the varieties of lipids comprising biomembranes are some whose primary function appears to be to modify membrane fluidity. These molecules act to keep bilayers in a stable state despite changes in environmental conditions, but are not themselves bilayer formers. Examples are certain of the sterols and very likely some of the hopanes and carotenoids. Early studies revealed that cholesterol (Chol) condenses fluid phospholipid monolayers while fluidizing solid ones (Shah and Schulman, 1967). Subsequent DSC demonstrated that cholesterol suppresses bilayer phase transitions (Ladbrooke et ul., 1968). Sufficient cholesterol in a bilayer acts as a plasticizer, causing the bilayer to exist in a state intermediate between crystalline and fully fluid (for review, see Demel and de Kruyff, 1976). The suppression of the bilayer transition is a reflection of this state of intermediate fluidity. Figure 6, taken from a dilatometric study on DPPC bilayers (Melchior er al., 1980), demonstrates the action of cholesterol as a plasticizer. At temperatures above the phase transition of DPPC (42"C), increasing cholesterol content causes bilayers to condense. Below the phase transition, increasing cholesterol content causes the bilayers to expand. Increasing cholesterol content progressively eliminates the bilayer transition. The plasticizing effect of cholesterol on membrane bilayers has been demonstrated to be reflected in dynamic processes occurring in the bilayer. For example, in cholesterol-rich bilayers, simple processes such as the transbilayer diffusion of water (Bittman and Blau, 1972), or more complex processes such as protein-mediated sugar transport (Melchior and Czech, 1979), are enhanced relative to crystalline bilayers but reduced relative to fully fluid bilayers. Whereas on a bulk level cholesterol is considered to put bilayers into a state of intermediate fluidity, on a molecular level the details of cholesterol-phospholipid interaction are not well understood. In bilayers composed of mixed phospholipids, cholesterol is not necessarily uniformly dispersed. I n studies on model bilayers composed of two lipids of the same class, such as PC or PE, but with sufficiently different melting points to produce two peaks in the calorimeter,

283

MEMBRANE PHASE TRANSITIONS

: E

.99

6 E

Mole Percent Cholesterol FIG. 6 . The apparent partial specific volume (V,) of pure and cholesterol-containing DPPC bilayers over the temperature range of 0 to 50°C. Va is plotted against mol% cholesterol (X,)at half-degree temperature intervals extending from 0 ' (bottom) to 50°C (top). The concentration of bilayer cholesterol in the'dilatometer runs used to construct the plots are indicated by the 15 arrows along the X , axis at X, = 0, 2, 5, 7, 10, 15, 17, 20, 23, 25, 29, 33, 40, 45, and 50. Experimental points at these concentrations are connected by straight lines. Since vertical cuts at the arrows reproduce the experimental volume-temperature curves, the vertical spacings between the lines are a measure of the coefficient of expansion at various cholesterol concentrations. (From Melchior et d., 1980.)

cholesterol associates with the lower melting point lipid (de Kruyff et ul., 1973a, 1974; Verkleij et ul., 1974; van Dijck ef al., 1976). That is, it appears to be frozen out of the solid crystalline region of the bilayer. However, even at temperatures above both calorimeter peaks, where bilayers are completely fluid, cholesterol shows a preference for specific lipid classes (Demel er ul., 1977). The order of affinity of cholesterol for three major classes of lipids is sphingomyelin > PC > PE. The biological relevance of these observations is complicated, though, by studies demonstrating that in mixtures of lipids that show a single broad transition, there does not appear to be a preferential association of choles-

284

DONALD C. MELCHIOR

terol with one lipid species over another (Calhoun and Shipley, 1979; Lange et ul., 1979). Because of a potential for preferential association, the presence of cholesterol may promote lipid heterogeneity within a bilayer by producing patches of cholesterol-rich regions containing specific lipids. Crystallization would not occur in such patches, and lipid-protein association might be altered. Not all types of fluidity-modulating lipids present in prokaryotes show the characteristic plasticizing effect of cholesterol. The effects of sterols, hopanoids, and carotenoids in microbial membranes are discussed in detail by Ourisson and Rohmer and by Razin in this volume.

V.

PATCHING

A. Fluid Bilayers Demixing of phospholipids is the rule during crystallization in bilayers, and although in some cases immiscibility or phase separation occurs in the solid state at low temperature (Fig. 4), such temperature-dependent demixing is the result of the crystallization process. In the liquid state, at temperatures above any orderdisorder transitions, the lipids in systems such as those illustrated in Figs. 3 and 4 are miscible in all proportions. Although transient associations no doubt occur between different lipids of miscible fluid systems, there are no actual phase separations into liquid domains. In membrane bilayers, a more permanent heterogeneity in the liquid state may be introduced by a variety of factors, including the interaction of cholesterol with specific phospholipid classes (Demel et a / . , 1977), by intrinsic protein immersed in the bilayer (Boggs et a / . , 1977), and by electrostatic binding of acidic phospholipids by ions and proteins (Galla and Sackman, 1975; Birrell and Griffith, 1976; Ohnishi and Ito, 1974; Papahadjopoulos et a / . , 1975a,b). However, at least two cases have been reported of separation of two liquid phases within bilayers, where phase separation is a result of the intrinsic properties of the lipids and not due to an added extrinsic factor. In one, fluid regions containing between 25 and 40% lecithin are formed in a fluid phosphatidic acid bilayer in the absence of ions (Galla and Sackman, 1975). In the second system a phase diagram has been worked out (Wu and McConnell, 1975). At temperatures higher than 50"C, the dielaidoyl lecithin-dipalmitoyl phosphatidylethanolamine binary system displays the behavior characteristic of partial immiscibility of liquids, such as butanol-water or phenol-water. Although both the lipids and the temperatures at which liquid-liquid phase separation occurs are rather unnatural in the dielaidoyl lecithin-dipalmitoyl phosphatidylethanolamine system, it is possible that the effect may take place in membranes. A puzzling effect occurs, for example, in the endoplasmic reticulum of the protozoan 7etrahjvnena pyrijorrnis (Wunderlich et al., 1975). At temperatures below 17"C, freeze-etch electron microscopy reveals the emergence of

285

MEMBRANE PHASE TRANSITIONS

smooth patches on the fracture faces. This change at about 17°C is accompanied by changes in the fluorescent intensity of 8-anilino-1-napthalene sulfonate, the motion of spin-labeled stearic acid, the partition of 4-doxyldecane, and the amplitude of the NMR signals arising from hydrocarbon chains. However, no hint of the usual order-disorder crystallization could be found by scanning calorimetry. Two environments of different fluidity were suggested, both by the physical studies and by the freeze-etch electron microscopy. An instance of separate coexisting liquid lipid phases occurs in the cholesterol-containing membrane of Mycoplusmu cupricolum (Melchior and Rottem, 1981, 1982). This prokaryote, in addition to several other mycoplasmas, has a membrane rich in long-chain cholesterol esters (CE) (Rottem, 1980). To understand the physical state of these membrane CE, it will be useful to discuss the interaction of CE, phospholipids, and cholesterol in terms of general phase behavior. Long-chain CE have a solubility in lipid bilayers of less than 5 mol%. When present in excess of this, they form a separate phase (Small, 1970). The relatively complex miscibility behavior of phospholipids, cholesterol, and cholesterol esters can be described by the type of phase diagram shown in Fig. 7 .

WATER

CE

I PHASE crvstal

I1 I PHASE. oily liquid

80

60

40

20

FIG. 7 . The three-component system of egg PCiCholicholesteryl linoleate at constant water content. The tetrahedron at the upper left shows the position of the section containing the fourcomponent system with 70% water by weight. This section is shown enlarged and is dealt with as the three-component system PCICholICE. In this illustration the three apexes are labeled PL for the phospholipid (egg phosphatidylcholine), C for cholesterol, and CE for cholesterol ester. Region I consists of one phase, PC bilayers containing varying amounts of Chol and CE (shown schematically in the upper right). Region I I is an oily CE phasc containing up to 8 weight percent Chol. In region 111 two phases are present, PC bilayers saturated with CE and Chol and an oily phase of CE. Region IV contains three invariant phases, PC bilayers saturated with CE and Chol, an oily CE phase saturated with Chol, and Chol crystals. (From Small and Shipley, 1974.)

286

DONALD L. MELCHIOR

This figure illustrates the egg PC/cholesteroI/cholesteryl linoleate/water system at 37°C and atmospheric pressure (Small and Shipley, 1974). Using this formalism, a proper representation of a four-component system requires a tetrahedron as shown in the upper left corner. Since we are concerned with lipid systems i n excess water, a simplification can be made by taking a triangular section parallel to the base of the tetrahedron at 70 weight percent water. According to the Gibbs phase rule, at a given temperature and pressure, I, = c - p , where v is the degree of freedom of the system, c is the number of components comprising the system, and p is the number of phrases present in the system. In this example, since the water content is fixed, we have in effect a three-component system and v = 3 - p . As shown in Fig. 7, the egg PC/cholesteroVcholesteryl linoleate system at 70 weight percent water is divided into four major regions. Region I contains only one phase, the bilayer, and therefore v = 2 . The composition of these bilayers has two degrees of freedom. As indicated in the phase diagram, the bilayers can incorporate up to about 68 weight percent Chol and up to a few percent CE. In region 111, two phases are present and v = 1. One phase is PC bilayers saturated with CE and Chol, and the other phase is an oily CE phase. Region I1 has only two components and one phase and, therefore, v = 1 . This region consists of an oily CE phase containing up to 8 weight percent Chol. Region IV contains three phases, v = 0, and there are no degrees of freedom. The composition of each of these phases is fixed. The phases are PC bilayers saturated with Chol and CE, oily CE saturated with Chol, and crystalline cholesterol. Although CE are not very soluble in lipid bilayers, they are, as mentioned, found in substantial quantities in the membranes of several of the mycoplasmas (Razin et al., 1980). Recent studies have shown these CE to be tightly associated with the mycoplasma membrane, but not intimately associated with the bulk of the membrane protein. Using DSC, it was demonstrated that the majority of these CE exist as fluid patches or “pockets” coexisting with the Chol/phospholipid membrane bilayer (Melchior and Rottem, 1981, 1982). The ratios of CE, Chol, and phospholipids found in these mycoplasma membranes fall into region 111 of Fig. 7, which predicts the coexistence of two lipid phases as found experimentally. The fluid CE pockets may be located in the hydrophobic core of the membrane bilayer or may be attached to either side of the bilayer. The CE in these pockets appear to be relatively pure, since they can crystallize upon low-temperature incubation in a manner characteristic of pure CE (Small, 1970; Tall and Robinson, 1979).

6. Membrane Proteins Although crystallization of bilayers may occasionally have little or no effect upon the random distribution of proteins, as a general rule intrinsic membrane

MEMBRANE PHASE TRANSITIONS

287

proteins are frozen out of the advancing crystalline regions produced during membrane crystallization. This effect is seen by freeze-fracture electron microscopy as the appearance of patches nearly or entirely free of intramembrane particles, when membranes are incubated before quenching at temperatures within or below the transition. Figure 8 shows this phenomenon in the cytoplasmic membrane of E . c d i W3110 (van Heerikhuizen et a / . , 1975). These membranes are fluid at 37°C and crystalline at 0°C. When quenched from 37°C (Fig. 8A), they display a random distribution of particles, whereas when quenched from 0°C (Fig. 8B) they display patches of aggregated membrane particles and particle-free patches. The temperatures at which patching occurs, as well as the ratio of the areas of smooth to particulate regions, have been shown in E.coli to correlate roughly with the cytoplasmic membrane transition, although the onset of aggregation can occur below the high-temperature end of the transition (Schechter et a l . , 1974). The correlation of protein patching with the bilayer transition of the inner membrane of E.coli has been studied by various physical techniques in addition to X-ray diffraction (Kleeman et al., 1974; Haest et ul., 1974; Verkleij and Ververgaert, 1975). Temperature-induced patching of intramembrane particles has been reported in

FIG.8. The effect of temperature on particle distribution in the cytoplasmic membrane of E . co/i W3110. (A) Incubation of membranes at 37°C before freezing results in a random distribution of particles. Bar = 0.5 pm. ( B ) Incubation of membranes at 0°C before freezing produces extensive particle-free patches. Bar = 0.5 p m . (C) Cytoplasmic membrane vesicles with low and high particle density obtained by breakage of EDTA-lysozyme spheroplasts at 0-4°C in a Ribi press. The sample was equilibrated and frozen from 25°C to ensure a random distribution of particles in all vesicles. Bar = 0.2 p m . (From van Heerikhuizen et d., 1975.)

288

DONALD L. MELCHIOR

FIG. 88 and C.

(See legend p. 287)

the membranes of many prokaryotes in addition to the inner and outer membranes of E . coli (Schechter et al., 1974; van Heerikhuizen e t a / . , 1975; Verkleij et al., 1976), for example, membranes of S. fueculis (Tsien and Higgins, 1974; Haest et al., 1974), Mycwplasma mycoides subsp. cupri (Rottem et al., 1973a), A . laidlawii (Tourtellote et al., 1970; Verkleij el al., 1972), V . parvula and

MEMBRANE PHASE TRANSITIONS

289

A . lipolyticu (Verleij et a l . , 1975), and the blue-green alga, Anucysfis niduluns (Verwer e f a l . , 1978). Although proteins are probably most frequently displaced

from ordered regions of the bilayer by moving laterally and accumulating at high concentration in aggregated regions, particle-free patches can occur without an obvious increase in particle concentration in the remaining areas (Tsien and Higgins, 1974; Duppel and Dahl, 1976). In this case, evidently particles are removed from the fracture faces by moving normal to the membrane surface. Thus the most common effect of bilayer crystallization is to produce protein-free regions with conservation of proteins into concentrated regions or to produce patching with loss of particles. That patching is a result of the order-disorder transition, and not a result of temperature itself, has been demonstrated in A . laidlawii by growing cells in media enriched in saturated fatty acids at constant temperature (Tourtellotte et d.,1970). Membranes from palmitate-supplemented cells, which have a transition extending well above growth temperature, showed patching at 37"C, whereas cells grown in oleate with transitions well below 0°C showed no patching. The formation of clear patches in membranes can be induced by factors other than temperature, for example, pH (Copps et ul., 1976). Thermally induced patching ordinarily implies lateral phase separation and free diffusion of both lipids and intrinsic proteins. Presumably the particles seen by freeze-fracture are excluded from the more solid portions of the membrane (which consists of lipids enriched in saturated fatty acids) and collect in fluid pools of lower melting point lipids. Dramatic evidence for the correctness of this presumption has been provided by the actual physical separation of particledepleted regions from particle-enriched areas (Fig. 8 C ) . By mechanical disruption of spheroplasts of wild-type E . coli W31 10 cells at ice temperatures followed by isopycnic-gradient centrifugation, van Heerikhuizen e f d . (1 975) were able to isolate a low-density population of vesicles devoid of intramembranous particles. The phospholipid/protein ratio of the protein-depleted membranes was four or five times greater than that of whole cytoplasmic membranes, whereas the fatty acids of their lipids were considerably more saturated. One protein with an apparent molecular weight of 26,000 was concentrated in the low-density fraction, where it comprised 50% of the total protein. NADH oxidase and succinic dehydrogenase were excluded from the smooth patches, but D-lactate dehydrogenase was not excluded and even appeared to be concentrated. By using a gentler method, osmotic lysis at 4"C, both particle-enriched and particle-depleted vesicles have been isolated from cytoplasmic membranes of an E . coli fatty acid auxotroph grown on linolenic acid (Letellier and Schechter, 1976; Letellier et ul., 1977). X-Ray diffraction showed that the particle-rich membranes, whose lipids were greatly enriched in unsaturated fatty acids, crystallized at lower temperatures than the smooth membranes. The preference of some proteins for the solid phase is quite remarkable, and offers evidence for specific lipid-protein association. These preferences can be

290

DONALD L. MELCHIOR

demonstrated in model systems (Kleeman et al., 1974). In mixed bilayers containing dielaidoyl and dipalmitoyl lecithin, or dimyristoyl and distearoyl lecithin, the erythrocyte protein glycophorin prefers fluid regions. However, glycophorin remains randomly distributed in pure dimyristoyl or dipalmitoyl lecithin both above and below the crystallization temperature. Magnesium -calcium ATPase from rabbit sarcoplasmic reticulum, on the other hand, shows a more pronounced incompatibility with the solid phase. It is excluded from pure dimyristoyl lecithin bilayers at low temperatures. Apparently the partition coefficients of proteins in membranes can depend upon rather subtle changes in protein conformation; bleached rhodopsin is randomly distributed in solid dimyristoyl lecithin bilayers, but unbleached rhodopsin is excluded (Chen and Hubbell, 1973). Thilo, Traiible, and Overath have considered the functional consequence of proteins partitioning between fluid and crystalline regions of the membrane (Thilo et al., 1977; Overath and Thilo, 1978). Using protein partitioning as a model, they tested the temperature dependence of sugar transport in E . coli. This work was the outcome of careful studies on the transport rates of P-glucoside and P-galactoside in the E . coli fatty acid auxotroph T105, whose membrane transition was varied by supplementation with different fatty acids. The membrane transition was characterized by fluorescence, and careful effort was expended to measure transport rates above and below the transition. According to this model (Fig. 9), individual carrier molecules sense changes in the state of their lipid environment, which influences their rate of transport. Transport proteins partition between fluid- and ordered-membrane regions, and the overall rate of transport is the sum of the transport rates of the carriers in the fluid and ordered domains. Thus the transport proteins act as membrane probes and their overall activity shows the first appearance of fluid membrane regions and the final disappearance of crystalline membrane regions. Not all membranes having order-disorder transitions show intramembrane protein patching. This is the case for S. aureus, B . subtilis, Bacillus cereus, and Bacillus megaterium, whose lipids contain almost exclusively branched chains (Haest et u / . , 1974). Lack of protein patching is definitely correlated with the presence of branched chains because A . laidlawii (Haest et a l . , 1974) and E . coli (Legendre et a / ., 1980) membranes, which ordinarily display protein patching during the membrane transition, do not do so if enriched in branched-chain fatty acids. From X-ray diffraction studies (Haest et al., 1974; Lengendre et al., 1980) it appears that fatty acids in membranes rich in branched-chain fatty acids are more loosely packed in the crystalline state than in membranes lacking branched-chain fatty acids. This looser packing is suggested as the reason that membrane particles are not squeezed out of crystalline regions of membranes rich in branched-chain fatty acids. Studies using phospholipase A, on A . laidlawii enriched with various fatty acids are in agreement with these X-ray observations

291

MEMBRANE PHASE TRANSITIONS

I

f

4

LO"

30"

31

32

'VC

200

h z l

I

-II

.

I

I

33

(TEMPERATURE).'

34 x

35

36

lo3 [OK-']

Fic,. 9. The distribution of carrier proteins between tluid- and ordered-membrane regions. The course of the membrane transition in E . c d i fatty acid auxotroph T I 0 5 supplemented with trans.19-16:I fatty acids is shown at the top of the figure as the ratio of fluid t o total membrane area. The solid curves at the bottom of the figure arc calculated p-glucoside transport rates for different distribution constants, K . of carrier proteins panitioning between fluid and crystalline bilayer regions (shown schematically in the center of the figure). The open circles in the lower pan ofthe figure show the experimentally determined irr vii.o temperature dependence of P -nitrophenyl pwglucopyranoside (NphGlu) hydrolysis. The best fit between theory and experiment was obtained for K = IS. (From Thilo cr ( I / . , 1977.)

292

DONALD L. MELCHIOR

(Bouvier e t a / ., 1981 ;Op den Kamp, 1982). Phospholipase A2 has no access to PG in palmitate- and elaidate-enriched A . luidlawii membranes in the crystalline state due to the tight packing of the membrane lipids. In contrast, the presence of branched-chain fatty acids in A . laidlawii results in a sufficiently loose packing of lipids below the membrane phase transition, so that even in the crystalline state, phospholipase A2 is able to penetrate into the bilayer and hydrolyze PG. Consistent with the action of cholesterol as a bilayer plasticizer, temperaturedependent protein aggregation does not occur in membranes rich in cholesterol (Rottem er ul., 1973b; Duppel and Dahl, 1976). Mycoplasma mycoides ordinarily incorporates sufficient amounts of cholesterol into its membrane to eliminate the order-disorder transition, and membrane proteins remain randomly dispersed upon low-temperature incubation. If the organism is adapted to grow on low levels of cholesterol, so that its membrane is almost devoid of the sterol, the membrane crystallizes at 4°C and extensive patching takes place (Rottem el a / ., 1973b). It is worthwhile noting that patching does not always occur in wild-type E . c-oli (Kleeman and McConnell, 1974). Since the organism contains neither cholesterol nor branched-chain fatty acids-both of which, as described, inhibit patching-the lack of patching in these studies must have been a result of the fatty acid distribution. Freeze-fracture is thought to split the membrane bilayer along its midplane, so that freeze-fracture electron microscopy reveals only intrinsic proteins that are deeply embedded in the lipid matrix. Little is known of the distribution of surface proteins, or of proteins that have limited bilayer penetration, during bilayer crystallization. A study on A . luidlawii (Wallace and Engelman, 1978) suggests that although the distribution of exposed protein is affected by lateral phase separation, the spatial distribution of some surface proteins may respond differently than intrinsic membrane proteins to the order-disorder transition. Ferritin-labeling was used to visualize surface proteins of A . tuidluwii in the electron microscope and the order-disorder transition was characterized by X-ray diffraction. As expected, intrinsic proteins visualized by freeze-fracture were found to be dispersed above the membrane transition and to patch progressively as the temperature was lowered through the transition. In contrast, the ferritinlabeled proteins appeared to form patches only at temperatures partially within the transition.

VI.

BIOLOGICAL CONSEQUENCES OF MEMBRANE STATE

There is no doubt that thermotropic transitions have given considerable insight into both structure and function of biomembranes and will continue to do so. It is also tempting to speculate that thermotropic order-disorder transitions play a direct role in the life of the cell. If growth temperature normally were to coincide

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with the temperature range of a transition, the membrane would exist in a partly fluid and partly crystalline state, and lateral phase separation might serve a physiological purpose. However, the information currently available indicates that this is not the case. Although bulk transitions can sometimes occur at growth temperatures, there is evidently no physiological necessity for them to do so. Completion of the membrane melt below growth temperature is common in gram-positive bacteria, such as M . lysodeikticus. Yersiniu enterocoliticu grown at 37°C has its membrane fully melted by 8°C (Abbas and Card, 1980). In B . stearothermophilus, a thermophilic bacterium, the membrane can be completely melted for at least 20°C below growth temperature, and in the extreme thermophile, T . uquaticus, melting is completed about 40°C below growth temperature (McElhaney and Souza, 1976; Melchior and Steim, 1976). Although the bulk melt in wild-type E . coli W945 sometimes is not finished until a few degrees above growth temperature (see Fig. 5a; it begins at about -2O"C), in many cases it is completed 5-10°C below growth temperature. Escherichia coli fatty acid auxotrophs can be forced to a transition that terminates 50°C below growth temperature (Baldassare et ul., 1976). In A . luidluwii cells the transition temperature can be profoundly shifted without affecting the temperature coefficients of growth (Tourtellotte, 1972; McElhaney, 1974), or absolute growth rates at the optimal growth temperature, provided that the transition is not high enough to occur at the temperature of growth. If the transition is too high, growth ceases. The lack of evidence for a unique physiological role of bulk transitions at the temperature of growth does not imply that such phenomena might not be important in specialized regions. Microcrystalline regions might exist, for instance, to a very limited extent even well above the bulk melt of the membrane, and related transitions might be triggered by ions, pH changes, and so forth (Trauble, 1971). But the bulk thermotropic transition and lateral phase separation, as seen by experimental methods now employed, appear to be unnecessary for the life of the cell at growth temperature. On the contrary, it is evidently an effect to be avoided. It is accompanied by a variety of usually undesirable physiological events, and it is clear that living systems take pains to lower their transition range to acceptable temperatures. Physiologically, membrane transitions reveal themselves most obviously by their effects on growth. Cells do not proliferate at temperatures below their transitions (Steim rt ul., 1969; Overath et ul., 1970; Tourtellotte, 1972; McElhaney, 1974; Petit and Edidin, 1974; Thilo and Overath, 1976). At temperatures below the order-disorder transition, where ordinarily fluid membranes are converted to the solid state, viscoelastic properties are drastically altered. In this condition, mechanical compliance is greatly reduced and cells can become osmotically fragile (Tourtellotte, 1972; van Zoelen et d.,1975). In the ordered state, the passive permeability barrier provided by the bilayer can lose its integrity. Leaks of erythritol and intracellular potassium are produced in E . cot! fatty

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acid auxotrophs either by quickly quenching the cells to low temperature or by the mechanical stress of filtration at low temperatures (Haest et ul., 1972). The temperature at which leakage begins depends on the fatty acid composition of the membrane lipids. This behavior is mimicked by liposomes prepared from cell lipids. Similar results have been reported for the passive leakage of o-nitrophenyl galactoside (ONPG) into E. coli unsaturated fatty acid auxotrophs (Steim, 1972). On quick quenching, increased leakage of ONPG into cells invariably begins around the low-temperature end of the calorimetrically observed transition. Both the temperature of the order-disorder transition and its attendant leak can be varied by varying the exogenous fatty acids supplied to the cells. This increased leakage, which reveals itself as an upward swing in Arrhenius plots at low temperature, is not affected by inhibitors of active transport, and occurs to the same extent in cells induced for permease, uninduced cells, and mutants free of permease. ONPG passes into the cells, since there is no release of P-galactosidase activity into the incubation medium. Again the lesion is transient and disappears within a few minutes. The effect in K1060 cells supplemented with oleate is illustrated in Fig. 10. In curve a, the cells were held at room temperature before adding ONPG at lower temperatures; in curve b, the same preparation of cells was first incubated for 3 minutes at 0°C before adding ONPG at higher temperatures. The transient increased leakage, which begins at about 15"C, coincides with the low-temperature end of the calorimeter peak. The fact

"C 25

5

5

15

2-

I-

u) 2

0

w

1-

.5-

3.3

3.4

I/T

3.5

3.6

x lo3

FIG. 10. Passive leakage of ONPG as a function of temperature (T = K ) through membranes of E . coli auxotroph K1060 cells supplemented with oleate. On quick quenching, increased passive leakage of ONPG into cells begins near the low-temperature end of the calorimetric transition (curve a). Curve b represents the same preparation of cells first incubated for 3 minutes at 0°C before adding ONPG at higher temperatures. This increased leakage is most likely a result of microscopic fissures able to heal themselves on a time scale of minutes. (From Steim. 1972.)

MEMBRANE PHASE TRANSITIONS

295

that this leakage occurs only below the bulk transition emphasizes that barrier integrity is maintained by only a small proportion of fluidity. Such leakage is most likely a result of microscopic fissures that can be induced by low-frequency mechanical deformation or simply shrinkage upon cooling. The cracks, which are not large enough to permit the passage of large proteins, are able to heal themselves on a time scale of minutes because the ordered membranes are not perfectly rigid. Some insight into the structure of crystalline membranes at temperatures just below their order-disorder transitions have come from studies of pure dipalmitoyl lecithin bilayers by Lee (1977a), who points out the likelihood of grain boundary defects. Such boundaries, which are commonly recognized to occur in other crystalline solids (Ubbelohde, 1965), occur at interfaces between differently oriented crystal domains, and necessarily give rise to disorder in those regions (Lawaczeck et al., 1975). In bilayers, grain boundaries might be expected to provide sites for increased permeation of ions or small molecules, although it seems unlikely that they are responsible for the cell leakages described earlier in this section because such leaks are transient and increase as temperature falls more and more below the order-disorder transition. Although it is very likely that grain boundaries or other lattice defects occur in crystalline lipid bilayers, it is not clear how extensive they may be, especially in naturally occurring mixtures of lipids. Another kind of boundary effect may occur within the temperature range of membrane melting, where both fluid and solid phases are present. The boundaries in this case are not between domains in the solid state, but at the interface between the fluid and solid regions within the bilayer. In these interfacial regions, the arrangement of lipid molecules would differ from either crystalline or fluid regions. A number of model systems (Papahadjopoulos et a!., 1973; van Dijck et a / . , 1975; Marsh et al., 1976; Blok et af., 1975; Nichols and Miller, 1974) show an increased permeability to small molecules in the neighborhood of the order-disorder transition, as might be expected if lipid conformation were deranged at the liquid-solid interface. Permeability decreases again at temperatures above and below the region of melting. Since increases in permeability in the neighborhood of the order-disorder transition occur in a number of different model systems, the leakage may arise from the same cause in all cases. The details differ, however, from system to system. In dipalmitoyl phosphatidylglycerol liposomes, sodium leakage reaches a maximum rate at the midpoint of the bilayer phase transition (Papahadjopoulos et al., 1973), whereas in equimolar mixtures of dimyristoyl phosphatidylglycerol and dimyristoyl lecithin, leakage rates of potassium peak at the beginning of the transition. In dimyristoyl lecithin liposomes prepared by sonication, Tempo leakage peaks at the upper end of the transition (Marsh et u l . , 1976), which is broadened and lowered in temperature by sonication (Sheetz and Chan, 1972; Melchior and Steim, 1976; Faucon and Lusson, 1973). Elevated permeability during melting does not seem

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to occur ordinarily in cells (McElhaney et al., 1973). The heterogeneous mixture of lipids in natural membranes, which includes an appreciable amount of unsaturated species, may behave quite differently from the lipids used in model studies. Although a totally crystalline membrane will not support cell growth, cells are known to be capable of growing well within the temperature range of the transition, where much of the membrane is crystalline. The point within the transition at which cell division ceases has been carefully measured with A . laidlawii and E . coli. The most elegant work has been carried out with A . laidluwii, which is particularly well suited for such experiments because its transition temperature can be immensely varied by diet. Acholeplusmu luidlawii shows changes in gross morphology and growth that correlate with the melting point of the fatty acid incorporated into its membrane lipids (Razin et al., 1966; McElhaney and Tourtellotte, 1969; Tourtellotte, 1972). Raised on oleate or other low-melting acids, the cells are filamentous and growth is rapid, but a diet of palmitate or stearate can cause swelling and eventual lysis. Growth ceases even before lysis, but incorporation of saturated fatty acids into the lipids continues until lysis, so that the membrane transition in badly swollen cells can occur entirely above growth temperature. As temperature decreases, the melting points of the fatty acids required for growth also decrease. The swelling experienced by cells with high transitions is osmotic in origin (Tourtellotte, 1972), and at low temperatures cells enriched in high-melting fatty acids become fragile and no longer behave as osmometers (van Zoelen et al., 1975). Rough correlations of growth and morphology with calorimetry (Steim er al., 1969) reveal that filamentous shapes are associated with transitions that are complete below growth temperature, swollen cells with transitions that encompass growth temperatures, and lysis with transitions above growth temperature. As fatty acids of higher melting point are incorporated, the calorimetrically detectable membrane transition rises, but growth continues even when much of the membrane is in the ordered state. This conclusion has also been reached by deuteron-resonance experiments (Oldfield er al., 1972) and is consistent with ' T - N M R and Tempo partitioning (Metcalfe et ul., 1972). More precise correlations of growth with fatty acid composition and membrane transitions observed by differential thermal analysis (DTA) reveal that the absolute growth rates at optimal growth temperatures and the apparent temperature characteristics of growth (the slope of an Arrhenius-type plot) are independent of fatty acid supplement above the transition until the growth temperature is lowered to about the midpoint of the transition (McElhaney, 1974). At this point, the temperature characteristic changes abruptly until, close to the lowtemperature end of the melt, growth ceases entirely. Judged by areas under the thermal analysis peaks, regardless of the fatty acid supplement, growth does not stop until about 90% of the membrane is crystalline. Thus growth continues at a reduced rate at temperatures below the approximate midpoint of the melt until only about 10% of the membrane remains fluid.

MEMBRANE PHASE TRANSITIONS

297

The same results are obtained in E . coli (Steim, 1970; Melchior and Steim, 1976). Ordinarily, wild-type cells have a very broad transition entirely o r almost entirely below growth temperature. Figure 5 , curve a is typical. Grown in the presence of 3-decynoyl-N-acetylcysteamine (DNAC), which inhibits unsaturated fatty acid synthesis (Kass, 1968), the membranes undergo a sharpened transition at elevated temperatures, shown in Fig. 5 , curve c . By varying the concentration of DNAC and correlating growth with calorimetry, one can determine the extent of transition compatible with growth. For sublethal concentrations of DNAC, cell division continues, even though the majority of the calorimeter peak is above growth temperature, until the thermogram in Fig. 5, curve c is obtained. Judged from areas under peaks, the membrane is again about 5-10% fluid and 95-90% ordered. In the state characterized by Fig. 5 , curve c the cells have been maintained by serial passage for 100 generations, but higher concentrations of DNAC elevate the transition even more, and growth ceases. DNAC-treated cells grown in oleate grow normally and have lower transition temperatures. Identical results, with cell division ceasing at about 90% crystallinity, were obtained with fatty acid auxotrophs fed elaidate. During membrane assembly, newly synthesized lipids and proteins may be inserted into the fluid portions of the bilayer and, after lateral diffusion, take their position in the membrane. New membrane would no longer be formed when the transition temperature rises so high that fluid sites are no longer available (Tsukagoshi and Fox, 1973). This could happen before completion of the order-disorder transition seen by physical methods, since the fluid regions remaining when cell division stops could be distributed above and through islands of crowded protein aggregates, or could be sprinkled randomly about the membrane in many small patches in inappropriate places, or could even exist on one side of the bilayer. At temperatures below the bulk bilayer transition, ordinarily fluid membranes are converted to an ordered state. And as previously discussed, not only are membrane proteins put into abnormal environments, but entiremembrane viscoelastic properties are drastically altered. A concept related to transitions is the idea that cells may find it advantageous to control the viscosity of their membrane bilayers by proper choice of fatty acids, even above transitions when the membranes are in an entirely fluid state, so that constant viscosity is maintained at any growth temperature (Sinensky, 1974). For example, in unsaturated fatty acid auxotrophs of E . coli, the dependence of the activation energies of some membrane-bound enzymes on the fatty acids that the membranes contain (Mavis and Vagelos, 1972) could reflect a viscous effect. Although it is certainly true that under normal conditions cells manipulate the fatty acid composition of their membranes in order to suppress the temperature of transition and maintain a fluid state, and though it may be true that viscosities in fluid membranes of many organisms have similar magnitudes, a true homeostatic control of fluidity as such does not appear to occur or does not appear to be necessary. The data already discussed suggest that variations in

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DONALD L. MELCHIOR

membrane viscosities over a wide range have little effect on the rate of growth of A . laidlawii. Absolute growth rates of A . laidlawii at optimal growth tempera-

tures and the apparent temperature characteristics of growth are independent of fatty acid supplement above the transition until the growth temperature is lowered to about the midpoint of the transition. Though absolute growth rates and temperature characteristics of growth are identical, it is unlikely that viscosities at 36°C are also identical in membranes greatly enriched in isostearate (where the transition begins at 20°C and terminates about 5°C above growth temperature), and in membranes rich in straight-chain heptadecanoic acid (where the transition starts at 0°C and terminates about 20°C below growth temperature) (McElhaney, 1974). Furthermore, the temperature characteristics of growth routinely remain unchanged well into the bulk transitions, where one might expect rather drastic viscosity changes. It has been shown (Sinensky, 1974) that apparent viscosities seen by methyl-1 2-nitroxylstearate in wild-type E . coli membranes are similar in magnitude, provided that the electron spin resonance (ESR) spectra are taken at the temperature of growth. This may be a special case, however, since transitions in wild-type E . coli are broad and ordinarily terminate in the neighborhood of growth temperature. At that temperature ESR patterns might indeed resemble one another and would be drastically different at any lower temperature, since at lower temperatures the membranes would be undergoing a transition. Thus what would appear to be a homeostatic control of viscosity might merely be a reflection of a more fundamental process, the cellular control of transition temperatures. Nevertheless, there is some evidence from A . laidlawii, B . stearorherinophilus, and Y . enterocolirica, indicating that although rigorous control of fluidity above a transition is not especially advantageous to growth, there might be an upper limit to the fluidity that cells will tolerate. In studies on A . laidlawii (McElhaney, 1974), optimal growth temperatures were 36°C for all fatty acid supplements, independent of the transition temperatures observed by DTA, except for oleate and linoleate. For these two supplements, with respective transition midpoints at -13 and -19"C, optimum growth occurred at 34 and 32"C, respectively. Stearate membranes were a special case, since their transition was so high that growth was abnormally slow even at 38°C. Thus at 37"C, slow growth in stearate correlates with a very high transition, whereas slow growth in oleate and linoleate correlates with a very low transition. For intermediate transitions, growth is faster and constant. Bacillus stearotherrnophilus wild-type cells (Reizer, 1978) increase the melting temperature of their membranes as growth temperature is increased. These cells adjust the membrane transition so that they grow near, but slightly above, its upper end. As in other organisms, an increased transition temperature is brought about by incorporation of higher melting fatty acids into membrane lipids. In the case of wild-type B . stearothermophilus, this is accomplished primarily by an increase in palmitate and stearate

299

MEMBRANE PHASE TRANSITIONS

relative to the lower melting branched-chain and unsaturated fatty acids. All these phenomena are illustrated by Fig. 11 (Reizer, 1978). A mutant of B . stearothermophilus, TS- 13, cannot increase the temperature of completion of its transition beyond 40°C (McElhaney and Souza, 1976). The membranes of TS-13 cells are fully melted at 40°C. From 42 to 52°C this mutant grew nearly as well as wild-type cells. When growth temperature was raised still further, cell growth ceased abruptly at about 6 0 T , 20" above the completion of the membrane transition. Since the wild-type cells grew normally in this temperature range, this may again represent an upper limit on the degree of membrane fluidity compatible with cell growth. Another possible example of an upper limit to membrane fluidity is seen in Y . enterocolitica (Abbas and Card, 1980). When grown at 3 7 T , this organism has a membrane transition extending from -18 to 8"C, whereas cells grown at 22°C have a transition extending from -24 to 4°C. When 37°C cultures were shifted to 45"C, good growth was observed. However, when 22°C cultures were shifted directly to 45"C, they failed to grow.

VII.

BIOLOGICAL CONTROL

A fluid or at least partially fluid lipid bilayer seems to be essential for cellular function. Abnormally high transition temperatures reflect abnormally crystalline membranes and are associated with cell leakage, changes in active transport and

0.50

0.75

M o l e F r a c t i o n (C,,:,+

0

C,8:o)

FIG. 1 I , The effect of growth temperature on the membrane fatty acid composition and phase transition of B . stearothertnr~pkilus.As growth temperature (@) is decreased, the temperature of the upper (X)and lower (0)ends of the transition is lowered. This results from a reduction in the mole fraction of high-melting-point fatty acids (palmitate and stearate) in favor of lower melting point fatty acids, mostly branched-chain. In all cases the transition is complete at growth temperature. (From Reizer, 1978.)

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DONALD L. MELCHIOR

some membrane-associated enzymatic activities, prolonged generation times, and eventual loss of viability and even cell death. Since transition temperatures depend primarily on the fatty acid composition of membrane lipids, low transition temperatures are assured by the biosynthesis of appropriate fatty acids or their selection from exogenous sources. In addition to straight-chain saturated fatty acids, to maintain low transition temperatures membrane lipids must also contain some fatty acids possessing lower melting points. Furthermore, the composition must be responsive to temperature in such a way that the membrane is totally or almost totally fluid at the temperature of growth. This necessity requires a control mechanism, optimally one that senses temperature and the physical state of the membrane and directs the incorporation of proportionally more unsaturated or other low-melting fatty acids into membrane lipids as the temperature decreases. Such control is seen in higher organisms (Irving et a/., 1956; Johnston and Roots, 1964; Rose, 1967). but is particularly important in prokaryotes (Marr and Ingraham, 1962) and other microorganisms in which the membrane can crystallize near growth temperature. For example, Melchior et al. ( 1 970) found by calorimetry that the temperature range of melting in A . luidlawii B , grown i n ordinary tryptose medium at 37"C, was in the neighborhood of growth temperature. At 37°C the membranes were mostly fluid, but at 25°C the membranes of cells grown at 37°C became almost completely crystalline. However, if the same organism was grown at 25°C the melting range was shifted down so that again the membranes were mostly fluid at growth temperature. A similar phenomenon has been observed calorimetrically in other prokaryotes, such as E . ~ ~ (Steim, d i 1972), B . steurotherrnophilus (Reizer, 1978), and Y . enrerocolitica (Abbas and Card, 1980). In addition to actively maintaining a fluid bilayer, it has recently been proposed that cells regulate their membrane lipid class composition in order to maintain them in a stable bilayer conformation (Wieslander et d.,1980). Although the detailed mechanism or mechanisms for temperature modulation of membrane fatty acid composition have not been worked out in any organism (Fulco, 1973). it has become clear that control can take place at several, possibly interrelated, levels. In some cases, desaturase activity appears to be governed by the solubility of oxygen, which serves as an eventual electron acceptor (Brown and Rose, 1969). In others, such as B . rneguterium (Fulco, 1970), enzyme synthesis is affected by temperature. Fatty acid desaturase is not synthesized in this organism at 35°C but is strongly induced at 20°C. Temperature also has a direct effect on the desaturase protein itself, which, once synthesized at low temperature, undergoes rapid irreversible inactivation at higher temperatures. Direct temperature effects on activity have also been found in E . coli, which produces monoenoic fatty acids via dehydration of the growing acyl chain within the fatty acid synthetase system itself. Deprived of glycerol in order to uncouple phosphatidic acid synthesis from fatty acid synthesis, E . coli accumulates large

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301

quantities of free fatty acids, which become proportionately more unsaturated as temperature is decreased (Cronan, 1975). A reversible dependence of P-hydroxy-decanoyl thioester dehydrase on temperature may be implicated. In addition to effects of temperature on the biosynthesis of unsaturated fatty acids, another level of control is apparently at the site of phospholipid synthesis in the membrane. Temperature-dependent selection of saturated and unsaturated fatty acid CoA by membrane-bound acyltransferase, which catalyzes the esterification of glycerophosphate, has been demonstrated by Sinensky (1971). Presented with a mixture of oleoyl- and palmitoyl-CoA, cell-free E . coli acylCoA:glycerophosphate acyltransferase produces increasingly greater proportions of unsaturated lysophosphatidic acid at lower temperature. The acyltransferase apparently possesses a “preprogrammed” selective temperature response. It is the molecular nature of this temperature “program,” and its possible interrelationships with fatty acid biosynthesis, that we shall consider in more detail. Crucial to the understanding of the temperature-dependent selection process at the membrane level is the realization that fatty acids seem to be selected on the basis of melting point, a thermodynamic property that only indirectly reflects molecular structure. Although unsaturation is the usual route to low melting point, the same goal is attained in many organisms by employing structural alternatives, such as branched chains in many gram-positive bacteria (Wakil, 1970). This is demonstrated in Fig. 11 for B . stearotherrnophilirs, which when grown at progressively lower temperatures reduces the mole fraction of its membrane straight-chain saturated fatty acid content. Saturated fatty acids, palmitate and stearate, are replaced by lower melting point fatty acids, primarily branched-chain. In this manner B . steurothermc~philusis able to keep the onset of its membrane transition below its growth temperature (Reizer, 1978). Another route for membranes to attain lower temperature transitions is to incorporate shorter chain fatty acids into their membrane lipids. A striking example of this occurs in the psychrophilic Microcvccus cryophilus (Russell, 197 1). This prokaryote has in its membrane a very high percentage of the monounsaturates, octadecenoic and hexadecenoic acids. When its growth temperature is reduced from 20 to O’C, the total membrane content of these fatty acids does not change but the ratio of the high melter (octadecenoic acid) to the low melter (hexadecenoic acid) goes down by a factor of 4. Yet another strategy used by prokaryotes to introduce lower melting point fatty acids into their membranes at reduced growth temperature is by the substitution of lower melting point unteiso-branched-chain fatty acids for higher melting point iso-branched-chain fatty acids. I n a study using four temperature-range variants of B . meguteriurn over a temperature span of 65”C, it was found that as growth temperature was lowered the dominant change in membrane fatty acid content was the substitution of anteiso- for iso-branched-chain fatty acids (Rilfors er ul., 1978). The ratio of the higher melters to lower melters decreased progressively with decreasing

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growth temperature, and at 5°C was about 12 times lower than at 70°C. Chain shortening also occurred with decreasing growth temperature. If the fatty acids are classified into long- and short-chain categories, there is a progressive decrease in the long-chain to short-chain ratio with decreasing temperature, the ratio being five times smaller at 5°C than at 70°C. A convincing argument that the principal consideration of fatty acid selection is thermodynamic rather than structural is based on the fact that a given organism, if forced to do so, will choose any low-melting exogenously supplied fatty acid to accomplish its goal of lowering transition temperatures. The best illustration is again A . laidlawii (McElhaney and Tourtellotte, 1969; Melchior et al., 1970; Tourtellotte, 1972), which lacks desaturase activity and, as pointed out earlier, incorporates large amounts of exogenous fatty acids into its membrane lipids. Fatty acids of progressively lower melting points are required as the growth temperature is decreased. Cis-unsaturates serve the purpose admirably even at the lowest temperatures, but growth is also normal if cis-unsaturates are replaced by branched-chain or cyclopropane fatty acids or by elaidate, an unnatural trans-unsaturated compound. Unsaturated fatty acid auxotrophs of E . coli show similar behavior, and will accept elaidate or even bromostearate (Schairer and Overath, 1969; Fox e t a / . , 1970). If in fact the temperature-sensing selection mechanism within the membrane is thermodynamically determined and depends on melting point, which is a bulk phenomenon, rather than on the chemical structure of the lipid, it is difficult to imagine it to be based on enzyme specificity. The binding of substrates to enzymes reflects the molecular structure of the ligand, and interaction occurs on a one-to-one basis, so that strictly thermodynamic properties have no meaning in such interactions. In accord with this thermodynamic point of view, it is proposed that the temperature program of acyltransferase in A . Iuidlawii, and in some other organisms as well, is an innate property of the bilayer in which the enzyme is embedded rather than a property of the protein itself. Some rather strong evidence supports this novel suggestion. In A . laidlawii cells, the pattern of esterification of palmitate and oleate from the incubation medium into the membrane polar lipids closely parallels the physical state of the membrane bilayer as determined calorimetrically (Melchior and Steim, 1976, 1977). Furthermore, the physical binding of free fatty acids to lipid bilayers formed from total extracted membrane lipids shows the same temperature dependence shown by the enzymatic process in live cells. This effect is seen in Fig. 12. Calorimetry of membranes grown in tryptose at 37°C produce the profile seen in Fig. 12, curve b, which, as the integral of the raw calorimeter peak, is a rough measure of the extent of membrane melting. The membranes are mostly fluid, but not completely fluid, at growth temperatures. Figure 12, curve a is a plot of the ratio of palmitate to oleate, both exogenously supplied, which are incorporated into membrane lipids when aliquots of cells grown at 37°C are briefly exposed to lower temperatures.

MEMBRANE PHASE TRANSITIONS

303

0

c

0

.o

" Q)

c X

0

20

40

ternperature('C)

Frc. 12. The membrane bilayer as a selector of fatty acids. Correlations between (curve a) the palmitate/oleate ratio incorporated into membrane lipids of A . luidlawii as a function of temperature; (curve b) the extent of the membrane transition; and (curve c) the palmitate/oleate ratio of fatty acids physically bound to bilayers of extracted membrane lipids as a function of temperature. Both incorporation and binding curves reflect the state of the bilayer, and are identical within experimental error. (From Melchior and Steim, 1977.)

The close correlation of the two curves could suggest that the conformation of the acyltransferase protein is somehow affected by the extent of fluidity of the membrane so that its affinity for palmitate increases relative to oleate with increasing temperature. However, hypothetical changes in enzyme specificity need not be involved. Fatty acid binding by liposomes formed from total membrane lipids, shown in Fig. 12, curve c , mimics the temperature-dependent selectivity of real cells. The agreement between the extent of melt and the pattern of uptake is not fortuitous, since changing the temperature range of melting produces a parallel change in both incorporation and binding. The ability to act as a temperature sensor and selector may be a general property of any phospholipid bilayer. Although lecithin is not found in A . luidlawii, bilayers prepared from lecithin mixtures showed similar correlations between the extent of bilayer melt and selective fatty acid binding. The consistent correlations between the physical state of the membrane bilayer, the binding of fatty acids by extracted lipids, and the incorporation of fatty acids into membrane lipids in live cells provide a new mechanism, based upon thermodynamic principles, for the temperature program of acyltransferase

304

DONALD L. MELCHIOR

activity. As temperature is lowered, an increased amount of oleate relative to palmitate is accepted by the bilayer, where it is acted upon by the resident acyltransferase. If the cells are allowed to grow at the lower temperature, the membrane transition will be shifted to lower temperatures as well. The detailed response to temperature will depend upon the shape of the bilayer binding curve. The acyltransferase proteins, although membrane-bound in A . laidluwii (M. Tourtellotte, personal communication) as they are in E . coli, are not required to sense the physical state of the membrane or to distinguish between different fatty acids. They may accept and use essentially any fatty acid molecules presented to them. The temperature-programmed selectivity, and hence the control of membrane-transition temperatures, resides in the bilayer itself. From another point of view, the acyltransferase enzymes are not simply proteins but proteins embedded in a bilayer. The catalytic function is assumed by the protein and the selectivity by the lipids. Similar considerations might also account for positional specificity, since the melting point of the acid esterified to the @carbon is usually lower than that on the a-carbon of the phospholipid (Hildebrand and Law, 1969; McElhaney and Tourtellotte, 1970; Okuyama et ul., 1976). Although the model just suggested is based upon the uptake of exogenous fatty acids by A . fuidluwii, the same model might be generalized to explain at least some temperature effects on the composition of membrane lipids synthesized from endogenously biosynthesized fatty acids (Melchior and Steim, 1978, 1979). That an interrelationship exists between selectivity at the membrane level and the synthetase system is demonstrated in E . coli cells, which produce longer chain fatty acids when uncoupled from phospholipid synthesis than when lipid synthesis is allowed to take place normally (Cronan, 1975). The intermediary for linking synthesis and desaturation of fatty acids with lipid synthesis in the membrane may be the bilayer as a temperature-dependent selective sink. Sumper and Trauble (1973) have in fact verified that long-chain acyl-CoA molecules will bind to and dissolve in dimyristoyl lecithin bilayers and E . coli membranes, where they are free to diffuse about and encounter appropriate membrane-bound enzymes. Furthermore, the efficacy of bilayers as an acceptor for fatty acyl-CoA is known to depend upon environmental conditions. Phosphatidic acid becomes an increasingly effective acceptor as ionic strength is increased, presumably because of charge neutralization by counter ions (Sumper, 1974). Since environmental temperature also modifies the efficacy of bilayers as acceptors (Melchior and Steim, 1976, 1977), such modification may provide the basis for temperature-dependent control. Consider the regulation of desaturase activity, and suppose that a desaturase exists that competes with the bilayer for the saturated end product of fatty acid synthetase. If at low temperatures the bilayer is in a relatively crystalline state that does not readily bind saturated fatty acids, the substrate will be operated upon instead by the desaturase. The resulting unsaturated fatty acid can now

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easily enter the bilayer and be incorporated into the membrane lipids. As the bilayer becomes more fluid, it more successfully competes with the desaturase for saturated fatty acid, and the spectrum of fatty acids entering the membrane reservoir shifts toward increasing saturation. A case of control of fatty acid unsaturation by the thermodynamic state of the membrane has been reported in T . pyrifr,rmis (Martin c'/ m l . , 1976; Kasai ot m l . , 1976). A second thermal effect that might be explained by the concept of the bilayer as a temperature-programmed sink is the shortening of biosynthesized fatty acids at lower growth temperatures. In addition to the previously described case of M . cryophilus, shortening occurs in many organisms, including A . IuidIm'ii deprived of exogenous sources of fatty acids (M. Tourtellotte, personal communication). The progressive tendency already demonstrated in A . lmicllaw~iifor the bilayer to accept relatively more unsaturated than saturated fatty acids as temperature is lowered is again central to the argument. If in fact thermodynamic properties rather than specific molecular structure are the predominant factor affecting acceptability of a fatty acid derivative by the bilayer, one might expect that shorter chain molecules, like unsaturated long-chain ones, would at low temperature be more acceptable than saturated long-chain ones. Long-chain saturated molecules that are not accepted could be desaturated before being accepted. An example of fatty acid synthesis linked to the properties of a bilayer acceptor is provided by in virro studies on the fatty acid synthetase of Mycobacteriutn smrgmatis (Odriozola and Bloch, 1977). This enzyme system is unusual in that it produces a bimodal product pattern of fatty acid acyl-CoAs, short-chain-length C16-C18 CoAs and longer chain length C,,-C2s CoAs. In experiments on the effect of added dimyristoyl lecithin bilayers upon the chain lengths of the fatty acids synthesized by this system, the effect of the bilayers was found to be slight in the temperature range below and near the transition temperature, but very marked above it. At higher temperatures, and with no added lecithin bilayers, a large portion of C24-C28fatty acids are ordinarily synthesized. However, in the presence of dimyristoyl lecithin bilayers at temperatures above the lipid's transition temperature, these long-chain acids were not synthesized. It appears that the fatty acid sink provided by the melted-lipid bilayers allows an earlier chain termination for the fatty acid synthetase end product. An alternative possibility for linking fatty acid chain length to the properties of a bilayer acceptor, this time through enzyme inhibition, is suggested by the work of Sumper (1974). Dimyristoy1 lecithin, acting as a fatty acid-CoA sink, reverses the inhibition of fatty acid synthesis from acetyl-CoA in a system containing fatty acid synthetase and acetyl-CoA carboxylase from yeast. The promotion of synthesis by added lecithin apparently arises from competitive reversible binding of palmitoyl- or stearoyl-CoA by the lipid bilayers and acetyl-CoA carboxylase. Furthermore, fatty acid chain length depended on inhibition of carboxylase by palmitoyl-CoA. Increased inhibition led to an increased rate of synthesis of fatty acids of shorter

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chain lengths. Although Sumper directed his attention toward an explanation for the chain-shortening effect of anaerobiosis, the same point of view can be extended to thermal effects if the temperature-dependent properties of the bilayer sink are kept in mind. At lower temperatures, as relatively more longer-chain saturated CoA molecules are excluded from the bilayer and accumulate, acetylCoA carboxylase inhibition by the accumulated longer-chain compounds would cause a shift toward the biosynthesis of shorter chains. From the aforementioned studies it appears that in some cases the physical state of the membrane bilayer can provide a temperature-sensitive mechanism to control the types of fatty acids incorporated into membrane lipids. Thus it is postulated that membranes may possess the ability to “self-control” their physical state. From one point of view, it is proposed that the bilayer be considered a temperature-programmed acceptor. Accordingly, the types of fatty acids incorporated at any temperature into membrane lipids by resident membrane enzymes are proposed to be those that enter the membrane-bilayer phase. These could partition into the bilayer phase from free solution or micellar aggregates, from a cytoplasmic enzyme, or from a carrier protein. Once the fatty acid or its derivative has entered the bilayer, a transfer mechanism involving lateral diffusion in two dimensions within the plane of the membrane would carry it to the subsequent membrane-bound enzyme. Such lateral diffusion in two dimensions within the membrane, combined with free diffusion in three dimensions within the cytosol, permits much faster transfer of molecules from the cell cytoplasm to a small target on the cell membrane than is provided by free diffusion alone (Sumpcr and Trauble, 1973; Adam and Delbriick, 1968). If, conversely, attention is focused on fatty acids or fatty acid derivatives that are excluded from the membrane bilayer but play a role in regulating the biosynthesis of fatty acids by cytoplasmic enzymes, it is proposed that the bilayer can be looked on as a tPmperururr-programmed sink. In this type of regulation, control would reside in the temperature-sensitive ability of the bilayer to selectively remove end products from the fatty acid synthetase system or compete f o r these products with such enzymes as desaturases, chain-elongation enzymes, or acetyl-CoA carboxylases. This type of control, which involves a multienzyme complex and competing acceptors, clearly differs from classical feedback inhibition (Bloch, 1977). For the feedback mechanism postulated here, end products need not bind to the enzyme component catalyzing the committed or earliest step of the pathway. However, the wasteful accumulation of intermediates for which no alternative routes are available is avoided as effectively as by conventional feedback. The functions of the membrane bilayer as acceptors and sinks are interrelated, of course, and in a certain sense separating the two functions is an artificial imposition done for the sake of clarifying this proposed mechanism. Taken together, the two functions-properly emphasized for the appropriate case at

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hand-provide a sensitive, unified means to control the fatty acid composition and hence the physical state of biological membranes, whether fatty acids are supplied exogenously in the growth medium or are endogenously biosynthesized by the organism. ACKNOWLEDGMENTS The author is grateful to Professor J . M. Steim for reading this article in manuscript. He would like to thank Genevieve D. Goditt for her help in preparing this manuscript and gratefully acknowledges the permission of other authors for use of their illustrations. D. L. M. is the recipient of an American Diabetes Association, Inc., Research and Development Award. REFERENCES Abbas, C . A , , and Card, G. L. (1980). The relationship between growth temperature, fatty acid composition, and the physical state and fluidity of membrane lipids in Yersinia enterocolitica. Biochim. Biophys. Actu 602, 469-477. Adam, G., and Delbruck, M . (1968). Reduction of dimensionality in biological diffusion processes. In “Structural Chemistry and Molecular Biology” (N. Davidson and A. Rich, eds.), pp. 198-215. Freeman. San Francisco. Ashe, G . B., and Steim, J . M. (1971). Membrane transitions in gram-positive bacteria. Biochim. Biophvs. Actu 233, 810-814. Baldassare, J . J . , McAfee, A. G., and Ho, C . (1973). A spin label study of E . coli membrane vesicles. Biochem. Biophys. Res. Commuri. 53, 617-623. Baldassare, J . J . , Rhinehan, K. B., and Silbert, D. F. (1976). Modification of membrane lipid: Physical properties in relation to fatty acid structure. Biochemistry 15, 2986-2994. Bevers, E. M., Singal, S . A., Op den Kamp, J. A. F., and van Deenen, L. L. M . (1977). Recognition of different pools of phosphatidylglycerol in intact cells and isolated membranes of Achofeplrrsma Luidluwii. Biochemistry 16, 1290- 1295. Bevers, E . M., Op den Kamp. J . A.F., and van Deenen, L. L. M. (1978). Physical Chemical Properties of Phosphatidylglycerol in Membranes of Acholeplusma laidlawii. E u r . J . Biochem. 84, 35-42. Bevers, E. M . , Wang, H. H . , Op den Kamp, J . A. F., and van Deenen, L. L. M . (1979). On the interaction between intrinsic proteins and phosphatidylglycerol in the membranes of Acholeplasma laidlnwii. Arch. Biochem. Biophys. 193, 502-508. Birrell, G . B., and Griffith, 0. H. (1976). Cytochrome c induced lateral phase separation in a diphosphatidylglycerol-steroidspin label model membrane. Biochemisrry 15, 2925-2929. Bittman, R., and Blau, L. (1972). The phospholipid-cholesterol interaction. Kinetics of water permeability in liposomes. Biochemistry 11, 4831 -4839. Blazyk, J . F., Melchior, D. L., and Steim. J . M. (1975). An automated differential scanning dilatometer. Anal. Biochem. 68, 586-599. Bloch, K. (1977). Control mechanisms for fatty acid synthesis in Mycobacteriurn smegmaris. Adv. Enzymol. 45, 1-84. Blok, M. C . , van der Neut-kok, E . C. M., van Deenen, L. L. M . , and de Gier, J . (1975). The effect of chain length and lipid phase transitions on the selective permeability properties of liposomes. Biochim. Biophys. Actu 406, 187-196. Boggs, J . M., Wood, D. D.. Moscarello, M. A . , and Papahadjopoulos, D. (1977). Lipid phase separation induced by a hydrophobic protein in phosphatidylserine-phosphatidylcholinevesicles. Biochemr.\t~y 16, 2325-2329.

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Nikaido, H., and Nakae, T . (1979). The outer membrane of Gram-negative bacteria. A t h . Mic.rob. Physiol. 20, 164-250. Odriozola, J. M., and Bloch, K . (1977). Effects of phosphatidylcholine liposomes on the fatty acid synthetase complex from Mvcohnctrrirrm smegmutis. Biochim. Biophys. Acru 488, 198-206. Okuyama, H., Yamada, K . , Ikezawa, and Wakil, S. J . (1976). Factors affecting the acyl selectivities of actyltransferases i n Eschrrichiu coli. J . Biol. Ckrm. 251, 2487-2492. Oldfield, E., Chapman, 0 . .and Derbyshire, W . (1972). Lipid mobility in A(holepla.sma membranes using deuterium magnetic resonance. Chem. Phys. Lipids 9, 69-81. Onishi, S . , and Ito, T . (1974). Calcium-induced phase separations in phosphatidyl-serine - phosphatidylcholine membranes. Riochemistn 13, 881 -887. O p den Kamp. J . A. F. (1982). The lipid phase transition of phosphatidylglycerol in Acho/ep/usmu l ~ i d l a ~membranes ii studied with phospholipase A2. R r v . Infrcf. Dis. 4, Supplement 3 Overath, P., and Thilo, L. (197X). Structural and functional aspects of biological membranes revealed by lipid phase transitions. MTP I r i t . Rev. Scr.: Biochem., S r r . Two 19, 1-44. Overath, P . , and Trauble, H . (1973). Phase transitions in cells, membranes, and lipids of Eschrrichio c d i . Detection by fluorescent probes, light scattering, and dilatometry. Biochemisrn 12, 2625-2634.

Overath, P., Shairer, H. V., and Stoffel. W. (1970). Correlation of in iivu and iti virro phase transitions of membrane lipids in 6sc.herichiu c,o/i. Proc. Natl. Acad. Sci. U . S . A . 67, 606-612.

Overath, P., Hill, F. F., and Lammek-Hirsch, 1. (1971). Biogenesis of E. c d i membrane: Evidence for randomization of lipid phase. Nurure (London), NPW B i d . 24, 264-267.

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Overath, P., Brenner, M.. Gulik-Krzywicki, T., Schechter. E., and Letellier, L. (1975). Lipid phase transitions in cytoplasmic and outer membranes of Eschcr-ichia coli. Biochim. Biophys. A C ~ U 389, 358-369. Papahadjopoulos, D.,Jacobson, K.. Nir, S., and Isac. T (1973). Phase transitions in phospholipid vesicles. Fluorescence polarization and permeability measurements concerning the effect of temperature and cholesterol. Biochim. Bioph\s. Acru 311, 330-348. Papahadjopoulos, D., Moscarello. M.. Eylar. E. H., and Isac, T. (1975a). Effects of proteins on thermotropic phase transitions of phospholipid membranes. Bioc.hiru. Biophy.5. A m r 401, 317-335. Papahadjopoulos, D., Vail, W . J . , and Moscarello, M. (1975h). Interaction of a purified hydrophobic protein from myelin with phospholipid membranes. Studies on ultrastructure, phase transitions and permeability. J . M e m h . Biol. 22, 143-164. Petit, V. A , , and Edidin, M. (1974). Lateral phase separation of lipids in plasma membranes: Effect of temperature on the mobility of membrane antigens. Science 184, 1183-1 185. Phillips, M. C., Ladhrooke, B. D., and Chapman, D. (1970). Molecular interactions in mixed lecithin systems. Eiochitn. Biophw. Acrrr 196, 35-44. Phillips, M. C . , Hauser. H . , and Paltauf. F. (1972). The inter- and intramolecular mixing of hydrocarbon chains in lecithiniwater systems. Chetn. Ph\.s. Lipids 8, 127-133. Poo, M.-M.. and Cone, R. A. (1974). Lateral diffusion of rhodospin in the photoreceptor membrane. Nature (Londoir) 247, 438-441. Razin, S . (1969). Structure and function of mycoplasma. Atinu. Re\’. Micruhiol. 23, 317-356. Razin, S. (1975). The mycoplasma membrane. Prog. Sut$ Menrbr, Sci. 9, 257-312. Razin, S. (1978). The mycoplasmas. Micruhiol. Rev. 42, 414-470. Razin, S . , Tourtellotte, M. E . , McElhaney, R. N . , and Pollack, J . D. (1966). Influence of lipid components of Myc~opI“r.smuluiclluwii membranes on osmetic fragility of cells. J . Bac[eriul. 91, 609-616. Razin, S . , Kutner. S., Ephrati, H., and Rottem, S. (1980). Phospholipid and cholesterol uptake by mycoplasma cells and membranes. Biochim. B i o p h ~ sActa 598, 628-640. Reinert, J . C . , and Steim, J . M . (1970). Calorimetric detection of a membrane-lipid phase transition in living cells. S(~ierrce168, 1580-1582. Reizer, J . (1978). Thermotropic properties and function of the membrane of a therniophilic bacillus. Ph.D. Thesis, Hebrew University, Jerusalem, Israel. Rilfors, L . , Wieslander, A . , and Stahl. S . (1978). Lipid and protein composiiion of membranes of Bacil1u.c megurerium. Variants in the temperature range 5 to 70°C. J . Bacteriol. 135, 10431052. Rose, A. H , , ed. (1967). “Thermobiology.” Academic Press, New York. Rottem, S . (1980). Membrane lipids of mycoplasmas. Biochim. Biophys. Acru 604, 65-90, Rottem, S . , and Samuni, A. (1973). Effect of proteins on the motion of spin-labeled fatty acids in mycoplasma membranes. BkJChittl. Biophvs. Acru 298, 32-38. Rottem, S . , Hubbell, W . L., Hayflick, L., and McConnell, H. M . (1970). Motion of fatty acid spin labels i n the plasma membrane of mycoplasma. Biochim. Biuphys. Acm 219, 104-1 13. Rottem, S . , Yashouv, J . , Ne’enian, Z . . and Razin, S. (1973a). Cholesterol in mycoplasma membranes. Composition, ultra-structure and biological properties of membranes from M w u plasma Mycoides var. m p r i cells adapted to grow with low cholesterol concentrations. Biochim. Biophys. Actu 323, 495-508. Rottem, S . , Cirillo, V . P.. de Kruyff, B.. Shinitzky, M., and Razin, S . (1973b). Cholesterol in mycoplasma membranes, correlation of enzymic end transport activities with physical state of lipids in membranes of Myc~op/a,st~ru rnycnides var. cupri adapted to grow with low cholesterol concentrations. Riochitrr. Biophys. Actu 323, 509-5 19. Russell, N. J . (1971). Alteration in fatty acid chain length in Micrococcus cryophilus grown at different temperatures. Biochim. Bioph\s. Acra 231, 254-256.

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Sackmann, E., Trauble, H., Galla, H . , and Overath, P. (1973). Lateral diffusion, protein mobility, and phase transitions in Escherichiu coli membranes. A spin label study. Biochemistry 12, 5360-5369. Schairer, H. V . , and Overath, P. (1969). Lipids containing rruns-unsaturated fatty acids change the temperature characteristic of thiomethylgalactoside accumulation in Escherichia c d i . J . M o l . B i d . 44, 209-214. Schechter, E . , Gulik-Krzywicki, T . , and Kaback, H. R. (1972). Correlations between fluorescence, X-ray diffraction, and physiological properties in cytoplasmic membrane vesicles isolated from Escherichiu coli. Biochim. Biophys. Aetu 274, 466-477. Schechter, E., Letellier, L., and Gulik-Krzywicki, T . (1974). Relations between structure and function in cytoplasmic membrane vesicles isolated from an Escherichiu culi fatty-acid auxotroph. Eur. J . Biochem. 49, 61-76. Shah, D. O., and Schulman, J . H. (1967). Influence of calcium, cholesterol, and unsaturation on lecithin monolayers. J . Lipid Res. 8 , 215-226. Sheetz, M. P., and Chan, S. 1. (1972). Effect of sonication on the structure of lecithin bilayers. Biochemisrry 11, 4573-4581. Shimshick, E. J . , and McConnell, H. J . (1973). Lateral phase separation in phospholipid membranes. Biochemisrn 12, 2351 -2360. Shipley, G . G . , Green, J . P., and Nichols, B. W . (1973). The phase behavior of monogalactosyl, digalactosyl, and sulphoquinovosyl diglycerides. Biochim. Biophys. Acru 311, 53 1-544. Shipley, G . G . , Avecilla, L. S . , and Small, D. M. (1974). Phase behavior and structure of aqueous dispersions of sphingomyelin. J . Lipid Res. 15, 124-131. Sinensky, M. (1971). Temperature control of phospholipid biosynthesis in Escherichiu coli. J . Bacteriol. 106, 449-455. Sinensky, M. ( 1974). Homeoviscous adaptation-a homeostatic process that regulates the viscosity of membrane lipids in Escherichiu coli. Proc. Nutl. Acud. Sci. U.S.A. 71, 523-525. Small, D. M. (1967). Phase equilibria and structure of dry and hydrated egg lecithin. 1. Lipid Res. 8, 551-557. Small, D. M. (1970). The physical state of lipids of biological importance: Cholesterol esters, cholesterol, triglycerides. Adv. Exp. Met/. B i d . 7, 55-83. Small, D. M., and Shipley, G. G . (1974). Physical-chemical basis of lipid deposition in atherosclerosis. Science 185, 222-229. Smith, I. C. P., Butler, K. W . , Tulloch, A. P., Davis, J . H . , and Bloom, M. (1979). The properties of gel state lipid in membranes of Achnlep/usma laidlutvii as observed by ‘H NMR. FEBS Lett. 100, 57-61. Sreim, J . M. (1968). Spectroscopic and calorimetric studies of biological membrane structure. In “Molecular Association in Biological and Related Systems’’ (R. F. Could, ed.), pp. 259-302. Am. Chem. Soc., Washington, D.C. Steim, J . M. (1970). Thermal phase transitions in biomembranes. Liq. Cryst. Ordered Fluids 1, 1-11. Steim, J . M. (1972). Membrane transitions: Some aspects of structure and function. In “MitochondriaiBiomembranes” (S. A. van den Berg, P. Brost, L. L. M. van Deenen, J . C. Riemersma, E. C. Slater, and J . M. Tager, eds.), pp. 185-196. North-Holland Publ., Amsterdam. Steim, J . M., Tourtellotte, M. E., Reinert. J . C., McElhaney, R. N . , and Rader. R. L. (1969). Calorimetric evidence for the liquid-crystalline state of lipids in a biomembrane. P r w . Natl. Acud. Sci. U . S . A . 63, 104-109. Stockton, G . W., Johnson, K . C., Butler, K. W., Polnaszek, C. F., Cyr, R., and Smith, I . C. P. (1975). Molecular order in Acholeplusmu luidluwii membranes as determined by deuterium magnetic resonance of biosynthetically incorporated specifically labelled lipids. Biochim. Biophys. Aclu 401, 535-539.

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Sumper, M. (1974). Control of fatty-acid biosynthesis by long-chain acyl CoAs and by lipid membranes. Eur. J . Biochetir. 49, 469-475. Sumper, M . , and Traiible, H. (1973). Membranes as acceptors for palniitoyl CoA in fatty acid biosynthesis. FEBS Lett. 30, 29-34. Tall, A. R., and Robinson. A. (1979). Absence of liquid crystalline transitions of cholesterol in reconstituted low-density lipoproteins. FEBS Lrrt. 107, 222-226. Tanford, C . (1973). "The Hydrophobic Effect: Formation of Micelles and Biological Membranes. '' Wiley, New York. Tecoma, E. S . , Sklar, L. A , . Simoni, R . D . , and Hudson, B. S . (1977). Conjugated polyene fatty acids as fluorescent probes: Biosynthetic incorporation of parinaric acid by Escherichitr c,o/i and studies of phaae transition$. Bioi.hi,i)iiury 16, 829-835. Templin, P. R. (1956). Coefficient of volume expansion for petroleum waxes and pure n-paraffins. /rid. E n g . Cheni. 48, 154-161. Thilo, L . , and Overath, P. ( 1976). Randomization of membrane lipids in relation to transport system assembly in Esc.hericliiu coli. Biochctnistry 15, 328-334. Thilo, L. Trauble, H., and Overath, P. (1977). Mechanistic interpretation of the influence of lipid phase transitions on transport functions. Brot~hetnistry16, 1283- 1290. Tourtellotte. M. E. (1972). Mycoplasina membranes, structure and function. Ir, "Membrane Molecular Biology" (C. F. Fox and A. 0. Keith, eds.), pp. 439-470. Sinauer Assoc., Stanford, Connecticut. Tourtellotte, M. E.. Branton, D., and Keith, A. (1970). Membrane structure: Spin labeling and freeze etching of Mye~op/us~uo ltridluwii. Proc. Nirtl. Acud. Sci. U.S.A. 66, 9OY-916. Trauble, H. (1971). Phase transitions in lipids. Biornrmhrcuirs 3, 197-227. Triuble, H., and Overath, P. (1973). The structure of E d w r i c A i r i i d i membranes studied by fluorescence measurements of lipid phase transitions. Biochitn. Biophys. A r m 307, 491 -5 12. Triuble, H . , and Sackmann. E. (1972). Studies of the crystalline-liquid crystalline phase transition of lipid model membranes. 111. Structure of a steroid-lecithin system below and above the lipidphase transition. J . A m . Chcwi. SO(,.94, 4499-45 10. Tsien, H . C . , and Higgins. M. L. (1974). Effect of temperature on the distribution of membrane particles in StrL.l"o(,oc.i.ic.\ ./iircdis as seen by the freeze-fracture technique. J . Bu(,trrio/. 118, 125-734.

Tsukagoshi, N.. and Fox, C. F. (1973). Transport system assembly and the mobility of membrane lipids in E.sc~/ierichiucoli. Biot.hrwtisrr;y 12, 2820-2822. Ubbelohde, A. R . (1965). "Melting and Crystal Structure.'' p. 325. Oxford Univ. Press. London and New York. Ulmius. J . , WennerstrAm. H.,Lindblom, G . , and Avidson, G . (1977). Deuteron nuclear magnetic resonance'studies o f phase equilibria in a lecithin-water system. Biochernr.\try 16. 57425745.

Untracht. S . H . , and Shipley, G . G . (1977). Molecular interactions between lecithin and sphingomyelin. J . Bi$. ChivJi. 252, 4449-4457. van Dijck, P. W. M . . Ververgaert, P. H . J . T . , Verkleij. A. J . , van Deenen, L. L. M . , and d c Gier, J . (1975). Influence of CaY' and Mg'+ on the thermotropic behavior and permeability properties of Iiposomes prepared from dirnyristoyl phosphatidylglycrrul and mixtures of dimyristoy1 phosphatidylcholine. Hioc.hirrr. B h p h k r t r 406, 465-478. van Dijck, P. W . M.. de Kruyff. B., van Deenen, L. L. M., de Grier, J . , and Demel. R. A . (1976). The preference o f cholesterol for phosphatidylcholine in mixed phosphatidylcholinephosphatidylethanolamine bilayers. E i o ~ ~ h i r iRi .i o / ~ h , ~k. tt. u 455, 576-587. van Heerikhuizen, H., Kwak, E . , van Bruggen, F. J . . and Witholt, B. (1975). Characterization of a low density cytoplasmic membrane subfraction isolated from Eschrrichiu coli. Biochi~rr. Biophys. Ac,tu 413, 177-191. van Zoelen, E. J. J., van der Neut-kok, E . C, M., de Gier, 1 . . and van Deenen, L. L. M . (lY75).

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Osmotic behavior of Acholeplasma laitllawii E cells with membrane lipids in liquid-crystalline and gel state. Eiochim. Eiophys. Acto 394, 463-469. Verkleij, A. J . , and Ververgaert, P. H. J . T . (1975). The architecture of biological and artificial membranes as visualized by freeze etching. Annu. Rev. Phys. Chem. 26, 101-122. Verkleij, A. J., Ververgaert, P. H. J. T., van Deenen, L. L. M., and Elbers, P. F. (1972). Phase transitions of phospholipid bilayers and membranes of Acholepfusma laidlawii B visualized by freeze fracturing microscopy. Eiochinr. Eiophys. Acta 288, 326-332. Verkleij, A. J . , Ververgaert, P. H. J. T., de Kruyff, B . , and van Deenen, L. L. M. (1974). The distribution of cholesterol in bilayers of phosphatidylcholines as visualized by freeze fracturing. Eiochirn. Eiophys. Acru 373, 495-501. Verkleij, A. J., Ververgaert, P. H. J . T . , Prins, R. A . , and van Golde, L. M. C. (1975). Lipid phase transitions of the strictly anaerobic bacteria V e i l l o n e h Purr& and Atiuerovibrio lipolyrico. J . Eucteriol. 124, 1522- 1528. Verkleij, A. J . , Lugtenberg, E. J . J., and Ververgaert, P. H. J. T. (1976). Freezeetch molphology of outer membrane mutants of Escherichici coli K 1 2 . Eiochim. A i o p h ~ s Arta . 426, 581 -586. Ververgaert, P. H. J . T . , Verkleij, A. J . , Elberts, P. F., and van Deenen, L. L. M. (1973). The distribution of cholesterol in bilayers of phosphatidylcholines as visualized by freeze fracturing. Biochim. Eiophys. Acru 373, 495-501, Verwer, W., Ververgaert, P. H. J. T . , Leunissen-Bijvolt, J . , and Verkleij. A. J . (1978). Particle aggregation in photosynthetic membranes of the blue-green alga Aiiucystis nidulurrs. Eiochirn. E i o p h y ~ ACIU . 504, 23 1-234. Wakil, S. J., ed. (1970). “Lipid Metabolism.” Academic Press, New York. Wallace, B. A , , and Engelman, D. M . (1978). The planar distribution of surface proteins and intramembrane particles in Acholeplasma laidlawii are differentially affected by the physical state of membrane lipids. Eiochim. Eiophys. A m 508, 431 -449. Wieslander, A , , Ulmius, J . , Lindblom. G . . and Fontell, K. (1978). Water binding and phase structures for different Acholeplasma laidlawii membrane lipids studied by deuteron nuclear magnetic resonance and X-ray diffraction. Biochim. Eioph?/.s. Acta 512, 241 -253. Wieslander, A , , Christiansson, A , , Wlater, H., and Weibull, C. (1979). Fractionation of membranes from Acholeplasmci laidlawii A on the basis of their surface properties by partition in twopolymer aqueous phase systems. Eiochim. Eiophys. Actu 550, 1-15. Wieslander, A,, Christiansson, A , , Rilfors, L . , and Lindblom, G . (1980). Lipid bilayer stability in membranes. Regulation of lipid composition in Acholephsma laiclluwii as governed by molecular shape. Eioch~mistry19, 3650-2655. Wu, S. H . , and McConnell, H. M. (1975). Phase separation in phospholipid membranes. Eiochemistry 14, 847-854. Wunderlich, F., Ronai, A., Speth, V., Seeling, J . , and Blume, A. (1975). Thermotropic lipid clustering in Tetrcihymena membranes. Biochemistry 14, 3730-3735. Yeagle, P. L., Hutton, W. C., and Martin, R. B. (1978). Sphingomyelin multiple phase behavior as revealed by multinuclear magnetic resonance spectroscopy. Eiochemistv 17, 5745-5750.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 17

Effects of Membrane Lipids on Transport and Enzymic Activities RONALD N . McELHANEY Depurrment of’Biochrmistr.v Universitj of Alhertu Edmonton, Alhrrtu, Ctrrttrtlu

Introduction . . . . . . . . . . . . . . . . . . . . . Relevant Properties of Membrane Constituents . . . . . . . A , Membrane Lipids . . . . . . . . . . . . . . . . B . Membrane Proteins . . . . . . . . . . . . . . . . 111. Arrheniua Plots of Membrane Transport Systems and Enzymes IV. Studies of Cells and Membranes . . . . . . . . . . . . A . CellGrowth . . . . . . . . . . . . . . . . . . . B. Chemotaxis . . . . . . . . . . . . . . . . . . . C. DNA Synthesis . . . . . . . . . . . . . . . . . D. Protein-Mediated Transport Processes . . . . . . . . E. Membrane-Associated Enzyme Activities . . . . . . . V. Studies of Isolated Membrane-Bound Enzymes . . . . . . VI. Conclusions . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . I.

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INTRODUCTION

The generally accepted fluid-mosaic model of membrane structure proposed by Singer and Nicolson ( 1 972) regards biological membranes essentially as two-dimensional solutions of oriented globular proteins in a fluid lipid bilayer phase. Thus proteins are free to diffuse laterally in the plane of the membrane unless constrained by their interactions with other proteins, either within the membrane itself or outside the membrane proper, including proteins functioning as cytoskeletal structural elements. The differential lateral mobility of proteins within the lipid bilayer can lead to a locally heterogeneous or “mosaic” two31 7

Copyright 0 1982 by Academic Press. Inc All nghts of reproduction in any form reserved. ISBN 0-12-153317.4

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dimensional organization of the membrane. Moreover, Israelachvili ( 1 977) has pointed out that thermodynamic and packing considerations suggest a coupled organization of lipids and proteins within the membrane, in which both lipid and protein may deform or cluster in order to accommodate each other most favorably. Although a refined fluid-mosaic membrane model seems applicable to most membrane systems, certain membranes, which have a low lipid/protein ratio and very extensive interactions between membrane proteins, may be more accurately characterized as two-dimensional, quasi-crystalline arrays of proteins with lipids filling the interstices between the protein molecules. In such membranes, almost all the lipid present undergoes continuous interaction with the membrane protein, and the lateral mobility of both constituents is quite restricted. A microbial example of such a membrane is the purple membrane from Halobacterium halobium (for review, see Stoe
II. RELEVANT PROPERTIES OF MEMBRANE CONSTITUENTS

A. Membrane Lipids The structure of microbial lipids, and their organization and disposition in microbial membranes, has been reviewed in preceding chapters, as has the thermotropic phase transition behavior of these compounds. For the purposes of this article, we need only consider that in most microbial membranes two different types of lipid normally exist simultaneously. The majority of the membrane lipids at any given instant are normally not in direct contact with integral membrane proteins, but instead are interacting exclusively with other membrane lipids in the bilayer. These lipids, which we shall term the bulk-phase lipids, have orientational and motional properties that are the same, or nearly the same, as those of a protein-free lipid-water dispersion existing under the same conditions. On the other hand, lipids in direct contact with the hydrophobic surfaces of

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integral membrane proteins clearly have their orientational and motional properties altered by the presence of the protein. The exact nature of this perturbed lipid, which has been termed boundary (Jost et ul., 1973), halo (Trauble and Overath, 1973; Stier and Sackmann, 1973), or annulus (Warren ef nl., 1975) lipid, is still a matter of some controversy (for a review, see Chapman et ul., 1979). In particular, some workers have suggested that this boundary lipid layer is only a single lipid molecule thick (Jost et a / . , 1973), whereas others have suggested that the extent of perturbation extends to three (Marcelja, 1976) or even six to seven layers (Curatolo et ul., 1978) around an integral membrane protein. Moreover, some workers have suggested that this boundary lipid shell contains rigid or immobilized lipid and is relatively long-lived (Jost et a / . , 1973; Warren et ul., 1975). However, recent physical studies have suggested that only the lipids close to the membrane protein surface are significantly perturbed, that these lipids are orientationally disordered and not highly immobilized, and that there is a relatively rapid exchange between the bulk and boundary lipid phases (van Zoelen et ul., 1978; David et a l . , 1979; Gally et ul., 1979, 1980; Kang et al., 1979a,b; Susi et a / . , 1979). Moreover, evidence exists that the boundary lipid may undergo a liquid-crystalline-like to gel-like “phase transition ” that is less cooperative (Marcelja, 1976) and that takes place at a lower temperature than the disorder-order transition of the bulk lipid phase (van Zoelen e t a / ., 1979; Susi et a/., 1979). Whatever the nature of this boundary lipid, it is important to realize that membrane enzymes and transport systems may be responding primarily to alterations in the physical properties of the boundary lipid, rather than to the bulk lipid phase, although of course the properties of one phase influence those of the other. This is an important consideration, since many of the physical techniques that are utilized to monitor membrane lipid fluidity and phase state may preferentially respond either to changes in the boundary or the bulk lipid phase. For example, spectroscopic techniques that employ artificial probes to measure membrane lipid physical properties may tend to report preferentially on boundary layer lipids, since some evidence exists that fatty acid spin labels and certain fluorescence probes tend to be localized at lipid domain boundaries (see Schreier et ul., 1978; Mely-Goubert and Freedman, 1980).

6. Membrane Proteins Singer and Nicolson ( 1 972) have proposed that membrane-associated proteins can be classified as either peripheral or integral. Peripheral (also called extrinsic) proteins are those that can be removed from membranes without recourse to the use of detergents, chaotropic agents, or organic solvents. Peripheral membrane proteins are usually (but not always) isolated free of endogenous lipid, and their removal from tile membrane normally does not alter the basic structure or most of

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the properties of the membrane itself. Peripheral proteins are considered to be surface localized, associating with the lipid bilayer only through electrostatic interactions with the lipid polar head groups, and/or with other membrane proteins. Integral (also called intrinsic) proteins, on the other hand, can only be removed from the membrane by use of the lipid bilayer-disrupting agents mentioned already, and are normally isolated with variable amounts of endogenous lipid remaining associated with them. Integral membrane proteins are considered to interact with the membrane lipids by hydrophobic as well as by electrostatic interactions, because a portion of the protein penetrates into or through the lipid bilayer. Both integral and peripheral proteins normally have an asymmetric distribution in the intact membrane. Determination of the primary structures of several integral membrane proteins has indicated that these proteins often consist of polar and nonpolar domains, and thus these proteins have an amphiphilic character, just as do the membrane lipids. In the case of certain integral proteins, such as mitochondria1 cytochrome h:, and cytochrome bs reductase, the bulk of the protein consists of a single polar domain in contact with the aqueous environment, and a smaller “hydrophobic tail” that penetrates partway into the lipid bilayer core and serves to anchor these proteins in the membrane. On the other hand, the erythrocyte integral membrane protein glycophorin has a three-domain structure, consisting of polar Cand N-terminal regions separated by a hydrophobic internal sequence. The very hydrophobic protein bacteriorhodopsin appears to consist of seven transmembrane a-helices connected by short polar sequences, the Lu-helices being largely buried in the lipid bilayer. The amphiphilic nature of some membranous enzymes and transport proteins having quaternary structure may arise from the presence of both polar and nonpolar subunits. This has been demonstrated for mitochondrial, chloroplast, and bacterial ATPases (for review, see Haddock and Jones, 1977). Other proteins may contain one or more covalently bound fatty acyl groups, whose presence also imparts an amphiphilic character to the protein molecule. Only in the case of bacteriorhodopsin has the detailed tertiary structure of an integral membrane protein been determined to high resolution (for review, see Stoeckenius, 1980). For more detailed summaries of information on the structure and disposition of integral membrane proteins, the reader is referred to reviews by Guidotti ( 1977) and by Singer ( 1977).

111.

ARRHENIUS PLOTS OF MEMBRANE TRANSPORT SYSTEMS AND ENZYMES

Investigation of the temperature dependence of chemical and enzymic reactions is an essential tool for understanding the molecular mechanisms of these processes. In chemical kinetics the effects of temperature on the rate constants

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characterizing a chemical reaction are analyzed in terms of an empirical activation energy (E;,)according to the Arrhenius equation, which may be written as logk=-

E R .-I 2.3 R T

where k is the rate constant of interest, R the gas constant, and T the absolute temperature. In practice the numerical value of E is determined from the slope of a plot of log k versus l/T (the Arrhenius plot). For the great majority of chemical reactions, and for chemical reactions catalyzed by most soluble enzymes, Arrhenius plots are linear over the accessible temperature range, although in both cases exceptions are known (see references in Han, 1972, and Gardiner, 1977). Thus the enthalpies of activation of these processes, which differ from their apparent activation energies only by a quantity equal to RT (about 0.6 kcal/mol), are essentially invariant with temperature. For membrane-associated transport systems and enzymes, however, nonlinear Arrhenius plots are quite commonly obtained (for reviews, see Linden and Fox, 1975; Sandermann, 1978). In some cases Arrhenius plots consisting of a relatively sharp break between two (or sometimes more) straight-line segments are reported, whereas in other cases Arrhenius plots of membrane-associated functions seem to consist of smooth curves. Normally even abrupt changes in the apparent activation energies of membrane transport or enzymic processes are not accompanied by a significant change in the reaction rate, although in some cases actual jump discontinuities in Arrhenius plots of membranous enzymes have been reported (for example, see Raison, 1973). It has been proposed that true jump discontinuities can only arise as a thermodynamic consequence of a phase change (Kumamoto et al., 1971). However, a break or change in slope in an Arrhenius plot, which is not accompanied by a marked change in reaction rate, can arise from a number of causes (Han, 1972). It is important to remember that an Arrhenius plot of an enzyme-catalyzed reaction will normally be linear only if a single species of catalyst is responsible for the chemical process under study and only if one particular step in the overall reaction is rate limiting over the entire temperature range examined. In biological membranes, several enzymes or transport systems may simultaneously participate in a given process. Moreover, membrane enzymic reactions and transport processes are generally complex multistep processes, and each partial reaction in the overall process may have a different temperature dependence (and a different lipid dependence; see Sandermann, 1978). Dixon and Webb (1964) and Han ( 1 972) have discussed the effects of temperature on the rate of enzymic reactions generally and have suggested experimental strategies to recognize and correct for some of the more trivial effects of temperature. These effects can include such things as unrecognized temperature-induced changes i n the pH of aqueous buffers, in solution viscosity, and in substrate-binding affinity (K,,,). In addition,

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Han (1972) has analyzed other factors that may produce nonlinearity in Arrhenius plots of enzymic reactions, and classified them into two categories: ( 1 ) thermodynamic factors, including all secondary equilibrium reactions that modify the elementary process being catalyzed; and (2) kinetic factors, attributed to changes in the rate-limiting step occurring within the experimental temperature range. Although the above treatments were developed for soluble enzymes, they apply to membrane enzymes and transport systems as well. In much of the work reviewed in subsequent sections, it appears that the complexities just noted have been largely ignored, with the result that at least some of the conclusions reached must be regarded as tentative. A few attempts have been made to develop a quantitative and systematic analysis of the mechanistic basis for nonlinear Arrhenius plots in membranous systems, taking into account the unique properties of these systems due to their existence in a lipid environment, the fluidity and phase state of which can also vary with temperature. Wynn-Williams (1976) has proposed that the sudden change in the apparent activation energy of membrane enzymes could be due to the simultaneous presence of pure lipid and of enzyme-lipid phases in the membrane. If enzyme activity depends on the composition of the enzyme-lipid phase, the temperature dependence of lipid solubility in the enzyme-lipid phase can lead to a sudden change in the apparent enzyme activation energy within the lipid phase transition temperature range, without an enzyme activity discontinuity. This is a consequence of the fact that the actual enthalpy of the “activated state” of the enzymic reaction is no longer equal to the slope of the Arrhenius plot of enzyme activity within the phase transition range. If this treatment proves valid for membrane enzymes and transport systems, it removes a theoretical difficulty, since it is no longer necessary to assume that a marked change in the activation enthalpy is exactly compensated for by a change in the activation entropy at the break temperature. Thilo et a l . (1977) and Silvius and McElhaney (1980a,b) have also proposed mechanistic explanations for membrane lipid phase transition-induced changes in the slopes of Arrhenius plots of several transport systems and of a membrane enzyme, respectively; these proposals will be discussed in detail later. Finally, Silvius and McElhaney (i980a) have systematically derived the rate-temperature relationships for a variety of physical models of membrane rate processes in order to predict the Arrhenius plot shape appropriate to each. Interestingly, only a few models predict Arrhenius plots with the “biphasic linear” form most commonly reported in studies of membrane enzymes and transport systems. Instead, most models predict Arrhenius plots consisting of smooth curves. However, many of the models yield plots that can be fit to two intersecting straight lines with a quite modest experimental error, particularly if the slope change around the “break” temperature corresponds to a change in apparent activation energy of less than 15-20 kcalimol. These findings indicate a need for a rigorous analysis of Arrhenius plot data in terms of graph

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shapes other than sets of intersecting straight lines and for a cautious interpretation of the physical basis of Arrhenius plot “breaks.” The need for accurate determinations of the true maximum rates of the membrane-associated process of interest, and at a large number of experimental temperatures, for the valid interpretation of Arrhenius plots, has been emphasized recently by several groups (Silvius et al., 1978; Londesborough and Varimo, 1979; Sprague et al., 1980). Moreover, the fitting of Arrhenius plot data points by eye, as is usually done, may lead to controversies over whether the author’s subjective representation is the most correct one. In particular, a tendency to draw two straight lines through data points that actually fall on a single, continuously curving line is often evident. Recently Cook and Charnock (1979) and Sprague er al. (1980) have developed statistical methods of assessing the goodness of fit of Arrhenius plot data points by various types of curves. To utilize these approaches effectively, however, a number of determinations of the rate of the process of interest at each experimental temperature must be available, and the variance between replicate measurements must be determined. Unfortunately, in very little of the present literature is this information provided. Thus in some published studies, the assignment of the exact position of the Arrhenius plot “break” (sometimes reported to the tenth of a degree!), and even the existence of a single, sharp slope change in the plot, can be debated.

IV.

STUDIES OF CELLS AND MEMBRANES

There have been a large number of studies published on the dependence of the rates of specific membrane transport and enzymic processes, as well as of several more complex physiological functions, on membrane lipid composition and physical properties. A number of these studies have employed more or less intact biological membranes, either in the form of the microbial cell itself, or in the form of derived resealed membrane vesicles or membrane fragments. These studies share the common problem of having to cope with the great compositional complexity of the microbial membrane, which may contain several hundred different proteins and at least three different classes of polar lipids. Moreover, each glyco- or phospholipid in the membrane may occur in a number of different molecular forms, depending on the type and distribution of fatty acid chains esterified to their glycerol backbones. Thus it may be difficult to establish in studies of intact membranes that the transport or enzymic process of interest is being catalyzed only by a single protein or system of proteins. In addition, although the fatty acid composition and, to a lesser extent, the polar head-group composition of the membrane lipids can often be altered, the question of whether the functional protein of interest interacts with all, or with only a portion of the many molecular species of lipid present, must be considered in the interpretation

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of the results obtained. Although successful studies of intact microbial membranes demand great care, the possibility in favorable cases of studying the operation of a single, relatively unperturbed protein-mediated process functioning in its “natural” lipid environment clearly makes such studies of great importance. A number of investigations, concerned with the effect of alterations in the fluidity and phase state of the lipids on a variety of functions taking place in intact microbial membranes, will be reviewed in this section.

A. Cell Growth Perhaps the most fundamental and comprehensive indicator of normal cellular function in general, and of normal membrane function in particular, is growth, a physiological parameter that can usually be easily and accurately measured with a minimum of perturbation to the microorganism under study. Thus it is not surprising that a number of workers have studied the relationship between the fluidity and phase btate of microbial membrane lipids and the rate of cell growth. The two microorganisms most extensively studied in this regard have been Acholeplusrna laidluwii B and the unsaturated fatty acid (UFA) auxotrophs of Escherichia coli, since their membrane fatty acid compositions can be readily manipulated. The pioneering work in this area was that of Overath et a / . (1970), who utilized a double mutant of E . coli unable to synthesize or degrade UFAs, but that can incorporate various exogenous cis- and trans-unsaturated or cyclopropane fatty acids into its membrane phospholipids. The temperature range of growth, as well as the rates of growth within the permissible temperature range, were correlated with the physical properties of isolated phosphatidylethanolamines (PEs) spread as monolayers at the air-water interface. When cells were enriched in various cis-UFAs, the minimum growth temperature of 10°C did not seem to be determined by membrane fatty acid composition. However, cells enriched in elaidic acid showed a greatly elevated minimal growth temperature of 37”C, which correlated reasonably well with the liquid-expanded to condensed transition temperature of the PE films. The optimum and maximum growth temperatures also varied slightly and in parallel with monolayer phase transitions, being reduced from their “normal” values in cells enriched in lowmelting fatty acids. Arrhenius plots of relative growth rate versus reciprocal temperature generally yielded biphasic curves (except for elaidate-enriched cells), with slopes corresponding to a temperature characteristic of growth of 15-16 kcal/mol at higher temperatures, but with much greater slopes at lower temperatures. The position of the “breaks” in the slopes of these Arrhenius plots correlated to some degree with the PE film transition temperatures. Overath et a / . (1970) concluded that the E. coli membrane lipids must be in a liquid-like

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state for proper membrane function and that there is an upper limit of fluidity compatible with growth. Moreover, from parallel studies of respiration and thiomethylgalactoside efflux, these investigations also established that membrane lipid fluidity and phase state also influence the function of membranebound proteins. The relationship between the phase state of the membrane lipids and the growth of E . cwli was subsequently studied by three different laboratories, each employing different physical techniques to measure the lipid phase transition and different environmental manipulations to limit the amount of cis-monoUFAs in the membrane lipids (Thilo and Overath, 1976; Uehara e t a / . , 1977; Jackson and Cronan, 1978; Akutsu rt ul., 1980). Each study demonstrated that E . coli was unable to grow when the amount of gel-state lipid in the cytoplasmic membrane exceeded about 50%. In addition, these investigators showed that domains of ordered lipid cannot be required for normal growth, since E . coli UFA auxotrophs supplemented with various cis-UFAs such as oleate grow "normally" (at wild-type rates) at temperatures near 37"C, some 20°C above the upper boundary of the gel to liquid-crystalline phase transition. These findings have recently been confirmed in an E . coli mutant unable to synthesize or degrade UFAs, but capable of sustained growth with only endogenous saturated fatty acids (of reduced chain length compared to wild-type cells) in its membrane lipid ( G . Pluschke and P. Overath, personal communication). Parallel studies with wild-type cells, which are able to alter their fatty acid compositions in response to variations in environmental temperature, showed that the membrane lipid phase transitions ended more than 10°C below the growth temperature when cells are grown at either 25 or 37"C, indicating that E . coli prefers to grow with its lipids entirely in the liquid-crystalline state. Confirmation that wild-type E . coli adjust the phase transition temperature of their inner-membrane lipids so that they exist entirely in the fluid state at the growth temperature has recently been provided by Davis ei al. (1979), Gally et ul. (1979, 1980), and Nichol et ul. (1980), and by Nakayama et ul. (1980), using 'H-nuclear magnetic resonance (NMR) and X-ray diffraction techniques, respectively. Similar conclusions were reached from calorimetric studies of the relationship between the phase state of the membrane lipids and the growth temperature of Micrococcus lysodeikticus (Ashe and Steim, 1971) and Bacillus stearothermophilus (McElhaney and Souza, 1976). The earliest study relating the physical state of the membrane lipids to cell growth was that of Steim e t a / . (1969) using the simple, cell wall-less prokaryote A . luidluwii B and differential scanning calorimetry (DSC) to monitor the phase state of the membrane lipids. These investigators reported that A . luidlawii B was capable of growth when its membrane lipids contained fatty acyl groups giving rise to gel to liquid-crystalline membrane lipid phase transitions from
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increasing levels of incorporation of stearate into the membrane lipids drove the phase transition temperature above the temperature of growth. These workers were the first to demonstrate that the membrane lipids must exist predominantly in the liquid-crystalline state to support normal function. McElhaney (1974a,b) subsequently published more detailed studies of the relationship between the fluidity and phase state of the membrane lipids, as determined both by fatty acid composition and environmental temperature, and the temperature range within which A . laidluwii B cells can grow and on growth rates within the permissible temperature ranges. The gel to liquid-crystalline membrane lipid phase transition temperatures of isolated plasma membranes were determined by differential thermal analysis (DTA). McElhaney found that the minimum growth temperature of 8°C is not defined by the fatty acid composition of the membrane lipids when cells are enriched in fatty acids giving rise to gel to liquid-crystalline membrane lipid phase transitions occurring below this temperature. The elevated minimum growth temperatures of cells enriched in fatty acids giving rise to lipid phase transitions occurring at higher temperatures, however, are clearly defined by the fatty acid composition of the membrane lipids. The optimum and maximum growth temperatures are also influenced indirectly by the physical state of the membrane lipids, being significantly reduced for cells supplemented with lower melting UFAs, suggesting the existence of an upper limit on membrane lipid fluidity that is compatible with the growth of this organism. The temperature coefficient of growth at temperatures near or above the midpoint of the lipid phase transition is 16 to 18 kcal/mol, but this value increases abruptly to 40 to 45 kcal/mol at temperatures below the phasetransition midpoint. Both the absolute rates and temperature coefficients of cell growth are similar for cells whose membrane lipids exist entirely or predominantly in the liquid-crystalline state, but absolute growth rates decline rapidly and temperature coefficients increase at temperatures where more than half of the membrane lipids become solidified. Cell growth ceases when the conversion of the membrane lipid to the gel state approaches completion, but growth and replication can continue at temperatures where less than one tenth of the total lipid remains in the fluid state. Interestingly, only a single fatty acid supplement (unreiso-heptadecanoic acid) of the many tested permits this organism to grow over its entire potential growth temperature range of 8-44"C, suggesting that the ability of most microorganisms (but not A . luidluwii B) to alter their fatty acid composition in response to changes in environmental temperature serves to extend the range of temperature over which growth can occur. Except for the fact that A . luidluwii B seemed able to grow normally with a somewhat higher proportion of ordered lipid in its membrane, these results are fully in accord with the observations of others on the relationship of growth and membrane lipid phase state in UFA auxotrophs of E . coli. The studies reviewed here firmly establish that the gel to liquid-crystalline

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membrane lipid phase transitions provide a “lower boundary” for cell growth, and that bacteria able to vary their fatty acid compositions with the growth temperature prefer to grow with exclusively liquid-crystalline lipid in their inner membranes. However, the relationship between the maximum degree of membrane lipid “fluidity” (within the liquid-crystalline phase state) that is compatible with growth is less well understood. The initial observations of Overath et al. (1970) on E . coli and of McElhaney (1974a,b) on A . laidlawii, that incorporation of low-melting fatty acids reduces the optimum and maximum growth temperatures, suggest that growth cannot continue at a temperature too far above the membrane lipid phase transition temperature. Baldassare et al. (1 976) provided additional support for this view. These workers utilized multiple mutants of E . coli defective in totul fatty acid biosynthesis to investigate the maximum amounts of palmitic acid and several UFAs that can be incorporated into the membrane lipids and still maintain relatively normal growth. DSC and electron spin resonance (ESR) measurements were made to determine how modifications in the fatty acid composition affected the membrane lipid phase transition. Baldassare et d.(1976) found that cells grown with cis-vaccenic acid could grow at nearly normal rates when over 90% of the total phospholipid fatty acid consisted of cis-UFAs. Such cis-monoUFA-enriched cells exhibited a sharp gel to liquidcrystalline phase transition centered at -13°C. The fact that E . coli cell membranes can apparently function almost normally at temperatures some 50°C above the lipid phase transition midpoint temperature indicates that a relatively high degree of membrane lipid fluidity can be tolerated by this organism. However, the fact that not all the saturated fatty acids in the membrane phospholipids could be replaced by UFAs suggests that cells so highly enriched in UFAs might be growing near an upper limit of tolerable lipid disorder at 37°C. Recent studies on the effect of exogenous fatty acid incorporation on the growth of A . luidlawii B rendered totally auxotrophic for fatty acids by selective inhibition of de n o w fatty acid biosynthesis and chain elongation also provide support for the existence of an upper limit for membrane lipid fluidity (or disorder) compatible with cell growth. Silvius and McElhaney (1978) demonstrated that A . laidlawii B could grow relatively normally at 37°C when supplemented with several single exogenous n-saturated, methyl iso- and nnteiso-branched or trans-monounsaturated or cyclopropane fatty acids; under these conditions the exogenous fatty acid typically made up 95-99 mol% of the total membrane lipid fatty acyl groups. However, short-chain n-saturated and branched-chain fatty acids, cis-cyclopropane, cis-polysaturated, as well as some cis-monounsaturated fatty acids, would not support significant growth. Silvius et a/. (1980a) also showed that the gel to liquid-crystalline membrane lipid phase transition midpoint temperatures of these normally growing cells ranged from -14.9 to +36.7”C, indicating that a broad range of lipid fluidities are compatible with normal membrane function. Although there appeared to exist a minimum chain-

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length requirement for cell growth (fatty acids of less than 14 carbon atoms were always unsuitable), which could not be completely explained on the basis of the thermotropic phase behavior of membrane lipids enriched in short-chain fatty acids, nevertheless the inability of methyl anreiso-branched and of cisunsaturated and cyclopropane fatty acids to support growth could be rationalized by assuming that fatty acids giving rise to membrane lipids having a phasetransition temperature much below - 15 to -20°C would produce a hyperfluid state at 37°C. Indirect evidence in support of this hypothesis is provided by the observations that in A . laidluwii B (McElhaney, 1974a,b) retaining the ability to biosynthesize endogenous saturated fatty acids, and in E . coli (Baldassare et ul., 1976) cells totally dependent on exogenous fatty acids, the minimum phasetransition midpoint temperatures that can be achieved by enrichment in UFAs are about -19°C and -13”C, respectively. Moreover, a good (but not a perfect) correlation exists between the growth-promoting ability of a series of cisoctadecenoic acid position isomers and their gel to liquid-crystalline transition temperatures in A . laidluwii B cells whose endogenous fatty acid metabolism has been inhibited (Silvius and McElhaney, 1978; Silvius et d.,1980a); in particular, cis-octadecenoates with the double bond located near the center of the hydrocarbon chain, and having the lowest phase-transition temperatures, are unable to support growth, whereas cis-octadecenoates with the double bond located on either side of the center, and that have moderate phase transition temperatures (about -10” to +30”C) support fair to good growth. Although it seems clear that an upper limit for membrane fluidity or disorder exists for functional biological membranes, we still have no good quantitative measure of this maximum fluidity. Rigorous studies with several nonperturbing spectroscopic techniques, sensitive to orientation and motion over a range of time scales, are clearly required. There have been several studies published that are at variance with previously discussed work on the relationship between membrane lipid fluidity, phase state, and cell growth. The first of these is the study of Esser and Souza (1974) of B . stearorhermophilus, in which “lateral phase separations” were monitored in spheroplast membranes and in membrane lipid-water dispersions by means of ESR, using a 5-doxy1 stearic acid spin probe intercalated into the bacterial membranes or into membrane lipid-water dispersions. Wild-type cells, which could alter their fatty acid composition in the characteristic way in response to changes in growth temperature, were found to increase the temperature at which the lateral phase separations were detected so the “change of state” in the membrane phospholipids occurred at the growth temperature. A temperaturesensitive mutant, unable to alter its fatty acid composition at higher temperatures, could survive only up to the temperature of the upper boundary for the lateral phase separation noted at its (reduced) optimal growth temperature. These data were interpreted to indicate that the maximum and minimum growth tempera-

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tures of this thermophilic microorganism are determined by the onset and completion temperatures of the phase separations of the particular membrane lipid compositicn synthesized, and that the simultaneous presence of both solid- and fluid-lipid domains is required for membrane function and assembly. McElhaney and Souza ( 1976) reinvestigated the relationship between membrane lipid phase state and cell growth in B . sreart,fhermo~hi/ususing DTA to detect membrane lipid phase transitions in spheroplast membranes and in membrane lipid-water dispersions. In the wild-type strain, the temperature range over which the broad gel to liquid-crystalline membrane lipid phase transition occurs shifts in response to fatty acid compositional changes such that the u p p r boundary of this transition is always near but below the temperature of growth. Thus the membrane lipids of this organism exist entirely in the liquid-crystalline state of the growth temperature, as has been reported for other bacteria and mycoplasmas. The mutant strain, which has lost the ability to increase the proportion of high-melting fatty acids in its membrane lipids in response to increases in environmental temperature, is also unable to increase its phase-transition temperature and to grow at elevated temperatures. The rate of increase in cell growth with environmental temperature in the mutant strain is normal until the environmental temperature exceeds the upper boundary ,and midpoint of the gel to liquid-crystalline phase transition by about 18- 19°C and 28-3OoC, respectively, at which point cell growth abruptly ceases. This latter result again suggests that an upper limit on the degree of membrane lipid fluidity compatible with cell growth exists, and implies that the processes of “homeoviscous adaptation” (Sinensky, 1974) and/or “homeophasic adaptation” (Silvius et d., 1980b), by appropriate fatty acid compositional shifts, serves to extend the growth temperature range in wild-type cells. Janoff et a / . (1979, 1980a) studied purified cytoplasmic and outer membranes of wild-type E . co/i cells grown at various temperatures, employing ESR spectroscopy of an intercalated 5-doxy1 stearic acid spin probe and fluorescence polarization of 1,6-diphenyl- I ,3,5-hexatriene (DPH), respectively. Apparent discontinuities in ESR and fluorescence polarization spectral parameters were ‘interpreted” to be due to gel to liquid-crystalline membrane lipid phase transitions. The position of the lipid order-disorder transition of the cytoplasmic membrane was reported to be invariant with growth temperature, such that the cytoplasmic membrane lipids exist in a mixed-phase state at low growth temperatures but only in the fluid state at higher growth temperatures. In contrast, the outer membrane lipid phase transition increased with increasing growth temperature, such that its lipids exist in the gel plus liquid-crystalline states over the entire growth temperature range. These studies, plus a later investigation purporting to show that anesthetics lower the phase-transition temperature of the outer membrane only while also lowering the maximum growth temperature (Janoff et a l . , 19XOb), were interpreted as evidence that adaptive changes that

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maintain a mixed-phase state in the outer membrane, but not in the cytoplasmic membrane, determine the growth temperature range of this organism. It is unlikely that the actual gel to liquid-crystalline phase transitions of the bulk membrane lipids in either the outer or cytoplasmic membranes of E . coli were being accurately recorded by the ESR and fluorescence-polarization techniques used by Janoff and co-workers. For one thing, the 5-doxy1 stearate and DPH probes report very different lower boundary temperatures for the outer membranes, and moreover, the latter probe reports a much lower transition for the outer membrane lipids than for the outer membrane itself. For another, the reported phase transition midpoints and temperature ranges do not agree well with those determined for E . coli cytoplasmic and outer membranes by a variety of nonperturbing physical techniques, such as DSC (Baldassare et ul., 1976; Jackson and Cronan, 1978), X-ray diffraction (Nakayama et ul., 1980), and 2H-NMR (Davis et ul., 1979; Gally et al., 1979, 1980; Kang et al., 197913; Nichol et al., 1980). Also, since it is well known that the fatty acid composition of cytoplasmic membrane lipid changes with increasing temperature, such that the saturatedlunsaturated fatty acid ratio is markedly increased, it is difficult to understand how the lipid phase transition temperature of this membrane could remain constant, if the cooperative melting of the bulk membrane lipid fatty acyl chains were indeed being monitored. It seems clear that, just as was the case for the ESR study of B . stearothermuphilus by Esser and Souza (1974), the ESR and fluorescence probes employed by Janoff and co-workers are not monitoring the bulk-lipid order-disorder transition, at least not for the cytoplasmic membrane. Until the nature and biological relevance of the spectral parameter changes reported are clarified, these studies will remain essentially uninterpretable, and as such do not pose a creditable challenge to the consensus view of the relationship between cell growth and membrane lipid fluidity and phase state. Since ESR spectroscopy, and to a lesser extent fluorescence spectroscopy, are so commonly used in biological membrane studies, the reader should be aware of the fact that a substantial body of evidence indicates that fatty acid spin probes can become localized in nonrepresentative local domains and can perturb both lipid-water model systems and biological membranes (Seelig and Seelig, 1974; McElhaney and Souza, 1976; Taylor and Smith, 1980; for reviews, see Keith et al., 1973; Schreier et a / . , 1978), as can some fluorescence probes such as DPH (Mely-Goubert and Freedman, 1980). Moreover, a recent theoretical analysis indicates that the motional parameters characteristic of such probes in biological membranes may respond to the approximate midpoint but not to the boundaries of broad membrane lipid phase transitions (Silvius and McElhlaney, 1980a). Extreme caution in the interpretation of data gathered by these techniques is thus in order. For additional information on the possible role of alterations in membrane lipid fatty acid and polar head-group compositions in microbial adaptation to

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growth in extreme environments-particularly extremes of temperature-the reader is referred to several recent reviews on this subject (McElhaney, 1976; Esser, 1979).

6. Chemotaxis Several groups have studied the relationship between membrane lipid fluidity and phase state, and the chemotactic response in E . coli. Lofgren and Fox (1974), using a simple capillary assay, concluded that fluid membrane lipid is required for chemotaxis but not for motility. However, Miller and Koshland (1977), in a much more extensive study in which tumbling frequency, swimming velocity, attractant response times, and cell viability were measured, demonstrated that neither sensory nor motility functions are dependent on the existence of a fluid membrane. Miller and Koshland attribute the results of Lofgren and Fox to the loss of viability of elaidate-enriched E . coli cells below 30°C and its effect on the capillary assay employed in this study.

C. DNA Synthesis Fralick and Lark (1 973), using 3-decynoyl-N-acetylcysteamine (3-DNAC) to inhibit UFA biosynthesis in wild-type E . coli, reported that UFA synthesis is required for the initiation of DNA synthesis in this organism. In contrast, Thilo and Vielmetter (1977), using a UFA auxotroph of E . coli, found that neither the initiation nor the propagation of DNA synthesis required UFAs or a membrane containing fluid lipids. Thilo and Vielmetter attributed the results of Fralick and Lark to unrecognized metabolic or structural disturbances induced by the presence of 3-DNAC, and indeed evidence for the inhibition of processes other than UFA biosynthesis by this compound have been reported (Kass and Bloch, 1967; Nunn and Cronan, 1974). However, the observation by Fralick and Lark (1973), that the inhibitory effects of 3-DNAC can be reversed by the addition of exogenous unsaturated (but not saturated) fatty acids, seems difficult to rationalize on this basis. Further studies seem necessary to resolve this apparent conflict.

D. Protein-Mediated Transport Processes I . SUGAR TRANSPORT The original investigations of the relationship between phospholipid biosynthesis and the induction of a functional lactose transport system were those of Fox (1969) and Hsu and Fox (1970); in the former study, phospholipid synthesis was inhibited by the starvation of an E . c-oli UFA auxotroph for UFAs, and in the

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latter study by starvation of a glycerol auxotroph for glycerol. In both cases inhibition of phospholipid synthesis resulted in a marked reduction of the ratio of P-galactoside transport induction to P-galactosidase induction, suggesting that, although the products of lactose operon were being produced in the absence of phospholipid biosynthesis, the assembly of a functional transport system required normal phospholipid synthesis. Nunn and Cronan (1974) and Weisberg et a / . (1975), however, using a generally similar UFA glycerol-starvation approach with the appropriate auxotrophs, have reported that the induction of lactose transport is not preferentially inhibited in the absence of UFA or phospholipid synthesis, respectively. Overath et a / . (1971), employing 3-DNAC to inhibit UFA biosynthesis in a wild-type strain, also found normal rates of P-galactoside transport induction initially, although after half a generation the cells became leaky, indicating membrane damage. Similarly, Robbins and Rotman (1972) reported that inhibition of UFA biosynthesis in E . co/i by 3-DNAC resulted in less than a 50% inhibition of lactose transport induction, if assayed before general cellular damage due to UFA deprivation was evident, although the induction of methylgalactoside transport was markedly inhibited by this treatment. Finally, Mindich (1971) and Willecke and Mindich (1971) have demonstrated that the lactose permease in Sfaphylococcus aureus and citrate transport in Bacillus suhtilis, respectively, can be induced in the absence of phospholipid synthesis, although the lactose permease system functions with a reduced efficiency (30-50% of normal) under such conditions. It thus appears that in general, the induction and assembly of functional lactose and at least some other transport systems can occur in the absence of UFA or phospholipid synthesis. This is reasonable in view of the fact that the growth studies reviewed earlier, as well as estimates of the amount of “boundary lipid” present in plasma membranes (Steim et a / . , 1969; Jost et al., 1973; Baroin et al., 1979). indicate that most membranes contain an “excess” of unperturbed, bilayer-phase lipid that at any given instant is not interacting directly with membrane protein; this “nonboundary” lipid would presumably be available for the solvation of newly synthesized integral membrane proteins, even in the temporary absence of continuing glycerolipid synthesis. In fact, McIntyre and Bell (1975) and McIntyre et a / . ( 1977) have demonstrated that a 60% increase in the protein/phospholipid ratio of the cytoplasmic and outer membranes can be induced by the inhibition of phospholipid synthesis in glycerol auxotrophs of E . coli before cell growth ceases. Tsukagoshi and Fox (1973a), utilizing an E . Cali UFA auxotroph supplemented with various fatty acids, have also reported that the induction of lactose transport is abortive in cells maintained at temperatures below the membrane lipid gel to liquid-crystalline phase transition temperature. This conclusion was again based on the decreased ratio of P-galactoside transport induction to P-galactosidase induction observed below characteristic temperatures, which depended on the fatty acid composition of the cell. Although this conclu-

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sion may be valid, one should be aware of the fact (discussed earlier) that E . coli cells are unable to grow at temperatures below their lipid phase transition midpoints, and, at least in the case of elaidic acid-enriched cells, rapidly lose viability (Overath et a / . , 1970; Esfahani et ul., 1971a; Tsukagoshi and Fox, 1973b; Miller and Koshland, 1977). Since Tsukagoshi and Fox (1973a) induced their cultures under these conditions, it is possible that the reduced ratio of P-galactoside transport to P-galactosidase induction observed may simply reflect a differential decline in membrane function generally (in relation, for example, to soluble protein biosynthesis), which accompanies a loss of viability. Also, since E . coli cells are known to become “leaky” below their lipid phase transition temperatures (Overath et al., 197 1 ; Haest rt al., 1972; Thilo et al., 1977), one must also consider the possibility that the transmembrane electrochemical proton gradient could become at least partially collapsed under these conditions, due to an inhibition of substrate oxidation, a defective coupling of substrate oxidation to proton pumping, an excessive proton passive leakage, or a combination of two or more of these factors. Since the electrochemical proton gradient is known to drive E . coli transport systems, either directly or indirectly (for a review, see Rosen and Kashket, 1978), a dissipation of this gradient could result in an inhibition of the transport function even in the presence of completely integrated and potentially fully functional membrane transport systems. The tacit assumption made in most studies of the role of lipids in microbial transport-that transport rates reflect only the behavior of the transport system itself-is at present largely unsubstantiated, since very little work has been done on the effect of alterations in membrane lipid fluidity and phase state on membrane energization. The association of the lactose transport system with the lipids of the cytoplasmic membrane was also studied by Wilson and Fox (1971) by taking advantage of the fact that the rate-temperature profile of lactose transport is dependent on the UFA supplement provided to UFA auxotrophs of E . coli. As first detnonstrated by Schairer and Overath (1969), and later by Wilson et ul. (1970), Arrhenius plots for transport could be fit (at least approximately) by two straight lines, the lower temperature line having a considerably greater slope than the higher temperature line, as illustrated in Fig. 1 . The “break” or inflection temperature observed in each Arrhenius plot depended on the fatty acid composition of the UFA auxotroph in such a way as to suggest that the break was related to the phase transition temperature of the membrane lipids. Thus the activitytemperature profile of the lactose transport system reflected the physical properties of its lipid environment in the membrane. By growing cells in the presence of one UFA before the induction of the lactose transport system and then shifting to a second UFA upon induction, Wilson and Fox (1971) reported that the rate-temperature profile of transport reflected the fatty acid present during the induction period, even though in some cases this was not the major fatty acid

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RONALD N. McELHANEY

400 -

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FIG. 1 . The temperature dependence of P-galactoside transport in an unsaturated fatty acid auxotroph of E . cufi grown on oleic acid- or linoleic acid-supplemented media. The rate of P-galactoside transport was measured as nmoles of 0-nitrophenol (ONP), released from the intracelMar hydrolysis of the P-galactoside transport system substrate 0-nitrophenolgalactoside, per hour by 2 x IOY cells. (From Wilson et ul., 1970.)

present in the total membrane lipid. They thus concluded that newly synthesized transport proteins are preferentially associated, in a relatively long-lived manner, with newly synthesized lipid. In contrast, Overath er al. (1971), using a generally similar experimental approach, found that the rate-temperature profiles of the lactose transport system reflected the average membrane lipid fatty acid composition and not that of the phospholipids synthesized during induction of the transport system, suggesting a relatively rapid randomization of the membrane phospholipids. In a later paper, Tsukagoshi and Fox (1973b) presented data tending to confirm the findings of Overath and co-workers, and ascribed their initial observations to an inadequate characterization of the lactose transport rate-temperature profiles. However, Tsukagoshi and Fox (1973b) then reported that if transport induction is carried out at 25°C rather than 37"C, triphasic Arrhenius plots are observed, with one break corresponding to the UFA present during growth at 37°C and the second break corresponding to the fatty acid present during induction at 25°C. These authors concluded that newly synthesized transport proteins are indeed preferentially associated with newly synthesized lipid, but that the relatively rapid randomization of the membrane lipid phase at 37°C simply precludes its observation, although at lower temperatures (25°C) randomization is not complete and some preferential association is still observable in the rate-temperature profiles. This conclusion appears to be a

EFFECTS OF MEMBRANE LIPIDS

335

tenuous one, however, for a number of reasons. First, there is uncertainty about the validity of transport measurements performed at temperatures below the phase transition temperature, because E . coli cells lose viability and become leaky under these conditions. Second, it is far from clear that the experimental data presented are uniquely fit by the three straight-line segment Arrhenius plots drawn; in fact, the experimental points actually fall on smooth, continuously curving lines. Third, later studies from the same laboratory report the presence of two or even three breaks in Arrhenius plots of lactose transport in cells cultured on only a single fatty acid (Linden and Fox, 1973; Linden et al., 1973a,b). Finally, recent 2H-NMR and saturation-transfer ESR studies of E . coli (Davis et al., 1979; Gally et a l . , 1979, 1980; Kang et al., 1979b; Nichol et al., 1980) and A . laidluwii B membranes (Stockton et ul., 1977; Smith et al., 1979; Davis et al., 1980; Rance et ul., 1980), and of several reconstituted membrane proteinlipid model membrane systems (Baroin et al., 1979; Kang et ul., 1979a), have demonstrated that rapid exchange (exchange rate greater than 103/second) must be occurring between boundary- and bulk-lipid populations, even at temperatures near 0°C. Thus, unless the lactose transport system is quite atypical, no preferential association of newly synthesized membrane lipids with newly induced transport proteins should be detectable in rate-temperature profile experiments of the type described, irrespective of the temperature at which induction was performed. Although it seems that the biogenesis of the lactose transport system is not dependent on concomitant phospholipid synthesis in general, nor upon the synthesis of specific molecular species of phospholipid in particular, it is well established that the function of the lactose transport system is dependent in some manner on the gel to liquid-crystalline membrane lipid transition, despite the discrepancies reported in the number and position of the Arrhenius plot breaks by Fox and co-workers (Wilson and Fox, 1971; Linden and Fox, 1973; Linden et al., 1973a,b) and by Overath and co-workers (Schairer and Overath, 1969; Overath and Trauble, 1973; Sackmann et a!., 1973). Thilo et ul. (1977) have recently provided a reasonably explicit and physically plausible explanation for the observed temperature dependence of P-galactoside (and P-glucoside) transport in a UFA auxotroph of E . coli, based on a recent reinvestigation of the transport rate-temperature profiles, paying particular attention to the determination of transport rates at low temperatures. These workers now observe triphasic Arrhenius plots consisting of two linear regions of similar and relatively shallow slopes occurring at both high and low temperatures, separated by a linear region of much steeper slope at intermediate temperatures (see Fig. 2 ) . The first fairly gradual, downward change in slope generally occurred between the lipid phase transition midpoint and upper boundary, as determined by a fluorescent probe, whereas the second, upward change in slope correlated well with the lower boundary of the phase transition. Thilo et al. (1977) interpreted these results in

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RONALD N. McELHANEY

-

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FIG. 2 . The temperature dependence in vivo of the rate of P-galactoside transport, measured as 0-nitrophenyl P-c-galactopyranoside hydrolysis, for palmitelaidic acid-( trans-A9-16:I ) supplemented cells (upper part) in comparison with the lipid phase transition as measured by N-phenyl-lnaphthylamine fluorescence in whole cells (lower part). (From Thilo et al., 1977.)

terms of a lateral partitioning of these transport proteins between the fluid- and ordered-lipid domains of the E . coli membrane, with these sugar transport proteins exhibiting a 10- to 25-fold higher activity in the liquid-crystalline than in the gel domains, but functioning by similar mechanisms in both lipid phases (hence the similar activation enthalpy values exhibited at temperatures both above and below the lipid phase transition). The apparent lateral partition coefficient varied with the membrane lipid composition (which would explain why the exact relationship of the upper temperature inflection to the position of the phase transition often appears to be variable), but in all cases the partitioning of the transport proteins into the fluid parts of the membrane appeared to be favored. The conclusion that the lactose transport system retains appreciable activity in a gel-state lipid environment should be accepted with caution, however, despite the authors’ great care in determining transport rates at low temperatures, since these cells become highly permeable to the lactose analogue utilized to measure transport at

EFFECTS OF MEMBRANE LIPIDS

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temperatures near the lower boundary of the lipid phase transition. It is thus unclear whether the rates of hydrolysis of the lactose analogue, which are only 15-30% above the high background levels observed at these low temperatures, accurately reflect the activity of the lactose transport system functioning via its “normal” mechanism. The concept of a selective partitioning of the lactose transport proteins into the fluid lipid domains of the membrane within the lipid phase transition temperature range has been supported by the studies of Therisod et ul. (1977) and of Letellier et al. ( 1977). These workers utilized the fluorescence changes purportedly accompanying the energy-dependent biridirig (but not the transport) of dansyl galactoside to study the number of functional lac carriers as a function of temperature and of the membrane lipid phase transition. They concluded that the changes in slope observed for Arrhenius plots of lactose transport are due prirnarily to a change in the ~ u m h e offuncfionul r curriers with temperature, rather than to a change in the rate at which a constant number of luc carriers translocate substrate. Shechter and co-workers confirmed that the luc-carrier proteins do indeed segregate preferentially into the liquid-crystalline as opposed to the gellipid domains, but found that about half of the carrier proteins in the fluid domains are nonfunctional, whereas all the carrier proteins in the ordered domains are fully functional! This latter observation is quite curious, since reconstitution studies with a large variety of membrane enzymes and transport systems have almost uniformly demonstrated a requirement for fluid lipid for activity (for review, see Sandermann, 1978). Moreover, Overath et al. (1979) have recently presented evidence that, contrary to the original reports, dansyl galactosides are in fact transported by E . coli and that the fluorescence increase observed upon energization of cytoplasmic membrane vesicles is due at least in part to a nonspecific binding of dansyl galactoside to the membrane. Overath and coworkers thus maintain that since transport and nonspecific binding, as well as possible specific binding to the luc-carrier proteins, can all induce changes in the fluorescence of dansyl galactoside, the studies of Shechter and co-workers could not really distinguish between changes in the number of luc carriers that are functional and the rate at which these carriers are transporting dansyl galactoside. Thus the original partition hypothesis of Thilo ef a / . ( 1 977) remains a viable one pending further clarification of the nature of the interaction of dansyl galactosides with the E . coli cytoplasmic membrane. Teather ef (11. (1980) have recently constructed an E . coli strain that, in addition to being auxotrophic for UFAs, contains a multicopy plasmid coding for the Y gene product of the lac operon, the lactose permease protein. Transport rates i n this lactose permease-overproducing strain are 6- 10 times higher than in normal cells. Using this strain, P. Overath and co-workers (personal communication) recently demonstrated that substrate hinrfing to the lactose-carrier protein in the membrane is not affected by the membrane lipid phase transition. That is, the

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RONALD N. McELHANEY

temperature dependence of the dissociation constant of lactose binding is linear, exhibiting no breaks in the region of the lipid phase transition, and the number of binding sites also remains constant over the physiological temperature range. These findings suggest that changes in the rate of lactose translocation across the membrane, and not in the number of functional lactose permease molecules, are responsible for the characteristic Arrhenius plot shapes observed for E . coli cells of varying fatty acid composition. Interestingly, however, no “breaks” or upward inflections in the slope of the Arrhenius plot of lactose transport at the lower boundary temperature of the lipid phase transition could be detected in this lactose carrier-enriched strain, in contrast to the earlier study using a “normal” UFA auxotroph (Thilo et al., 1977). Instead, Arrhenius plots that are curved at the higher temperatures and become linear and more steeply sloping at the lower temperatures are observed. This behavior is not that predicted by the partition hypothesis put forward by Thilo et ul. (1977). The dependence of the rate-temperature profile of the /?-glucoside transport system of E . coli on the phase state of the membrane lipids has often been studied in parallel with the /?-galactoside transport system by Fox and co-workers (Wilson et al., 1970; Wilson and Fox, 1971; Linden and Fox, 1973; Linden et ml., 1973a,b) and by Overath and co-workers (Thilo et ul., 1977). In all cases the P-glucoside and the lactose transport systems exhibited almost identical behavior. Thus the /?-glucoside transport system responds to the order-disorder transition of the membrane lipids just as does the lactose transport system previously discussed. The transport of glucose in E . coli takes place via the phosphoenolpyruvate sugar phosphotransferase system, in contrast to the transport of /?-galactosides and /?-glucosides, which are driven by the electrochemical proton gradient across the cytoplasmic membrane (Rosen and Kashket, 1978). Shechter et al. (1974) demonstrated that the rate-temperature profiles of glucose transport, into cytoplasmic membrane vesicles prepared from an E . coli UFA auxotroph, are not dependent on the fatty acid composition or on the phase state of the membrane lipids. The lack of a “break” in the region of the membrane lipid phase transition was also shown by Rottem et ul. (1973b) for a-methylglucoside uptake in a Mycoplasma mycoides var. Capri strain adapted to grow with low levels of cholesterol (Rottem et al., 1973a), although the apparent activation energy for a-methylglucoside uptake was much higher than in the native (cholesterol-rich) strain. These workers also reported that the rates and apparent activation energies for a-methylglucoside phosphorylation were the same for isolated membranes of each strain. Since a-methylglucoside transport into M . mj~coidesvar. Capri also occurs via the phosphoenolpyruvate (PEP)-dependent sugar phosphotransferase system (for review, see Cirillo, 1979), these observations imply that vectorial, group-translocation transport systems may function without “mobile carrier” components that are sensitive to the phase state of the membrane lipid bilayer, in

E f FECTS OF MEMBRANE LIPIDS

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contrast to most “classical ” active transport systems. Alternatively, the different types of energy coupling mechanisms operating in the two types of transport systems may explain their different responses to the membrane lipid gel to liquid-crystalline phase transition. Interestingly, however, Arrhenius plots of a-methylglucoside eff2ux from M . rnycoides var. cupri showed breaks at temperatures corresponding to those of the lipid phase transitions. Evidence for the role of phosphatidylglycerol (PG) in the function of the E . coli PEP-sugar phosphotransferase system in membrane vesicles was apparently provided by Milner and Kaback (1970), who reported that phospholipase D treatment inhibited the vectorial phosphorylation of a-methylglucoside without inhibiting the efflux of intravesicular a-methylglucoside phosphate. It was found that phospholipase D treatment specifically hydrolyzed PG and that after phospholipase D treatment, membrane vesicles could resynthesize PG with a concomitant return in their ability to take up a-methylglucoside. The transport of proline by these same vesicles was only slightly inhibited by phospholipase D treatment. However, Long and Dittmer (1974) reported that, under the experimental conditions employed by Milner and Kabac (1970), the hydrolytic activity of phospholipase D was negligible due to a lack of Caz+, which is required to activate this enzyme. Although no hydrolysis of E . coli membrane phospholipids occurred, an inhibition of a-methylglucoside uptake by membrane vesicles was again observed. When Ca2+was added to stimulate phospholipase D activity, the majority of phospholipid hydrolyzed in the membrane vesicles was PE. Thus the inhibition of sugar transport in membrane vesicles by phospholipase D does not appear to be due to a specific hydrolysis of PG, but is caused by some other component present in the enzyme preparation. Confirmation that PG is required for the in vivo function of the E . coli PEP-sugar phosphotransferase system will apparently require additional experimental work. Read and McElhaney ( 1 975) studied the effect of variations in the fatty acid composition and cholesterol content of the A . laidlawii B membrane on the rate-temperature profile of glucose uptake into intact cells. Glucose transport in this organism occurs via an electrochemical potential-driven, active-transport process and not via the PEP-sugar phosphotransferase system (Cirillo, 1979). These workers reported that the rate of glucose uptake (at 37”C, for example) increases as the calorimetrically determined gel to liquid-crystalline membrane lipid phase transition temperature decreases. Moreover, the presence of cholesterol reduces the rate of glucose uptake for each fatty acid enrichment tested. These results indicate that the absolute rate of glucose transport increases with the increasing fluidity of the membrane lipids and suggest that the glucose carrier protein(s) interacts intimately with the membrane lipids. In contrast to transport rates, the apparent activation energy for glucose uptake (above the phase transition midpoint temperature) was found to be independent of membrane lipid fatty acid composition and cholesterol content, suggesting that the apparent

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RONALD N. McELHANEY

activation energy is determined by glucose binding to the carrier protein at the membrane surface, or at least by some process not influenced by the fluidity of the membrane lipids. Accurate estimates of glucose transport rates at temperatures below the lipid phase transition midpoint temperatures could not be obtained due to the mechanical fragility and leakiness of the cells under these conditions.

2. AMINOACIDTRANSPORT Holden et a / . (1975) studied the effect of a pantothenic acid deficiency in Lactobacillus plaritarurn on the initial rates and steady-state accumulation levels of a number of amino acids. Pantothenate deficiency in L . plantarum results in a reduced membrane phospholipid content, presumably due to an inhibition of de iiovo fatty acid biosynthesis. The steady-state accumulation capacity for all amino acids tested was markedly reduced in pantothenate-deficient as compared to control cells, apparently due to a markedly increased rate of passive leakage of the accumulated amino acids in the membrane lipid-deficient cells. Initial rates of amino acid uptake, however, were either unchanged (asparagine, alanine, lysine) or were actually increased (glutamic acid, aspartic acid, or leucine). These findings demonstrate that a reduction of membrane lipid content heterogeneously affects the operation, or possibly the biogenesis, of amino acid transport systems. A convincing explanation for the observation that a reduction in membrane lipid content results in an incwasa in the rate of operation of some transport systems has yet to appear. The influence of the fatty acid composition and phase state of the membrane lipids on the rates and temperature dependence of proline uptake by membrane vesicles prepared from E . coli UFA auxotrophs have been studied by Esfahani e f (11. (I971 b) and by Shechter et a / . (1974). Both groups utilized X-ray diffraction techniques to monitor the phase state of the membrane lipids. Esfahani e / a / . (1971b) reported single Arrhenius plot breaks at 26, 19, and 14°C for elaidic, oleic, and linolenic acid-enriched membrane vesicles, respectively. The apparent activation energy for proline transport was independent of fatty acid composition above the Arrhenius break temperature, but varied slightly below it. Shechter and co-workers (1974) also reported Arrhenius plots with single breaks, but at temperatures of 38, 2 2 , and 19°C for elaidic, oleic, and linolenic acid-enriched vesicles, respectively. Moreover, the apparent activation energy of proline transport was reported to vary markedly both above and below the break temperature in this latter study. Thus the agreement between these two studies of proline transport is only fair, as is the agreement between either study and investigations of the temperature dependence of the p-galactoside and /3-glucoside transport systems reviewed earlier. However, in both studies the rate-temperature profile of the proline uptake system correlated at least qualitatively with the membrane lipid order-disorder transitions detected by X-ray diffraction.

EFFECTS OF MEMBRANE LIPIDS

34 1

Rosen and Hackette ( 1972) demonstrated that the rate-temperature profiles of arginine and glycine transport into cells of an E . coli UFA auxotroph were influenced by the UFA supplementation employed. Both transport systems produced Arrhenius plots with single breaks at about 30 and 13°C for elaidic and oleic acid-supplemented cells, respectively. These break temperatures are in reasonable agreement with those reported by some investigators for the lactose transport system, but only in fair agreement with the values for proline transport just discussed. One should note that the arginine transport system in E . coli is an osmotic shock-sensitive system that is dependent on the presence of a periplasrnic binding protein for optimal function, whereas the glycine transport system is an osmotic shock-insensitive system that does not have a periplasmic binding protein component; there is some evidence that the mechanism of energization of these two types of systems is different, with the former being driven by ATP hydrolysis and the latter directly by the transmembrane electrochemical proton gradient (see Rosen and Kashket, 1978). Rosen and Hackette (1972) did not report fatty acid compositions or phase transition temperatures in their study, so a more detailed comparison with other rate-temperature investigations is difficult. We ( M . 0. Eze and R. N . McElhaney, unpublished observations) have recently studied the temperature dependence of the osmotic shock-sensitive glutamine and osmotic shock-insensitive proline transport systems, and the response of these transport systems to variations in the fatty acid composition and phase state of the membrane lipids in intact cells of an E . c d i UFA auxotroph. The thermotropic phase behavior of the E . c d i lipids in the cytoplasmic membrane fraction was determined by DTA. Arrhenius plot breaks for glutamine uptake occur at 29, 23, 17, 14, and
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RONALD N. McELHANEY

less pronounced, variation in the response of the system to the degree of conipletion of the lipid phase transition. A convincing molecular explanation for these observations is not yet at hand. Holden et o/. (1978) studied the initial rates of uptake of a number of amino acids in an E . coli UFA auxotroph enriched in various UFAs. These workers found that proline and threonine transport rates (measured at about 21°C) were much more depressed by enrichment with trans-UFAs (relative to cis-vaccenic acid enrichment) than were lysine and asparagine transport rates. Aspartic acid and leucine initial uptake rates exhibited intermediate degrees of inhibition by the trans-UFA enrichment. Lipid enrichment with very low-melting fatty acids also reduced relative initial transport rates for alanine (markedly) and for arginine (moderately), but appeared not to affect, or even slightly stimulate, asparagine uptake. Although differential changes in the relative number of functional transport systems in response to alterations in fatty acid composition might explain these results, the temperature-activity profiles of proline and lysine transport rates, in cells enriched in cis-vaccenic or palmitelaidic acids, suggested that nonuniform alterations in transport rates must also be involved. In general it appeared that binding protein-dependent (osmotic shock-sensitive) amino acid transport systems were less sensitive to fatty acid cornpositional alterations than were binding protein-independent (osmotic shock-insensitive) transport systems. Again the heterogeneous response of various amino acid transport systems to membrane lipid compositional alterations was noted. Relatively little work has been done on the effect of in vivo alterations in the membrane lipid polar head-group composition on membrane transport processes. One of the first studies performed was that of Beebe (1972), who studied the initial rates of uptake of a large number of amino acids and some other metabolites into a PE-deficient mutant of B . subtilis. In this mutant, PE was reduced from its normal levels of 7-8% to less than 1.0% of the total membrane lipids (Beebe, 1971). The depletion of PE had no statistically significant effect on the initial rates of uptake of the neutral amino acids tested (except glycine), but reduced initial uptake rates of a variety of other amino acids by various degrees ranging from slight to marked. The uptake rate of aspartic acid, however, actually doubled in the PE-deficient B . subtilis mutant. The initial uptake rates of glucose and acetate were also unaffected, but the rates of uptake of pyruvic acid and of several bases and nucleotides were substantially reduced. Beebe (lY72) suggested that PE must play a specific role in some transport systems but not in others. Ohta et ( I / . (1977) utilized hydroxylamine, an inhibitor of the enzyme phosphatidylserine (PS) decarboxylase, to alter the phospholipid head-group composition of E . coli. In hydroxylamine-treated cells, PS increased from trace quantities to up to 20% of the total membrane phospholipids, with a concomitant decrease in the levels of PE, the major phospholipid in this organism. The initial

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rates of uptake (at 25°C) of serine and glutamate were higher in the hydroxylamine-treated than in the control cells, whereas the uptake activities for alanine and phenylalanine were unchanged and that for lysine was reduced. Membrane vesicles prepared from hydroxylamine-treated cells also displayed higher initial rates of uptake of serine, glutamate, proline, and threonine than did vesicles prepared from control cells, whereas the uptake rates of alanine, phenylalanine, lysine, and glycine were the same in both vesicle preparations. All of the above effects were reversed when the phospholipid composition of hydroxylamine-treated cells was permitted to return to normal. The various amino acid-transport systems thus respond in a quite heterogeneous way when a portion of the neutral phospholipid PE is replaced by negatively charged PS in the E . coli cytoplasmic membrane. Johnson PI u l . ( 1 980) utilized pantoyl lactone to alter the phospholipid (and fatty acid) composition of M . lysodeikticus and Erwinia tarotovoru. Micrococcus Iysodrikticus cells grown in the presence of pantoyl lactone contain elevated levels of diphosphatidylglycerol (DPG), markedly increased levels of lysophospholipids, and markedly reduced levels of PG; reduced levels of methyl antriso-pentadecanoic acid and increased levels of methyl iso-palmitic acid are also observed after growth for 12 hours in the presence of this compound. The net effect of these changes on the physical state of the membrane lipids was to increase the phase transition midpoint temperature of the cell membranes, as measured by DSC, from about 10 to 16°C. Despite pronounced changes in the relative proportions of these phospholipid classes, the uptake rates of alanine, aspartic acid, and glutamic acid (apparently measured at room temperature) were not affected. Similar observations were reported for E . carotovora. The ratetemperature profiles for the transport of these amino acids were not determined in this study. 3.

ION T R A N S P O K I

Little work has been done on the relationship between membrane lipid fluidity and phase state, and ion transport activity in microorganisms. Cho and Morowitz (1972) studied the effect of growth temperature on the rates of K+ influx and efflux, and on K+ steady-state levels, in A . laidlawii B. These workers found that the rate of K+ uptake (measured at 37°C) in cells grown at 37°C was 2.5-fold higher than for cells grown at 25"C, whereas the rate of K + efflux (at 15°C) and the steady-state K+ levels (at 37°C) were independent of the temperature of growth. They thus concluded that K+ influx in this organism is sensitive to the fluidity and phase state of the membrane lipids, whereas K+ efflux and K+ accumulation are not. However, these conclusions rest on the assumption that altering the growth temperature of this organism will also alter the fatty acid composition, and thus the fluidity and phase state of its membrane lipids, as is

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true for most bacteria. However, other studies have shown that the fatty acid composition (McElhaney, 1974a; Saito and McElhaney, 1977; Saito ef a / . , 1977, 1978) and phase transition temperature (Melchior et d.,1970; McElhaney, 1974a,b) of the membrane lipids of A . Icridlawlii B are essentially growth-temperature invariant. Therefore, these studies d o not appear to provide any information o n the possible effects of membrane lipid physical state on K+ translocation in this organism. Le Grimellec and Leblanc (1978) first studied the growth characteristics, intracellular K+ content, and ability to extrude protons of native M . mycoiu’cs cells grown on medium supplemented with cholesterol and either palmitic plus oleic acids or elaidic acid, and of a strain adapted to grow on low levels of cholesterol in the presence of elaidic acid; cholesterol accounts for 20--25% of the total membrane lipid in the native strain and less than 2% in the adapted strain. Native organisms grown on cholesterol-rich medium exhibited identical growth characteristics, intracellular K+ contents, and medium-acidification properties, irrespective of fatty acid supplementation. In contrast, cholesterol-deficient organisms were unable to grow below pH 6.5 (instead of pH 5.2 as in the native strain), and exhibited lowered intracellular K+ levels and a reduced ability to extrude protons. Moreover, K+ passive permeability was drastically increased in the adapted strain, although K+ remained in equilibrium with the (reduced) transmembrane potential, and the intracellular Na+ content increased. Replenishing cholesterol in membranes of cholesterol-deficient cells resulted in a recovery of native growth characteristics, intracellular Kf level, and acidification potential. These authors suggested that cholesterol depletion produces its characteristic effects by inducing an increase of proton permeability, which in turn reduces the transmembrane electrochemical proton gradient that can be generated by this organism, thereby reducing intracellular K+ accumulation and limiting growth at lower pH values. The changes observed in K+ passive permeability did not appear to be involved in determining intracellular K+ levels. This study is an important one in that it demonstrates that lipid-dependent changes in the energy state of a cell can affect transport processes. Using the same organism, Le Grimellec and Leblanc (1980) investigated the temperature-activity relationship of Kt active influx, Mg’+-ATPase activity, transmembrane potential, and membrane lipid composition. Arrhenius plots of the initial rates of K+-exchange influx in the native strain enriched in palmitic plus oleic acids gave a linear relationship with an apparent activation energy of 9 kcal/mol (see Fig. 3). On the other hand, the native strain enriched with elaidic acid produced a biphasic linear Arrhenius pIot with a discontinuity at about 28-30°C; above this discontinuity, the apparent activation energy was 24 kcal/ inol and below it about 40 kcal/mol. Finally, the adapted strain grown in the presence of elaidic acid exhibited a biphasic linear Arrhenius plot with a break at about 23°C; above the break temperature, the apparent activation energy was

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FIG.3. Arrhenius plots of "K active influx an a function of the membrane lipid composition qf M . i i l ~ c ~ o i r / c , var. ,s u p r i celk. 0, Cells grown in cholesterol plus oleic and palmitic acids; A, cells grown in cholesterol and elaidic acid; and 0 , cells grown without cholesterol but with elaidic acid. (From Le Grimellec and Leblanc. 1980.)

7 kcalimol, and below about 44 kcal/mol. A broad endothermic lipid phase transition, occurring between 20 and 48"C, was observed by DSC for membranes from the cholesterol-deficient strain, whereas no phase transitions could be detected with this technique i n membranes of the native strain, irrespective of fatty acid composition. However, DPH steady-state fluorescence polarization results suggest the presence of a "phase separation" between 29 and 31°C in both palmitic plus oleic acid and in elaidic acid-containing native-strain membranes. Thus the rates of active K + influx appear to be sensitive to the phase state, and possibly to the fluidity, of the membrane lipids. In contrast, Arrhenius plots of Mg'+-ATPase activity and of transmembrane potential did not exhibit shurp discontinuities or breaks (see Fig. 4), and the activity-temperature profiles of the native and adapted strain were not significantly different. These workers thus concluded that the absolute Mg'+-ATPase activity and its temperature dependence, as well as the temperature dependence of the transmembrane potential difference, were not affected by the order-disorder phase transition of the membrane lipids. Therefore, the observed alterations in the K' influx rates with membrane lipid composition must reflect the dependence of the Kf carrier itself on the physical properties of the membrane lipids.

RONALD N. McELHANEY

346

A

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FIG. 4. ( A ) Arrhenius plots of membrane ATPase activity as a function of the membrane lipid composition of M . rriwwitles var. ctrpri cells. Symbols as in Fig. 3. (B) A plot illustrating the effect of temperature on the transmembrane potential of M . mycoides var. ctrpri cells grown with cholesor with cholesterol and elaidic acid (0). (From Le Grimellec terol and oleic and palmitic acids (0) and Leblanc, 1980.)

4 . OTHER TRANSPORT SYSTEMS Magnuson and co-workers have studied chlortetracycline transport in S. LZUreus (Dockter and Magnuson, 1974, 1975) and in Bacillus meguteriutn (Dockter er ul., 1978), and related it to the lipid order-disorder phase transition of the cell membrane of these organisms. In S. uureus, the Arrhenius plot of initial rates of

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antibiotic transport was biphasic with a fairly pronounced break at a temperature of 27°C. Culturing S. uureus at 37°C in the presence of exogenous oleic acid, or at 10°C without exogenous fatty acid supplementation, increased the UFA content of the cell membrane and reduced the Arrhenius plot break temperature by 8-9°C. Although the existence of membrane lipid phase transitions was not actually demonstrated by an independent physical technique, these workers suggested that the movement of chlortetracycline was facilitated by the more fluid lipid state existing above the presumed phase transition. In B . rneguterium, Arrhenius plots of initial rates of antibiotic uptake were interpreted to be triphasic, with breaks occurring at 20 and 9.5”C. Correlative ESR and fluorescence probe experiments indicated that the break temperatures apparently observed for chlortetracycline transport corresponded to the upper and lower boundaries of the gel to liquid-crystalline “lateral phase separation ” region. However, a careful examination of the Arrhenius plot of antibiotic transport reveals that in fact the experimental points fall on a single, slightly curving line. Thus the assignment of discrete break temperatures seems quite arbitrary, and for this reason the significance of this study is unclear.

E. Membrane-Associated Enzyme Activities 1 . OXIDATION-REDUCTION ENZYMES

One of the first studies of the relationship between the activity of a membrane enzyme and the fluidity and phase state of the lipids of a microbial membrane was that of Esfahani et a / . ( 1 97 1 b), who studied the activity-temperature profile of the succinic acid dehydrogenase (actually succinic acid-dichloroindophenol reductase) in membrane vesicles prepared from a UFA auxotroph of E. coli. These workers reported that Arrhenius plots of this enzyme exhibited breaks at characteristic temperatures for each fatty acid composition tested. However, in contrast to the behavior exhibited by Arrhenius plots for proline transport in these same vesicles, the succinate dehydrogenase break temperatures did not correlate with the gel to liquid-crystalline phase transition of the membrane lipids as monitored by X-ray diffraction. Esfahani et a / . ( 1 97 I b) thus concluded that the activity of this enzyme is influenced in some manner by the fatty acid composition of the membrane lipids, but not by the lipid phase transition per se. However, since the absolute activity of this enzyme was quite similar in vesicles having very different fatty acid compositions, and since the changes in the apparent activation energy values at the “break” temperature were quite modest, it is not clear that the experimental points are best fit by two straight lines, particularly as the experimental error of the enzyme-activity determinations was not given. Esfahani et a / . (1972) did establish in a subsequent study, however, that the succinic dehydrogenase activity, present in membranes of UFA auxotroph of E .

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coli, is indeed dependent on and modulated by phospholipids. These workers found that removal of about 50% of the phospholipid from isolated membranes with aqueous acetone resulted in a 70% loss of succinic dehydrogenase activity that, however, could be restored by incubation with the E . cofi total membrane lipids. Arrhenius plots of the (reduced) activity remaining after delipidation were linear in the temperature range 10-37°C. Arrhenius plots of succinic dehydrogenase activity in lipid-reconstituted membranes, however, exhibited single breaks, located at 19°C if oleate-enriched E . coli lipids were used for relipidation, or at 28°C when elaidate-enriched lipids were employed. The total cell lipid fraction could be replaced with the phospholipid fraction plus coenzyme Q, although neither component could restore activity alone. Phospholipids from elaidic acid-grown cells, when utilized for relipidation, produced a somewhat lower enzyme activity than did phospholipids from oleic acid-grown cells. This work demonstrated that the presence of an inflection in the Arrhenius plot of succinic acid dehydrogenase was dependent on the presence of sufficient levels of phospholipid, and in this latter study a good correlation between the inflection temperature and the membrane lipid phase transition temperature was observed. Morrisett c't ul. (1975) studied the activity-temperature profiles of NADH oxidase and of D-lactate oxidase in an E . coli UFA auxotroph enriched in elaidic acid. The Arrhenius plot for NADH oxidase activity exhibited two abrupt changes in slope, occurring at 27 and 32"C, whereas the Arrhenius plot of D-lactate oxidase activity also exhibited two breaks, but at 31 and 36°C. In both cases the slope of these plots showed significant decreases at each of these intlection temperatures as the temperature was raised. Pyrene excimer fluorescence and spin-labeled fatty acid ESR results indicated that the beginning, midpoint, and end of a single lipid order-disorder transition occurred at 25.529.0"C, 30.0-3 I .0"C, and 33.0-35.5"C, respectively (a second membrane structural change was also observed to occur over the temperature range 9.52 1 .0"C). Thus the NADH oxidase appeared to respond to the beginning and midpoint of the lipid phase transition, whereas the D-lactate oxidase responded to the midpoint and upper end of that transition. However, because the results obtained by these fluorescence and ESR techniques are suspect, as discussed earlier, and because the transition temperatures reported in this study are significantly lower than those obtained in X-ray diffraction, DSC, or TEMPO partitioning studies (see Cronan and Gelmann, 1975, and the work reviewed earlier), it is more likely that the abrupt changes in the apparent activation energies of both enzymes actually lie below the gel to liquid-crystalline phase transition midpoint. Nevertheless, this study again establishes the heterogeneous response of membrane enzymes to the membrane lipid phase transition, and suggests that different enzymes may preferentially interact with lipid domains that differ at least slightly in their physical properties. Haest et ul. (1974) studied the temperature dependence of NADPH oxidase

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and succinate-dichlorophenolindophenolreductase in isolated membranes from B . suhtilis and S. ciureiis. The B . siihriiis enzymes both exhibited pronounced breaks in their Arrhenius plots at about 20°C, which is near the lower boundary of the gel to liquid-crystalline membrane lipid phase transition as detected by DSC, which extends from 20 to 50°C. The S. m r e w NADPH oxidase also showed a definite Arrhenius plot break at about I Y C , whereas the succinatedichlorophenolindophenol reductase exhibited a less-pronounced slope change at about 20°C. In the latter case, the changes in the apparent activation energies of these enzymes occur just below and near the lipid phase transition midpoint, respectively. The phase transition in the S . uiireids membranes, as detected by DSC, extends from about 4 to 30°C. Esfahani et d.(1977) investigated the interaction of phospholipids with certain respiratory enzymes of the E . c d i membrane by use of a delipidationrelipidation procedure. E . cwli membranes were first treated with cold 90% acetone, which removed about 60% of the phospholipids and resulted in the loss of 90-9570 of the u-lactate, NADH, and succinate oxidase activities. The initial enzyme activities could be largely restored by incubation of the lipid-depleted membranes with an aqueous dispersion of coenzyme Q and an appropriate phospholipid. The three oxidase activities exhibited differences in their responses to the structure of the phospholipid head group. The only phospholipid that could significantly activate n-lactate oxidase was cardiolipin (CL), all other phospholipids being largely ineffective. The activity of N A D H oxidase, in contrast, could be restored by dispersions of coenzyme 0," plus PG, PS, or C L , although the neutral phospholipids phosphatidylcholine (PC) and PE were ineffective. Succinate oxidase was activated best by C L , followed by PS, PE, PG, and PC. Interestingly, in the absence of coenzyme Qlo, C L was the only phospholipid that could restore appreciable activity to these three enzymes. The apparent K , values of these lipid-reconstituted oxidases were also shown to be significantly altered by the nature of the phospholipid employed. This study would appear to demonstrate a specificity toward the structure of the polar group of phospholipids in the segment of the E . c d i electron-transport chain from the dehydrogenase through coenzyme Q. and a fairly specific interaction between the various protein components of this chain and certain phospholipids. Some caution should be used in accepting these conclusions, however, since the substantial amounts of phospholipid that remained associated with the acetone-treated E . c d i membrane may have altered the apparent phospholipid specificity of these enzymes. Moreover, since the fatty acid compositions of the various phospholipids used for reconstitution varied considerably, the observed differences in the ability of the various phospholipids to restore enzyme activity may not have been due solely to differences in polar head-group structure. Nachbar and Salton (1970) have also reported that the NADH dehydrogenase activity of M . Iy.socfc~ikricusmembranes is reduced by phospholipid extraction, and Mavis ct NI. (1972) have shown that

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the NADH oxidase of the E . coli membrane is inhibited by phospholipase C treatment. George-Nascimento et ul. (1976) studied the binding and reconstitution of a purified and delipidated preparation of D-lactate dehydrogenase, prepared from wild-type E . coli cells, employing isolated membranes of an E . i d i UFA auxotroph lacking this enzyme activity. These workers reported that the binding of D-lactate dehydrogenase to the membranes was not significantly affected by the fatty acid composition of the vesicle membrane lipids or by the temperature at which the constitution was performed. Thus enzyme binding appeared to be independent of the fluidity or phase state of the E . coli membrane lipids. In contrast, the reconstitution of D-lactate oxidase activity at 25"C, after binding to the membrane vesicles, was markedly influenced by the fatty acid composition of the lipids. The efficiency of reconstitution was greatest for oleate-enriched membranes, intermediate for linolenate-enriched membranes, low for palmitelaidateenriched membranes, and almost negligible for membranes enriched in elaidic acid. These results suggest that the activity of this enzyme is highest in membranes where the lipids are completely in the liquid-crystalline rather than the gel state at the reconstitution temperature, and also that the activity is reduced in membranes whose lipids exist too far above their characteristic phase transition temperature (i.e., whose lipids may be too fluid). George-Nascimento ef ul. ( 1976) also demonstrated that the membrane vesicle-reconstituted D-hCtate oxidase activities regained their characteristic fatty acid composition-dependent rate-temperature profiles; in membrane vesicles enriched in oleic or linolenic acids, linear Arrhenius plots were obtained over the temperature range 15-40°C, whereas in membrane vesicles enriched with palmitelaidic acid, a biphasic Arrhenius plot with a break in slope at about 30°C was presented. However, a careful inspection of the actual data points in this latter Arrhenius plot suggests that they could be just as well fit by a single curving line rather than by two straight-line segments of differing slopes. Silbert and co-workers have explored the effect of a high membrane lipid fluidity on the activity of several membrane respiratory enzymes, using a mutant of E . coli defective in total fatty acid synthesis (Davis and Silbert, 1974; Baldassare el al., 1977). When such a mutant is enriched to levels greater than about 85% in cis-vaccenic acid, there is a progressive inactivation of membrane-bound NADH, ~-a-glycerol-3-phosphate, succinate, and D-lactate oxidases. The dehydrogenase activities associated with each of these oxidases, however, were not affected. In addition, the temperature dependence of NADH oxidase activity was markedly reduced in cells containing only small quantities of saturated fatty acids. The permeability of the E . coli cytoplasmic membrane to K+ and to a lactose analogue was also markedly increased in cis-vaccenate-enriched cells. Identical changes in enzyme activity could be produced in vitro by the transfer of E . coli membrane lipids containing cis-monoUFAs from liposomes to isolated

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membranes so as to produce enrichments in cis-monounsaturates comparable to those obtained it7 vivo by growth on cis-vaccenate. Moreover, the oxidase activities could be restored to near-normal levels by the incubation of cis-vaccenic acid-enriched isolated membranes with liposomes containing lipids with normal or wild-type fatty acid composition. These studies convincingly demonstrate that the presence of phospholipid molecules possessing both a saturated and an unsaturated fatty acyl chain is necessary for the optimal activity of some, but not all, membrane enzymes, as well as for certain other membrane functions (including cell growth). Physical studies of E . coli cells highly enriched in cis-UFAs suggest that the inability of phospholipid molecules containing two unsaturated fatty acyl chains to support normal membrane function may be due to their high fluidities, and also possibly to a tendency to undergo "quasi-crystalline clustering" at higher temperatures (Baldassare et u l . , 1976).

2.

LIPID M E T A B O I J C

ENZYMES

Mavis and Vagelos ( 1 972) investigated the temperature dependence of the activities of three membrane-associated enzymes, including two involved in phospholipid biosynthesis, in a UFA auxotroph of E . coti. Arrhenius plots of glycerol-3-phosphate acyltransferase activity were essentially identical in membranes enriched in any of a number of cis-mono- or polyUFAs exhibiting linearity at temperatures below about 15°C and a gradual decrease in slope above about 15°C. In membranes enriched in trans-monoUFAs, the region of linearity extended up until about 20"C, whereupon the slope decreased more markedly above this temperature than was the case for the cis-unsaturated-enriched membranes. In contrast, Arrhenius plots of 1-acylglycerol-3-phosphateacyltransferase were all linear over the temperature range 0-37"C, regardless of the fatty acid composition of the membrane. However, membranes enriched in trans-UFAs exhibited a higher slope than those enriched in the cis-UFAs. Although the apparent specific activities of this enzyme also varied considerably with the fatty acid composition of the E . c d i membranes, the actual apparent specific activity values of membranes enriched in the various UFAs studied were not reported. Finally, Arrhenius plots of glycerol-3-phosphate dehydrogenase were also linear over the temperature range 0-37"C, with virtually identical slopes being observed with membranes containing any of a variety of cis- or trans-UFAs. The unique response of each of these three membranous enzyme activities to variations in membrane lipid fatty acid composition indicates a heterogeneity of membrane proteins with respect to their lipid dependencies. Although none of the three enzymes responded specifically to the bulk membrane lipid phase transition, the activity of two acyltransferases was obviously influenced by the physical properties of membrane lipids. Mavis and Vagelos (1972) suggested that these enzymes may selectively associate with lipids of the appropriate fatty acid

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composition so a s to maintain relatively normal rate-temperaturc profiles in the facc of alterations in the fatty acid composition of the bulk lipids, and/or that these acyltransfcrases might interact with lipids in their immediate environment s o as to modulate their fluidities and phase transition properties. The responses of the E . coli glycerol-3-phosphate and 1-acylglycerol-3phosphate acyltransferases (as well as several other membranous enzymes) to phospholipase C treatment was investigated by Mavis et al. (1972). These workers demonstrated that the rate and extent of phospholipid hydrolysis was essentially identical in isolated cytoplasmic membrane and in sonicated membrane phospholipid-water dispersions. In both cases PE and PG were completely hydrolyzed to diglyceride and phosphorylated alcohol by phospholipase C, whereas C L was partially resistant to hydrolysis. The glycerol-3-phosphate acyltransferase (and NADH oxidase) were progressively and completely inactivated in parallel with phospholipid hydrolysis. The 1-acylglycerol-3-phosphate acyltransferase was also progressively inactivated to about 50% of initial activity during hydrolysis of the first half of the membrane phospholipid, but did not lose additional activity as phospholipid digestion continued to near completion. Glycerol-3-phosphate dehydrogenase and succinate dehydrogenase were not affected by phospholipase C hydrolysis of 95% of the membrane phospholipid. These results apparently demonstrate the heterogeneity of membranous enzymes with respect to their dependence on the presence of intact membrane phospholipids, and suggest that lipid-protein interactions do not affect the susceptibility of phospholipids to phospholipase hydrolysis. This latter result is at variance with the observation of Bevers et ul. (1977), who showed that phospholipase A and C hydrolysis of the PG associated with the A . luidlnwii B Mg”-ATPase takes place at a much slower rate than does the hydrolysis of the bulk-phase PG. Moreover, the inability of Mavis et a / . (1972) to restore the activity of the acyltransferases by the addition of E . c d i PG is somewhat disturbing, and suggests the possibility that the products of phospholipid hydrolysis (probably the diglycerides) may have inhibited enzyme activity. Alternatively, phospholipid hydrolysis may have irreversibly reduced the stability of these enzymes, as has been demonstrated by Bevers r t (11. (1977) for the A . laidluwii B Mg’+ATPase and by many other workers for various nonmicrobial membranous enzymes (see Sandermann, 1978), making reconstitution of activity impossible. Ishinaga et cil. (1976) studied the level and temperature dependence of glycerol-3-phosphate acyltransferase in membrane particles derived from wildtype E . coli grown at 17 or 37°C and from a UFA auxotroph enriched in elaidic acid. It was found that the activity of this enzyme in 17°C-grown wild-type membranes and in elaidic acid-enriched membranes was significantly lower than in membranes derived from wild-type cells grown at 37”C, implying that the reduced fluidity of the former membranes may be responsible for the reduced activity of this enzyme. Support for this hypothesis was provided by experiments

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in which various phospholipids derived from membranes of wild-type cells grown at 37°C were added to elaidate-enriched membranes. The added PE had no effect on acyltransferase activity and CL produced a slight inhibition of activity, but PG produced a 2- to 4-fold enhancement of enzyme activity, indicating that this enzyme is relatively specific for the phospholipid polar head group. When various molecular species of PG were tested, it was found that the disaturated PG inhibited enzyme activity at both 17 and 37”C, whereas the I-saturated-24sunsaturated and I ,2-di-cis-unsaturated PGs increased activity markedly at both temperatures, the former being more effective at 37°C and the latter at 17°C. These results indicate that this enzyme requires a particular class of phospholipid, which exists in the liquid-crystalline state but which is not hyperfluid, for maximal activity. However, the Arrhenius plot of acyltransferase activity, which in elaidate-enriched membranes exhibited slope changes at 22 and 31°C. was not altered after incubation with the various E . c d i PGs, except that the absolute activity at all temperatures was elevated. Ishinaga et 01. (1976) suggested that the phase behavior of the endogenous elaidic acid-containing phospholipids dominate thc rate-temperaturc profile but do not determine the activity of this enzyme when exogenous PG is added, although no explanation for how this could come about was put forward. It is at least possible that the changes in slope observed in the Arrhenius plot may be due to the intrinsic properties of the acyltransferase protein and not to the physical state of its membrane lipid environment, as is apparently the case for the Ca‘+ ,Mg’+-ATPases and for the succinate cytochromc c oxidoreductases of Rhodospirillum ruhrum and Rhodopscwiornonus sphueroidPs (Kaiser and Oelze, 1980) and for a number of nonmicrobial membranous enzymes (see Sandermann, 1978). Whatever the difference in the shapes of the Arrhenius plots obtained by these workers and earlier by Mavis and Vagelos (1972) was ascribed by Ishinaga et u l . (1976) to a failure to compensate for the temperature-induced pH changes in the TrisHCI buffer employed by Mavis and Vagelos in assaying acyltransferase activity. Since the activity of many enzymes is markedly influenced by pH, small changes in the pH of a reaction mixture with temperature can produce appreciable changes in activity, which, if unrecognized, may be erroneously ascribed to a direct effect of temperature on the enzyme itself, or, in the case of membranous enzymes, to an effect of temperature on the fluidity or physical state of the membrane lipids that may indirectly modulate enzyme activity. Rottem et al. (1977) studied the temperature dependence of the membranebound, long-chain fatty acyl-CoA thioesterase activity in A . laidlawii, strain PG8, enriched in palmitic, elaidic, or oleic acids. In addition, these investigators determined the “phase transition temperature” of the lipids in each of these membranes by ESR using intercalated 12-doxy1 stearic acid as the spin probe. The absolute activity of this enzyme was found to be quite similar i n all membrane preparations, irrespective of fatty acid composition, and the Arrhenius plot

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of thioesterase activity in oleate-enriched membranes was linear over the temperature range of 5-40°C. The Arrhenius plots of thioesterase activity in elaidateand palmitate-enriched membranes, however, exhibited moderate increases in slope below about 12 and 18"C, respectively. The spin label, on the other hand, revealed apparently sharp phase transitions at about 26 and 35°C for elaidate- and palmitate-enriched membranes, respectively, and no phase transition in the physiological temperature range for membranes enriched in oleate. The ESR transitions reported in this study lie near the midpoint temperatures of the gel to liquid-crystalline membrane lipid phase transitions of the closely related A . laidlawii B strain of similar fatty acid compositions, as determined by DSC (Steim ct ul., 1969), DTA (McElhaney, 1974a,b), and X-ray diffraction (Engelman, 1971). The Arrhenius plot break temperatures, on the other hand, lie near the lower boundary of this transition. The thioesterase may thus be associated in the membrane with the lipid molecular species having the lowest phase transition temperatures. Alternatively, the effect of the lipid phase transition on the thioesterase activity may be due to the reduced solubility of the enzyme substrate, fatty acyl-CoA, in the gel-state lipid phase, rather than to a lipid transition-induced conformational change in the enzyme molecule itself. 3. ADENOSINE TRIPHOSPHATASES

De Kruyff et (11. (1973) studied the temperature dependence of three membrane-bound enzymes in A . luidlawii B cells of varying fatty acid and sterol compositions. For NADH oxidase and p-nitrophenylphosphatase, Arrhenius plots were linear over the temperature range 5-35°C and no breaks or slope changes were observed. Moreover, the absolute activity of these enzymes in isolated membranes did not vary with the fatty acid and sterol composition. De Kruyff and co-workers thus concluded that NADH oxidase and p-nitrophenylphosphatase, despite being integral membrane proteins, were unaffected by the fluidity and phase state of the membrane lipids. In contrast, the Mg"-ATPase seemed to exhibit biphasic linear Arrhenius plots in membranes enriched in relatively high-melting fatty acids, with the slopes increasing about 2.5-fold below the break temperature, but no breaks in membranes enriched in low-melting fatty acids. The Arrhenius plot break temperatures always fell within a few degrees of the lower boundary of the gel to liquid-crystalline lipid phase transition as measured by DSC. When grown in the presence of cholesterol, the Arrhenius break temperatures were reduced by 6-7"C, as was the lower boundary of the lipid phase transition. Treatment of cholesterol-enriched membranes with the polyene antibiotic filipin, which specifically complexes cholesterol and withdraws it from interaction with the membrane glycerolipids, reversed the effect of cholesterol incorporation on both ATPase activity and the

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lipid phase transition. Finally, the incorporation of epicholesterol instead of cholesterol had no effect on either the ATPase activity-temperature profile or the membrane lipid phase transition. De Kruyff et ul. (1973) concluded that the activity of the A . luidlawii B Mg‘+-ATPase was markedly influenced by the phase state of the membrane lipids and that, within the phase transition temperature range at least, this enzyme preferentially associated with the lipid molecular species having the lowest phase transition temperatures. Qualitatively similar results were subsequently presented by Hsung et al. (1974). Bevers et ul. (1977) studied the phospholipid requirement of the same three membrane-bound enzymes studied earlier by de Kruyff et al. ( I 973), by employing various phospholipases to degrade the PG selectively in A . luidluwii B membranes. These workers found that the complete hydrolysis of PG, the only phosphatide present in the membrane of this A . luidluwii strain, by phospholipases A,, C, or D had no effect on the activity of NADH oxidase or of p-nitrophenylphosphatase. Similarly, phospholipase A, and C hydrolysis of about 90% of the membrane PG could occur without effect on Mg”-ATPase activity, but hydrolysis of the final 10% of this phospholipid, which proceeded at a slower rate, resulted in a marked and progressive loss of activity. Interestingly, the complete conversion of PG to phosphatidic acid by phospholipase D treatment did not affect Mg2+-ATPase activity. The inactivated MgZi-ATPase in PG-depleted membranes could be reactivated by adding PG, phosphatidic acid (PA), or PS but not by PC, PE, or by any of the A . laidluwii glycolipids. These results indicate that this enzyme requires small amounts of a diacyl phosphatide bearing a net negative charge for optimal activity. Phospholipid reconstitution experiments demonstrated that the fatty acid composition of both the residual PG present in the membrane and the added phospholipid determine the activation energy of the Mg”+-ATPase and the Arrhenius plot break temperature. In these reconstitution experiments, a preferential association of this enzyme with the more fluid phospholipid species did not seem to occur, in contrast to the suggestion made earlier by de Kruyff rr a / . (1973). One should note that the ability to restore Mg’+-ATPase by adding exogenous phospholipid to membranes containing less than 2% of their original PG is gradually Iost with time. This result probably means that a certain minimal amount of phospholipid is required for stabilization of this enzyme in an active conformation. The dependence of the kinetic parameters of the A . luidlawii ATPase on the fluidity and phase state of the membrane lipids has recently been investigated in more detail by McElhaney and co-workers. These investigators showed that the K, of this enzyme for ATP is markedly temperature dependent, the K, value increasing roughly 10-fold as the temperature is increased from 5 to 40°C (Jinks el a/., 1978; Silvius ef u/., 1978, 1979). This temperature dependence of substrate-binding affinity appears to be an intrinsic property of the enzyme molecule, since it is not affected by the fatty acid composition, cholesterol

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content, or phase state of the membrane lipids. Moreover, Silvius P / (11. (1978) demonstrated that this and other enzymes with a temperature-dependent K , can yield a variety of Arrhenius plot artifacts, most notably erroneous plot “breaks,” if enzyme activity is assayed at a fixed substrate concentration; in fact, the positions of the Arrhenius plot “breaks” previously reported by de Kruyff et ul. ( 1973) were actually significantly undcrestirnated (by 7- 10°C) because ATPase activity was becoming substrate-limited at the higher assay temperatures. These investigators also demonstrated that the Mg’+-ATPase of A . luidluwii B is strongly and specifically stimulated by Na+ (Silvius et u l . , 1978; Jinks et ul., 1978), and therefore that this enzyme should be denoted as a Na+ ,Mg”-ATPase. The results of initial studies of the activity-temperature profile of the A . IaiciluKii Na+,Mg”-ATPase, under conditions where the true ,,/I of the enzyme was being measured, produced the classic biphasic linear Arrhenius plots for membranes enriched with fatty acids giving a lipid phase transition in the physiological temperature range, except that the break temperatures now occurred slightly below but generally near the phase transition midpoint temperature (as measured by DTA), instead of at the lower boundary of the transition (Jinks et al., 1978; Silvius ef u l . , 1979). Interestingly, the Arrhenius plots of ATPase activity in membranes enriched in low-melting fatty acids appeared to be curved downward, instead of being linear as previously reported (de Kruyff et n l , , 1973; Hsung et a l . , 1974). A subsequent study, in which more accurate ATPase activity values at additional temperature points were determined, confirmed that, in membranes containing exclusively liquid-crystalline lipid in the physiological temperature range, Arrhenius plots of Na+ ,Mg’+-ATPase activity are clearly nonlinear (slope gently downward; see Fig. 5). The trtnperuturc depcndrric~eof the ATPase activity is not dependent on membrane lipid fatty acid

1C

32

34

3.6

10’lT

FIG.5 . Arrhenius plots ofthe Na’ , MgL+-ATPase activity in fatty acid-homogeneous membranes of A . luidluivii B cells grown with avidin plus u~irriso-pentadecanoicacid ( 0 )or cis-vaccenic acid (0). The gel to liquid-crystalline membrane lipid phase transition in these membranes occurs below 0°C. The ATPase activity measured was arbitrarily scaled to permit a comparison of temperature dependencies. (From Silvius and McElhaney, 1980h.)

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EFFECTS OF MEMBRANE LIPIDS

. I

3 2

34

.

..

.

36

1O"T

FIG.6 . Arrhenius plot of the Nat, M$-ATPaae activity in fatty acid-homogeneous membranes of A . Iuicllawii B cells grown with avidin plus i.wheptadecanoic acid. The gel to liquid-crystalline membrane lipid phase transition midpoint ( 6 . ) of 28.8"C is indicated. The phase transition range is 22.5 to 32.5"C. (From Silviua and McElhaney, 1980b.)

composition as long as the lipids exist in the fluid state. The uh.solute ucti\i!\. of this enzyme, however, does vary significantly with fatty acid composition, but there is no discernible relation between enzyme activity and lipid fluidity per se (Silvius ef a / . , 1980b). If a gel to liquid-crystalline phase transition occurs within the physiological range, however, a gently curving biphasic o r triphasic Arrhenius plot was observed, in which the Na+,Mg"-ATPase activity falls off more steeply with decreasing temperature than would otherwise be the case (see Fig. 6). No effect of the lipid phase transition on the ATPase activity was noted until about half of the membrane lipid had been converted to the solid state, and some ATPase activity remained at temperatures considerably helow the lower boundary of the lipid phase transition, although eventually all ATPase seemed to be lost. (Note that the upward inflection at low temperatures seen in Fig. 6 appears to be due to another ATPase, which is "unmasked" when the Nat ,Mg'+-ATPase is inhibited.) These results suggest that the A . InidlaLcVi Na+,Mg'+-ATPase is active only in association with liquid-like boundary lipids, and that the ATPase hydrolytic reaction exhibits a significant heat capacity of activation in this case. This enzyme appears to become progressively inactivated

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RONALD N. McELHANEY

when its boundary lipids undergo a liquid-like to solid-like “phase transition” that is driven by the liquid-crystalline to gel phase transition of the bulk membrane lipid phase, but which is less cooperative and which takes place over a lower temperature range than does the bulk lipid transition. The lateral aggregation of intramembranous protein particles normally observed as the bulk lipid enters the gel state is apparently not responsible for the loss of ATPase activity, since membranes enriched in methyl ;so-branched fatty acids give Arrhenius plots indistinguishable froin those described above, despite the fact that no significant clustering of intramembranous particles occurs in iso-branched acidenriched membranes. These results suggest that the familiar “biphasic linear” Arrhenius plots commonly reported for many membrane enzymes and transport systems may in fact have a more complex shape, analysis of which can furnish useful information regarding the behavior of the enzyme molecule in its membrane environment (Silvius and McElhaney, 1980b). Rottem et CJI. ( 1 973b) studied the activity-temperature profiles of the membrane-bound Mg” -ATPase in isolated membranes from the native (highcholesterol) and adapted (low-cholesterol) strains of M . rnycoides var. cupri. In the native strain, Arrhenius plots of Mg’+-ATPase activity were linear over the temperature range 1 O-40cC, and no discrete membrane lipid phase transitions could be detected by fluorescence-polarization, ESR, or DSC measurements in these membranes, presumably due to their high cholesterol content. In the adapted strain, however, Arrhenius plots of Mg“-ATPase were biphasic, showing a marked increase in slope below a characteristic break temperature. In membranes from the adapted strain grown on palmitic plus oleic acids, the break temperature was 18-2272; in adapted-strain membranes enriched in palmitic and elaidic acids, the break temperature shifted upward to 30-32°C. The break temperatures observed in the adapted strain were near or just below the lower boundary of the membrane lipid phase transition as detected by the three different physical techniques just mentioned. At temperatures above the break temperature, the apparent specific activity of this ATPase was always considerably ( 2 - to 5-fold) higher in the adapted than in the native strain. These results were interpreted as indicating that the Mg2+-ATPase of this organism generally requires liquid-crystalline lipid, and in a particular state of fluidity, for optimal function. One should note, however, that Le Grimellec and Leblanc (1980) have recently reported that Mg’+-ATPase Arrhenius plots in both native and adapted strains of this organism are quite similar, being best described as gently curving (see Fig. 4) and without any sharp changes in slope of the type reported by Rottem et ul. ( 1 973b). A convincing explanation for this discrepancy in results has yet to be advanced. Sineriz et al. (1973) studied the allosteric properties of the membrane-bound Cd+-ATPase of a UFA auxotroph of E . coli. The Hill coefficient of Na+ inhibition ranged from 1.4 in cis-vaccenic acid-enriched membranes to 2.8 in linolenic

359

EFFECTS OF MEMBRANE LIPIDS

acid-enriched membranes, indicating that the allosteric interaction of this enzyme with Na+ occurs in all membranes regardless of their fatty acid composition. However, a positive correlation between the value of the Hill coefficient and the double bond index of the membrane lipids was found (see Fig. 7). Since in general membrane lipid fluidity should increase as the number of double bonds present in the hydrocarbon chain of the membrane lipids increases, these workers proposed that the allosteric behavior of this enzyme is modulated by membrane lipid fluidity. In a subsequent study, Moreno ef ul. (1974) reported that the membrane-bound (Ca’+)-ATPase exhibited cooperativity toward both Na+ and ATP at 3 6 T , whereas the solubilized form of the enzyme, which behaved as a peripheral membrane protein and did not require lipid for activity, did not show cooperative behavior toward either ligand at this temperature. In contrast, at 19°C the membrane-bound enzyme exhibited no cooperativity for interaction with Na+ or ATP, whereas the soluble enzyme showed cooperativity in the appropriate ligand concentration ranges. The allosteric behavior characteristic of the membrane-bound enzyme at both temperatures was regained when the soluble enzyme was reconstituted. Farias er d.(1975) later presented evidence that a Na+-triggered “folded-unfolded” conformational transition occurred in the soluble Ca2+-ATPase, and that the interaction between the enzyme subunits in the folded or compact state might be reduced, leading to a lack of cooperativity. Whether or not such a conformational change in the soluble enzyme molecule is physiologically significant, and whether it also occurs in the membrane-bound form of the enzyme, remains to be determined, as does the mechanism by which lipids exert their characteristic effects. Regulation of the allosteric behavior of a

I

I

I

0.25 0.75 1.25 1.75 Double bond index/Sat u ra t e d

FIG.7. Scattergrani of the correlation between the values of I I (Hill coefficient) for Na+ inhibition of the Ca”-ATPase and the membrane lipid double bond index per saturated fatty acid chain ratio of E . coli unsaturated fatly acid auxotrophs grown on a variety of unsaturated fatty acids. Cells grown with glucose on L broth medium containing: (a) vaccenic; (b) oleic; (c) palmitoleic; (d) linoleic; and (e) linolenic acid. Cells grown without glucose on the same medium and supplemented with: ( f ) vaccenic; and (g) linolenic acids. (From Farias r f a / . , 1975.)

360

RONALD N. McELHANEY

number of membranous enzymes from nonmicrobial sources by changes in membrane lipid composition has been reported (for a review, see Farias et ul., 1975).

4.

OFHER

ENZYMES

The rate-temperature profiles of the enzyme uridine diphosphategalactoselipopolysaccharide galactosyltransferase was studied by Beacham and Silbert (1973) in a UFA auxotroph of E . c d i . These investigators reported that the absolute activity of this enzyme was generally greater when the E . coli cell envelope fraction was enriched with low-melting fatty acids, that is, with shorter chain versus longer chain fatty acids, with UFAs having a double bond at the center of the chain rather than nearer the methyl terminus, and with cis- rather than trans-UFAs. Arrhenius plots of galactosyltransferase activity were linear over the temperature range 5-37°C in the trans-UFA-enriched cell envelopes, but were interpreted as showing one or more breaks at temperatures of 18-23°C for the three cis-monoUFAs studied. However, the data points for the cis-UFAenriched cell envelope can be well fit by smooth curves exhibiting a continuous decrease in slope with increasing temperature, and thus the existence of breaks is debatable. In any case, the activity of this galactosyltransferase was not markedly altered by the lipid transition per se, but its absolute activity and the manner in which this activity varies with temperature is clearly influenced by the nature of the fatty acyl chains of the membrane phospholipids. It is of interest to note that this enzyme is probably an extrinsic membrane protein (Endo and Rothfield, I969a). Weppner and Neuhaus ( 1 979) have investigated the interaction of lipid with the phospho-N-acetylmuramyl-pentapeptidetranslocase, the enzyme that catalyzes the first membrane reaction in peptidoglycan synthesis in membrane fragments of S. uureus. Two distinct discontinuities, one at 22°C and another at 3 0 T , were observed in the Arrhenius plot of the uridine monophosphate (UMP) exchange reaction catalyzed by this enzyme; at each of these discontinuities a marked upward jump in the rate of the reaction and a decrease in its apparent activation energy occurred as the temperature was increased. Changes in slope (but not actual discontinuities) were also reported for the Arrhenius plot of the transfer activity, again at 22 and 30"C, although these breaks, if real, were much less marked than seen for the exchange activity. Perylene fluorescence anisotropy and ESR measurements of 12- and 16-doxy1 stearic acid probes intercalated into the membranes of this organism indicated that a membrane lipid phase change occurs over the temperature range 16-22°C to 30°C. Since these values correlate with the discontinuities observed for the translocase activity measurements, Weppner and Neuhaus proposed that the physical state of the membrane lipids has a significant effect on the catalytic activity of this enzyme. This effect could occur either by alteration of the conformation of the enzyme

EFFECTS OF MEMBRANE LIPIDS

361

molecule, or by a change in the conformation or mobility of the hydrophobic substrate, undecaprenyl phosphate, or by both mechanisms. It should be noted, however, that the DSC measurements of Haest pi ( I / . (1974) indicate that the lower and upper boundaries of the gel to liquid-crystalline phase transition in S. u u r w s membranes are 4 and 3 I 0 C , respectively. Thus the translocase may respond to the phase transition midpuinr and upper boundary instead of the phase transition lower and upper boundaries, as suggested by Weppner and Neuhaus (1979). In a subsequent study, Lee et ul. (1980) showed that treatment of S. uureirs membranes with ti-butanol results in an increase in membrane lipid fluidity and a stimulation of translocase activity. An earlier study of the fluorescence properties of a dansylated derivative of undecaprenyl diphosphate pentapeptide, the product of the translocase-catalyzed reaction, was interpreted to suggest that this lipid intermediate is immobilized within a hydrophobic environment close to the membrane surface, and that its motion is not affected by changing the fluidity of the lipid matrix (Weppner and Neuhaus, 1978). However, the addition of n-butanol to S. uureus membranes was later found to increase the motion and the polarity experienced by the dansylated reaction product (Lee p t ul., 1980). Thus it remains unclear whether lipid-translocase or lipid-substrate and lipid-product interactions are more important for determining the overall rate of enzymic activity. The effects of pressure, and of detergent and phospholipase treatment, on the Arrhenius plot behavior of the partially purified, particulate nitrogenase from Azotobacter vinelandii was studied by Ceuterick et uf. (1978). At atmospheric pressure, the Arrhenius plot of nitrogenase activity appeared to be biphasic and linear, exhibiting a break at about 22"C, with the activation energy below this temperature increasing over 3-fold. As the pressure was increased up to 400 atmospheres, the break temperature progressively increased at the rate of 2°C per 100 atmospheres, with no significant changes occurring in the apparent activation energies above o r below the break temperature. A similar rate of increase with increasing pressure in the phase transition temperature for a variety of natural and synthetic phospholipid vesicles was also demonstrated. Treatment of the nitrogenase with Triton X-100 o r DOC produced preparations in which a linear Arrhenius plot was observed, with no break at 22°C. Moreover, the apparent activation energy of the detergent-treated enzyme over the entire temperature range studied corresponded to the value observed below 22°C for the detergentfree enzyme. Treatment of the nitrogenase with phospholipase A also abolished the Arrhenius plot break and increased the apparent activation energy. A biphasic linear Arrhenius plot could be reintroduced by incubating phospholipase-treated enzyme preparations with certain lipids at 40°C. Dipalmitoyl PE and PG produced Arrhenius plot breaks at about 27"C, whereas E . coli PE and an Azotobacter lipid extract produced breaks at 21°C. Thus there was no obvious relationship between the break temperature and the lipid phase transition temperature. Sev-

362

RONALD N. McELHANEY

era1 synthetic PCs and egg PA did not reinduce an Arrhenius plot break. Ceuterick ct ul. (1978) concluded that the nitrogenase exhibited a specificity for the polar head group of the phospholipid and also a requirement for a “fluid” lipid, although in fact dipalmitoyl PE and PG both exist in the solid state, even at the enzyme incubation temperature of 40”C! These workers suggested that a pressure- or temperature-induced change in the phase state of the boundary lipids triggers a conformational change in this enzyme, which is in turn responsible for the altered temperature dependence of its activity. However, since the nitrogenase was not pure nor was it completely free of endogenous lipids, the interpretation of certain of the findings reported must remain tentative. It does seem clear, however, that membrane lipids are in some way responsible for the breaks observed in the Arrhenius plots of this enzyme.

V. STUDIES OF ISOLATED MEMBRANE-BOUND ENZYMES Many difficulties stemming from the compositional complexity of intact microbial membranes can be circumvented by the isolation and purification of the membrane protein of interest, followed by its functional reconstitution with single lipids of defined fatty acid and polar head-group composition. The reconstituted enzyme activity may then be employed as a sensitive and obviously relevant indicator of the nature of the lipid-protein interactions necessary to maintain the enzyme (or transport protein) in an active, functional state. Despite the obvious appeal of this classical biochemical approach, membrane proteinlipid reconstitution experiments have their own pitfalls. The isolation and complete purification of integral membrane proteins particularly is often a difficult task, especially in a form free of endogenous lipid and yet which can be fully reactivated by appropriate amphiphilic compounds. Integral membrane proteins are normally solubilized by detergents, chaotropic agents, or organic solvents, which of course destroy the native membrane structure and organization, and which may partially but irreversibly denature the protein of interest. Moreover, these solubilizing agents, particularly the detergents, may be difficult to remove completely from the protein, once isolated. Although a protein may regain some activity upon reconstitution with phospholipids or other amphiphilic compounds, it is usually hard to judge whether or not the level of activity is that expected for the protein of interest, assuming that it has regained full function. Moreover, although the conformation of the active site of the reconstituted protein may be relatively “normal,” the conformation of those peptide domains that interact with the lipid may be subtly altered compared to the “native” protein. Nevertheless, studies of reconstituted lipid-protein systems are certainly required for a complete understanding of lipid-protein interactions at the molecular level, and such studies are reviewed in this section.

EFFECTS OF MEMBRANE LIPIDS

363

A number of membrane-associated enzymes, particularly those that behave as integral membrane proteins, have been shown to have an absolute requirement for amphiphilic lipids or detergents for activity. Other membrane-associated enzymes, particularly those that behave as peripheral membrane proteins, may have their activity modulated by lipids or detergents but may retain appreciable activity in the complete absence of these compounds. Thus it is useful to differentiate between lipid-requiring and lipid-modulated enzymes. In some cases, whether or not an enzyme exhibits a requirement for lipid depends on the method of its isolation, and on the substrate and conditions employed in the assay of its activity. Thus certain membranous enzymes, particularly those that may be loosely associated with the membrane, may not require lipid when acting on a water-soluble substrate, but may require lipid when acting on a water-insoluble compound. The effectiveness of various lipids and detergents in stimulating of activity of lipid-requiring enzymes is often markedly dependent on the concentration of the amphiphilic activator employed, and high concentrations of these compounds may completely inhibit activity. Generally speaking, detergents exhibit their maximum activation at concentrations near or just above their critical micellar concentrations. Phospholipids, on the other hand, typically show maximum activation at concentrations of 0.1-2 mM. which is far in excess of their very low critical micellar concentrations (around lo-''' M for dipalmitoyl PC; see Tanford, 1973). Since the phospholipid concentration giving maximum stimulation of the activity of a particular enzyme may vary greatly with the chemical structure of the phospholipid tested, a range of concentrations should be employed in studies where a comparison of the relative effectiveness of a series of different phospholipids is attempted. In many of the studies summarized in the table, only a single phospholipid or detergent concentration was tested, so conclusions about the relative effectiveness of various activators are tentative. In general, lipid-requiring enzymes do not exhibit a high degree of specificity with regard to the chemical structure of their phospholipid or detergent activators. Sometimes detergents are as effective in the activation of enzyme activity as are phospholipids. For example, of the many membrane-associated enzymes involved in phospholipid biosynthesis of E . coli, only diglyceride kinase and glycerol-3-phosphate acyltransferase require phospholipid (in addition to the appropriate substrates) for maximal activity. All other enzymes are fully active in detergent extracts and are not further stimulated by the addition of phospholipid (see Snider and Kennedy, 1977). Moreover, in only a relatively few cases is the detergent specificity of lipid-requiring enzymes very great. More typically, however, phospholipids, or a mixture of phospholipids and detergents, provide the highest degree of reactivation. There appear to be no known membranous enzymes that have a strict structural

364

RONALD N . McELHANEY

requirement for particular phospholipid fatty acyl groups. However, the fatty acid composition of the phospholipid activator is important in the sense that it determines the fluidity and phase state of the phospholipid at a given temperature. In almost every case, phospholipids having fatty acyl chains that are “melted” at the temperature at which enzyme activity is assayed are far superior activators as compared to gel-phase phospholipids. In a few cases highly unsaturated phospholipids have been shown to provide a definite but suboptimal stimulation of enzyme activity as compared to their less-unsaturated analogues, possibly due to their “hyperfluid” condition. One should remember that the presence of the enzyme itself, as well as any lipophilic substrates or detergents present in the enzyme assay medium, may alter the thermotropic phase properties of the phospholipid activator. Thus a particular phospholipid may actually exist in at least a partially fluid state in the assay medium at temperatures below the gel to liquid-crystalline phase transition temperature of the pure phospholipid-water dispersion. Lipid-requiring enzymes normally exhibit some degree of phospholipid polar head-group specificity for activation, but in most cases the degree of specificity is not great. Usually a number of different phospholipids will provide some stimulation of activity, although in general negatively charged phospholipids are superior activators of membranous enzymes. Among microbial enzymes, a strict requirement for a single phospholipid (PG) has been demonstrated only for the enzyme I1 complex of the E . coli PEP-sugar phosphotransferase system (Kundig and Roseman, 1971). However, only E . coli membrane lipids were tested (PE, PG, CL) and only at a single concentration; also, SDS alone would support some activity. Among nonmicrobial membrane-associated enzymes, a strict phospholipid requirement for activation has been convincingly demonstrated only for the mitochondria1 P-hydroxybutyrate dehydrogenase, which is significantly stimulated only by PC among the natural lipids tested (Gazzotti el a/., 1975; Grover et u l . , 1975). However, chemically similar analogues can substitute for PC to some degree. Often, but not always, mixtures of two or more different phospholipids produce a higher level of enzyme activity than does any single phospholipid alone. For this reason, testing mixtures of phospholipids, as well as single components, is to be recommended when the phospholipid requirements of isolated membranous enzymes are being studied; also, this approach more closely mimics the situation in the intact membranes, where the enzyme is presumably exposed to a large variety of different phospholipid molecular species. Since the fatty acid composition of the phospholipids being tested may also influence their potency as enzyme activators, it is important to compare phospholipids of similar fatty acid composition when the degree of polar headgroup specificity is being investigated. Phospholipids and detergents can apparently activate membrane-associated enzymes by a variety of different mechanisms. Some peripheral enzymes are

EFFECTS OF MEMBRANE LIPIDS

365

stimulated by amphiphilic compounds indirectly via the “solubilization ” of water-insoluble substrates, which in the absence of these dispersing agents form a separate nonpolar phase on which the water-soluble enzyme acts poorly. The addition of phospholipids or detergents solubilizes these substrates by the formation of mixed micelles, upon which the enzyme can now act. Similarly, amphiphilic lipids may solubilize the lipophilic products of membrane-bound or soluble enzymes, thereby relieving the inhibition of enzyme activity that may be produced by the accumulation of these products. Membrane enzymes that are also integral membrane proteins tend to aggregate in aqueous medium, due to the interaction of their now-exposed hydrophobic domains. Such aggregated enzymes are typically inactive and another function of phospholipids and detergents appears to be to “solubilize” and disperse these enzyme molecules. However, simple solubilization by itself is often not sufficient to produce enzyme activation, since many detergents that are effective solubilizing agents for a particular enzyme are poor or completely ineffective activators, indicating that these agents normally must also play another role in the activation process. The stabilization of lipid-requiring or of lipid-modulated membranous enzymes in an active conformation seems to be one of the key roles of phospholipids, in particular. Although enzyme conformational changes upon phospholipid addition have been directly demonstrated in only a relatively few cases, a variety of indirect evidence indicates that phospholipids (and detergents i n some cases) restore the active conformation of the enzyme, which seems to be lost upon removal from its native membrane environment. This indirect evidence includes a higher binding affinity for substrates and/or for ligand cofactors, an increased thermal stability, and an alteration in the susceptibility of the enzyme molecule to inactivation by proteases or inhibitors. upon reconstitution with the appropriate phospholipid. Phospholipids appear to bind to discrete, high-affinity sites on certain peripheral membrane-associated or soluble enzymes, and to act as allosteric effectors. In the case of certain enzymes that contain polypeptide subunits, such as the bacterial, mitochondrial, and chloroplast F,,-F, Ca’+Mg’+ATPases, lipid may interact with only a portion of the enzyme complex (see Haddock and Jones, 1977). In complex multistep enzyme-catalyzed reactions, only certain partial reactions may exhibit a lipid dependence. There is evidence that both the viscotropic and the interfacial properties of the phospholipidenzyme complex can be important in enzyme activation. A summary of lipid-requiring or lipid-modulated microbial enzymes is presented in the table, along with an indication of the lipids or detergents that can act as enzyme activators, and the probable mechanisms by which these compounds stimulate enzyme activity. For a more thorough treatment of the regulation of microbial and nonmicrobial membrane enzymes by lipids, the reader is referred to a recent excellent review by Sandermann (1978).

LISTING OF

ISOLATED

Enzyme (source) Oxidation-reduction enzymes Malate-vitamin K reductase ( Mycobacterium phlei) Malate dehydrogenase (Mycobacrerium avrutn) o-lactate dehydrogenase ( E . coli) L-lactate dehydrogenase ( E . coli)

MEMBRANE-BOUND MICROBIAL Esz\MriS Protein type

WHOSt

ACTIVITYIS

D E P E N D t , N T ON, OR IS MODU1.ATED B Y ,

Lipid activator(s)

Activator function(s)

Peripheral

Various phospholipids

Vitamin K solubilization, enzyme dispersion

Peripheral

Various phospholipids, some detergents Various phospholipids, lyso-PC, Triton X - l o 0 Various phospholipids, detergents (negatively charged ) Various phospholipid (esp. CL), several detergents Various phospholipids, neutral lipids, detergents Various phospholipids, nonionic detergents, fatty acid esters Various phospholipids

Enzyme conformation stabilization (FAD binding) Enzyme dispersion

Integral Integral

NADH dehydrogenase ( E . coli)

Integral

Pyruvate oxidase ( E . coli)

Peripheral

Malate oxidase (E. cofi)

Peripheral

Pyridine nucleotide transhydrogenase ( E . coli)

Integral

Enzyme dispersion,

LIPIDS

References

Asano and Brcdie ( 1963); Asano pf a / . ( 1965);Imai and Brodie ( 1973) Tobari ( 1 964); Imai and Tobari ( 1977) Tanaka rr a / . (1976); Fung et a / . (1979) Kirnura and Futai (1978)

conformational stabilization Enzyme dispersion, conformational stabilization

Dancey and Shapiro (1977)

Allosteric activators, promote ligand binding

Cunningham and Hager ( 1 97 la,b) O’Brien et a / . (1977); Schrock and Gennis (1977) Narindrasorasak er a/. (1979)

Allosteric activators, promote ligand binding ?

Houghton e t a ! . (1976)

Lipid metabolic enzymes Diglyceride kinase ( E . t d i )

Glycerol-3-phosphate acyltransferase ( E . coli) Fatty acid synthetase (Mycohactrriuui smeginufis) Adenosine triphosphatases F,,-F, Ca'+,Mg'+-ATPase ( E . coli) F(,-F, Ca'+ ,Mg'+-ATPase (thermophilic bacterium) F,, Ca'+ ,Mg'+-ATPase (thermophilic bacterium) F, Ca'+,Mg"-ATPase ( E . coli) F, Ca2',Mg'+-ATPase ( M . lyodeikticus) Peptidoglycan biosynthetic enzymes Isoprenoid alcohol phosphokinase (S. aureus)

Integral

Integral

Peripheral (soluble)

Various phospholipids plus a detergent (esp. Triton X-100) Mixtures of acidic and neutral phospholipids, or PG PC liposomes

1

Schneider and Kennedy ( 1973, IY76)

Snider and Kennedy (1 977); Kito e t a / . (1978) Solubilization of product (palmitoyl CoA)

Odriozola and Bloch (1977) (see Sumper and Trauble, 1973)

F,,-integral F, -peripheral

Various phospholipids. lyso-PC, detergents

F<,-integral F,-peripheral

Various phospholipids

Integral

Several phospholipids

Nieuwenhuis et a/ (1974); Hare ( 1975); Bragg and Hou ( 1 976) Kagawa and Racker ( 1 966); Sone el a / . (1975); Yoshida et a / . (1975) Okamoto et a / . (1977)

Peripheral

E . wli,other phospholipids None

Peter and Ahlers (1975); Bragg and Hou (1978) Munoz er al. (1969)

Peripheral

Integral

Various phospholipids (esp. PG and CL), also some detergents and polar lipids

Enzyme dispersion, interfacial regulation, substrate and product solubilization

Higashi et a/. (1970); Higashi and Strominger ( 1970); Sandermann ( 1972. 1974, I976a,b); Gennis and Strominger (1976a,b); Gennis el al. (1976) (continueti)

(Continued)

Enzyme (source) Isoprenoid alcohol phosphokinase ( L . plantarum) Pentapeptide translocase ( Micrococcus lureus) Pentapeptide translocase ( S . aureus) Pentapeptide translocase ( E . coli) N - Acetylglucosaminyltransferase ( E . coli) Polymerase ( E . coli) Other enzymes Galactosyltransferase (Salmonella tyhimurium)

Mannosyl- I -phosphorylundecaprenol synthetase ( M lysodeikticus) PEP-sugar phosphotransferase system, enzyme I1 complex (E. coli) Bacteriorhodopsin ( H . halobium)

Protein type

Lipid activator(s)

Activator function(s)

References

Integral

Various phospholipids

Kalin and Allen (1980)

Integral

Umbreit and Strominger (1972)

Integral

Various phospholipids, unknown neutral lipid Various phospholipids. some detergents Various phospholipids

Integral

Several phospholipids (?)

Integral

Several phospholipids (?)

Peripheral

Various phospholipids (esp. PE)

Integral

Various phospholipids and detergents

Integral

PG (or E . coli extract)

Integral

Asolectin, various PC's

Integral

Pless and Neuhaus (1973) Geis and Plapp (1978); Taku e t a / . (1980) Taku et a!. ( I 980) ?

Taku e f al. (1980)

Substrate solubilization, enzyme-conformational stabilization

Rothfield and Pearlman (1966); Rothfield and Home (1967); Weiser and Rothfield (1968); Endo and Rothfield (1969a,b); Romeo e f 01. ( 1 970a,b); Beadling and Rothfield (1978) Lahav er al. (1969)

Enzyme dispersion, conformational stabilization Conformational stabilization. other?

Kundig and Roseman ( I 97 1 )

Racker (1 973); Racker and Stoeckenius (1974); Hwang and Stoeckenius (1977); Huang e t a / . ( 1980)

369

EFFECTS OF MEMBRANE LIPIDS

VI.

CONCLUSIONS

It is clear from studies of intact cells and membranes that the fluidity and physical state of the membrane lipids can have a marked effect on the activity of membrane enzymes and transport systems. However, although some plausible hypotheses concerning the general mechanism by which lipids produce their characteristic effects have emerged, much work remains to be done to verify and extend these concepts. At the molecular level, very little is known about how membrane proteins respond to alterations i n the physical properties of membrane lipids. Studies of isolated and lipid-reconstituted membrane enzymes and transport proteins have demonstrated that lipids can function to solubilize water-insoluble substrates or products, to solubilize and disperse the enzyme or transport protein, and to provide a proper environment for stabilizing the protein of interest in an active conformation. Both the fluidity and interfacial properties of the lipids employed in reconstitution studies can be important in determining the activity of membrane proteins and the regulation of this activity by ligand binding. However, until the nature of lipid-protein interactions at the molecular level is better understood, and until the secondary and tertiary structures of integral membrane proteins in a lipid milieu have been determined, the molecular mechanisms by which lipids modulate the function of membrane proteins will remain obscure. ACKNOWLEDGMENT The author's own work was supported by Research Grant MT-4261 from the Medical Research Council of Canada. REFERENCES Akutsu, H., Akamatsu, Y . , Shinbo, T . , Uehara. K., Takahashi, K . , and Kyogoku, Y . (1980). Evidence for phase separation in the membrane of an osmotically stabilized fatty acid auxotroph of E . cofi and its biological significance. Biochim. Eiophps. Acta 598, 437-446. Asano, A., and Brodie, A. F. (1963). Oxidative phosphoryfation in fractionated bacterial systems. XII. The properties of malate-vitamin K reductase. Biochem. Biuphps. Res. Co/iimioi. 13, 423-427. Asano, A , , Kaneshiro, T . . and Brodie. A. F. (1965). Malate-vitamin K reductase, a phospholipid. 895-905. requiring enzyme. J . Riol. C ' h e r ~ 240, Ashe, G . B., and Steim, J . M. (1971). Membrane transitions in Gram-positive bacteria. B i o c h i r ~ . B i o p h j ~ Artu . 233, 810-814. Baldassare, J . J . , Rhinehart, K. H.. and Silbert, D. F. (1976). Modification of membrane lipid: physical properties in relation to fatty acid structure. Biorhemistrx 15, 2986-2994. Baldassare, J . J . , Brenckle, G. M., Hoffman. M., and Silbert, D. F. (1977). Modification of membrane lipid. Functional properties of membrane in relation to fatty acid structure. J . B i d . Chem. 252, 8797-8803. Baroin, A , , Bienvenue, A , , and Devaux, P. F. (1979). Spin-label studies of protein-protein interac-

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tions in retinal rod outer segment membranes. Saturation transfer electron paramagnetic resonance spectroscopy. Eiochemisrn 18, 1151-1 155. Beacham, 1, R., and Silbert, D. F. (1973). Studies on the uridine diphosphate-galactose: lipopolysaccharide galactosyl-transferase reaction using a fatty acid mutant of Escherichiu coli. J . Biol. Chern. 248, 5310-5318. Beadling, L., and Rothfield, L. I . (1978). Modulation of the conformation of a membrane glycosyltransferase by specific lipids. Pruc. Nut/. Aeud. Sci. U . S . A . 75, 3669-3672. Beebe, J . L. (1071). Isolation and characterization of a PE-deficient mutant of Bucillus srtbtilis. J . Bacreriol. 107, 704-71 1 . Beebe, 1. L. (1972). Transport alterations in a PE-deficient mutant of Bucilius subtilis. J . B u c t ~ r i o / . 109, 939-942. Bevers, E. M., Snock, G. T., Op den Kamp, J . A. F., and van Deenen, L. L. M. (1977). Phospholipid requirement of the membrane-bound M$+-dependent adenosine triphosphatase in Acho/ep/asmr luidiawii. Eiochiin. Biopliys. Actu 467, 346-356. Bragg, P. D., and Hou, C. (1976). Solubilization of a phospholipid-stimulated adenosine triphosphatase complex from membranes of Escherichiu coli. Arch. Eiochmi. Eiophys. 174, 553561. Bragg, P. D., and Hou, C. (1978). Binding of the CP,Mg'+-activated adenosine triphosphatase of Esl-herichin coli to phospholipid vesicles. Cun. J . Biochrrrr. 56, 559-564. Ceuterick, F., Peeters, J . , Heremans, K.. De Smedt, H., and Olbrechts, H. (1978). Effect of high pressure, detergents phospholipase on the break in the Arrhenius plot of Azorcihacter nitrogenase. b u r . J . Eiodwrn. 87, 401-407. Chapman, D., Gomez-Fernandez, J . C.,and Goni, F. M . (1979). Intrinsic protein-lipid interactions. Physical and biochemical evidence. FEES Lett. 98, 21 1-223. Cho, H. W., and Morowitz. J. H. (1972). Characterization of the plabma membrane of Mycq~krstnu Iuidluwii. VIll, Effect of temperature shift and antimetabolites on K' transport. Biochirn. Biophys. Act0 274, 105-1 10. Cirillo. V. P. (1979). Transport systems. /ti "The Mycoplasmas" (M. F. Barile and S. Razin, eds.), Vol. I , pp. 323-349. Academic Press, New York. Cook, D. A , , and Charnock, J . S . (1979). Computer-assisted analysis of functions which may be represented by two intersecting straight lines. J . Phurmacol. Methods 2 , 13- 19. Cronan, J . E., and Gelmann, E. P. (1975). Physical properties of membrane lipids: Biological relevance and regulation. Bacreriol. Rev. 39, 232-256. Cunningham, C. C., and Hager, L. P. (1971a). Crystalline pyruvate oxidase from Escherichia c d i . 11. Activation by phospholipids. J . Biol. C h m . 246, 1575-1582. Cunningham, C. C., and Hager, L. P. (1971 b). Crystalline pyruvate oxidase from Escherichiu C O / i . I l l . Phospholipid as an allosteric effector for the enzyme. J . E i o l . Chem. 246, 1583-1589. Curatolo, W., Verma, S. P., Sakuid, J . D., Small, D. M., Shipley, G. G., and Wallach, D. F. H. (1978). Structural effects of myelin proteolipid apoprotein on phospholipids: A raman spectroscopic-study. Biochemi.rtn 17, 1802-1807. Dancey, G . F., and Shapiro, B. M. (1977). Specific phospholipid requirement for activity of the purified respiratory chain NADH dehydrogenase of Escherichiu coli. Biochirn. Biophys. Actu 487, 368-377. Davis, J . H., Nichol, C. P., Weeks, G . , and Bloom, M. (1979). Study ofthe cytoplasmic and outer membranes of Esdterichiu coli by deuterium magnetic resonance. Biochemistr? 18, 2 10321 12. Davis. J. H.. Bloom, M., Butler, K . W.. and Smith, I . C. P. (1980). The temperaturedependenceof molecular order and influence of cholesterol in Aclioleplusmu laidltrwii membranes. Biochirn. Biopli,vs. Actu 597, 477-491. Davis, M. B . , and Silbert. D. F. (1974). Changes in cell permeability following a marked reduction

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Index

B

E

Bacteria, see also Prokaryotcs regulation of membrane lipid synthesis control of phospholipid acyl-group composition, 208-222 rate of phospholipid synthcsi,, 222-226 Bacteria living in extreme environments acidic lipids of phosphoglycolipids. 67-68 phospholipids. 66-67 sulfolipids. 68-69 apolar lipids fatty acids. 49-5 1 isopranyl glycerol ethers, 5 1-56 glycolipids of glycosyldiacylglyccrols,62-63 isopranyl glycerol ether glycosides. 64-65 other polar lipids. 65 tetrahydroxybacteriohopane glycosides, 63-64 neutral lipids of isoprenoid derivatives. 56-61 other components. 62

Environments, organisms and, 46-49 Enzyme(s), isolated membrane-bound, studies of, 362-368 Enzyme activities, membrane-associated adenosine triphosphatase, 354-360 lipid metabolic enzymes, 35 1-354 others. 360-362 oxidation-reduction enzymes, 347-35 1 Eukaryotes, unicellular, sterols and, 157- I58

C Cell(s), studies of. 323-324 chemotaxis, 331 DNA synthesis, 33 I growth, 324-331 membrane-associated enzyme activities, 347-362 protein-mediated transport processes, 33 1 347 Cholesterol, uptake by mycoplasma membrane. 185- 191

G Gram-negative bacteria, lipopolysaccharides of isolation and purification, 82-1 14 selected aspects of biology, 114-129

L

Lipid(s) of bacteria living in extreme environments acidic lipids, 66-69 apolar residues, 49-56 glycolipids, 62-65 neutral lipids, 56-62 membrane maintenance of asymmetry, 256 relevant properties of, 3 18-3 I9 of prokaryotcs apolar chains, 2-5 nonextractable lipids. 12 nonpolar lipids, 11--12 other polar lipids, 9-1 1 polar lipids with 1,2-di~adylsn-glyceroI backbone, 5-9

382

INDEX

Lipid(s) ( m n r . ) transbilayer distribution in cytoplasmic membranes cholesterol, 253-256 glycolipids, 244-245 phospholipids, 245-253 in microbial membranes, assessment of, 236-238 in outer membrane lipopolysaccharides, 239-240 phospholipids, 240-243 translocation between outer and cytoplasmic membranes, 243-244 Lipopolysacharrides of Gram-negative bacteria isolation and purification of, 82-83 structure and biosynthesis of core, 90-97 structure and biosynthesis of lipid A , 971 I4 structure and biosynthesis of 0 chains, 83-90 selected aspects of biology of endotoxic and immunogenic properties of lipid A, 114-1 18 fate of lipid A io expcl-imental animals. 123-129 physicochemical and structural prerequisites for biological activities, I 18- 123

M Membrane( s) Arrhenius plots of transport systems and enzymes, 320-323 bilayer transitions examples, 275-282 general properties, 267-269 lateral phase separation, 269-274 biological control of, 299-307 cytoplasmic, transbilayer distribution of lipids in cholesterol, 253-256 glycol ipids, 244-245 phospholipids, 245-253 maintenance of lipid asymmetry, 256 outer, transbilayer distribution of lipids in lipopolysaccharides, 239-240 phospholipids, 240-243

translocation of lipids between outer and cytoplasmic membranes, 243-244 patching fluid bilayers, 284-286 membrane proteins, 286-292 prokaryotic fluidity modulating lipids. 282-284 lipid phases and, 264-267 relevant propertick of constituents lipids, 3 18-3 19 proteins, 3 19-320 state, biological consequence5 of, 292-299 studies of associated enzyme activities, 347-362 cell growth, 324-33 I chemotaxis, 33 I DNA synthesis, 33 I protein-mediated transport processes, 33 1-347 transbilayer distribution of lipids in, assessment of, 236-238 Membrane phospholipid control of composition conversion of unsaturated acids to cyclopropane derivatives, 219-222 fatty acid chain length, 218-219 positional distribution of acyl moieties, 208-2 I I saturated: unsaturated ratio, 21 1-218 regulation of rate of synthesis candidate regulatory sites, 222 coordination with macromolecular synthesis, 223-225 interrelationships with cellular energy metabolism, 225-226 Mycoplasma membranes cholesterol uptake by cholesterol donors, 185-186 location of sterols in membranes, 190-191 membrane components involved in cholesterol uptake, 186- 190 role of sterols new concepts of, 197-198 regulation of membrane fluidity, I91 -193 structural features of required sterols. 193- I97 why are sterols required'?, 198-200

INDEX

383 P

Polyterpenoids ah phylogenetic precursors of sterols, l(>7I77 of prokaryotcs, 158- I67 Prokaryotes, .see ~ r l s oBacteria biological consequence5 of membrane state, 292-299 distribution of lipids in. 12-13 actinomycetes and related organisms. 28-3 1 budding and/or appendagcd bacteria, 16- I7 cyanobacteria, 14 endospore-forming hacteria. 27-28 gliding and sheathcd bacteria, 16 Gram-negative aerobic rods and cocci, 18-20 Gram-negative anaerobic bacteria, 21 -23 Gram-negative anaerobic cocci, 23 Gram-negative chemolithotrophic bacteria. 23-24 Gram-negative cocci and coccobacilli, 23 Gram-negative facultatively anaerobic rods, 20-21 Gram-positive cocci, 25-27 Gram-positive, non-spore-forming rods. 28 methane-producing bacleria, 24 mycoplasma, 3 1 phototrophic bacteria, 14- IS rickettsia, 3 1 spiral and curved bacteria, 18-20 spirochetes, 17- I8 lipids and phylogeny. 3 1-34 membrane biological control of, 299-307 fluidity modulating lipids, 282-284 membrane bilayer transitions examples, 275-282 general properties, 267-269 lateral phase separation, 269-274 membrane patching fluid bilayers, 284-286 membrane proteins, 286-292 polyterpenoids as phylogenetic precursors ol sterols archaehacterial polyterpenes. 176- 177

carotenoids as precursors of cyclic polyterpenoids, 175- I76 general features of common polyterpene biogenetic pathway, 167-170 hopanoids as precursors of sterols, 170 I75 polyterpenoids of, IS8 distribution of, 159-162 functional equivalence to sterols, 166- 167 structural regularities in membrane polyterpenoids, 162-166 regulation of membrane fluidity, lipid phases and. 264-267 sterols of absence of, 155- I56 apparent exceptions, 156- I57 case of unicellular eukaryotes, 157-158 structure of lipids apolar chains, 2-5 nonextrdctable lipids, I2 nonpolar lipids, 11-12 other polar lipids, 9- 1 I polar lipids with I ,2-diradylsn-glyceroI backbone, 5-9 Protein(s), membrane, relevant properties of, 3 19-320

S Stcrols o f prokaryotes absence of, 155- I56 apparent exceptions, 156- 157 case of unicellular eukaryores, 157-158 role in mycoplasma membrane, 19 1-200

T Transport processes, protein-mediated amino acids, 340-343 ions, 343-346 other, 346-347 sugars. 33 1-340 Tricyclohexaprenol, as putative prokaryotic triterpene and putative phylogenetic precursor of hopanoids, 177-178

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