Advances in Microbial Physiology Volume 4

Advances in

MICROBIAL PHYSIOLOGY

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Advances in

MICROBIAL PHYSIOLOGY Edited by A. H. ROSE School of Biological Sciences Bath University England

and

J. F. WILKINSON Department of General Microbiology University of Edinburgh Scotland

VOLUME 4 1970

ACADEMIC PRESS - LONDON and NEW YORK

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U.S. Edition published by ACADEMIC PRESS INC. 111 FIFTH AVENUE

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10003

Copyright 01970 By ACADEMIC PRESS INC. (LONDON) LTD.

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Contributors t o Volume 4 PATRICIA H. CLARKE, Department of Biochemistry, University College, Gower Street, London, England. A, J. GBIFFITHS, Department of Microbiology, University College of South Wales and Monmouthshire, Cathays Park, Cardiff, Wales.

F. M. HAROLD, Division of Research, National Jewish Hospital and Department of Microbiology, University of Colorado School of Medicine, Denver, Colorado, U.X.A. K.

Department of Experimental Biology, Roswell Park Memorial Institute, Buffalo, New York 14203, U.S.A.

PAIaEN,

J. SOMMERVILLE, Protozoan Genetics Unit, Institute of Animal Genetics, Edinburgh University, Edinburgh 9, Scotland.

D. W. TEMPEST, Microbiological Research Establishment, Porton, Nr. Salisbury, Wiltshire, Enghnd.

E. D. WEmBma, Department of Microbiology, Indiana University, Bloomington, Indiana 47401, U.S.A.

BEVERLY WILLIAMS,Department of Experimental Biology, Roswell Park Memorial Institute, Buffalo, New York 14203, U.X.A.

V

ERRATUM LLAdvances in MierobiaI Physiology,” volume 3, pp. 95 and 97; in Tables 2 and 3 of this article, organism 3 should be designated 1-1 and not Escherichia coli 1-1, and organism 5 designated 2-1 and not Escherichia coli 2-1.

Contents Contributors to Volume 4

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v

Biosynthesis of Secondary Metabolites: Roles of Trace Metals. E. D. WEINBERG

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I. Introduction 11. Characteristics of Secondary Metabolites A, ChemicalNature B. Kinetics of Synthesis . C. Proposed Functions . 111. Specific Macromolecules as Secondary Metabolites IV. Trace-Metal Roles in Secondary Metabolism A. Unique Requirements and Tolerances . B. Proposed Sites of Action V. Prospects and Conclusions . VI. Acknowledgement References .

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1 2 2 5 13

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24 31 36 39 39

Antimicrobial Agents and Membrane Function. F. M. HAROLD I. Introduction . 11. Structure and Functions of Microbial Membranes A. Permeability Barriers . B. Transport Systems . C. Electron Transport and Generation of ATP D. Membrane, Wall and Nucleus: An Integrated Unit 111. Compounds which Disorganize Lipoprotein Membranes A. Organic Solvents . B. Detergents . C. Reversible Membrane Disorganization? D. Peptide Antibiotics. E. Basic Polypeptides and Proteins F. Polyene Antibiotics and Membrane Sterols. IV. Proton Conduction and Uncoupling of Oxidative Phosphorylation

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46 47 47 4s 49 51 53 54 55 57 58 60 61 63

viii

CONTENTS

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V. Alkali Metal Ionophores A. Valinomycin B. Enniatins . C. Gramicidins . D. Macrotetralides : Nonactin and its Homologues . E. Nigericin, Monensin and other Carboxylic Polyethers F. Other Ionophores . . VI. Inhibitors of Energy Transfer and of the Respiratory Chain A. ATPase and Energy Transfer . B. Inhibitors of the Respiratory Chain . C. Interaction of Heavy Metals with the Membrane VII. Bacteriocins : Antibiotics which Interact with Specific Membrane Receptors VIII. Summary and Prospect IX. Acknowledgements . References .

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68 69 74 74 76 78 80 81 81 86 90 91 93 95 96

Encystment in Amoebae. A. J. GRlFFlTHS I. Introduction . 11. Experimental Approaches Employed in the Study of Encystment A. MixedCnltures . B. AxenicCultures . C. Replacement Technique D. Measurement of Encystment . 111. Structural Changes During Encystment A. General. B. The Cyst Wall . C. TheGolgiBody D. Autolysosomes . E. Mitochondria. F. Other Cytoplasmic Organelles . G. The Nucleus and Nucleolus H. FoodReserves I . Time-Course of Structural Changes IV. Physiology of Encystment . A. Encystment in Mixed Cultures . B. Encystment in Axenic Cultures . C. Induced Encystment V. Biochemical Aspects of Encystment . A. Respiratory Metabolism B. Fate of Major Cell Components. . C. Enzymesynthesis . D. Control of Encystment by Metabolites VI. Excystment .

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106 107 107 107 107 108 109 109 109 113 114 115 115 115 116 116 117 117 118 119 123 123 124 124 125 126

CONTENTS

VII. Resistance and Function of Cysts VIII. Concluding Remarks IX. Acknowledgements References

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ix

126 127 127 128

Serotype Expression in Paramecium. J O HN SOMMERVILLE

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I. Introduction A. Theorganism . B. Serotypes . . 11. Structure of i-Antigen Molecules A. General Properties . B. Subunits . C. Relationship between Different i-Antigens . D. Hybrid Molecules . E. Secondary Antigens 111. Cellular Location of i-Antigens A. Nature of Surface Association . B. Internal Sites. IV. Genetics of Serotype Expression A. Nuclei and Chromosomes. B. i-Antigen-Determining Genes . C. Regulation of Gene Expression. V. Function of i-Antigens. VI. Formation of i-Antigens A. Synthesisinvivo B. Synthesis i n vitro C. Transportation . VII. Serotype Transformation A. Induction Kinetics . B. Nuclear Activity . C. Regulation VIII. Conclusions IX. Acknowledgements References .

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132 133 133 134 134 135 139 141 143 144 144 145 147 147 148 150 157 158 159 163 164 165 166 170 172 175 176 176

The Aliphatic Amidases of Pseudomonas aeruginosa. PATRICIA H. CLARKE

I. Microbial Amidases

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A. Enzymes Hydrolysing Amide Bonds . B. Aryl and Aliphatic Amidases

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179 179 180

CONTENTS

X

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C. Amide Hydrolases and Transferases D. Pseudomonad Amidases . 11. The Amidase of Pseudomonas aeruginosa 8602. A. General Properties of the Amidase System B. Regulation of Synthesis . C. Enzyme Characteristics . 111. Amidase Mutants A. Regulator Mutants B. Mutants Producing Altered Enzyme Proteins IV. Genetic Analysis V. Genetic Homology among Pseudomonas spp. . VI. Acknowledgements References .

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182 183 183 183 184 192 196 196 206 217 218 221 221

The Place of Continuous Culture in Microbiological Research. D. W. TEMPEST I . Introduction . 11. Microbial Growth in a Closed System: The “Batch Culture” 111. Microbial Growth in an Open System: The Continuous-Flow Culture . IV. The Chemostat as a Research Tool. A. Use of a Chemostat in Studies of Bacterial Cation Metabolism B. Use of a Chemostat in Studies ofBacteria1 Cell-Wall Synthesis C. Use of a Chemostat in Studies of Microbial Enzyme Synthesis V. Some Inadequacies of Continuous Culture as a Research Tool VI. Operational Problems . A. Foaming B. Wall Growth . VII. Conclusions . VIII. Acknowledgements References

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223 224 228 230 232 238 241 245 246 247 247 248 249 249

Catabolite Repression and Other Control Mechanisms in Carbohydrate Utilization. KENNETH PAIGEN and BEVERLY WILLIAMS

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I . Introduction A., The Control of Carbohydrate Utilization B. The Choice of Alternative Substrates C. Historical Review 11. Catabolite and Transient Repression

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252 252 252 253 254

xi

CONTENTS

A. Quantitative Factors . 254 B. Occurrence of Catabolite Repression 256 C. Environmental Conditions Which Produce Catabolite Repression 271 D. Occurrence of Transient Repression . 276 111. The Mechanisms of Transient and Catabolite Repression . 281 A. Transcriptional or Translational Control 281 B. Repression in Regulatory Mutants 285 C. Models of Repression 29 1 D. Identity of the Effector . 293 IV. Catabolite Inhibition 298 A. Definition and Properties. 298 B. Examples of Catabolite Inhibition 300 C. Mechanism of Catabolite Inhibition 302 V. Control of Inducer Concentration . 303 A. Gratuity 303 B. Long-Term Adaptation . 304 C. Inducer Entry 305 D. Effector Synthesis . 306 E. Summary 308 VI. Diauxie . 308 VII. Acknowledgements . 311 References 311 Note Added in Proof . 319 Additional References 323

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Author Index Subject Index

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Biosynthesis of Secondary Metabolites : Roles of Trace Metals EUGENE D. WEINBERU Department of Microbiology, Indiana University, Bloomington, Indiana 47401, U.X.A.

. ..

Study of the rare and curious often brings t o light general phenomena which may be exaggerated in the rare but overIooked in the commonplace. Arthur T.Henrici (1939).

I. Introduction . 11. Characteristics of Secondary Metabolites . A. Chemical Nature. B. Kinetics of Synthesis . C. Proposed Functions . 111. Specific Macromolecules as Secondary Metabolites IV. Trace-Metal Roles in Secondary Metabolism . A. Unique Requirements and Tolerances . B. Proposed Sites of Action V. Prospects and Conclusions . VI. Acknowledgement . References .

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1 2 2 5 13 18 24 24 31 36 39 39

I. Introduction “Amongst the metabolic activities of micro-organisms, those usually classed as secondary are far from trivial; they can contribute to a fuller understanding of microbial biology” (Bu’Lock, 1967). The many thousands of final products of secondary metabolism comprise an extraordinary bestiary of organic compounds; yet in recent years, several unifying principles concerning their formation have been discovered. In this essay, I will (1) summarize the general characteristics and possible functions of secondary metabolites, ( 2 ) propose the inclusion in this category of an additional class of substances, and (3) suggest reasons for the need of secondary metabolism for unique quantities of trace metals. Secondary metabolites are defined as natural products that have a restricted taxonomic distribution, possess no obvious function in cell growth, and are synthesized by cells that have stopped dividing. The Dedicated t o Professor Stewart A. Koser on the occasion of his 75th birthday and in recognition of his distinguished career in teaching and research. 1 1

2

EUGENE D. WEINBERQ

systematic study of such metabolites began in 1922 when, following the suggestion of Sir Prederick Gowland Hopkins, Raistrick (1931) and his colleagues initiated their classic investigations on the chemical activities of fungi. I n the decades of the 1940s and 1950s) the attention of most natural-product scientists was directed toward those secondary metabolites that are toxic to pathogenic micro-organisms. I n the decade of the 1960s) interest shifted somewhat to those secondary substances that have unusual pharmacological effects on tissues of plants or animaIs. Nevertheless, during the past thirty years, many plant and microbial physiologists as well as natural-product chemists continued to extend the nonapplied studies of the Raistrick group. Thus, in addition to knowledge obtained for antibiotics, toxins, and growth promoters, there also is now considerable understanding of the biogenesis and chemical nature of numerous secondary metabolites that have no known pharmacological activities. Information gained from examination of both secondary metabolites that have medicinal or poisonous effects as well as those that are pharmacologically inactive has contributed much to our general awareness of the chemosynthetic functions of non-proliferating microbial cells, A leading investigator in this field is J. D. Bu’Lock (1961, 1967); he has provided extensive experimental data, considerable theoretical insight, and, not least, the name : secondary metabolism.

11. Characteristics of Secondary Metabolites A. CHEMICAL NATURE “Perhaps the most striking fact arising from (our) observations”, noted Raistrick (1931)) “is the extraordinary specificity of the different mould products. These highly specific products are only produced in some cases by a single species and in others by .. . a very few species”. The great chemical diversity of secondary metabolites is illustrated by a listing of some of the classes of compounds in which these substances are found (Table 1). The majority of these classes do not contain primary metabolites (substances formed during cell multiplication). Of the secondary metabolites that consist entirely or partially of peptides, many contain D- as well as L-amino acids ; often, amino acids and derivatives not found in proteins are present (Perlman and Bodanszky, 1966). Unusual chemical linkages are common ;consider the cyclic pentapeptide and dodecadepsipeptide features, respectively, of the malformins (Takeuchi et al., 1967) and of valinomycin (MacDonald, 1967); the large lactone rings of the macrolides (Vangk and Majer, 1967) and polyenes (Birch, 1967); the bridged ring systems of the gibberellins (Wood,

BIOSYNTHESIS O F SECONDARY METABOLITES

3

1967); and the triply unsaturated bonds of the polyacetylenes (Anchel, 1967). TABLE1. Classes of Organic Compounds in which Secondary Metabolites are Found Amine sugars Anthocyanins Anthraquinones Aziridines Benzoquinones Coumarins Diazines Epoxides Ergoline alkaloids Flavonoids Glutaramides Glycosides Hydroxylamines Indole derivatives

Lactones Macrolides Naphthalenes Naphthaquinones Nucleosides Oligopeptides Perylenes Phenazines Phenoxazinones Phthaldehydes Piperazines Polyacet ylenes Polyenes Pyrazines

Pyridines Pyrones Pyrroles Pyrrolines Pyrrolizines Quinolines Quinolinols Quinones Salicylates Terpenoids Tetracyclines Tetronic acids Triazines Tropolones

In addition to being chemically diverse, secondary metabolites exist typically as members of closely related chemical families. There are, for example, at least three naturally occurring ochratoxins, three neomycins, four malformins, four tyrocidines, five mitomycins, eight aflatoxins, ten bacitracins, ten polymyxins, and more than twenty actinomycins. Often, cells of a single microbial strain can synthesize more than one member of a family. I n such cases, the final yields of the various members can be shifted by appropriate precursor pressure. In the absence of either exogenous phenylaIanine or tryptophan, the ratio of tyrocidines A : B: C synthesized by Bacillus brevis is 1: 3 : 7 ; if either L- or D-phenylalanine is provided, the main component formed is tyrocidine A. If L- or D-tryptophan is fusnished, component D predominates; when both phenylalanine and tryptophan are supplied, each of the four components is synthesized (Mach and Tatum, 1964; Fig. 1). Nevertheless,some components (e.g.the L-psoline units in the tyrocidines (Weinberg, 1967b),the D-cysteine residues in the malformins (Takeuchi et al., 1967),and the sarcosine moieties in the actinomycins (Katz, 1967) are invariant. Even in positions in which substitutions can occur, the permissible amino acids are chemically similar and of identical optical configuration. Despite the immense array of final products, large numbers of related families of secondary metabolites are assembled from quite similar and, indeed, commonplace precursor units. Biogenesis often can be traced from relatively few initiator primary metabolites such as acetate,

4

EUGENE D. WEINBERG

pyruvate, malonate, mcvalonate, shikimate, prephenate, amino acids, or purines, or a t least from transient or intermediate secondary metabolites such as orsellinic acid or 6-methylsalicylic acid (Woodruff, 1966; Bu'Lock, 1967; Mitscher, 1968). I n fact, production of the final, as well as the intermediate, products requires enzymatic steps very similar to those of primary metabolism. Nonetheless, a t least a few synthases that are absent during primary metabolism are essential for biogenesis of specific terminal secondary metabolites; prior to the appearance of the latter, a brief period of de novo RNA and protein synthesis is required (Section II.B.l, p. 5 ) . B

A L-orn

L-Val

L-tyr

f

L-leu

f

D-phe

--f

1 1

1 L-phc

L-glu t- L-asp f-- D-phe rjH2 iH2

D-phc

+--

D

C --f

u-try

1

L-PI'O

L-pro

L-try

L-try

1

_j

u-try

L-pro

L-try

D-phe

FIG.1. Structural formulae of tyroeidines A, B, C and D. -+ indicates a C-N bond; asp, an aspartate residue; glu, a glutamate residue; leu, a leucine rosidue; orn, an ornithine residue ; pho, a phenylalanine residue ; pro, a proline residue ;try, a tryptophan residue; tyr, a tyrosine residue; and val, a valine residue. Reproduced with permission from Weinberg (1967b).

To some extent, a structure-taxonomyrelationship exists. For example, nearly all strains of Aspergillus niger produce malformin but, of eleven other species in the A . niger group, only four are positive (Curtis and Tanaka, 1967). Of eight species of aspergilli outside the A . niger group, none is active. To complicate matters, of twelve non-aspergillus fungi (contained in eleven genera), two produce considerable quantities of malformin (Curtis and Tanaka, 1967). Of 77 strains in 13 species of Aspergillus tested for aflatoxin formation, 34 of 38 of A.Jluvus, and four of A . parasiticus are positive. The remaining 35 isolates as well as 44 non-aspergillus fungi (in six genera) are negative (Wilson et al., 1968). Of 14 strains of aspergilli studied, four produce a t least three different kinds of secondary metabolites (aflatoxin, kojic acid, tremorgen) ; four others synthesize the first two, four the last two, and the remaining two strains form only kojic acid (Wilson, 1966). Twenty three strains (in biotypes A through F) of Pseudomonas Jluorescens and five strains of P. multivoruns were examined for ability

BIOSYNTHESIS OF SECONDARY METABOLITES

5

to produce pyrrolnitrin (Elander et al., 1968). One of eight strains of biotype D, three of seven of E, and four of the strains of P. multivorans are positive; the other 15 strains are inactive. I n the case of all specific secondary metabolites, moreover, “strain degeneration” is quite common. Selection of organisms in the population that cannot synthesize detectable quantities of a given metabolite is achieved easily ; indeed, “degeneration” can be of considerable nuisance in the industrial use of micro-organisms.

B. KINETICS OF SYNTHESIS 1 . Onset

In batch cultures, biogenesis of secondary metabolites begins subsequent to cessation of cellular multiplication; after a period of time, it is terminated even though the cells remain viable. I n continuous cultures, secondary metabolism does not occur in “cell generator” vessels in which balanced growth is continuously maintained. Industrial microbiological processes have been described in which the cells are passed from the “generator” into tubular “reactors” and traverse slowly through the latter during the time in which they are synthesizing the desired metabolite; thereafter, though still alive, they are discarded (Reusser, 1961; Hough and Brown, 1968). If the rate of dilution in the cell generator vessel is lowered, the culture can shift into secondary metabolism in the same container (Bu’Lock et al., 1965; Pirt and Callow, 1960; Zacharias and Bjorklund, 1968).I n a continuous culture of Penicillium urticae, for example, balanced growth can be maintained at a high dilution rate; at a lower rate, after a lag period, production of 6-methylsalicylic acid occurs. When addition of fresh medium is stopped, the latter compound is then converted to the variety of final secondary metabolites typical of the species (Bu’Lock et al., 1965). I n contrast, the yield of primary metabolites parallels the yield of cells over a wide range of dilution rates (e.g. Holmstrom, 1968). In natural environments, there occur complex series of events that possess features of both batch and continuous cultures; thus, the kinetics of production of a specific secondary metabolite are difficult to predict. Moreover, unlike the situation in the majority of laboratory fermentations in which pure cultures are employed, secondary metabolites formed in nature are subject to degradation by enzymes of neighbouring microbial species (Pramer, 1958). In batch cultures of unicellular organisms, such as algae, yeasts, and bacteria, secondary metabolites begin to be formed in the late portion of the exponential phase of growth; the small quantities produced at that time are attributed to cells that are no longer dividing. Likewise, in

6

EUGENE D. WEINBERG

filamentous organisms, small amounts of secondary metabolites appear while the batch cultures are still increasing in contents of nucleic acids and protein. I n these systems, cells within the filaments that are no longer dividing are believed to be responsible. If the sole criterion for growth is increase in dry weight, misleading results are obtained in those cases in which true growth has been superceded by increased lipid synthesis ; non-dividing cells are capable of concurrently forming lipid and secondary metabolites (Bu’Lock, 1967). However, if growth is correctly monitored by measuring increases in nucleic acids, protein, and cell number, it becomes quite clear that the bulk of secondary-metabolite biogenesis occurs after these parameters are no longer increasing, namely, in the early portion of the stationary phase. Although “true” secondary metabolites are not detected during the early and middle stages of the phase of balanced growth, LLp~eudo’7 secondary substances formed in large quantity by a small number of industrially important strains are synthesized in low yield during the growth phase. These materials are exemplified by riboflavin, cyanocobalamin, glutamic acid, and citric acid, which, during growth, have normal physiological functions. They are produced by a very wide taxonomic range of organisms and are, of course, true primary metabolites. I n the industrially useful strains, control mechanisms have been so damaged that, during the stationary phase, the quantities of materials produced are analogous to those of secondary metabolites. As mentioned previously, evidence is available that enzymes necessary for true secondary metabolism are synthesized during the late phase of cell growth and immediately prior to the formation of secondary substances. As an example, data on the appearance of the enzymatic activities responsible for synthesis of dipicolinic acid by Bacillus megaterium are plotted in Fig. 2 (Bach and Gilvarg, 1966). Similarly, enzymes specific for synthesis of streptidine from myo-inositol are repressed in actively growing cultures (Walker, 1967). The most detailed studies have been made with phenoxazinone synthase which is needed for the production of actinocin, the chromophoric portion of actinomycin (Marshall et al., 1968). I n actively growing cultures, synthesis of this enzyme apparently is prevented by catabolite repression and, even with cells from older cultures, glucose and galactose but not fructose can suppress its formation. Chloramphenicol and puromycin are likewise inhibitory; actinomycin is inactive because cells from older cultures cannot assimilate this compound. During the period in which phenoxazinone synthase is formed, the levels of such other enzymes as kynureninase, several amino acyl-t-RNA-activating enzymes, and N1O-formyl-H,-folate-methionyl-t-RNA transformylase remain unchanged or decrease (Marshall et al., 1968).

7

BIOSYNTHESIS OB SECONDARY METABOLITES

In a bacitracin-forming system, production of postulated synthases is suppressed by both actinomycin and chloramphenicol (Weinberg and Tonnis, 1967). The drugs are inactive if added after bacitracin synthesis has begun. Likewise, edeine is formed if inhibitors of protein synthesis are added after the synthases for production of the antibiotic have been produced (Kurylo-Borowska, 1967).I n Penicillium urticae, in which the intermediate secondary metabolite, 6-methylsalicylic acid, is converted to gentisyl alcohol which in turn is oxidized to patulin, a

i

120 -

3

I

.-cVI

c

+ c

I

90-

&Dipicolinic acid 1 production

a -

Y

._ 3 60u ._

-2

k-

30 -

l

._ U

10'Oo8

40.006

0 0

0 .C ._ 0 0 ..-a

n

0Tirne.(hr.)

FIa. 2. Dipicolinic acid synthesis in extracts of Bacillus megaterium harvested a t different stages during growth in batch culture. Substrates were sodium pyruvate and aspartate semialdehyde. Dipicolinic acid was determined spectrophotometrically by the increase in absorbance at 269 nm. Reproduced with permission from Bach and Gilvarg (1966).

more complex situation obtains (Bu'Lock and Shepherd, 1968). Inhibitors of protein synthesis, added at any time during the stationary phase, permit the formation of 6-methylsalicylic acid but not of the latter two compounds. Synthases for production of salicylate appear to be formed prior to the stationary phase of growth and are stable whereas those for gentisyl alcohol and patulin are produced by non-growing cells and are unstable (Bu'Lock and Shepherd, 1968). It is generally assumed that formation of synthases needed for secondary metabolism is suppressed during the growth phase by catabolites of such nutrients as glucose. I n the bacitracin system, for example, the secondary metabolite does not appear until one hour after glucose has been exhausted from the medium (Bernlohr and Novelli, 1960). I n the

8

EUGENE D. WEINBERG

neomycin fermentation, glucose not only prevents formation of synthases but also, if added after they have been formed, has the additional effect of promptly inhibiting their activity (Maxon and Chen, 1966). I n some industrial fermentations, after the initial source of carbon has been consumed for cell growth, a slow supply of the same carbohydrate or a second less readily utilized carbon compound is provided for the desired secondary metabolic process (Vining and Taber, 1963). I n others, the fully grown organisms are transferred to a replacement medium deficient in a limiting nutrient other than the utilizable carbon source. Such procedures favour the orderly shift into secondary metabolism and diminish the possibility of abrupt death and autolysis that might otherwise occur at the cessation of balanced growth (Vining and Taber, 1963). 2. Duration and Cessation

After the synthases have been formed, the arithmetic rate of production of secondary metabolites consistently is linear with respect to time. However, the duration of synthesis of the metabolites varies with the nature of the producing organism and its environment. Generally, the period of secondary metabolism ranges between one-half t o slightly more than twice that needed for the phase of balanced growth. The shorter portion of this range contains some, but not all, bacterial systems; the longer portion contains bacterial as well as all of the filamentous microbial systems examined. Examples of bacterial systems in which secondary metabolism is brief include a bacitracin fermentation in which growth, synthase formation, and bacitracin production require, respectively, seven, three, and four hours (Weinberg and Tonnis, 1966, 1967). Similarly, edeine is formed in five hours following an eight-hour growth stage (Kurylo-Borowska, 1967; Fig. 3) and, in a polymyxin system, secondary metabolism requires 14 hr after a 27-hr period of growth (Daniels, 1968). Examples of bacterial systems in which secondary metabolism requires the same or more time bhan cell multiplication include the production of (1)diphtheria toxin for 17 hr after a 20-hr growth phase (Pappenheimer, 1965; Fig. 4); ( 2 ) staphylococcal enterotoxin for 13 hr after a growth phase of 5 hr (McLean et ul., 1968; Fig. 5) or for 9 hr after growth of 9 hr (Markus and Silverman, 1969; Fig. 6) ; and (3) bacitracin for 11 hr after a 9-hr growth stage (Bernlohr and Novelli, 1960). Among actinomycetes and moulds, typical ratios of the duration of vegetative growth as compared with that of secondary metabolism are, for actinomycin (Katz, 1967; Fig. 7), erythromycin (Stark and Smith, 1962), mitomycin (Kirsch, 1967), streptomycin (Homer, 1967), ergoline alkaloids (Taber and Vining, 1963), and kojic acid (Kitada et al., 1968), respectively, 1:2, 1:2.4, 1:1.5, 1:2, 1:1.25, and 1:2.

9

BIOSYNTHESIS O F SECONDARY METABOLITES

Typically, the duration of production of secondary metabolites is considerably less than the longevity of the respective cultures. The

FIG.3. Time-course of growth (measured as absorbancy of cultures) and of production of edeine by cells of Bacillus brevis. Reproduced with permission from Kurylo-Borowska (1967).

-i .

-0

E

2 _1

3-

v

W

1-

-25 Toxin production

20

$

c 3

2 h 0

c

-15

C

-lo

:

-5

2

0

FIG.4. Time-course of growth and of synthesis o f toxin by cells of Corynebacterium diphtheriae. The toxin contents of culture filtrates are given as the number of flocculating doses per ml o f filtrate (Lf/ml.). A flocculating dose is the smallest amount that will form a visible precipitate with an appropriate a,mount of antitoxin. Reproduced with permission from Pappenheimer (1965).

abrupt cessation of secondary metabolism in viable cells conceivably could occur because ( 1 ) the proportion of producing to non-producing cells suddenly decreases as would occur when a population undergoes

EUGENE D. WEINBERG

Toxin production

800

W

.$

400

c ._

-8

x

e 2

300

c

W

-% 0

+-

I -7 a I

Time (hc)

FIG.5. Time-course of growth (measured as the turbidity of the culture) and of synthesis of enterotoxin by cells of XtaphyZococcw aureus. Changes in the pH value ofthe culture are also shown. ReproducedwithpermissionfromMcLeanetaZ. (1968).

200

160

-

,E

120

5m

9

C ._ x 0 c

80 e

z r

8 %

-0

w

4

8 12 Time ( h c )

16

40

71"

3

3

1

FIG.6. Time-course of growth and of synthesis of enterotoxin by cells of StaphyZoaureus. Changes in the pH value of the culture are also shown. Reproduced with permission from Markus and Silverman (1969). coccus

11

BIOSYNTHESIS OF SECONDARY METABOLITES

differentiation, (2) the secondary metabolite exerts end-product inhibition on the activity of a key synthase, (3) molecules of the synthase decay and cannot be reformed because of end-product repression, and/or (4)precursors of the secondary metabolite become depleted. Specific evidence for or against any of these proposals is lacking. I n the case of proposal ( l ) , cells of those microbial strains that form spores or conidia do cease production of secondary metabolites when they begin

p

Dry 1 weight2

C

I

1.6 -

-

or

1.2-

L

c ._

- 40

- 20

0

1

h

i

4

5

6-0

Time (days)

FIG.7. Time-course of growth and of synthesis of actinomycin by cells ofstreptomyces antibioticus. Reproduced with permission from Kcttz (1967).

to show morphological signs of differentiation. But cells of species that do not differentiate also abruptly terminate their formation of secondary substances. Proposals (2) and (3) are unlikely because of the bizarre chemical nature of secondary metabolites; indeed, the reason for their unusual structures may be that of precluding their ability to exert regulatory controls. I n some cases, of course, control mechanisms for primary metabolites indirectly affect the yield of specific secondary substances. For example, by inhibiting its own synthesis, lysine also causes a diminution in formation of a penicillin precursor, a-aminoadipic acid (Demain, 1966). Other examples of regulation of secondary metabolism are described in an excellent review by Demain (1968).

12

EUGENE D. WEINBERC

However, in some fermentations, the accumulating product does, in fact, suppress attainment of the yield that would be achieved by its continuous removal or neutralization. I n such cases, the metabolite is believed to act by altering unfavourably the pH value or the availability of essential metal ions. For example, inclusion of salts of magnesium or calcium is desirable in the tetracycline fermentations; the cations saturate the metal-binding sites of the metabolites and thus prevent the latter from poisoning the producing organisms (Hockenhull, 1963). Although proposal (4) is perhaps the most reasonable of the group, it has not yet been possible to extend the duration of secondary metabolism by continuous supply of probable precursors without also continuously supplying new cells of the proper age. But, it may be argued, even with such well-known secondary metabolites as the penicillins (Abraham and Newton, 1967; Demain, 1966), we have insufficient knowledge of the correct balance of the necessary precursors and intermediates. This defect should be overcome as increasingly more secondary-metabolite syntheses are achieved in cell-free systems. 3. Yield, Location and Stability

The total yield of a single secondary metabolite in highly selected strains employed in industry can be equivalent t o as much as 5.20% of the dry weight of cell protein in the culture. I n strains of less interest to the industrial microbiologist, an array of metabolites is formed; the total yield of these probably is of a magnitude similar to the above but quantitative information on this point is not yet available. The permissible range of incubation temperatures and amount of aeration are often narrower for maximum production of specific secondary metabolites than for growth and the ratios of members of a family (e.g. aflatoxins; Schindler et al., 1967) may vary with the temperature of incubation. I n general, secondary metabolites enter the extracellular milieu as they are formed. Usually, more than 850h of the total yield (in chloramphenicol, 99.99% ; Gottlieb, 1967) accumulates in culture supernatants. The small amounts remaining in the cells are associated with walls and membranes rather than with soluble cytoplasm. Exceptions to the generalization that secondary metabolites are discarded by intact viable cells occur in the case of those spore-forming strains (e.g. basidiomycetes ; Bu’Lock, 1967) in which some of the substances are subsequently incorporated into the developing spore coats. Possible locations of dipicolinic acid in spores of bacilli have been reviewed by Murrell(l967). I n those fermentations in which the pharmacologically important substance is not the final product of secondary metabolism, conditions must be adjusted to retard alteration of the desired material to a less

BIOSYNTHESIS OF SECONDARY METABOLITES

13

active compound. I n the mitomycin system, for example, more than 90% of the active material is lost if it is permitted t o remain in contact with mycelial enzymes in non-aerated vessels (Gourevitch et al., 1961). Penicillin is inactivated by the rise in pH value above 7.5 in “senescent” cultures (Hockenhull, 1963). I n the aflatoxin fermentation, 50-75% of the active compounds disappear during a period of several days (Stubblefield et al., 1967); this inactivation is associated with release of a nonenzymatic factor from disrupted mycelia (Ciegler et al., 1966). C. PROPOSED FUNCTIONS During the past three decades, many suggestions have been made concerning the possible functions of secondary metabolites. These can be grouped into three categories: ( 1 ) general functions that apply to all secondary metabolites, ( 2 ) specialized functions that apply to specific secondary substances and which pertain to the physiology or anatomy of the producing cells, and (3) specialized functions that apply to specific substances thab affect the physiology or anatomy of cells of other organisms. 1. General Functions

The proposed general functions applicable to all secondary metabolites include (1)waste products of cellular metabolism, ( 2 )reserve food-storage materials, (3) breakdown products derived from cellular macromolecules, and (4)“safety-valve” shunts of very low molecular-weight precursors into innocuous products. The first of these proposed functions is untenable because secondary metabolites accumulate neither during balanced growth nor during the later stages of the stationary phase. The second is unlikely because secondary substances are excreted as they are formed and generally are not further metabolized by the celIs that have produced them. Distinctions between shunt metabolites and actual energy-storage compounds have been summarized by Wilkinson (1959). The third suggestion has been disproved by demonstrating that secondary metabolites are formed de novo after macromolecular synthesis has been halted. Mach et al. (1963),for example, observed that the amino-acid sequence in tyrocidine is not a peptide fragment derived from pre-existing protein by showing that [U-14C] tyrosine, [2-14C] ornithine, [3,4-3H] proline, and [U-14C]aspartic acid are incorporated into the antibiotic by whole cells whose protein synthesis has been blocked by chloramphenicol. The fourth proposal (Woodruff, 1966), favoured by the present reviewer, considers the process of secondary metabolism t o be much more important t o the organism than specific products. Briefly, it maintains

14

EUGENE D. WEINBERQ

that, despite efficient control mechanisms of repression and end-product inhibition, microbial cells that enter the phase of non-balanced growth are faced with death because of the accumulation of very high levels of such precursors as acetate, malonate, pyruvate, specific amino acids, or specific purine nucleotides. Secondary metabolism relieves this crisis by effecting conversion of the precursor(s) to innocuous endproducts that cannot suppress their own formation and which need have no other value or function to the producing cells. Among predictions generated by this proposal are : (1)cultured microbial, plant, or animal cells rendered unable either to differentiate or to engage in secondary metabolism (by genetic or environmental manipulation) should die early in the stationary phase o€ growth, and ( 2 ) differentiating microbial, plant, or animal cells should not excrete large amounts of terminal secondary metabolites because their accumulating precursors and intermediate secondary substances are being shunted into useful molecules involved in cellular and tissue development. Indirect evidence concerning the first prediction has been obtained by showing that, in non-differentiating microbial cultures in which secondary metabolism is suppressed by unfavourable trace-metal environments (Section IV.A, p. 24), death rapidly ensues (Leonard et al., 1958; Steenbergen et al., 1969; Weinberg and Goodnight, 1969). However, the possibility that the unfavourable trace-metal balance has merely distorted rather than completely aborted secondary metabolism has not been eliminated. Evidence concerning the second prediction has been reported by Bernlohr and NovelIi (1960, 1963) who observed that environmental conditions that permit a high proportion of cells of Bacillus licheniformis to sporulate inhibit the production of bacitracin. 2. Specialized Functions that Aflect Producing Cells

The possibility that some secondary metabolites can be functional in the cells that produce them has been suggested by several investigators. Yoshida et al. (1966) have speculated that antibiotics such as actinomycin might be made normally in small amounts in growing cells where they might serve as low molecular-weight repressors selectively to inhibit macromolecular synthesis or function. These authors point out that the huge production of an antibiotic in non-growing cells may be analogous to the uncontrolled or derepressed synthesis of such wellrecognized primary metabolites as vitamins or amino acids in stationaryphase cells, and that the defective controls occur only in relatively few strains of highly selected micro-organisms. However, Woodruff (1966) is convinced that every microbial strain can produce measureable amounts of some antibiotic substance but he does acknowledge that,

BIOSYNTHESIS O F SECONDARY METABOLITES

15

in many cases, laboratory manipulations (nutritional and mutagenic) are required to obtain such quantities. Growing cells often can be shown to be sensitive to the same kind of antibiotic that they would produce later in their stationary phase (an obvious exception is penicillin), but there presently exists no evidence that growing cells require or produce these compounds for balanced replication. It is possible, of course, to obtain streptomycin-dependent mutants but these are not derived from organisms that synthesize streptomycin. Furthermore, such enzymes as phenoxazinone synthase and dipicolinate synthase needed for biogenesis of true secondary metabolites are not detected until late in the growth phase of the cultures (Marshall et al., 1968; Bach and Gilvarg, 1966). If some antibiotic secondary metabolites are, in reality, primary substances, the number of molecules per growing cell would be expected to be low. It will be difficult to determine if these are actually being formed in small amount by each of many growing cells or in large quantity (astrue secondary metabolites) by each of a few non-growing cells in the population under study. The polypeptide lipid-soluble antibiotics of molecular weight 500-1500 that are produced by species of actinomycetes and Bacillus have also been suggested to be functional for their producing cells. Mach and Tatum (1964) proposed that peptides similar to tyrocidine and gramicidin might have important regulatory roles provided they could bind to cell membranes. Many investigators have observed that the valinomycin (Hunter and Schwartz, 1967) and nigericin (Pressman, 1968) classes of secondary metabolite mediate transport of monovalent cations across both natural and synthetic lipid membranes. This action may occur because these ionophorous compounds increase the pore size of the membranes or because they transport the cations as a drug-metal complex. Although most of these studies have been performed with mitochondria, erythrocytes, or artificial membranes, valinomycin has been shown to accelerate uptake of potassium ions by Azotobacter ; cells of Xtreptomyces strains that produce the compound have not yet been examined. Paulus (1967) believes it unlikely that the peptide antibiotics play a role during balanced growth but he proposed that they may be essential during sporulation. He has observed that polymyxin is removed from the culture medium during a late stage in the spore-forming process and is bound to cellular material. Similarly, bacitracin is assimilated from the culture medium by sporulating cells (Bernlohr and Novelli, 1960, 1963). The primary antimicrobial action of polymyxin (Sebek, 1967) and bacitracin (Weinberg, 1967a) is alteration of cell membranes, and Paulus (1967) suggested that polymyxin and other polypeptide antibiotics may be required to participate in the cellular autolysis that permits release of free spores.

16

EUGENE D. WEINBERC:

Halvorson (1965) proposed that antibiotics formed early by presporulating cells of Bacillus spp. might combine with vegetative-cell DNA to prevent its functioning during sporulation; maintenance by the compounds of the cells in the spore-development stage has also been suggested by Schmitt and Freese (1968). Although several actinomycete antibiotics interfere with DNA structure or function, most Bacillus products affect membranes rather than nucleic acids. An exception is edeine which apparently suppresses activity of DNA polymerases (Kurylo-Borowska, 1967). The possibility exists, of course, that membrane alteration by Bacillus antibiotics impairs attachment of DNA to membranes with consequent prevention of transcription. Although it is unlikely that polypeptide antibiotics are incorporated into bacterial spore structures (Brenner et al., 1964; Snoke, 1964), there is no doubt that the secondary metabolite dipicolinic acid is an essential component of heat-resistant spores. I n basidiomycetes, such secondary substances as hispidin are converted to lignin-like polymers in the ripening fruit bodies ;in fruits of certain ascomycetes, polymers of perylenequinone serve in a structural capacity (Bu’Lock, 1967). Numerous other quinone and phenolic derivatives have been obtained from fungal reproductive structures (Bu’Lock, 1967). Such systems appear to be examples of evolutionary selection of populations that have found a use for their own secondary metabolites and perhaps are analogous to human communities that use their garbage for land-fill. The possibility that secondary metabolites are merely over-produced components of walls of vegetative cells has been suggested often but little evidence is available in favour of this idea. H. Koffler’s group (unpublished work cited by Hockenhull, 1963) tested this suggestion for streptomycin and observed that small quantities of streptidine and N-methyl-L-glucosamine associated with cell walls of Streptomyces griseus are merely absorbed secondary metabolites rather than true cell-wall components. On the other hand, streptidine has been reported to be an integral unit of cell walls of both a streptomycin-producing strain as well as of a non-producing mutant (BarabBs and Szabo, 1968). Secondary metabolites tend to be chemical caricatures of primary metabolites; thus it is not surprising if some secondary substances might contain molecular portions that are similar to, or even identical with, the units of walls or other structures in vegetative cells. Many secondary metabolites are strong metal-binding agents (Weinberg, 1957) and the quantity of trace metals available to the cells controls the yield of metabolite (Section IV.A, p. 24). However, some metalbinding metabolites that appear in large quantity during the stationary phase of growth are actually primary in nature since they are formed in amall amounts during balanced growth presumably to serve as metal-

BIOSYNTHESIS O F SECONDARY METABOLITES

17

transport and metal-storage ligands. For example, the sideramine, mycobactin, is formed by growing cells of Mycobacterium phlei in large quantity when iron is present in growth-limiting amounts ; however, if the concentration of metal is adequate for growth, the metabolite is produced in high concentration only during the stationary phase (Antoine and Morrison, 196s). I n the stationary phase, the concentration of iron in the medium has been lowered as a result of growth. If excess iron is added to the medium initially, very little mycobactin is synthesized by either growing or non-growing cells. Neilands (1957) proposed that the ability to produce and secrete strong iron chelators in low-iron environments might have survival value and has termed organisms that have this capability “autosequestric”. In studies on itoic acid synthesis, Peters and Warren (1968) suggested that the actual repressor or end-product inhibitor is the ligand-metal complex. As the concentration of environmental iron is increased, synthesis of this primary metabolite is decreased; in the presence of small amounts of ligand, lower quantities of iron are required to inhibit synthesis than in its absence. Yields of both primary and secondary metabolites as well as the detection of previously unknown substances often are enhanced by lowering the concentrations of iron and other trace metals in culture media (Schatz, 1961; see also Section IV.A, p. 24). Nisin, a polypeptide antibiotic of molecular weight 3290 (Gross and Morell, 1967),has been postulated to regulate the initiation and cessation of cell growth (Hurst and Dring, 1968). However, since nisin synthesis begins in early exponential growth-phase and parallels the formation of cell protein, this antibiotic clearly is a primary rather than secondary metabolite. Other primary substances that lie within the mainstream of cellular biosynthesis but yet have antibiotic potency have been described by Woodruff (1966). 3. Specialized Functions that Affect Other Organisms

The diversity of chemical structures of secondary metabolites is paralleled by the great variety of pharmacological activities and spectra of target cells possessed by this class of natural products. Unfortunately, we cannot readily estimate the true percentage of secondary metabolites that are pharmacologically potent because their discovery is so much easier (and more profitable) than that of their inactive congeners. Perhaps the percentage would be similar to that for synthetic organic molecules; in the latter case, approximately 10% of randomly conceived compounds may exhibit one or more types of biological activity (E. E. Campaigne, personal communication).

18

EUGENE D . WEINBERG

In earlier decades, the suggestion often was made that formation of secondary metabolites which poison or stimulate growth of neighbouring micro-organisms,plants, or animals confers a selective advantage on the producing micro-organisms. Weaknesses inherent in this suggestion include observations that (1) survival in nature of microbial strains that form innocuous secondary metabolites is as successful as that of strains that are toxigenic, (2) in some systems, potent secondary metabolites accumulate only in certain types of pure-culture fermentations and are not produced in nature, and (3) in other systems, potent secondary metabolites are formed in natural environments but alter the latter in ways detrimental to the toxigenic organisms. It is not likely that strains of Pseudomonas improve their fortunes by inducing a fatal hypoglycaemia in persons ingesting the triazines and unsaturated fatty acids formed by these bacteria. Similarly, it is difficult to understand how the neuromuscular or haemolytic phycotoxins, or the anaesthetic, analeptic, oestrogenic, hallucinogenic, parasympathomimetic, photosensitizing, sedative, teratogenic, tremorigenic, or anabolic hormonal mycotoxins could, by poisoning or stimulating plant or animal cell populations, improve the life or status of the algal or fungal cells that elaborate these fascinating molecules.

111. Specific Macromolecules as Secondary Metabolites Ordinarily, secondary metabolites are considered to be low molecularweight materials with an upper limit of about 1500. Occasionally, an antibiotic of slightly larger size (e.g. saramycetin with a molecular weight of 2100; Kirschbaum and Aszalos, 1967) is described. Unlike nisin (Section II.C.2, p. 14),the saramycetin peptide is probably a secondary rather than primary metabolite because it is formed by early stationaryphase cells (Berger et al., 1962) and is believed to possess a number of thiazolidine groups (Baudet and Cherbuliez, 1964). But there exists a group of high molecular-weight polypeptides that contain many of the attributes of typical secondary metabolites. Among these compounds are the well-studied toxins of Gram-positive bacteria and it may be observed in Table 2 that, in general, criteria established for secondary metabolites apply to the four materials listed. For example, Kindler et al. (1956) noted that “most of the botulinus toxin is formed within a short period of time early in the phase of decline” ;similar observations have been made not only for botulinus toxin (Gerwing et al., 1968) but also for tetanus toxin (Miller et al., 1960), diphtheria toxin (Pappenheimer, 1965; Fig. 4,p. 9), and staphylococcal enterotoxin (McLean et al., 1968; Markus and Silverman, 1969; Morse et al., 1969;Figs. 5,6).And, as is true for low molecular-weight secondary

TABLE2. Selected macromolecules that possess characteristics of secondary metabolites

Macromolecule

Molecular weight

Number Formed of by non- Restricted molecular growing taxonomic species cells distribution

Specialized function Released for producinto ing cell medium

Unique metal requirement for synthesis (see Table 3, P. 26)

Reference

E? %*

;

Latham et al. (1962); Miller et al. (1960); g Sterne and van Heyn- o r ingen (1965). m Sterne and van Heyningen (1965);Boroff et al. ‘4 (1968); Gerwing et al. (1968); Kindler et al. + (1956).

Tetanus toxin

67,000

One

Yes

Yes

None known

Yes

Requires iron

Botulinus toxin

10,000; 128,000150,000

Six

Yes

Yes

None known

Yes

Suppressed by EDTA

Diphtheria toxin

72,000

One

Yes

Yes

None known

Yes

Suppressed by iron

Pappenheimer (1965); Yoneda and Pappenheimer (1957).

Staphylococcal enterotoxin

34,00035,000

Four

Yes

Yes

None known

Yes

Requires iron

Bergdoll (1967); Casman m (1958); Morse et al. (1969); McLean et al. (1968) ;Markus and Silverman (1968).

E

B 2

#

M

20

EUGENE D. WEINBERG

metabolites (Bu’Lock and Powell, 1965),tetanus toxin can be produced in continuous culture provided that the rate of dilution is sufficiently lowered (Zacharias and Bjorklund, 196s).Tetanus toxigenesis is inversely proportional to sporulating ability (Sanada and Nishida, 1965);similarly, yields of such low molecular-weight secondary metabolites as bacitracin are highest in strains that sporulate poorly. A thorough discussion of possible relationships between sporulation and synthesis of exotoxins and antibiotics has recently appeared (Schaeffer, 1969). Another feature that two of the toxins referred to in Table 2 share with low molecular-weight secondary metabolites is that they are secreted into the medium by intact non-lysing cells. Yoneda and Pappenheimer (1957), for example, have observed that “protein (including diphtheria toxin) released during the phase of decline does not originate from phage-lysed cells and occurs without lysis of any important proportion of cells”. Enterotoxin is released into the culture without noticeable autolysis (Friedman and White, 1965);moreover, no toxin can be detected in sonic extracts prepared from staphylococci from either the exponential or stationary phase of growth (Markus and Silverman, 1969). On the other hand, release of botulinus toxin (Gerwing et al., 1968) and tetanus toxin (Miller et ab., 1960)does occur during the period of time in which the non-growing cells are lysing. Of 12 different inhibitors tested (Kindler et ul., 1956), only ethylenediamine tetraacetic acid suppresses botulinus toxigenesis without interfering with cell growth; the key metal rendered unavailable to the cells is probably iron (Weinberg, 1966) but media with sufficiently low iron contamination have not yet been examined in the botulinus system. The latitude permitted between the quantity of a specific trace metal required and the amount of that metal tolerated for toxigenesis in each of the other three systems in Table 2 is much narrower than the latitude for primary metabolism. I n systems in which low molecular-weight secondary metabolites are being formed, this phenomenon also is consistently observed (Section IV.A, p. 24). Additional reasons for considering substances such as staphylococcal enterotoxin to be secondary metabolites have been noted by McLean et al. (1968) and Markus and Silverman (1969).Each group has observed the typical change in pH value that occurs at the start of secondary metabolism (Figs. 5 and 6, p. 10) as well as differential effects of temperature and aeration on growth and toxigenesis. The former group reported that 3% NaCl depresses toxin formation much more than total growth, and the latter that 2% glucose suppresses toxigenesis in nongrowing cultures. And, as is true for low molecular-weight secondary metabolites, such inhibitors of protein synthesis as streptomycin and chloramphenicol (Markus and Silverman, 1968) cannot interfere with

BIOSYNTHESIS O F SECONDARY METABOLITES

21

toxin formation when added to cells in which synthases or metabolite precursors are already present. However, in addition to the dissimilarity in molecular weight, the substances in Table 2 differ from well established secondary metabolites in two respects : (1) the molecules listed in Table 2 appear to be typical polypeptide chains with no unusual amino acids or “odd” linkages, and ( 2 )multiple molecular species have not been discovered in either tetanus or diphtheria toxins. Spero et al. (1965), for example, have discussed various possible unusual configurations that might be possessed by the enterotoxin single-chain of amino acids but have concluded that no evidence exists for any of these. Of the 299 amino acids in enterotoxin, 42 are lysine and 55 aspartic acid (Spero et ul., 1965); a function of the toxin for the cells may be that of providing a convenient disposal package for these two primary metabolites. Other macromolecules formed in large quantity after most or all of cell growth has occurred, restricted in taxonomic distribution, and excreted into the medium, and which might thus be candidates for the secondary metabolite designation include the protective antigen of Bacillus anthracis (Gladstone, 1946), the D-glutamyl polypeptide capsular material of B. unthracis and B. subtilis (Leonard et al., 1958), streptolysin-S of group-A streptococci (Bernheimer, 1949; Fig. 8), the neurotoxin of Shigellu dysenteriae (Engley, 1952), and the vascular permeability factor of Vibrio cholerue (Evans and Richardson, 1968). Still other candidates include the parasporal crystalline toxin of Bacillus thuringiensis (Heimpel and Angus, 1960) and, perhaps, such enzymes unique to secondary metabolism as the synthases of phenoxazinone in Streptomyces untibioticus (Marshall et al., 1968) and of dipicolinic acid in Bacillus megaterium (Bach and Gilvarg, 1966; Fig. 2, p. 7); these three materials, of course, are retained within the cells. Production of most microbial extracellular enzymes parallels growth (Davies, 1963) and these are clearly primary metabolites. However, some degradative enzymes are formed in large amounts during the stationary phase. Examples include proteases of species of Myxobucter (Ensign and Wolfe, 1965), Bacillus (Spizizen, 1965), streptomycetes (Mizusawa et ul., 1966), and fungi (Jonsson, 1968; Fig. 9) and the CLamylase and ribonuclease of B. subtilis (Coleman, 1967). Since it is not yet clearly established that these enzymes differ from similar molecules formed by these and other organisms in low amounts during balanced growth, they should not presently be included in the group of macromolecular secondary metabolites. Also to be excluded from the category of secondary metabolites are such taxonomically restricted macromolecules as staphylococcal leucocidin (Gladstone and van Heyningen, 1957) and 6-haemolysin

22

EUGENE D. WEINBERG 0.8

r

-1800 800

+ C 0 ._ c

x

+

f 0.4t

cn C ._ x

OO

;

2

J i S Time (hr.)

6

+

O

FIG.8. Time-course of growth (measured as the turbidity of the culture) and of synthesis of streptolysin-S by cells of Streptococcus pyogenes. Reproduced with permission from Bernheimer (1949).

Time (hr.)

FIG.9. Time-course of growth and of synthesis of protease by cells of Entomophthora coronata. Reproduced with permission from Jonsson ( 1968).

(Yoshida, 1963), streptococcal M-protein (Hahn and Cole, 1963) and various polysaccharide capsular materials (e.g. Taylor and Juni, 1961). The first three of these compounds are produced by growing rather than

BIOSYNTHESIS O F SECONDARY METABOLITES

23

non-proliferating cells, and the polysaccharides are formed by both growing and stationary-phase cells. Although bacteriocins are produced by non-growing cells (Ivanovics, 1962), members of this heterogeneous macro- and multi-macromolecular group of substances should likewise not be considered to be secondary metabolites; their synthesis occurs in non-viable bacteria and they are released as the cells autolyse. A process in Bacillus subtilis apparently associated with secondary metabolism is that of transformation. I n continuous cultures, the rate of transformation is increased when the rate of growth is decreased

Time (hc)

FIG.10. DeveIopment of competence for transformation in cells of Bacillus subtilis. Data were obtained from Table 2 in Young and Spizizen (1961).

(Kretschmer and Bergter, 1966) and, in batch cultures, competency of transformation and transfection has a sharp peak between two and three hours after the cessation of exponential growth (Young and Spizizen, 1961;Wilson andBott, 1968; Figs. 10 and 11).Fifty times more manganese is required for transformation than for growth (Thorne and Stull, 1966) and the former process is more sensitive to copper than the latter (Anagnostopoulosand Spizizen, 1961). Moreover, provision of sufficient amino acids and manganese to permit sporulation decreases the number of transfectants by about 98% (Bott and Wilson, 1968);this observation is analogous to the incompatibility between sporulation and biogenesis of secondary metabolites mentioned previously (Section 1I.C.1, p. 13). The production by species of Bacdlus of all secondary metabolites thus far studied requires a more controlled concentration of manganese than is needed for vegetative growth (Section IV.A, p. 244, but it remains to

24

EUGENE D. WEINBERG

be determined if the role of this metal in competency is associated with synthesis of proposed essential polypeptides (Kammen et al., 1966a) or proteins (Kammen et al., 1966b) or is needed merely for maintenance of viability in stationary-phase cells (Leonard et al., 1958).For development of competence in Haemophilus inJluenxae, cessation of cell reproduction followed by a period of protein synthesis must occur (Stuy, 1962; Spencer and Herriott, 1965); however, the need for specific trace metals in this system has not yet been examined. 0.7

0

,

I

2

4

I

6 Time (hc)

I

I

I

8

10

12

FIG.11. Development of competence in cells of Bacillus subtilis. Reproduced with permission from Wilson and Bott (1968).

IV. Trace-Metal Roles in Secondary Metabolism

A. UNIQUE REQUIREMENTS AND TOLERANCES During the middle third of the twentieth century, investigators concerned with the biogenesis of large quantit,iesof specific natural products in non-growing microbial cells repeatedly made the observation that certain features of the trace-metal environment must be more rigidly controlled than is necessary for primary metabolism (these observations are summarized in Weinberg, 1962,1964,1966).Even the earliest workers in that period (e.g. Locke and Main, 1931) recognized that the metal concentrations critical for secondary metabolism neither stimulate nor depress vegetative growth.

BIOSYNTHESIS OF SECONDARY METABOLITES

25

Prior to the decade of the 19309, the yield of secondary and related metabolites varied uncontrollably because of random shifts in tracemetal contamination of organic nutrilites, water supply, and eluates from the walls of fermentation vessels. Mueller (1941) commented that “SO narrow is the zone (of iron concentration) in which (diphtheria) toxin is obtained and so sharp the peak of maximal production that this single uncontrolled factor must have played a greater role in any previous experiments than specific conditions supposedly under investigation”. Of the nine trace metals of biological interest (atomic numbers 23 through 30 and number 42), manganese, iron, and zinc are most important in secondary metabolism. Microbial products whose synthesis requires, or is inhibited by, quantities of trace metals different from the amounts that affect vegetative growth are listed in Table 3. For secondary metabolites or differentiated structures, as well as for synthesis of large quantities of some primary metabolites, manganese is the “key” metal for species of Bacillus, iron for other bacteria including actinomycetes, and zinc for all fungi and many actinomycetes. The concentrations of these metals critical for secondary metabolism are from one t o three log units higher than that of approximately 1 M-manganese, lo-’ M-zinc, and 2 x M-iron required for cell growth of all microorganisms. Moreover, primary metabolism generally tolerates quantities of each metal greater than IOP3 M ; this is approximately two log units higher than the amounts that often are inhibitory to secondary metabolism. In nearly all of the systems cited in Table 3, a t least five or six trace metals as well as magnesium and calcium have been examined. As may be seen in the Table, the majority of biosyntheses are affected by a single metal ion. Where more than one metal is listed as required, these are not interchangeable and each is required to obtain the maximum yield. Where more than one metal is listed as inhibitory, each suppresses the yield by itself. Magnesium and calcium are unable to substitute for any of the active trace metals. I n the large majority of data listed in Table 3, the synthetic media employed were sufficiently free of metal-binding agents and metal contaminations so that the concentrations of metals that are active are generally reproducible. I n each system cited, the authors presented evidence that vegetative growth is not altered by the concentration of metal that enhances or suppresses the yield of the particular product. In a number of systems, quantities of inorganic phosphate greater than that needed for growth suppress secondary metabolism presumably by depriving the cells of an essential metal. I n every secondary metabolic system for which sufficient data have been reported, the yield of the product varies linearly with the log of the concentration of the “key” metal. The linear relationship does not persist,

TABLE3. Microbial products whose yield is affected by concentrations of trace metals greater than those required for maximum growth Organism

Product or structure

I. Bacillus spp.

Metal concentration ( x 10-5 M )

Manganese

t 9

ua

Reference

Others

PRIMARY METABOLITES

Bacillus anthracis Bacillus anthracis Bacillus subtilis

3,4-Dihydroxybenzoic acid Coproporphyrin III 2,3-Dihydroxybenzoylglycine

Chao et al. (1967)

Fe-R-20" Fe-1-5.0" F-1-0.15

*

Chao et al. (1967) Peters and Warren (1968) M

$M

SECONDARY METABOLITES

(and Differentiated Structures) Bacillua anthracis Protective antigen

Bacillus lickenifoorrnis

Bacitracin

Bacillus licheniformis

Transformants

Bacillus subtilis Bacillus aubtilis

Bacillin D-glutamyl polypeptide My cobacillin Subtilin Transfectants Phage Spores

Bacillus subtilis Bacillus subtilis Bacillus subtilis Bacillus megaterium Bacillus megaterium

11. Bacteria other than Bacillus spp.

R-0.5" 1-2.0* R-0.07 1-4.0 R-20"

U

Weinberg and Tonnis (1966)

Iron

+I

Thorne and Stull (1966); AnagnostoM poulus and Spizizen (1961) w Foster and Woodruff (1946) Q Leonard et al. (1958)

Fe-R-0.5

Majumdar and Bose (1960) Jansen and Hirschmann (1944) Bott and Wilson (1968) Huybers (1953) Weinberg (1964); Kolodziej Slepecky (1962)

C~-R-0.03

Others

PRIMARY METABOLITES

Brevibacterium ammoniagew

5'-Inosinic acid

1

cu-1-1.0"

R-10" R-0.15 R-0.6 R-0.5 I-20* R-10* R-0.5

3

Wright et al. (1954)

Mn-1-0.0

15

Furuya et al. (1968)

and

Clostridium acetobutylicum Clostridium perfringens

Riboflavin Lecithinase

Cory nebacteriurn diphtheriae Escherichia coli

Coproporphyrin 2,3-Dihydroxybenzoylserine Mycobactin Salicylic acid Bacteriochlorophyll porphyrins Protease

1-0'75 1-0'03

Clarke (1958) Brot et al. (1966)

1-0'3 * R-4'0* R-0.2 1-0.2

Antoine and Morrison (1968) Ratledge and Winder (1962) Lascelles (1956)

Actinorubin Neurotoxin Toxin Alkyl-quinolinols Fluorescin Pyocyanine Pyrryldipyrryl methene Neurotoxin Enterotoxin Actinomycin Monensin Neomycin

R-2.0* R-3.0 1-0.7 1-2.0 1-0'3 R-0.3 R-0.3 1-2.0 1-0.6 R-10* R-lo* R-100 R-1.0 1-15

Mycobacterium phlei Mycobacterium s m e g m t i s Rhodopseudornonas spheroides Streptornyces sp.

1-2.5

1-10

CO-1-2.5 Zn-R-2.O* Mn-R-2.0*

Mn-R-10*

Hickey (1945) Murata et al. (1965)

Mizusawa et al. (1966)

SECONDARY METABOLITES

Actinomyces sp. Clostridium tetani Corynebacterium diphtheriae Pseudomonas aeruginosa Pseudornonm aeruginosa Pseudornonm aeruginosa Serratia marcescens Shigella shigae Staphylococcus aureus Streptomyces antibioticus Streptomyces cinnamonensis Streptomyces fradiae Streptomyces griseus Streptornyces griseus Xtreptomyces griseus

Candicidin Grisein Streptomycin

R-4*0* R-4.0 R-1.0

Streptomyces venezuelae Streptomyces verticillatus

Chloramphenicol Mitomycin

R-2.0* R-40*

Kelner and Morton (1947) Latham et al. (1962) Mueller (1941) Wensinck et al. (1967) Totter arid Mosely (1953) Kurachi (1958) Waring and Werkman (1943)

Zn-R-lo* Zn-R-0.1 Zn-1-1.0 Mn-1-10 Zn-R-4.0* Zn-R-0.3 Zn-1-20 Zn-R-2.0*

van Heyningen (1955) Casman (1958) Katz et al. (1958) Stark et al. (1968) Majumdar and Majumdar (1965) Acker and Lechevalier (1954) Reynolds and Waksman (1948) Chesters and Rolinson (1951) Gallichio et al. (1958) Kirsch (1967)

E

23

8w m 0 4

m

M c2 0

2 U

B

m

* z

z

1

0

5m v1

TABLE3--continued t.j

Organism

Product or structure

111. Fungi

Metal concentration ( x lO-5M)

Zinc

00

Reference

Others

PRIlMARY METABOLITES

Aspergillus niger

Citric acid

1-2.0

Candida guilliermondii Penicillium griseofulvurn Ustilago sphaerogena Ustilago sphaerogena

Riboflavin Mycelianamide Coproporphyrin Ferrichrome

R-0.3 1-0.2

Fe-1-6.0 Mn-1-0.02 Fe-1-0'1 F+I-40

Shu and Johnson (1948) Clark et al. (1965) Tanner et al. (1945) Bayan et al. (1962) Komai and Neilands (1968) Komai and Neilands (1966)

*

CO-R-0.3

M

SECONDARY METABOLITES

(and Differentiated Structures) Aspergillus jlavus Aflatoxin Aspergillus niger Malformin

d 0

R-0.5 1-20

Mn-R-0.1 Mn-1-1.0

Mateles and Adye (1965) Steenbergen and Weinberg (1968)

EM

Foster (1939)

8 9

Aspergillus niger Claviceps paspali Claviceps purpurea F w a r i u m vminfectum

Spores Lysergic acid Ergotamine Fusaric acid

Penicillium chrysogenum (and P. notatum) Penicillium coronata

Penicillin

Penicilliurn griseofulvum Penicillium urticae Penicillium urticae Penicillium urticae Pythium graminicola

Griseofulvin 6-Methylsalicylate Gentisyl alcohol Patulin Oogonia

R = required; I =inhibitory; values.

* quantities starred with asterisk are 100% end-pointvalues; those not starred are 50% end-point

Vesicles

I-loo* R-0.5 R-1.0 R-0.3 1-0.6 R-0.1 1-3.0 R-12 1-30 1-20" 1-0.1 * R-O.l* R-O.l* R-2*0*

Fe-R-2.0* Cu-1-1.0

Rosazza et al. (1967) Stoll et al. (1957) Kalyanasundaraxn and Devi (1955) Foster et al. (1943) Koffler et al. (1947) Sharp and Smith (1952)

*

Fe-I- 1* 5 Fe-R- 1.5 Mn-R-0.03

*

P 2

Saraswathi-

Grove (1967) Ehrensvilrd (1955) Ehrensviird (1955) Brack (1947) Lenney and Klemner (1966)

td M

$

29

BIOSYNTHESIS O F SECONDARY METABOLITES

of course, at concentrations of the metal that are either insufficient or toxic for cell growth. The extent of the linear relationship varies from a 2 .c

I .E

I .E I ,L

1.2 I .c

c .

T

0.E 0.i 0.E 0.5

P

0.4

-

0.3

Gx

0 ._ c

P +c

0.2

V

6 .o 0 I

0.16 0.14 0.12 0.10

0.08 0.07 0.06 0.05

0.04 0.03

002 1

/

6

100

200

300

400

I

I

I

500

600

700

Yield of metabolite (arbitrary units)

FIG.12. Examplcs o f the linear relationship between loglo o f the concentration of “key” metal and the yield of secondary metabolite. Curve 1 shows the relationship between bacitracin production and the concentration of manganese ion (Weinberg and Tonnis, 1966) ;curve 2, mycobacillin and manganese ion (Majumdar and Bose, 1960); curve 3, fluorescein and ferric ion (Totter and Mosely, 1953); curve 4, diphtheria toxin and fcrric ion (Mueller, 1941) ; curve 5, Shigella neurotoxin and ferric ion (van Hcyningen, 1955); curve 6, pyocyanine and ferric ion (Kurachi, 1958); curve 7, penicillin and zinc ion (Foster et al., 1943); curve 8, aflatoxin and zinc ion (Mateles and Adye, 1965).

span of metal concentrations of as little as one-half log unit to slightly over two log units ; the average figure for 17 different systems is approximately one unit. Examples are given in Fig. 12.

w

0

TABLE4. ‘‘Key’’metal control of cultural longevity Number of viable cells per ml on day : Organism and Reference

Metal concentration ( ~ 1 0 - 5AT)

I

zero

one-half

one

two

three

four

five

eight

( x 107)

Bacillus subtilis (asporogenic strain) Leonard et al. (1958)

MANGANESE

I

I

0.007 0.154

3 3

-

80 170

0.01 180

0.001 1.0

0.001

-

-

61.500

3

-

170

180

170

150

100

-

M

3M 2

M

(x107) Pseudomonm aeruqinosa

IRON

lTreinberg and Goodnight (1969)

0.02 0.20

I

0.2 0.2

-

490 575

0.01 0.0001 152

120

sterile 200

sterile 26

sterile 40

P 8

8

(x107)

Escherichia coli E. D. Weinberg and S. K. Simmons (unpublished data)

IRON

0.02 0.20

0.2 0.2

Torulopsis hornii Steenbergen et al. (1969)

ZINC

r

103 116

15 0.08

0.002 0.00007 stmerile sterile

sterile sterile sterile sterile

(~05) 0.1 1.0 10.0

7

0.1

-

0.1 0.1

-

200 190 190

200 105 52

190 42 10

182 16 3

150 5 1-1

105 0.8 0.1

BIOSYNTHESIS OF SECONDARY METABOLITES

31

The effects on cultural longevity of concentrations of trace metals greater than those needed for primary metabolism have been mentioned in Section II.C.l (p. 13). If cultural longevity is linked to some yet undetermined facet of secondary metabolism, the “key” metals that control the latter should be identical to those important in longevity; the data obtained thus far are in accord with this prediction. I n Table 4, it may be observed that manganese enhances longevity of cultures of non-sporulating Bacillus sp., iron affects longevity of Pseudomonas and Escherichia spp., and zinc suppresses longevity of cultures of Torulop8is sp. Metals tested in these systems and found to be inactive are: for Bacillus sp., calcium, zinc, and cobalt; for Pseudomonas and Escherichia spp., magnesium, calcium, manganese, cobalt, and zinc; and for Torulopsis sp., manganese and iron. I n the last system, copper and cadmium could substitute for zinc. Another interesting effect at the population level of “key” metal control of secondary metabolism was reported by Musilek (1963). Populations of Xtreptomyces griseus, subcultured in media containing sufficient iron to permit streptomycin production, degenerate (i.e. lose the capacity to form the metabolite) at the expected rate whereas those grown without iron fail to degenerate. Apparently, non-producing cells have a selective advantage over producers under conditions in which the latter synthesize the compound. Unfortunately, in this study, longevity was not monitored.

B. PROPOSED SITESOF ACTION 1. Non-“Key” Metals

Suggestionsconcerning the sites of action of trace metals that influence the synthesis of large quantities of primary metabolites, or that are listedin the columnlabelled “Others” inTable 3 (p. 26) for either primary or secondary metabolism, can be grouped in three categories. The first consists of systems in which the environmental concentration of iron regulates the extent of formation of primary metabolic ligands whose function is concerned with iron transport. Such systems, previously discussed in Section II.C.2 (p. 14), include the synthesis by procaryotes and eucaryotes of salicylates, hydroxamates, and hydroxybenzoates. Evidence that the site of action of iron (or the iron-ligand complex) involves suppression of activity rather than formation of synthases has been reported for the enzyme that catalyses the synthesis of 2,3-dihydroxybenzoylserine from 2,3-dihydroxybenzoic acid and serine (Brot et ul., 1966). Presumably, other classes of primary metabolites will be discovered that are concerned with transport of other trace metals, and their synthesis will be found to be controlled by the environmental concentrations of the respective metals.

32

EUGENE D. WEINBERG

A second category of sites of action of non-“key7’metals is concerned with primary metabolites that are porphyrins. Synthesis and excretion of large amounts of porphyrins by a wide variety of bacteria (reviewed by Lascelles, 1961) is suppressed by low concentrations of iron. These quantities of the metal are required for the production of haems and chlorophylls but the amounts of such compounds obtained are only 10% of the yield of porphyrins that would have accumulated in the absence of iron. Lascelles (1961) proposed that the metal activates enzymes that convert porphyrin precursors into haems and chlorophylls. The third category of possible actions of non-“key” metals comprises a miscellaneous group that pertains neither to metal transport nor to porphyrin metabolism. For example, the proportion of gentisyl alcohol to patulin formed by Penicillium urticae during secondary metabolism is determined by the iron concentration merely because the oxygenase needed to convert the former to the latter is activated by this particular metal (Bu’Lock and Shepherd, 1968). The synthesis of large amounts of a protease in the stationary-phase of growth by a thermophilic species of Streptomyces requires manganese; the metal is believed to activate an intracellular peptidase that in turn would liberate amino acids that could be used to form the protease (Mizusawa et al., 1966). 2. “Key” Metals

In as much as a single “key” metal is operative in secondary metabolism in every member of large taxonomic groups of micro-organisms, it is tempting to search for a unitary function of the metal. Numerous possible roles of the “key” metals have been suggested. These can be grouped temporally with respect to ribosomal formation of synthases of low molecular-weight secondary metabolites, or to ribosomal production of macromolecular (protein) secondary metabolites, namely, roles operative prior to transcription, during transcription or translation, or following translation. Relevant temporal questions include : (1) when is the “key” metal assimilated by the cells, (2) how late in the postexponential growth phase can the metal be provided and activity be obtained, and (3) how long must the metal be present in the cells during secondary metabolism to exhibit complete activity? I n my laboratory, Mr. K. Y. Lee (unpublished data) has observed that manganese, provided at the time of inoculation, is assimilated by cells of Bacillus megaterium within a 1-hr period at the start of secondary metabolism. We have previously found that the time of addition of a required “key” metal can be delayed for several hours beyond the point at which secondary metabolism would normally begin provided that the cells have not yet begun to die. I n some systems, however, the pH value

BIOSYNTHESIS OF SECONDARY METABOLITES

33

has become altered to a reaction unfavourable for secondary metabolism so that an adjustment of pH value is required at the time the metal is added (Weinberg and Tonnis, 1966). Delayed addition of the metal causes a corresponding delay in the appearance of secondary metabolites ; the elapsed time between addition and appearance remains constant (Weinberg and Tonnis, 1967). The duration of time in which inhibitory “key” metals are active is not known; in the case of a primary metabolic synthesis that occurs in large quantity in the stationary phase of growth (citric acid formation), production can be aborted at any time that the toxic non-“key” metal, manganese, is added (Clark et al., 1966). Attempts have not yet been made to answer the third temporal question; it is not known if the “key” metal can be removed from its functional site by the presently available synthetic chelating agents. The latter are not sufficiently specific for individual metals, and restoration to the cells of the other metals that had been removed by the chelator might not serve to permit structural and metabolic recovery. Hopefully, natural products that have a highly selective and very strong affinity for a single metal will eventually become available for this type of experiment. a. pre-transcription events. Suggested functions during this phase of secondary metabolism have been made primarily in Bacillus systems for the “key’)metal manganese. This metal predominates as an activator of decarboxylases, dehydrogenases, and hydrolases (Nason and McElroy, 1963; Williams, 1967) and increased levels of many such enzymes at the onset of presporulation in Bacillus have been described. For example, a manganese-activated protease (Stockton and Wyss, 1946) that is active in early stationary-phase cells might function to yield endogenous amino acids that have been postulated to stimulate new m-RNA synthesis (Balassa, 1964). I n Bacillus licheniformis, an arginase is induced when glucose is exhausted (or when cells are grown on other carbon sources; Laishley and Bernlohr, 1966) ; the L-ornithine derived from L-arginine might then be incorporated directly into a metabolite that is later released into the medium as bacitracin (Ramaley and Bernlohr, 1966). Arginases from a variety of sources are activated primarily by manganese (Spector, 1956).

In presporulating cells of Bacillus megaterium, the level of inorganic pyrophosphatase is increased, and manganese is required for maximum stabilization and activity of this enzyme (Tono and Kornberg, 1967). An additional electrophoretic species, found only in sporulating cells, is partially converted to the principal form by treatment with manganese. Unlike glutamine synthases of several other bacterial genera, that from presporulating cells of B. Zichenijormis requires manganese and cannot utilize magnesium (Hubbard and Stadtman, 1967). Moreover, 2

34

EUQENE D. WEINBERG

the concentration of manganese determines the sensitivity of the enzyme to ATP and nucleoside triphosphate concentrations, and the ratio of ATP to manganese determines the susceptibility of the enzyme to activation or inhibition by the nucleoside triphosphates (Hubbard and Stadtman, 1967).Theoretical aspects of metal control of enzyme activity have been discussed by Wyatt (1964). Instead of, or in addition to, activating “early” enzymes, the “key” metal might function as either a co-derepressor or a co-repressor of operator genes that control formation of secondary metabolic synthases or of macromolecular (protein) secondary metabolites. The ability of metals to cause subtle conformational changes in enzyme protein structure has been well illustrated by Stadtman and colleagues (e.g. Kingdon et al., 1968), and perhaps the “key” metals can similarly affect the structure and function of repressor proteins. Sardesai and Rao (1966) have suggested that, at low concentrations, iron acts as an inducer and, at high amounts, as a co-repressor of diphtheria toxigenesis. However, in a cell-free preparation, an iron porphyrin-bound protein has been observed to inhibit toxin formation; in this system, the metal is believed to act at the translational level rather than as a repressor (Sato and Kato, 1965). b. transcription and translation. When inhibitors of RNA and protein synthesis are added to postlogarithmic-phase cultures of B. Zicheniforrnis concurrently (or within two subsequent hours) with manganese, bacitracin formation does not occur (Weinberg and Tonnis, 1967). Unfortunately, this observation does not discriminate among the possibilities that the metal might function either during pre-transcription, transcription, or translation of bacitracin synthases. However, a similar experiment in a fungal system has yielded somewhat more definite information. Addition of inhibitors of protein synthesis, but not those of RNA synthesis, to 48-hr cultures of Ustilago sphaerogena simultaneously with zinc prevents the zinc-induced increase in activity of 6-aminolaevulinate dehydratase, indicating that the metal is required for the synthesis of this protein at the translational level (Komai and Neilands, 1968). Although this particular enzyme is concerned with formation of large quantities of a primary metabolite, several features of the system are analogous to secondary metabolism; for example, grown cultures are required, 300 times more zinc is needed for enzyme production than for growth, zinc can be added after growth has been completed, and manganese, cobalt, and nickel are inactive. Hopefully, the effect of the delayed addition of zinc and selective inhibitors of DNA and RNA functions on formation of secondary-metabolite synthases will be examined in a variety of fungal systems. The content of firmly bound metals in nucleic acids for several mammalian and algal sources has been surveyed by Wacker and Vallee (1959).

BIOSYNTHESIS O F SECONDARY METABOLITES

35

The ratio of micromoles of total metal to phosphate groups is 1 :50 in RNA and 1:150 in DNA. Typical values (microgram of metal per gram of RNA) for magnesium, chromium, manganese, iron, and zinc in rat liver- and algal-RNA are, respectively, 580 and 400, 102 and 76, 33 and 73,180 and 180,and 1300 and 650. The requirement for maintenance of a secondary helical structure in RNA of such trace metals as chromium, manganese, and zinc has been proposed (Fuwa et al., 1960). The conformation of tryptophan-t-RNA from Escherichia coli is altered by metals (Ishida and Sueoka, 1968) and aurin tricarboxylic acid, a strong mctal-binding agent, prevents attachment of viral m-RNA to E. coli ribosomes (Grollman and Stewart, 1968). Conformational changes in ribosomes of E . coli caused by zinc or nickel have been described (Tal, 1968). Unfortunately, comparative data are not available concerning the relative importance of individual trace metals in the structures of transfer-, messenger-, or ribosomal-RNAs in various species of bacteria and fungi, nor in RNA species extracted from primary and secondary metabolic stages of individual microbial cultures. Zinc deficiency has been observed to impair RNA and protein synthesis in Euglena gracilis (Wacker, 1962) and in Rhixopus nigricans (Wegener and Romano, 1963). Manganese is required for cell-free synthesis of RNA by polymerases of Micrococcus lyysodeikticus (Fox et al., 1964) and for in vivo RNA synthesis in Bacillus subtilis (Demain et al., 1964).Though it is tempting to propose that the action of the key metals in secondary metabolism is associated with a unique effect eit,her on RNA polymerases or on the structure or function of one of the three species of RNA, obvious problems remain unsolved. For example, why are the “key” metals not active on RNA formation or function during primary metabolism ; why might RNA synthesis or activity in Bacillus be controlled by manganese but in all other bacteria by iron; and why is the tolerated range of “key” metal concentrations so much narrower in secondary than in primary metabolism? c. post-translation events. Since the same “key” metal functions in the production of both small and large molecular-weight secondary metabolites, it is very unlikely that the unitary role (if any) of the metal would occur after translation. As previously stated, moreover, inhibitors of transcription and translation (added with the metal) prevent appearance of the metabolites. Nevertheless, a few observations are available concerning such a late role as activation by the “key” metals of synthases of non-protein secondary metabolites. I n these systems, it is quite possible that the “key” metal has both an early as well as a late function. I n B. licheniformis, manganese is required for activation of polyglutamate synthase; neither magnesium, calcium, zinc, nor cobalt can be substituted (Leonard and Housewright, 1963). I n iron-deficient cultures of

36

EUGENE D . WEINBERG

Pseudomonas aeruginosa, the accumulation of 2-alkyl-4-quinolinols is believed to result from diminished activity of iron-dependent tryptophan pyrrolase and kynurenate oxidase (Wensinck et al., 1967). In Pencillium cultures, the quantity of the “key” metal is important in determining the final proportion of secondary metabolites in at least two species. In cultures of Penicillium urticae grown in zinc-deficient medium, the intermediate metabolite, 6-methylsalicylic acid, predominates ; in media containing higher concentrations of zinc, such terminal products as gentisyl alcohol, toluquinol, and patulin accumulate (Ehrensvard, 1955). With Penicillium griseofulvum, fulvic acid is obtained instead of griseofulvin when Raulin-Thom rather than CzapekDox medium is used (Grove, 1967); the former medium contains ten times more zinc than the latter. With Alternaria tenuis, the synthase that catalyses formation of alternariol is suppressed by zinc and also by copper (Sjoland and Gattenbeck, 1966). Another type of possible late role is that of metal stabilization of the intermediate substrates. Bu’Lock (1967) has proposed that growing chains of malonyl units might be maintained in a suitable configuration until the growing loop of C2 units itself could occupy the co-ordination positions of the metal. In the simplest case, this would be obtained with four C2 units to yield orsellinic acid.

V. Prospects and Conclusions Many attempts have been made to obtain formation of secondary metabolites in cell-free systems. A number of these have been unsuccessful probably because (1)extracts were prepared at incorrect times during the growth cycle, (2) an incorrect mixture of soluble- and membranefractions were employed, or (3) a number of sequentially active enzymes are needed and not all of these were present in the cells at the time of fractionation. In the penicillin-N cephalosporin-C system, for example, active enzymes can be sedimented but unknown precursors must be supplied from the supernatant, and it is not clear to what extent normal mycelial permeability barriers have been altered in the precipitate (Abraham and Newton, 1967).A net synthesis of polymyxin in cell-free extracts could not be achieved, and that reported in low-speed centrifugates for circulin was probably due to unbroken cells (Paulus, 1967). Unsuccessful attempts have also been reported for cell-free synthesis of actinomycin, bacitracin, hadacidin, mycobacillin, and pyocyanine (Gottlieb and Shaw, 1967). On the other hand, cell-free systems have been described for synthesis of malformin (Yukioka and Winnick, 1966), gramicidin S (Bhagavan

BIOSYNTHESIS OF SECONDARY METABOLITES

37

et al., 1966; Spaeren et al., 1967), and edeine (Kurylo-Borowska and Tatum, 1966). The active extracts each consist of 105,000 g supernatants (or 11,000 g in the preparation of Spaeren et al., 1967) of cell sonicates from fully grown cultures. I n each case, the usual energy-generating components as well as known constituents of the metabolites must be supplied and the potency of each extract is resistant to RNAase. Cell-free synthesis of orsellinic acid in 90,000 g supernatants of Penicillium madriti has also been achieved (Gaucher and Shepherd, 1968). Acetyl-CoA and malonyl-CoA must be supplied to the extract, and activity can be sedimented by centrifuging at 210,000 g. Ten steps in the conversion of myo-inositol to streptidine are catalysed by enzymes from several types of 30,000 g supernatants of sonicates from mature mycelia of streptomycin-producing strains (Walker and Walker, 1967). Continued study of secondary metabolic functions in cell-free systems should provide knowledge concerning the mechanisms whereby unusual constituents and chemical linkages are constructed as well as identification of enzymes unique to secondary metabolism. Also, the precise sequence of precursors and of active enzymes can be elucidated and perhaps the reason for cessation of the various syntheses might be ascertainable. And finally, study of such systems might lead to a more precise determination of the site of action of the “key” metals. The formation of secondary metabolites in plant-cell cultures is receiving increasing attention (for bibliography see Staba and Jindra, 1968) and observations are similar to those made with micro-organisms. For example, in Datura stramonium suspension cultures, cusohygrine and pseudotropine are formed after growth has occurred and their appearance is enhanced by concentrations of manganese not required for vegetative growth. Large quantities of arginine accumulate in the cultures, and one of the functions of the metal might be that of activating an arginase (Staba and Jindra, 1968). I n mammalian-cell cultures, production of substances such as collagen by fibroblasts, pigment by iris pigment and melanoma cells, and myosin by muscle cells occurs when cell multiplication ceases; on the other hand, hyaluronic acid is formed by fibroblasts that are actively multiplying (Green and Todaro, 1967). Embryonic cartilage chondrocytes produce chondroitin sulphate under cultural conditions that prevent multiplication and encourage differentiation (Holtzer et al., 1960). Is the finite mortality of mammalian cell strains in non-differentiating cultures (Hayflick, 1965) due to their loss of ability to produce cryptic secondary metabolites ; conversely, have the indefinitely cultiviable cell lines retained or gained such activity? Perlman (1968) has stressed the importance of studying environmental cultural conditions needed for the synthesis of hormones and antibodies

38

EUGENE D. WEINBERG

by mammalian cells. He noted that knowledge of these requirements may lead to new chemotherapeutic use of “old” compounds or to development of new ones, and perhaps to identification of conditions for rejuvenation of aging or injured cells. I n micro-organisms, therapeutic (Weinberg, 1966) and diagnostic (Okami et al., 1963; Garibaldi, 1967) applications of knowledge of trace-metal control of secondary metabolism have been suggested. But, in addition to the desirable pursuit of such applied studies in plant, animal and microbial cell cultures, there remains the challenge of a number of fundamental questions about secondary metabolism. Among those that can be answered by in vitro laboratory experiments are : ( 1 ) does a “key” metal unitary function exist and why is this function

not operative during primary metabolism? ( 2 ) how is the time of uptake of the “key” metal controlled? (3) under the same environmental conditions, is the total quantity of

all secondary metabolites produced by closely related strains approximately equal? (4) why do secondary metabolic transcription and translation cease? Relevant ecological questions include : (1) in nature, must non-multiplying cells perform secondary metabolism (or differentiate) to remain alive? ( 2 ) do predilections of pathogenic micro-organisms for specific host tissues depend on environments that possess the correct available quantities of trace metals for secondary metabolism? (3) is the function of some factors of virulence that of adjusting the levels of available trace metals so that the pathogen can either multiply or perform subsequent aspects of secondary metabolism (e.g. Weinberg, 1966)? Questions more philosophical than the foregoing include : (1) what evolutionary factors caused the establishment of the tolerance of secondary metabolism to so narrow a quantity of trace metal, and why does the “key” metal differ among Bacillus spp., all other bacteria, and fungi? ( 2 ) are low molecular-weight secondary metabolites evolutionary forerunners of plant and animal hormones? (3) are protein secondary metabolites evolutionary forerunners of protein hormones and of antibodies? I n summary, secondary metabolites consist of low molecular-weight natural products that ( 1 ) are restricted in taxonomic distribution, ( 2 ) are synthesized for a finite period by cells that have stopped dividing, and (3) most probably function as convenient disposal packages of

BIOSYNTHESIS OF SECONDARY METABOLITES

39

excess primary substances. Viability is retained following completion of secondary metabolism whereas inability to form secondary substances or to differentiate can be lethal. Such high molecular-weight materials as protein toxins of Gram-positive bacberia possess many of the attributes of secondary substances. Secondary metabolism and cellular differentiation are accomplished within a range of concentrations of a “key” metal that is much narrower than that permitted for primary metabolism. The “key” metals for Bacillus spp., all other bacteria including actinomycetes, and fungi are, respectively, manganese, iron, and zinc. A unitary role of the “key” metals has not yet been established.

VI. Acknowledgement This work was supported in part by research grant HD-03038-01 from the National Institute of Child Health and Human Development, U.S. Public Health Service. REFERENCES Abraham, E. P. and Newton, G. G. F. (1967). I n “Antibiotics”, (D. Gottlieb and P. D. Shaw, eds.), Vol. 11, p. 268, Springer-Verlag, Berlin. Acker, R. F. and Lechevalier, H. (1954). A p p l . Microbiol. 2, 152. Anagnostopoulus, C. and Spizizen, J. (1961).J . Bact. 81, 741. Anchel, M. (1967). In “Antibiotics”, (D. Gottlieb and P. D. Shaw, eds.), Vol. 11, p. 189, Springer-Verlag, Berlin. Antoine, A. D. and Morrison, N. E. (1968). J . Bact. 95, 245. Bach, M. L. and Gilvarg, C. (1966).J . biol. Chem. 241, 4563. Balassa, G. (1964). Biochem. Biophys. res. Commun. 15, 236. BarabiLs, Gy. and Szabo, G. (1968). Can. J . Microbiol. 14, 1325. Baudet, P. and Cherbiiliez, E. (1964). Helv. chim. Acta 47, 661. Bayan, A. P., Nager, U. F. and Brown, W. E. (1962).Antimicrob. Agents Chemother. p. 669. Bhagavan, N. V., Rao, P. M., Pollard, L. W., Rao, R. K., Winnick, T. and Hall, T. B. (1966). Biochemistry, N.Y. 5, 3844. Bergdoll, M. (1967). In “Biochemistry of Some Food-borne Microbial Toxins”, (R. I. Mateles and G. N. Wogan, eds.), p. 1, MIT Press, Cambridge, Mass. Berger, J.,Sternbach, L. H., Muller, M., LaSaIa, E. R., Grunberg, E. and Goldberg, M. W. (1962). Antimicrob. Agents Chemother. p. 436. Bernheimer, A. (1949).J . exp. Med. 90, 373. Bernlohr, R. W. and NovelIi, G. D. (1960). Archs Biochem. Biophys. 87, 232. Bernlohr, R. W. and Novelli, G. D. (1963). A r c h Biochem. Biophys. 103, 94. Birch, A. J. (1967). I n “Antibiotics”, (D. Gottlieb and P. D. Shaw, eds.), Vol. 11, p. 228, Springer-Verlag, Berlin. Boroff, D. A., DasGupta, B. R. and Fleck, U. S. (1968). J . Bact. 95, 1738. Bott, K. F. and Wilson, G. A. (1968). Bact. Rev. 32, 370. Brack, A. (1947). Nelv. chim. Acta 30, 1. Brenner, M., Gary, E. and Paulus, H. (1964). Biochim. biophys. Acta 90, 401. Brot, N., Goodwin, J. and Fales, H. (1966). Biochem. biophys. Res. Commun. 25, 454.

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Antimicrobial Agents and Membrane Function FRANKLIN M. HAROLD Division of Research, National Jewish Hospital and Department of Microbiology, University of Colorado School of Medicine, Denver, Colorado, U . S. A . That which one man gains by discovery is a gain of other men. And these multiple gains become invested capital, the interest in which is all paid to every owner, and the revenue of new discoveryis boundless. It may be wrong t o take another man’s purse, but it is always right t o take another man’s knowledge, and it is the highest virtue to promote another man’s investigation. John Wesley Powell, Director, U.S. Geological Survey, 1886.

I. Introduction . 11. Structure and Functions of Microbial Membranes . A. Permeability Barriers B. Transport Systems . C . Electron Transport and Generation of ATP . . D. Membrane, Wall and Nucleus : An Integrated Unit 111. Compounds which Disorganize Lipoprotein Membranes . A. Organic Solvents . B. Detergents . C. Reversible Membrane Disorganization? . ‘$ D. Peptide Antibiotics . E. Basic Polypeptides and Proteins F. Polyene Antibiotics and Membrane Sterols . $ IV. Proton Conduction and Uncoupling of Oxidative Phosphorylation . V. Alkali Metal Ionophores . A. Valinomycin . B. Enniatins C. Gramicidins D. Macrotetralides: Nonactin and its Homologues . E. Nigericin, Monensin and other Carboxylic Polyethers . F. Other Ionophores . VI. Inhibitors of Energy Transfer and of the Respiratory Chain . A. ATPase and Energy Transfer . B. Inhibitors of the Respiratory Chain . C. Interaction of Heavy Metals with the Membrane . VII. Bacteriocins: Antibiotics which Interact with Specific Membrane Receptors . VIII. Summary and Prospect . IX. Acknowledgements . References .

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I. Introduction By 1940 it was clearly recognized that certain antibiotics and synthetic antimicrobial agents act at the level of the cytoplasmic membrane. Most of these, including the antibiotic tyrocidine (Hotchkiss, 1944) and the quaternary ammonium compounds (Domagk, 1935) brought about gross disruption of the osmotic barrier; they were indeed useful disinfectants, but offered little promise of revealing the molecular details of membrane structure or function. This bleak prospect has been transformed by the pioneering efforts of many investigators. Among the landmarks are the studies of Lardy and his associates on the use of antibiotics in the analysis of oxidative phosphorylation, the work of Lampen, Kinsky and Van Deenen on the interaction of polyene antibiotics with sterols and, more recently, the far-reaching discovery of ion conduction by Chappell, Mitchell, Mueller and especially Pressman. It has thus become clear during the past decade that antibiotics will prove to be as valuable in the analysis of membrane functions as they have in unravelling the complexities of macromolecule synthesis. The purpose of this review is to consider the interactions of antibiotics (and antimicrobial agents generally) with cellular membranes, and the application of these reagents to the study of membrane physiology in micro-organisms.However, much of the experimental material currently available refers to mitochondria and to artificial membrane systems, perhaps because biochemists were particularly alert to the selective effects of many antibiotics upon membrane functions. It proved necessary to limit the scope of this review in several respects. Antimicrobial agents which inhibit the synthesis of the cell wall and the replication of DNA were excluded even though the membrane participates in these processes. This article is restricted to those functions which appear to be intrinsically associated with membranes : impermeability to small molecules, active transport and the generation of metabolic energy. Because of my own interests examples were chosen from the bacteria more often than from the fungi. Finally, I have tried to select from the profusion of pharmacological agents which affect membranes, those compounds which promise to be of particular value in microbiology. These restrictions still leave a literature both voluminous and scattered, and I can but offer my apologies to those investigators whose contributions were overlooked. Some selection of references was unavoidable ; review articles and recent research papers were cited whenever possible, sometimes at the expense of prior reports. TWOgeneral sources of information on antibiotics deserve special mention. A monumental work by Korzybski, Kowszyk-Gindifer and Kurylowicz ( 1967) compiles chemical data and

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applications for every known antibiotic. But for those who wish to use antibiotics as chemical probes in the dissection of physiological processes, the indispensible reference work is the treatise edited by Gottlieb and Shaw (1967). Throughout the preparation of this review I have drawn heavily upon their work for information, insight, and models of useful scholarship. 11. Structure and Functions of Microbial Membranes Eucaryotic cells, such as fungi and algae, differ fundamentally from the procaryotic bacteria in the organization of membranous elements. I n the former, the plasma membrane serves as the main osmotic barrier and energy generation is the function of specialized organelles, mitochondria and chloroplasts. I n procaryotic cells, the division of labour is much less obvious. Not only transport and permeability but also oxidative and photosynthetic phosphorylation are attributes of the plasma membrane or, at least, of membranous elements which cannot yet be clearly separated from the plasma membrane (Salton, 1967; Lascelles, 1968). I n addition the plasma membrane is intimately involved in the biosynthesis of all cellular elements external to it, such as cell-wall mucopeptides, lipopolysaccharides, teichoic acids and exoenzymes ; it is the locus to which flagella are attached, and it apparently ensures the equal partitioning of the genome among daughter cells at division. The multiplicity of known and suspected functions of the bacterial cytoplasmic membrane suggests an intricate mosaic ; we may well discover that, far from being relatively simple, bacterial membranes are actually among the most complex.

A. PERMEABILITY BARRIERS Despite the complexity of some microbial envelopes, the plasma membrane appears to be in all cases the main osmotic barrier. I n Mycoplasma species, the membrane is directly exposed to the medium. I n Grampositive bacteria and in fungi, the cell wall shields the membrane but impedes the passage of only quite large molecules (Schemer and Gerhardt, 1964). The envelope of Gram-negative bacteria is more elaborate, and includes a lipopolysaccharide layer external to the plasma membrane. This also appears as a “unit membrane” in electron micrographs, and clearly constitutes a permeability barrier to certain compounds (Section II.D, p. 51), but the properties of sphaeroplasts and of plasmolysed cells leave no doubt that even here the main osmotic barrier resides at the inner, cytoplasmic membrane (Salton, 1967). Bacteria frequently contain internal membranous organelles. Some of these, such as the vesicular chromatophores (Lascelles, 1968), may constitute osmotic compartments in the intact cell. Whether mesosome4

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are segregated from cytoplasm and medium by a permeability barrier is not clear (Salton, 1967), and for the present we may retain the comfortable assumption that bacteria generally lack internal structural compartments. Microbial membranes contain proteins and lipids in roughly equal proportions. Characterization of membrane proteins is just beginning but considerable information on the phospholipids is available (see reviews by Gel’man et al., 1967; Goldfine, 1968; Kates, 1966; Lennarz, 1966). As is well known, sterols are major constituents of eucaryotic membranes but are absent from bacteria, except possibly for trace amounts (Schubert et al., 1968; de Souza and Nes, 1968). The role of sterols in membrane structure is not clear, but is usually considered to be the stabilization of phospholipid arrays ; in bacteria, carotenoids may play an analogous role. The traditional Danielli model of membrane structure continues to be a valuable guide to the design of experiments, and inspired the successful effort to prepare artificial, purely lipid membranes. Of these, the black bilayer membranes approximate most closely the properties of living membranes with respect to water permeability, electrical characteristics and perhaps overall structure. Phospholipid sphaerules, consisting of multiple concentric lipid shells within which solutes may be trapped, are another useful model albeit less realistic (see reviews by Lucy, 1968; Rothfield and Finkelstein, 1968; Tien and Diana, 1968). Nevertheless there is growing doubt that a phospholipid bilayer is an adequate representation of membrane structure. The evidence for and against the existence of subunits, and the relationship of the electron microscopists’unit membrane to the complexitiesof theliving membrane, have been thoroughly discussed in many recent reviews (Chapman and Wallach, 1968; Gel’man et al., 1967; Korn, 1966; Rothfield and Finkelstein, 1968; Salton, 1967; Wallach and Gordon, 1968). As is the case with other cellular membranes, observations on bacteria suggest that the triple-layered unit membrane seen in electron micrographs does not, in fact, correspond to a trilaminar phospholipid bilayer (Grula et ul., 1967) and that the conformation of membrane proteins is different from that predicted by the Danielli model (Lenard and Singer, 1966).

B. TRANSPORT SYSTEMS There is general agreement that the plasma membrane is fundamentally quite impermeable to most metabolites and nutrients of biological interest. Passage of nutrients across the membrane is mediated by specific transport systems; these may be coupled to a source of metabolic energy and can then accumulate their substrate in the cyDo-

ANTIMICROBIAL AGENTS AND MEMBRANE FUNCTION

49

plasm against substantial concentration gradients. The specificity of action and of genetic control which is characteristic of bacterial transport systems (Kepes and Cohen, 1962; Stein, 1967) is clearly a function of specific protein components. Proteins which bind transport substrates with high affinity have been isolated from the cytoplasmic membrane and from the periplasmic space, and their role in membrane transport is under intensive investigation (Pardee, 1968). Much less progress has been made with the mechanism of energy coupling. I n fermentative bacteria, as in red blood cells, ATP and phosphoenolpyruvate which can be derived from glycolysis are almost certainly the ultimate energy donors for “active” transport (Pardee, 1968; Skou, 1965; Stein, 1967). This is not necessarily true for aerobic bacteria. Studies on mitochondria have provided excellent evidence that ion transport can be energized, not only by ATP, but also by energy-rich states or intermediates which are generated by respiration prior t o the formation of ATP (Lehninger et al., 1967). Whether such entities can participate in membrane transport by micro-organismsis not known, nor is it clear how metabolic energy is employed to drive active transport. Students of membrane transport have come to distinguish two kinds of energy-requiring transport systems (Mitchell, 1967a; Stein, 1967): (1) Primary transport systems, in which translocation of the substrate is directly coupled to an enzymic process. The sodium- and potassiumdependent ATPase of mammalian cell membranes which mediates accumulation of K+ and extrusion of Na+ (Skou, 1965)remains the most familiar example. Enzymes performing similar functions appear to exist in bacteria as well (Section VI.A, p. 81). (2) Secondary or gradientcoupled transport makes use of concentration gradients estabIished by the primary transport systems to drive the accumulation of other metabolites. The accumulation of sugars and amino acids by the mammalian intestine, which requires sodium and is indirectly driven by the sodium pump, is a well established case. This concept is less familiar to microbiologists, yet it appears a priori likely that the accumulation of many nutrients in bacteria depends, not directly upon the splitting of ATP, but upon the utilization of gradients (H+, Na+ or K+, perhaps) established by a limited number of primary transport systems.

C. ELECTRON TRANSPORT AND GENERATION OF ATP ATP formation at the substrate level can occur in solution, but thus far at least, ATP generation linked to the respiratory chain appears to be obligatorily associated with membranous structures. Eucaryotic micro-organismshave mitochondria and chloroplasts which are structurally quite analogous to those of higher organisms and need not be

50

FRANKLIN M. HAROLD

discussed here. The situation in the bacteria is much more ambiguous.

It is well established that dehydrogenases and the electron carriers of respiration are found in the membrane fraction after disintegration of the cell (Gel’manet al., 1967; Salton, 1967; Smith, 1968). Some investigators have argued that the respiratory enzymes are localized in the mesosome but the balance of the evidence presently available does not favour this view. It may also be significant that the stalked particles seen in membranes of certain aerobic bacteria (Abram, 1965; Gel’man et al., 1967; Mufioz et al., 1969) and which are probably analogous to the PI particles of the inner mitochondrial membrane, are not confined to the mesosome. Morphological criteria do not permit us to differentiate the respiratory system from the plasma membrane, but chemical separation may be possible. Salton et al. (1968) have recently described the isolation from Micrococcus lysodeikticus of a membrane fraction which is depleted of lipids but still forms a continuous sheet and contains the bulk of the cytochromes and of succinate dehydrogenase. The enzymes and electron carriers of bacterial respiration have been the subject of recent reviews (Gel’man et al., 1967; Smith, 1968) which also summarize what is known concerning the mechanism of oxidative phosphorylation in bacteria. I n the last analysis, the hypotheses proposed for bacteria can be reduced to those now being vigorously debated by students of mitochondria. A quick sketch of this complex subject is necessary here, since so many of the antimicrobial agents to be considered below affect oxidative phosphorylation. 1. Chemical Coupling

I n the traditional view, free energy released at certain sites in the electron-transport chain is trapped in the form of energy-rich intermediates. A sequence of reversible chemical transformations, which includes both non-phosphorylated and phosphorylated intermediates, links the redox reactions of the respiratory chain to the ultimate product, ATP. Some, at least, of these hypothetical intermediates can themselves serve as energy donors for energy-requiring processes such as ion transport. The crux of the matter is the nature of the chemical intermediates, which have thus far eluded isolation and chemical characterization (see reviews by Chance et al., 1967; Pullman and Scliatz, 1967; Slater, 1966). 2. Chemi-Osmotic Coupling

The chemi-osmotichypothesis was deveIoped by Mitchell (see Mitchell, 1966,1967b for recent summaries)in an attempt to provide an alternative

interpretation which would not depend upon hypothetical chemical entities. He proposed that the electron-transport chain is so arranged as to generate H+ and OH- on opposite sides of the mitochondrial inner

ANTIMICROBIAL AGENTS AND MEMBRANE FUNCTION

51

membrane. The membrane is assumed to be relatively impermeable to protons and to ions generally. Consequently a proton-gradient is generated across the membrane, consisting of two components : a difference of pH value and/or a membrane potential. It is this protonmotive force which is called upon to synthesize ATP by reversing the ATPase reaction. The enzyme is assumed to be localized in the membrane in such a way that the active centre is accessible to OH- from one side, to Hf from the other side, and to water as such from neither side. Such a system could synthesize ATP from ADP and Pi by, in effect, withdrawing OH- to the “outside” (acidic and positively charged) and H+ to the “inside’) (alkaline and negatively charged). 3. Conformutional Coupling

It is also possible that the intermediates which intervene between the electron carriers and the first stable chemical product, ATP, are in fact energized conformational states of the mitochondria1 membrane itself. The most persuasive evidence in favour of this concept has come from Green’s laboratory (Green et al., 1968; Harris et ul., 1968). Electron micrographs depict most graphically the effect of substrates and inhibitors upon 6he gross structure of the cristae membranes. As the authors point out, structural transformations elicited by ATP are familiar from the contraction of muscle ;the reverse process should not be implausible. Much effort has been expended in attempts to decide among these alternatives, which are as pertinent to photosynthesis as they are to oxidative phosphorylation (Jagendorf, 1967;Vernon, 1968).Some of the arguments turn upon the effects of various antimicrobial agents and will be considered below, but a general survey of this sophisticated controversy would be out of place here. Significantly, the positions taken by the principals increasingly embody elements derived from the alternative hypotheses; perhaps in the end neither thesis nor antithesis will prevail, but a constructive synthesis.

D. MEMBRANE, WALLAND NUCLEUS: AN INTEGRATED UNIT The Gram stain divides bacteria into two broad classes which differ in many structural and physiological features. One of these is their response to antimicrobial agents. It has long been recognized that Gram-positive organisms are sensitive to anionic detergents and related compounds, to ion-conducting antibiotics, and to a miscellany of antibacterial agents such as actinomycin, whereas Gram-negatives are relatively resistant (for examples see sections 111, p. 53; Iv, p. 63; and V, p. 68). Gram-negative bacteria can be rendered sensitive b y conversion to sphaeroplasts, suggesting that the cell wall prevents access

52

FRANKLIN M. HAROLD

of the drug to the membrane. However, removal of the mucopeptide layer is not required. Treatment of Gram-negative bacteria with EDTA together with an organic cation (tris is most commonly used) suffices to render many Gram-negative bacteria sensitive to drugs which they normally resist (Brown and Richards, 1965 ; Leive, 1965 ; MacGregor and Elliker, 1958; Voss, 1967; Weiser et aZ., 1968). The effect of EDTA on Escherichia coli has been thoroughly studied by Leive (1965, 1968). EDTA and tris caused release of a large fraction of the cell-wall lipopolysaccharide and rendered the cells sensitive to actinomycin ; the cells also became relatively permeable to various substrates which are normally excluded. The morphological effects of EDTA are beautifully depicted in the electron micrographs of Birdsell and Cota-Robles (1967). I n the presence of tris-EDTA, the outer membrane which still surrounds lysozyme sphaeroplasts ruptures and peels back. Large sections of the inner, cytoplasmic membrane are exposed, but coils of the outer layer remain attached to one end. Such lysozyme-EDTA sphaeroplasts are highly sensitive to low concentrations of Brij 59, a non-ionic detergent to which the intact cells are largely resistant (Birdsell and Cota-Robles, 1968). A similar situation obtains in Pseudomonas. EDTA is toxic to some, but not all pseudomonads (Wilkinson, 1967). Cox and Eagon (1968) demonstrated release of lipopolysaccharide with formation of osmotically sensitive “osmoplasts”. These have no obvious abnormalities in permeability properties but lose pre-induced transport systems and are unable to form new ones (Eagon and Asbell, 1966; Asbell and Eagon, 1966). Evidently the lipopolysaccharide layer constitutes a permeability barrier whose disruption exposes the membrane to agents from which it was previously shielded. I n a deeper sense, we must regard wall and membrane as closely integrated components of the cell envelope such that the structure and function of each depends upon the other. Let us recall, for instance, that protoplast formation is accompanied by extrusion of mesosomes and loss of the capacity to divide, apparently because of the altered structural configuration of the envelope. The biosynthesis of cell-wall mucopeptide and lipopolysaccharides are functions of the membrane which will not be surveyed here, but a brief comment is desirable on those antibiotics whose effects are pleiotropic. Bacitracin is a good example. Weinberg (1967), in a lucid summary of the literature, records multiple biochemical effects of this antibiotic including inhibition of protein synthesis and of cell-wall formation as well as leakage of various constituents from the cells. He concluded that the primary target of bacitracin is the cell membrane. Very recently, Siewert and Strominger (1967) found bacitracin to be a specific inhibitor of one step in mucopeptide biosynthesis: it blocks

ANTIMICROBIAL AGENTS AND MEMBRANE FUNCTION

53

dephosphorylation of the lipid-pyrophosphate carrier. Sublethal concentrations of the antibiotic induce morphological changes in both cell wall and membrane of Mycobacterium phlei, including the loss of mesosomes (Rieber et al., 1969). It seems reasonable to conclude that bacitracin binds to the membrane, presumably to a specific site, and induces pleiotropic effects on both cell-wall synthesis and membrane structure, illustrative of their interdependence. Vancomycin is another example of an antibiotic which inhibits cell-wall synthesis by blocking a chemical reaction that involves membrane constituents (Jordan and Reynolds, 1967), and has diverse effects on membrane transport and oxidative phosphorylation. Finally novobiocin, an antibiotic now thought to interfere primarily with DNA polymerase (Smith and Davis, 1967), may bind to a siteon themembrane which could account for its multiple secondary effects on cell-wall synthesis and membrane processes (Brock, 1967).

111. Compounds which Disorganize Lipoprotein Membranes Exposure of bacteria to certain compounds, including organic solvents and detergents, destroys the osmotic barrier. This is readily recognized by release from the cells of small metabolites such as K+, phosphate, amino acids and sugars, and is generally lethal. At the same time, internal enzymes may be rendered accessible to substrates which do not normally pass across the membrane: assay of /3-galactosidase in the presence of toluene is a familiar application. Although it is customary to speak of the “destruction” of the osmotic barrier, the physical integrity of the cytoplasmic membrane is not necessarily impaired. For example, after exposure of E . coli to toluene, there is no gross change in membrane morphology, and enzymes remain sedimentable with the cells (Jackson and DeMoss, 1965). The immediate loss of selective permeability to small molecules reflects, then, not disintegration of the cytoplasmic membrane but a structural disorganization which modifies its permeability to a greater or lesser degree. What is the nature of this disorganization? The structural integrity of a membrane depends upon the orderly arrangement of both proteins and lipids, but its impermeability to small, water-soluble molecules must be attributed primarily to the lipid phase. This serves as a barrier because the hydrocarbon interior largely excludes water. Disorganization of a membrane by solvents or detergents implies a structural change such that this hydrophobic barrier is breached. Re-orientation of lipid molecules in a film or micelle may occur in a variety of ways so as to produce discontinuities and channels in the hydrophobic barrier. Phospholipids in water can exist in a variety of

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FRANKLIN M. HAROLD

phases: a lamellar phase consisting of phospholipid sheets with a hydrocarbon interior and polar head groups facing the water; cylindrical configurations, in which polar groups line the interior which is filled with water while hydrocarbon chains occupy the space between cylinders; globular micelles, and others (Lucy, 1968; Luzzati, 1968; Stein, 1967). Transitions from one phase to another are known to occur in phospholipid-water mixtures, and may be the physical basis of many phenomena described below.

A. ORGANICSOI.VENTS Chloroform and toluene are traditionally employed to keep solutions sterile and to disrupt permeability barriers. The time-course of events following exposure of E . coli to toluene was described by Jackson and DeMoss (1965). As little as 1.5 pl. of toluene per ml caused rapid cell death and loss of selective permeability. The general structure of the cells was unaffected, enzymes remained sedimentable and even the respiratory chain appeared to remain largely intact. Subsequent changes including temperature-dependent loss of protein, disaggregation of ribosomes and breakdown of RNA, may involve autolytic enzymes. Alcohols probably provide the best insight into the interaction of solvents with lipid membranes. n-Butanol and other alcohols disrupt certain lipoprotein membranes with release of water-soluble proteins (seeWallach and Gordon, 1968, for references),but at the concentrations employed in bacterial physiology (0.4 M ) such drastic effects are not evident (Gilby and Few, 1960b).More probably, alcohols disorganize the lipid structure by penetrating into the hydrocarbon region. I n their study of protoplast lysis, Gilby and Pew (1960b) found that equal degrees of lysis were produced by concentrations of alcohols having equal thermodynamic activities. The concentrations of alcohol in the lipid phase appears to be the critical quantity, and lysis may occur at a concentration which produces a surface pressure of about 34 dynes/ om. (Pethica, 1958). However, short-chain alcohols produce quantitatively greater changes in membrane organization than do the higher homologues (Bangham et al., 1965). Lysis of red blood cells and of Bacillus megaterium protoplasts (Kinsky, 1963; Fitz-James, 1968) by low concentrations of vitamin A may be a related phenomenon. Vitamin A penetrates and expands the surface area, of lecithin-cholesterol monolayers ;massive quantities of the vitamin accumulate in the film, apparently due to formation of a complex with lecithin. Penetration of vitamin A can be prevented by raising the surface pressure above 34 dynes/cm. (Bangham et al., 1964).It would be of interest to determine whether vitamin A accumulates in bacterial membranes, and with which component i t interacts.

ANTIMICROBIAL AGENTS AND MEMBRANE FUNCTION

55

B. DETERGENTS The use of detergents as disinfectants reaches back into the 1930s. As a general rule, cationic detergents are bactericidal to both Grampositive and Gram-negative organisms, anionic detergents primarily to Gram-positive, and non-ionic detergents have little effect on either. Although anionic detergents do attack lipopolysaccharide layers, it is the cytoplasmic membrane which is the primary target. Prolonged exposure to detergents leads to breakdown of macromolecules and other autolytic changes. The literature has been reviewed by Newton (1958), Salton (1968) and by Schulman et al. (1955).

CH3(CHz)loCHz--O--SO3N&

Sodium doclwyl sulphate

[

I

I;

C H ~ ( C H ~ ) I ~ C H ~ - N - C H ~Ur-

I

CH3 CH3

Cetyltrimethylammonium bromide

I I

CH2 CHz--N+(CH3)31-

ND 212, an azasteroid

Chlorhoxidine

FIG.1. Chemical structures of detergents.

Anionic detergents, exemplified by sodium dodecylsulphate (Fig. I ) , not only lyse protoplasts but solubilize isolated plasma membranes, suggesting gross disruption of the lipoprotein framework (Gilby and Few, 1960a; Razin and Argaman, 1963). Activity is a function both of the chain length and of the nature of the polar group (Gilby and Few, 1960a; Salton, 1968). Anionic detergents are employed in fractionating membranes into their constituent parts; in some cases, at least, reaggregation occurs when the detergent is removed by dialysis, with formation of sheet-like structures resembling the original membranes (Razin et al., 1965; Grula et al., 1967). However, early hopes that the dissociation yields membrane subunits have been abandoned. The detergents cleave protein from lipid and re-association upon dialysis appears to be quite random (Grula et al., 1967; Razin and Boschwitz, 1968; Rodwell et al., 1967; Salton, 1967).

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FRANKLIN M. HAROLD

Whereas intact bacteria are highly resistant to non-ionic detergents, protoplasts and isolated membranes are solubilized more readily. I n fact, non-ionic detergents such as Nonidet and Brij-59, appear to be reagents of choice for dissociating isolated membranes into fragments which retain enzymic activities (Birdsell and Cota-Robles, 1968; Salton et aZ., 1967). Cationic detergents exemplified by cetyltrimethylammonium bromide (Fig. 1) lyse protoplasts but do not disaggregate isolated membranes. Gilby and Few (1960a)proposed that the positively charged head associates with the phosphate groups of phospholipids, while the non-polar portion of the detergent penetrates into the hydrophobic interior of the membrane. Thus, both the basic head group and the alkyl chain influence the potency. The resulting distortion of the membrane could increase its permeability, exposing the protoplasts to osmotic lysis. The interaction of detergents with purely lipid artificial bilayer membranes has been studied by Seufert (1965). Anionic, non-ionic and cationic detergents all lowered the electrical resistance by several orders of magnitude. I n addition, anionic and non-ionic, but not cationic, detergents produced a resting potential if the membrane separated compartments of differing salt concentrations. The specific increase in permeability to cations was attributed to re-arrangement of the bilayer to produce localized water-filled pores lined with fixed negative charges ; these would preferentially pass cations. One wonders whether sublethal concentrations of detergents induce similar specific permeability changes in biomembranes. A broad range of structures can be loosely classified as cationic detergents. Examples include azasteroids, steroid analogues which containnitrogenin the nucleus (Smith et al., 1964; Varicchio et al., 1967); substituted guanidines (Weinberg, 1968)including the very potent bactericidal agent, chlorhexidine (Fig. 1; Davies et al., 1954; Davies and Field, 1968; Hugo and Longworth, 1964a, b, 1966); and the triphenylmethane dyes which were among the earliest chemotherapeutic agents (Browning, 1964). All share the combination of a positively-charged polar nitrogen group with a hydrophobic region. I n sufficient concentrations they disorganize membranes just as cetyltrimethylammonium bromide does, with release of osmoljtes and penetration of dyes excluded by the intact cells. In many cases, including cetyltrimethylammonium bromide (Salton, 1968; Schulman et al., 1955)it is possible to show quantitative correspondence between killing and the release of solutes, so that disruption of the membrane can safely be taken to be the lethal event. Sometimes, however, viability is decreased by concentrations of drug which cause little leakage (for examples, see Hugo and Longworth, 1964a; Varicchio et al., 1967),suggesting that the lethal event may be a

ANTIMICROBIAL AGENTS AND MEMBRANE FUNCTION

57

more subtle one ;we shall consider chlorhexidine again, in Section VI.A, p. 81. The relationship between chemical structure and activity has been studied in detail for the guanidines (Weinberg, 1968), dyes (Browning, 1964) and azasteroids (Smith et al., 1964; Varicchio et al., 1967) but has as yet given little insight into the reaction between drug and membrane ; this would appear to be a promising field for further inquiry. The response of micro-organismsto detergents is subject to some degree of genetic control. I n addition to the major differences between Grampositive and Gram-negative bacteria (Section II.D, p. 51), individual genes have been found to modify the sensitivity of E. coli to anionic detergents. Nakamura (1968) has mapped a gene which increases resistance to sodium dodecylsulphate and also to phenyl alcohol; conversely, some colicin-tolerant mutants are particularly sensitive to detergents (de Zwaig and Luria, 1967). Genetic alterations in membrane proteins have been postulated but not demonstrated, and it may be well to keep the lipopolysaccharide layer in mind (Section II.D, p. 51).

C. REVERSIBLE MEMBRANE DISORGANIZATION? The preceding sections have stressed the lethal effects of detergents and solvents, but there is every reason to expect, and to seek, reagents which distort membrane structure reversibly. Narcotics and local anaesthetics are thought to act in this manner upon mammalian cell membranes (Cuthbert, 1967). Sublethal concentrations of organic solvents may have reversible effects on membrane permeability. Phenethyl alcohol, structurally related to toluene, reversibly increases membrane permeability of E. coli (Silver and Wendt, 1967) and of Neurospora crassa (Lester, 1965); the membrane may in fact be the primary target of this reagent which has received much study as an inhibitor of macromolecule synthesis. The most promising of the compounds which increase membrane permeability reversibly appear to be the steroid diamines. Irehdiamine (Fig. 2) and the related malouetine are plant alkaloids which first attracted the attention of molecular biologists as inhibitors of bacteriophage growth in E. coli. Silver and Levine (1968a, b) subsequently found that concentrations around M induce rapid efflux of thiomethylgalactoside and K+ and also inhibit their uptake. The effects of irehdiamine on transport could be reversed by removal of the drug or by Mg2+,but loss of viability was apparently irreversible. These studies are incomplete and questions persist regarding the effects of the steroids on energy metabolism and on specific transport systems, but they are a valuable lead.

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FRANKLIN M. HAROLD

\/NHz

FIU.2. Chemical structure of irehdiamine, a steroid diamine.

Levallorphan, an analogue of morphine which inhibits RNA synthesis in E . coli (Simon and Van Praag, 1964)and induces breakdown of ATP (Greene and Magasanik, 1967),is also a possible candidate for reversible effects on membrane structure (E. J. Simon, personal communication).

D. PEPTIDE ANTIBIOTICS 1. Tyrocidines The observation that Bacillus species in mixed culture antagonize the growth of other Gram-positive bacteria goes back to the very dawn of bacteriology. The reason was found in 1940 when Dubos and Hotchkiss isolated tyrothricin (Hotchkiss, 1944) and thereby opened the antibiotic era. Tyrothricin proved to be composite, including members of the gramicidin (Section V.C, p. 74) and tyrocidine families. Hunter and Schwartz (1967b) have prepared a comprehensive review on tyrocidines. Tyrocidines kill sensitive organisms by disruption of the osmotic barrier ; small metabolites are released, but the cytoplasmic membrane is not solubilized. More prolonged exposure brings about breakdown of ribosomes and of nucleic acids (Mach and Slayman, 1966). Tyrocidines also lyse protoplasts, indicating direct action on the membrane. Unlike the related gramicidins, tyrocidines increase membrane permeability generally and are not specific cation conductors (Pressman, 1965; Graven et aH., 1966b). The chemical structure of the tyrocidines (Fig. 3) was clarified by Craig and his associates (for early references see Hunter and Schwartz, 1967b; Ruttenberg et al., 1965, 1966, Ruttenberg and Mach, 1966). They form a family of related cyclic peptides; the left half is invariant, but substitutions occur in the right half. A special case is the misnamed gramicidin-S, which resembles the tyrocidines in biological activity ; its structure is that of a dimer of the invariant half. All the biologically active tyrocidines bear a net positive charge. The evident similarity between the effects of tyrocidines and cetyltrimethylammonium bromide led Hotchkiss to conclude (1944) that “Tyrocidine is a bacterial detergent, unique only in its origin and complex chemical structure”. The relationship between activity and chemical

ANTIMICROBIAL AGENTS AND MEMBRANE FUNCTION

L-Leu

59

(~)L-DAB

--f

I

P

D-Phe

(~)L-DAB

t L-Pro -+L-Phe

7 u-Phc

\u-l’hc

L-Thr

(~)L-DAB

5

MOA Tyrocirline A

Polymyxin B1

WIG. 3. Chemical structures of some polypeptide antibiotics. Asp, indicates an aspartate residue ;DAB, a diaminoisobutyrate residue ;Glu, a glutamate residue ; Lue, a leucine residue; MOA, a 6-methyloctanoate residue; O m , an ornithine residue; Pho, a phenylalanine residue; Pro, a proline residue; Thr, a threonine residue; Tyr, a tyrosine residue; and Val, a valine residue. --f indicates a C-N linkage.

structure is far from clear. The tyrocidines are strongly surface-active, and tend to form aggregates which resemble lipid micelles. Aggregation may be related to antibiotic activity; an open-chain analogue of tyrocidine having the same amino-acid sequence does not aggregate and also is not an antibiotic (Ruttenberg et al., 1966). However, gramicidin-S, which also does not form aggregates, is very similar to the tyrocidines in its biological effects. Katchalsky and his associates (1964) have described synthetic peptides related to gramicidin-S. The corresponding linear decapeptide had about one tenth of the activity of the cyclic molecule, as had a random copolymer of the constituent amino acids in the proper ratios and steric configurations. Curiously, a copolymer of D-ornithine and L-leucine was more active against E. coli than gramicidin-S itself. It appears that basic residues are essential to antimicrobial activity ; hydrophobic residues serve t o anchor the molecule to the membrane, but the role of the peculiar mixture of D and L amino acids and of the overall conformation of the molecule remains to be clarified.

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FRANKLIN M. HAROLD

2. Polymyxins Bacillus polymyza and related strains elaborate a class of structurally I

allied antibiotics consisting of heptapeptide rings with a long side-chain terminating in methyloctanoic or in iso-octanoic acids (Fig. 3). Their activities and applications have been thoroughly reviewed by Sebek (1967). It is noteworthy that the polymyxins are in general more active against Gram-negative bacteria than against the Gram-positives. An impeccable series of experiments led Newton (see Newton, 1956, for a summary) to conclude that polymyxin B binds to the cytoplasmic membrane and breaches the osmotic barrier. Subsequent effects include inhibition of respiration, cytological changes especially in the nuclear region (Wahn et al., 1968; also Sebek, 1967) and release of ribosomal RNA (Nakajima and Kawamata, 1966). Massive quantities of the antibiotic are adsorbed by the cells, mostly to internal receptors made available by breakdown of the permeability barrier. Wahn et al. (1968) reported that E. coli cells treated with polymyxin display numerous blebs or projections all over their surface. It would now appear that these are not, as one might think, due to cytoplasm escaping through breaks in the membrane, but result from interaction of polymyxin with the cell wall (Koike et al., 1969). Morphological effects of polymyxin on the membrane were not visible. It is not clear why polymyxins act preferentially on Gram-negative bacteria. Association with the lipopolysaccharide layer may be a factor, but is evidently not obligatory as polymyxins do bind strongly to the plasma membrane itself (Newton, 1956; Koike et al., 1969). Little is known concerning the significance of either the specific amino-acid constituents or of the lipid side-chain. At the molecular level, the mode of action of polymyxins is probably to be sought in a fairly generalized reaction with phospholipids. Polymyxin B preferentially penetrates monolayers of phosphatidylethanolamine ; the increase in surface area or pressure may induce re-orientation of membrane lipids and breakdown of the permeability barrier (Few, 1955; Schulman et al.,1955; Newton, 1956).

E. BASICPOLYPEPTIDES AND PROTEINS '

Many basic polypeptides, including the histones and protamines, exhibit antimicrobial activities. The synthetic polylysines have received the most study (Katchalsky et al., 1964); they are bacteriostatic for E. coli at low concentrations but higher levels are bactericidal, inhibit respiration and induce leakage of amino acids. It is presumed that they disorient the cytoplasmic membrane as the cationic detergents do. However, polylysines agglutinate bacteria and alter their electrophoretic

ANTIMICROBIAL AGENTS AND MEMBRANE FUNCTION

61

mobility, and one wonders whether this surface-binding might not secondarily distort the membrane and render it leaky. Polylysine does bind to the exposed cytoplasmic membranes of protoplasts, enhancing their resistance to osmotic shock (Harold, 1964). In view of the relatively specific effects of polylysine on membrane phenomena in mitochondria (Johnson et al., 1967) and chloroplasts (Dilley, 19GS), its effects on protoplast permeability should be explored. In yeast, a variety of basic polypeptides and proteins induce gross membrane leakiness (Yphantis et al., 1967). The bactericidal action of serum proteins and of complement may also involve destruction of the permeability barrier, but this subject is outside the scope of this article.

F. POLYENE ANTIBIOTICS AND MEMBRANE STEROLS Thus far we have discussed compounds which penetrate into phospholipid membranes but apparently do not bind to any one receptor molecule. In the case of the polyene antibiotics there is very strong evidence that their effects on membranes result from specific association with sterols. Consequently polyenes have no antibacterial activity and, strictly speaking, come within the scope of this review only by grace of certain

OH

OH

OH

OH

OH

FIG.4. Chemical structure of filipin, a polyene antibiotic.

1Mycoplasmn species which incorporate sterols into their membrane and are thereby rendered sensitive to polyenes (Weber and Kinsky, 1965). As the literature through 1966 has been thoroughly surveyed in several recent reviews (Kinsky et uE., 1966; Kinsky, 1967; Lampen, 1966), only the main conclusions need be summarized here. The polyene antibiotics are a large and diverse class of compounds, which share certain structural features, namely a system of conjugated double bonds, and the general geometry of a ring. Few of their structures have been established; the tentative structure of one that is widely used, filipin, is shown in Fig, 4,

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but even this is called into question by the recent discovery (Bergy and Eble, 1968) that filipin is a complex of at least four related compounds. Polyenes are known to differ with respect to the size of the ring, the number of carboxyl groups and double bonds and the presence (and structure) of a carbohydrate moiety. Nevertheless, all appear to act upon the cytoplasmic membrane : the membrane is not destroyed, but is rendered leaky to small metabolites. Inhibition of growth, glycolysis and other processes follow secondarily. Polyenes cause graduated degrees of damage to yeast membranes, as measured by leakage. The most limited damage is done by succinyl perimycin, a synthetic derivative of the polyene antibiotic perimycin. This induces only the loss of K+ from yeast and all its effects, including the inhibition of growth, are reversed by high concentrations of K+ (Borowski and Cybulska, 1967). Other polyenes produce more generalized leakage. Nystatin induced loss of K+ and, more slowly, of cellular constituents such as phosphate, but inhibition of glycolysis could be annulled by K+ and NHL. Nystatin did not cause leakage of sorbose, except at high concentrations. Finally, filipin induced rapid leakage of most small metabolites such as K+, phosphate, sorbose and amino acids; inhibition of glycolysis could not be reversed even by a mixture of ions and cofactors, and several intracellular enzymes were exposed (ATPase, pyruvate decarboxylase) but even here the membrane was not physically disrupted. There is a general correlation between the size of the ring and the degree of membrane damage, the smaller rings having the more profound effects. However, by the criterion of fungicidal activity, the heptaene antibiotics are the most potent even though they have large rings and induce the least leakage The evidence for the role of sterols as specific receptors for polyene binding has been summarized (Kinsky et al., 1966; Kinsky, 1967; Lampen, 1966) and it seems superfluous to re-iterate it here. Suffice it to mention that at least one polyene antibiotic, filipin, does react with artificial phospholipid membranes which do not contain sterols (Sessa and Weissmann, 1967; Weissmann and Sessa, 1967). This, however, is seen only at very high concentrations of the antibiotic, some three orders of magnitude above the growth-inhibitory level, and reflects the nonspecific effects of a minor component of the filipin complex (Sessa and Weissmann, 1968).There is no doubt that sterols are the specificreceptors M . Sterols at physiological concentrations of the antibiotics, near 1 are clearly a necessary condition for polyene sensitivity, but apparently not a sufficient one. To what extent a membrane is perturbed by polyene antibiotics depends on the proportion of sterols to phospholipids (Kinsky et al., 1966; Demel et al., 1968; Kinsky et al., 1968; Sessa and Weissmann, 1968; Weissmann and Sessa, 1967).

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Insight into the molecular basis of the interaction between polyenes and membrane sterols has come particularly from the work of Kinsky, Van Deenen and their associates on monolayers and phospholipid bilayer membranes. The polyenes penetrate lipid monolayers which contain cholesterol and increase the surface pressure. The magnitude of this effect on monolayers correlates well with the degree to which various polyenes disrupt fungal membranes. The increase in surface pressure is large, quite out of proportion to the number of antibiotic molecules which penetrate into the monolayer, and hence it is argued that the polyenes induce a re-orientation of the sterol molecules, a molecular domino effect as it were (Kinsky et al., 1966; Demel et al., 1968).Visual evidence for this phenomenon was obtained by electron microscopy. Artificial membranes exposed to filipin show numerous “pits”, 125 A in diameter, which may be deposits of the polyene-cholesterol complex. Such pits were also seen in membranes of erythrocytes lysed by filipin (Kinsky et al., 1966, 1967a, b). Studies with lipid bilayer membranes are beginning to shed some light on the nature of the permeability changes induced by the polyenes. Whereas filipin ultimately disrupts the bilayers, nystatin and amphotericin B lower electrical resistance and increase membrane permeability, particularly to anions. It is unlikely that the antibiotics serve as lipid-soluble anion carriers ; more probably they induce the formation of pores or channels, a reaction which must involve the sterols (Finkelstein and Cass, 1968; Andreoli and Monahan, 1968). Whether the pits seen in electron micrographs correspond to these pores or channels, remains to be determined.

IV. Proton Conduction and Uncoupling of Oxidative Phosphorylation The concept that ions traverse membranes in association with lipidsoluble carriers is traditional in membrane physiology, but only recently has it been recognized that certain pharmacological agents exert their effects by serving as artificial ion carriers. To my knowledge, this possibility was first envisaged by Mitchell (1961a) in his proposal bhat uncouplers of oxidative phosphorylation render the mitochondria1 membrane permeable to protons. The general significance of this insight became apparent following the discovery by Pressman (Moore and Pressman, 1964; Pressman, 1965)that valinomycin and other antibiotics promote uptake of Kf by mitochondria, and has since been vigorously explored by many investigators. It would be difficult to exaggerate the importance of these discoveries to membrane physiology ; the realization that ion transport and energy generation are inextricably linked is but

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one of the benefits. The present section will consider proton-conducting agents, reserving the alkali-metal ionophores for Section V, p. 68. Historically, the concept of proton conduction is rooted in the discovery that certain compounds (of which 2,4-dinitrophenol is the most familiar) uncouple oxidative phosphorylation from respiration, and in fact arose out of efforts to explain the mechanism of this uncoupling. Dozens of uncouplers are now known, many of them excellent inhibitors of growth and uncouplers of oxidative phosphorylation in bacteria. Among these we should mention the salicylanilides which have been used

-

clo" OH

c1

Tetrachlorosalicylanilide

/Cl c1 Pentachloropheiiol

N=C--C--C=N I

NH I

Carbonylcyanide Tetrachlorotrifluoromethylbenzimidazole tnn-chlorophenylhydrazone .

FIG.5. Chemical structures of uncouplers of oxidative phosphorylation : Tetrachlorosalicylanilide (TCS); carbonylcyanide rn-chlorophenylhydrazone(CCCP) ; pentachlorophenol (PCP) and tetrachlorotrifluoromethylbenzimidazole(TTFB).

for many years as disinfectants in medicine and industry (Hodes and Stecker, 1968; Hamilton, 1969; Woodroffe and Wilkinson, 1966a, b). Halogenated salicylanilides are potent uncouplers of oxidative phosphorylation in mitochondria (Whitehouse, 1964; Williamson and Metcalf, 1967) and in bacteria (Hamilton, 1968). Other compounds which uncouple oxidative phosphorylation in both mitochondria and micro-organisms include penhiwhlorophenol, derivatives of carbonylcyanide phenylhydrazone containing chlorine (CCCP) or fluorine (FCCP), and tetrachlorotrifluoromethylbenzimidazole (Asano and Brodie, 1965; Beechey, 1.966; Bragg and Hou, 1968; Cavari et al., 1967; Heytler, 1963; Heytler and Prichard, 1962; Weinbach, 1957). Structures of some of these compounds are shown in Fig. 5. Uncouplers are routinely used in the analysis of energy-dependent processes in micro-organisms such as active transport and macromolecule synthesis. At the same time, the mechanism of uncoupling

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continues to be a touchstone for any hypothesis designed t o explain the mechanism of oxidative phosphorylation. Although our primary interest here centres on the effect of uncouplers on bacterial energy metabolism, the available evidence is almost entirely derived from mitochondria ; we shall assume in what follows that the same principles apply to both. In the traditional view, coupling between electron transport and phosphorylation involves chemical intermediates common to the two pathways. 2,4-Dinitrophenol and related compounds may bring about hydrolysis of such intermediates and thereby dissociate respiration from phosphorylation (Slater, 1966). I n the chemi-osmotic hypothesis (Mitchell, 1966, 1967b) such intermediates do not exist and ATP synthesis depends upon a gradient of p H value and of electrical potential across the membrane (Section II.C, p. 49). It is an essential postulate of the hypothesis that the mitochondrial membrane is relatively impermeable to protons and, indeed, to ions generally. Mitchell (1961a) pointed out that 2,4-dinitrophenol and many other uncouplers are lipid-soluble acids which could facilitate passage of protons across the membrane and thus collapse the proton gradient. Mitchell and Moyle also produced the first evidence that a number of familiar uncouplers including 2,4-dinitrophenol, CCCP and FCCP specifically catalyse passage of protons across the membranes of mitochondria and of bacteria (Mitchell, 1961b, 1966; Mitchell and Moyle, 1967a). The basis for their conclusion deserves a brief examination as it involves principles which we shall encounter again in subsequent sections. Upon addition of a pulse of hydrochloric acid t o an unbuffered suspension of mitochondria the p H value falls abruptly, then rises slowly as H+ passes into the interior of the organelle. The rate of titration of the inner compartment is limited by the permeability of the mitochondrial membrane, and can therefore be greatly accelerated by disrupting the membrane with detergents. Compounds such as 2,4-dinitrophenol and FCCP likewise accelerate titration of the inner compartment but do not disrupt the membrane and are considered to catalyse diffusion of protons (or OH-). Quantitative measurement of proton permeability in the presence and absence of a putative proton conductor requires a more sophisticated approach (Mitchell and Moyle, 1967a). Movement of protons into the mitochondria introduces positive electrical charges ;unless compensatory ion movements take place, a membrane potential builds up (inside positive) which inhibits further proton movements. Valinomycin, a highly selective I<+ conductor (Section V.A, p. &?), permits K+ to flow out and thereby relieves the electrical restrictions on proton movements. By this procedure it was determined that FCCP increased the proton conductance of the mitochondrial membrane by a factor of 160, sufficient 3

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t o account for uncoupling in terms of the chemi-osmotic hypothesis (Mitchell and Moyle, 1967a, b ; see also Mitchell, 1966, 1967b for discussions of uncoupling by proton conductors). I n photosynthesis, also, there is evidence for a close relationship between proton gradients and phosphorylation. Chloroplasts and chromatophores take up protonsinthelight ;the resultingproton gradient is dissipated by many uncouplers of photophosphorylation (Mitchell, 1966, 1967b; Jagendorf, 1967; Kaplan and Jagendorf, 1968; Schwartz, 1968).Finally, uncouplers of oxidative phosphorylation facilitate proton movements across membranes of cells which do not carry out oxidative phosphorylation, including red-blood cells (Harris and Pressman, 1967), Streptococcus fueculis (Harold and Baarda, 1968b) and anaerobic E . coli (Pavlasov&and Harold, 1969). I n all cases it appeared that only proton translocation was accelerated, not that of other ions. Perhaps the most compelling evidence comes from studies on phospholipid bilayer membranes. It has been established that a number of familiar uncouplers, including 2,4-dinitrophenol, CCCP and tetrachlorotrifluoromethylbenzimidazole, greatly increase the electrical conductance of such membranes to protons, and protons only (Bielawski et ul., 1966; Hopfer et ul., 1968; Liberman and Topaly, 1968). I n principle, proton conductors could act either as lipid-soluble carriers or else pass protons along the 7r-orbitals of molecules which themselves remain relatively fixed in the membrane. Liberman and Topaly (1968) concluded from their observations that a carrier mechanism was involved. Despite the evidence summarized above, by no means all investigators accept the thesis that uncoupling of oxidative phosphorylation is due to proton conduction. Chance et ul. (1967) have questioned the entire concept of a proton-impermeable mitochondria1 membrane. Weinbach and Garbus (1968a, b) have described experiments which suggest that uncouplers induce conformational changes in membrane proteins and they hold these responsible for uncoupling. On balance, it seems t o me that the reality of proton conduction has been established beyond reasonable doubt, but it should be stressed that this does not, ips0 fucto, validate the chemi-osmotic hypothesis. It is conceivable, for example, that proton conductors facilitate access of protons to labile, energy-rich intermediates of oxidative phosphorylation (Hopfer et ul., 1968). Alternatively, uncouplers may facilitate passive entry of protons which the organelle must then extrude by a proton pump driven by high-energy intermediates. A proton cycle would thus be set up, the net effect of which is the dissipation of metabolic energy and uncoupling of ATP synthesis (Harris et ul., 1967a).Whatever proves to be the correct explanation it is still true (with some limitations, Section V.E, p. 78) that “facilitating proton diffusion through the membranes has

ANTIMICROBIAL AGENTS AND MEMBRANE FUNCTION

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now become a sufficient reason for a chemical being an uncoupler, whether one accepts the chemi-osmotic hypothesis or not” (Jagendorf, 1967). A complication should be mentioned which applies particularIy to the effect of uncouplers on bacteria. I n mitochondria, low concentrations of uncouplers prevent phosphorylation but respiration itself is not inhibited and may even be stimulated. I n bacteria, compounds such as 2,4-dinitrophenol, pentachlorophenol and CCCP tend to inhibit respiration as well as phosphorylation, especially with certain substrates (see, for example, Asano and Brodie, 1965; Cavari et al., 1967; Smith, 1968). This effect, which can also be observed in mitochondria exposed to relatively high concentrations of uncouplers, is the basis of a hypothesis put forward by van Dam and Slater (1967).They suggest that uncoupling, as well as inhibition of respiration, are ultimately due to dissipation of energy by transport of the uncoupler itself. However, the inhibition of respiration may yet find an explanation as a secondary consequence of proton movements (see below). By whatever mechanism, proton conductors do uncouple oxidative phosphorylation and interfere with. production of ATP. It is therefore not surprising that uncouplers inhibit energy-dependent processes such as motility and active transport (for examples see Faust and Doetsch, 1969; Hamilton, 1968; Kepes and Cohen, 1962; Pardee, 1968; Stein, 1967; Winkler and Wilson, 1966) and reasonable to attribute the inhibition simply to lack of ATP. There is, however, a discrepancy. It has been known for some time that uncouplers inhibit phosphate uptake in yeast even under anaerobic conditions (Windisch and Heumann, 1960; Riemersma, 1968).Anaerobic growth of E.coli is sensitive to uncouplers (Kovai: and Kuiela) and other examples of this kind range from microorganisms t o the toad bladder (Galeotti et al., 1968; Klahr et al., 1968). Some insight into the basis of this unexpected phenomenon came from studies with a strain of Streptococcus faecalis which lacks cytochromes and appears to produce ATP exclusively via glycolysis. A number of uncouplers including tetrachlorosalicylanilide (TCS), CCCP, pentachlorophenol and also a novel antibacterial agent, tetramethyldipicrylamine (Meyer et al., 1967) inhibited energy-dependent transport of K+, phosphate and of certain amino acids. The uncouplers did not inhibit glycolysis, ATP synthesis, ATP turnover or even the utilization of ATP for the synthesis of macromolecules. It was therefore concluded (Harold and Baarda, 1968b) that the uncouplers specifically prevent the utilization of metabolic energy for active transport. I n a subsequent study (PavlasovB and Harold, 1969) this conclusion was extended to the accumulation of ,B-galactosides by E . coli under anaerobic conditions : TCS and CCCP had no effect on the ATP pool, nor did they inhibit the

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transport system as such, but they prevented accumulation of thiomethyl galactoside against a concentration gradient. A substantial body of evidence, (Harold and Baarda, 1968b, Pavlasovb and Harold, 1969 and unpublished results) warrants the conclusion that inhibition of active transport is a consequence of the fact that uncouplers facilitate passage of protons across the membrane. These experiments call into question the tacit assumption that uncouplers inhibit energy-dependent processes secondarily by interfering with ATP synthesis. Even under aerobic conditions, uncoupling of oxidative phosphorylation and inhibition of active transport may be consequences of a single primary effect, proton conduction. We may even legitimately ask whether inhibition of bacterial motility by uncouplers (Paust and Doetsch, 1969) is due to lack of ATP or hints a t more subtle phenomena. We do not know why proton conductors interfere with active transports even when glycolytic ATP is the ultimate energy donor. It is possible that a gradient of p H value across the cytoplasmic membrane is part of the machinery by which metabolic energy is coupled to membrane transport : many active transports may, in fact, be secondary translocations driven by a primary proton pump which maintains the p H gradient (Mitchell, 1963, 1966, 1967a; Chappell and Crofts, 1965, Harold and Baarda, 1968b; Pavlasovb and Harold, 1969). Other explanations are by no means excluded, and can be formulated in terms quite analogous to those employed above to explain the uncoupling of oxidative phosphorylation (Harold and Baarda, 196Sb). Be this as i t may, proton-conducting uncouplers raise insistent questions not only concerning the mechanism of oxidative phosphorylation but for membrane processes in general.

V. Alkali Metal Ionophores I n 1944 Hotchkiss reviewed what was known a t the time concerning the mode of action of the antibiotics tyrocidine and gramicidin. He concluded that tyrocidine disrupts the cytoplasmic membrane of sensitive cells and subsequent work has borne him out (Section III.D, p. 58). Gramicidin appeared to act in a different manner. It did not induce leakage of cell constituents but affected respiration and phosphate uptake much as did the recently discovered 2,4-dinitrophenol. Hotchkiss recognized that alkali-metal ions were involved in the effects of gramicidin, but the time was not yet ripe for further insight. Interest in gramicidin then languished, except for the demonstration that it does indeed uncouplq; oxidative phosphorylation in mitochondria and bacteria, and also photophosphorylation (for references see Hunter and Rchwartz, 1967c).

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A decade later, interest in polypeptide uncouplers revived as a result of Lardy's search for antibiotics which affect oxidative phosphorylation. One antibiotic, valinomycin, appeared to be the most potent uncoupler yet discovered (McMurray and Begg, 1959).Attempts to explain certain peculiar features of uncoupling by valinomycin led to the discovery (Moore and Pressman, 1964) that uncoupling required the presence of K+ and was related to the induction of K+ uptake by mitochondria. Within a year, gramicidin as well as a number of other antibiotics of very different structure had been found to induce cation uptake (Chappel1 and Crofts, 1965; Pressman, 1965). An amusing account of the discovery of cation conduction has been given by Pressman (1969). Studies in many laboratories have since revealed that the primary effect of these antibiotics is to facilitate passage of alkali-metal ions across lipid membranes. The antibiotics differ strikingly in specificity, ranging from valinomycin which is highly selective for Kf to the promiscuous gramicidins. These antibiotics are the first compounds to exhibit the solubility and specificity characteristics that ion carriers in natural membranes would be expected to possess (Pressman et al., 1967; Pressman, 1968),and they are thus both models and guides to future research on ion transport.

A. VALINOMYCIN Valinomycin, an antibiotic produced by certain strains of Xtreptomyces, inhibits the growth of a variety of fungi and Gram-positive bacteria.

Valinomycin

Enniatin

FIG.6. Chemical structures of certain depsipeptide antibiotics. Hydroxyisoval, indicates a hydroxyisovaleric acid residue ; N-methylval, an N-methylvaline residue; and val, a valine residue.

Its structure, determined by Shemyakin and his associates and confirmed by synthesis (Shemyakin et al., 1965; Shemyakin, 1965) is shown in Fig. 6. It is a cyclodepsipeptide consisting of three repetitions of the

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FRANKLIN M. HAROLD

sequence (D-valine-L-lactic acid-L-valine-D-hydroxyisovaleric acid). Like all of the antibiotics to be considered in this section, valinomycin is soluble in ethanol but virtually insoluble in water. Studies on its structure and mode of action through 1965were reviewed by Hunter and Schwartz (1967a). More current surveys of this rapidly moving subject will be found in Pressman’s articles (1968, 1969).We shall begin here with the effects of valinomycin on artificial lipid membranes and then consider interaction of the antibiotic with biological systems of increasing complexity. Valinomycin greatly increased the K+ permeability of both phospholipid spherules (Chappell and Crofts, 1966 ; Chappell and Haarhoff, 1967) and of black bilayer membranes. The latter system proved most informative. Valinomycin, in presence of K+,increased the electrical conductivity of the membranes by five orders of magnitude and also elicited an electrical potential across membranes separating compartments containing different concentrations of K+. Both phenomena are characteristic of the passage of charged particles across the electrical barrier, presumably a complex of the neutral valinomycin molecule with K+. Valinomycin did not induce anion permeability and discriminated sharply among the various cations ; K+was preferred t o Na+ by a factor of a t least 400. When present at equal concentrations, the sequence of ionic conductivities is : H+ > Rbf > Kf > Cs+ > Na+ > Li+. However, a t physiological p H values the concentration of H+ is very low and the antibiotic then acts as a highly selective carrier for K+ (Andreoli et al., 1967 ;Lev and Buzhinsky, 1967; Liberman and Topaly, 1968; Mueller and Rudin, 1967). The selectivity of valinomycin is independent of the composition of the lipid membrane and must thus reside in the antibiotic molecule itself (Mueller and Rudin, 1967; Pressman, 1968). A priori, several mechanisms were envisaged by which valinomycin could facilitate K+ movements. Valinomycin is a ring-shaped molecule ; several investigators suggested that cations are inserted into the hole and considered various factors, including the diameters of the ring and of the ion, which could determine the stability of the complex. The ring could act as a carrier or else a stack of valinomycin molecules could make up a K+-selective pore (Chappell and Crofts, 1965; Mueller and Rudin, 1967; Andreoli et al., 1967). Recent evidence favours the concept that valinomycin acts as a lipidsoluble carrier which shuttles K+ across membranes in the form of a valinomycin-K+ complex (Pressman et al., 1967; Pressman, 1968; Stein, 1968; Liberman and Topaly, 1968; Tosteson, 1968). The most compelling argument is the finding that valinomycin facilitates K+ diffusion across a bulk phase of lipid or organic solvent. The concentration dependence indicates that each valinomycin molecule carries a single K+ ion; Na’ fails to form a lipid-soluble complex with valinomycin,

ANTIMICROBIAL AGENTS AND MEMBRANE FUNCTION

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but Na+ and other cations do displace K+ from the antibiotic (Pressman et al., 1967; Pressman, 1968; Tosteson, 1968; Tosteson et ul., 1968). The three-dimensional structure of the valinomycin-K+ complex has just been established by use of optical rotatory dispersion, nuclear magnetic resonance and other spectroscopic techniques (Ivanov et ul., 1969; Haynes et ul., 1969). It is a kind of clathrate, with the K+ enfolded 1 within a cage that is held together by induced dipoles between K+ and ?' the oxygen atoms of valinomycin. The non-polar side chains are directed outwards to form a hydrophobic surface, resulting in a lipid-soluble molecule which can equilibrate with K+ a t the lipid-water interface. Shemyakin and his associates (1965) have synthesized numerous analogues and derivatives of valinomycin in an effort to establish the structural features required for antibiotic activity. Linear analogues were inactive, as were rings containing more or fewer repeating units. The hydroxyisovaleric acid moieties were essential, but considerable latitude was allowable in the hydrophobic amino acids. There was excellent correlation between antibiotic activity, the induction of K+ transport in mitochondria (Shemyakin et al., 1965; Pressman, 1965) and K+ conduction across phospholipid membranes (Mueller and Rudin, 1967), indicating that the antibiotic activity of valinomycin may well be a consequence of K+ translocations. With this background let us now turn t o a consideration of the biological effects of valinomycin. The interpretation is simplest for Streptococcus fueculis which generates ATP by glycolysis and lacks oxidative phosphorylation. Valinomycin was bacteriostatic for Xtrep. fuecalis and inhibition of growth could be reversed by raising the K+ content of the medium. The antibiotic specifically induced loss of K+ from the cells, by stoichiometric exchange for other cations present in the medium, especially Na+. I n agreement with findings made in other systems, valinomycin greatly incrcased membrane permeability to K+ but not to other ions or metabolites. It did not interfere in any way with the generation of ATP by glycolysis or with its utilization (Harold and Baarda, 1967). Cessation of growth is due to inhibition of protein synthesis, which requires K+ (Lubin, 1964; Harold and Baarda, 1967; 1968a). The effects of valinomycin on erythrocytes can also be understood in terms of facilitated K+ diffusion (Harris and Pressman, 1967; Tosteson et ul., 1967; Tosteson et ul., 1968). Valinomycin inhibits the growth of various aerobic bacteria and fungi (Hunter and Schwartz, 1967a; Shemyakin et ul., 1965), but I am aware of only a single paper describing its physiological effects. Pressman (1967) demonstrated that, in both Azotobucter and Mycobucterium phlei, valinomycin induced K+ uptake and stimulated respiration, just as in mitochondria. It was suggested that inhibition of growth is due to dissipation

'

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FRANKLIN M. HAROLD

of metabolic energy resulting from the induced K+ uptake, an interpretation derived from the extensive work of Pressman and his associates with mitochondria. This topic is both complex and controversial but a summary is essential since any future research on the effects of valinomycin on aerobic bacteria must take the mitochondrial system as its point of departure. There seems to be fairly general agreement on the basic facts. Valinomycin, at nanomolar concentrations, initiates net K+ uptake by mitochondria. This is an energy-requiring process which can be supported either by oxidation of a substrate or by exogenous ATP. UncoupIers of oxidative phosphorylation, including the proton conductor CCCP, reverse the direction of K+ movement, such that K+ runs out into the medium. Valinomycin also increases theflux of 42Kacross the membrane in both directions, i.e. it increases permeability of the mitochondrial membrane to KS (Chappell and Crofts, 1965, 1966; Harris et al., 1966, 1967a, 1967b; Hofer and Pressman, 1966; Pressman, 1968; Mitchell, 1967b; Mitchell and Moyle, 1967a, b). The net uptake of K+ is accompanied by ejection of H+ and/or the uptake of anions, and sometimes by swelling ; the magnitudes of these processes depend upon conditions (Chappell and Crofts, 1965, 1966; Pressman, 1965; Harris et al., 1966). Although originally described as an uncoupler of oxidative phosphorylation, this is clearly not the primary effect of valinomycin, since conditions can be found under which phosphorylation keeps pace with the increased respiratory rate (Hofer and Pressman, 1966; Harris et al., 1967a).Pressman, Harris and their associates (Cockrell et aZ., 1966; Harris et al., 1966; Pressman, 1968)have determined that as many as six K+ ions can be translocated per ATP molecule hydrolysed. Indeed, it has been possible to couple Kf efflux induced by valinomycin to the synthesis of ATP (Cockrell et aZ., 1967). Two fundamentally different hypotheses have been offered to rationalize the experimental results. These are shown schematically in Fig. 7, and are obviously related to the chemical coupling (a)and chemi-osmotic (b) hypotheses of oxidative phosphorylation. (a) Pressman and his collaborators (Harris and Pressman, 1969; Harris et aZ., 1967b; Pressman, 1968, 1969; Pressman et al., 1967)believe that the mitochondrial membrane contains an electrogenic cation pump. This pump tends to drive K+ into the organelle againsti its concentration gradient, at the expense of metabolic energy derived either from ATP or from the hypothetical energy-rich intermediate of oxidative phosphorylation. Now, mitochondria do not normalIy accumulate K+. Pressman has suggested that the physiological role of the pump is the transport of anions and that, in untreated mitochondria, K+ has no access to the pump. The effect of valinomycin is to breach the permeability barrier;

ANTIMICROBIAL AGENTS AND MEMBRANE FUNCTION

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this delivers K" to the pump which now carries out net K+ accumulation. Compensatory movements of protons, anions or both maintain electroneutrality. A cyclic flux of K+ is thus set up by valinomycin-in through the pump, out by several possible pathways which do not seem to be well defined-which would consume and dissipate the energy-rich intermediates and therefore uncouple oxidative phosphorylation. A. Respiratory Chain

4

X-I

ATP

Cations+ ___f

H+

B.

ATPase

Respiratory Chain

+----

+----

H+

H+

Cations+ ___3

FIG.7. Possible schemes depicting the relationship of cation transport t o energy goneration (see text for explanation). After Mitchell ( 1969).

(b) Mitchell's view (l966,1967b, 1969; Chappell and Crofts, 1965) is in essence the converse of that just outlined. The primary gradient is established by pumping protons out of the mitochondrion, either by operation of the respiratory chain itself, or by means of a proton pump. Since the mitochondria1 membrane is relatively impermeable to ions, proton movements are restricted by formation of a membrane potential -(inside negative). Valinomycin simply renders the membrane permeable to K+, and no specific interaction with a n ion pump is required: Kf accumulation results from movement of the ion down the electrochemical gradient produced by the proton pump. Uncoupling of oxidative phosphorylation is ascribed to the dissipation of the proton-motive force by K" movements. Both Mitchell's and Pressman's hypotheses have difficulties, which have been skilfully exposed by Pressman and Mitchell, respectively, as well as by others (for recent position statements see, Caswell, 1968; Harris and Pressman, 1969; Mitchell, 1967, 1969; Mitchell and Moyle, 1969; Poe, 1968; Pressman, 1968,1969; Slater, 1967). Clearly, what is a t issue here is the very nature of energy coupling in oxidative phosphorylation and the time does not seem ripe for final judgement.

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BRANKLTN M. HAROLD

B. ENNIATINS The enniatins are a family of cyclodepsipeptide antibiotics which inhibit the growth of Gram-positive bacteria and fungi. Both in structure (Fig. 6; Shemyakin et al., 1963; Shemyakin, 1965) and in their mode of action they resemble valinomycin but are less potent. Enniatins facilitate K+ diffusion across lipid bilayer membranes (Mueller and Rudin, 1967); an increase in permeability to thiourea has been reported (Lippe, 1968) but the effect is small. A stoichiometric complex of enniatin B with K+ has been isolated (Shemyakin et al., 1967a; Wipf et al., 1968). This is presumably the carrier, but its threedimensional structure is not yet known (for discussion see Lardy et al., 1967; Mueller and Rudin, 1967; Pressman, 1968). The sequence of ionpreference for complex formation is : Rb+ > K+ > Csf > Naf (Mueller and Rudin, 1967; Henderson et al., 1969). Shemyakin and his associates ( 1 963,1967a, b) have prepared numerous analogues and derivatives of the enniatins, which give some insight into the structural requirements for antibiotic activity. A regular sequence of amino acids and hydroxyamino acids is critical, as is the size of the ring. The enniatins which are active antibiotics induce K+ uptake by mitochondria (Lardy et al., 1967; Pressman, 1968) suggesting that, as with valinomycins, the induction of K+ permeability is a t the root of the antimicrobial effect. However, no studies on the mode of action of enniatins on bacteria appear to have been published. I n our hands (F. M. Harold and J. R . Baarda, unpublished observations) the effects of enniatin A (5 pg.ml.) on Strep. fuecalis were very similar to those of valinomycin. C. GRAMICIDINS The gramicidins are a family of closely related linear peptides. I n all, the terminal amino group is formylated and the carboxyl group is masked by ethanolamine, and substitutions in various positions give rise to the individual members. Gramicidin A, for example, has the sequence HCO -L-Val-Gly -L-Ala-D-Leu-L-Ala-D-Val-L-Val-D-Val- L - Try - D - LeuL-Try-o-Leu-L-Try-o-Leu-L-Try-NHCH,CH,OH. The structure and mode of action of gramicidins have been reviewed by Hunter and Schwartz (1967~). Although it had been known for twenty years that gramicidins uncouple oxidative phosphorylation, progress a t the molecular level had to await the discovery (Chappell and Crofts, 1965; Pressman, 1965) that gramicidins induce energy-dependent net uptake of cations by mitochondria. They also increase the permeability of the mitochondria1

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membrane to cations (Chappell and Crofts, 1965, 1966; Harris et al., 1967a). However, whereas valinomycin is highIy selective for K+, the gramicidins induce the uptake of Na+, Li+ and NH4+ as well as K+. Gramicidins increase the cation permeability of artificial lipid membranes, with little discrimination between cations (Chappell and Crofts, 1966; Henderson et al., 1969; Mueller and Rudin, 1967; Tosteson et al., 1968). Selectivity for K+ over Na+ was only by a factor of six, compared with 400 for valinomycin (Mueller and Rudin, 1967). The molecular basis of the interaction between gramicidins and cations is unknown. Models can be constructed in which one or perhaps two linear polypeptide molecules coil up around the cation (Lardy et al., 1967; Mueller and Rudin, 1967 ; Tosteson et al., 1968) but there appears to beno information as to carrier versus pore conduction. The increased cation permeability of the cytoplasmic membrane is sufficient t o account for the inhibition of growth of Strep. faecalis by lop7 M-gramicidin D. The antibiotic induces rapid loss of K+ from the cells by exchange for external Na+, Li+ or even NH4+ and thus halts protein synthesis for lack of Kf (Harold and Baarda, 1967). In addition, gramicidin partly inhibits glycolysis and the active transport of various metabolites. This apparently results from loss of K+ by exchange for H+; gramicidin thus acts to some degree as a proton conductor (Harold and Baarda, 1968b). A similar explanation was earlier advanced by Harris et al. (1967a) to explain aspects of the uncoupling of oxidative phosphorylation by gramicidin. The effects of gramicidin on erythrocytes can be understood in the same way (Bielawski, 1968; Scarpa et al., 1968), and account for the early observation that gramicidin induced slow, progressive haemolysis when injected into animals (Hotchkiss, 1944). There are surprisingly few data on the effect of gramicidin on energy metabolism of aerobic bacteria, apart from the original observations of Hotchkiss (1944) that K+ was required for the stimulation of respiration H) in Staphylococcus by this antibiotic. High concentrations ( inhibited motility of Pseudomonas fiuorescens but stimulated oxygen uptake (Faust and Doetsch, 1969). The relationship of cations to the mode of action of grainicidin is more explicit in the findings of Pressman and his associates (Pressman, 1965; Harris et al., 1967a) and of Chappell and Crofts (1965, 1966) with mitochrondria. I have already summarized the divergent interpretations which these two groups give to their results (Section V.A, p. 69). Yet a third hypothesis has been offered by Palcone and Hadler (1968), who regard the cation uptake induced by gramicidin as a consequence of a primary effect of the antibiotic on oxidative phosphorylation, rather than the other way around.

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FRANKLIN M. HAROLD

D. MACROTETRALIDES : NONACTIN AND

ITS

HOMOLOGUES

Certain streptomycetes produce yet another class of ion-conducting antibiotics, loosely referred to as the “nactins”. These are cyclic tetralactones which, like valinomycin, form lipid-soluble complexes with K+. The original member of this series appeared to lack antibiotic activity and was named nonactin. Subsequently, homologues containing additional methyl groups were isolated and designated monactin, dinactin and trinactin. Moreover, the “nactins” turned out to be potent inhibitors of Gram-positive bacteria after all (Meyers et al., 1965; see also the review by Shaw, 1967b). The structure of these compounds is shown in Fig. 8. The demonstration that the nactins, like valinomycin, induce ion transport and uncouple oxidative phosphorylation led to their recognition as ioiiophores (Graven et d.,1966b, c).

FIG.8. Chemical structure of monactin, a macrotetralide antibiotic.

The primary ion-conducting properties of the macrotetralides have been explored in considerable detail. Dinactin and monactiii induced both electrical conductivity and ion-diffusion potentials in phospholipid bilayers with a K+/Na’ selectivity ratio of 37 (Mueller and Rudin, 1967). A diffusible complex appears to be formed, containing K+ and the antibiotic in equimolar amounts. This carrier conducts Kf across a bulk lipid phase as well as across a membrane (Eisenman et al., 1968; Tosteson, 1968). Curiously, monactin and dinactin appear to complex sodium as well as K+; failure of the antibiotics to translocate Na+ as measured by electrical conductivity must thus be attributed to the differing properties of the two complexes (Tosteson, 1968). The conformation of the K+ complex of monactin was determined by X-ray crystallography (Kilbourn et al., 1967). As shown in Fig. 9, the molecule in the crystalline state is not planar but folds up into the general shape of a “tennis-ball seam”, with the K+ encaged in the middle. The polar oxygens are directed inwards while the lipophilic surface permits the complex t o traverse lipid barriers and equilibrate with K+ a t the interface (Eisenman et al., 1968; Tosteson, 1968).

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Very little work has been done on the antimicrobial effects of the nactins. Monactin inhibited growth of &rep. faecalis by inducing exchange of cellular K+ for Na+. The antibiotic had no direct effects on the generation and utilization of glycolytic ATP. A technical convenience is that, unlike valinomycin, monactin was readily removed by washing the cells, which restored their original impermeability to ions (Harold and Baarda, 1968a). The effects of monactin on red blood cells are also due simply to increased K+ flux (Tosteson et al., 1968).

FIG.9. Xpacc-filling model of the monactin-K omitted to show the central cavity).

complex (the cation has beon

I am not aware of any work with aerobic bacteria, but the effects of the nactins on mitochondria have been investigated in detail. The nactins induce ion transport, preferentiall? of K+; this is energy-dependent and may be accompanied by swelling. Under some conditions they uncouple oxidative phosphorylation and induce ATP hydrolysis (Graven et al., 1966b, c, 1967; Henderson et ak., 1969). The effects of the nactins are clearly similar to those of valinomyciii and their interpretation must be considered in much the same terms (Section V.A, p. 69).

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E. NIGERICIN,MONENSIN AND

OTHER

CARBOXYLICPOLYETHERS

Harned et al. (1951) originally described the antibiotic nigericin as a lipid-soluble monocarboxylic acid active against Gram-positive but not Gram-negative bacteria. Most remarkably, inhibition was reversed by addition of excess K+ to the medium. The structure of nigericin, established very recently by Steinrauf et al. (1968),is shown in Fig. 10. Studies in Lardy’s laboratory provided the first clue to its mode of action: nigericin was found to counteract the effect of valinomycin, i.e. to release K+ from the mitochondria (Graven et al., 1966a; Lardy et al., 1967).

FIG.10. Carboxylic polyether antibiotics.

Several antibiotics are now known which, like nigericin, are monocarboxylic acids. O f these the most important is monensin, a new antibiotic of considerable clinical promise (Haney and Hoehn, 1967). Its structure (Agtarap et al., 1967; Agtarap and Chamberlin, 1967) is shown in Fig. 10. Others of this general type include dianemycin, X-206 and X-537 ; scattered references indicate that their mode of action is similar to that of nigericin (Henderson et al., 1969; Pressman et al., 1967; Pressman, 1968, 1969). Nigericin induced increased permeability to both K+ and protons in various phospholipid membrane systems (Mueller and Rudin, 1967 ; Henderson and Chappell, 1967; Henderson et al., 1969; Tosteson et al., 1968) but this was not accompanied by electrical effects such as ionic potentials or increased conductivity. The antibiotic forms lipid-soluble complexes with Rb+ and K+, and can carry these even across a bulk solvent phase, but only a t high pH values (Pressman et al., 1967 ; Press-

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79

man, 1968, 1969).The proposed explanation is as follows : K+ binds only to the dissociated form of the antibiotic, and can traverse lipid barriers in this state; at lower pH values, the undissociated antibiotic acts as a proton carrier. Nigericin therefore catalyses electrically neutral exchange of K+ for H+ (and of K+ for other cations) which cannot be detected by electrical measurements. By contrast, let us recall that valinomycin forms a Ki- complex which bears a net positive charge; valinomycin therefore induces both ionic potentials and increased conductivity (Pressman, 1968, 1969; Pressman et al., 1967). Various members of this group differ significantly in ion selectivity. For example, nigericin prefers K+ to Na+, whereas monensin and dianemycin prefer Na+ (Pressman, 1968; Henderson et al., 1969). The structural basis of the selectivity may reside in the properties of the complex of cation and antibiotic, which is not a salt but an electrically neutral clathrate-in fact, a Zwitterion: it appears that only under conditions in which the carboxyl group is dissociated does the molecule fold over to enclose the cation. The protonated form has an entirely different conformation, perhaps that of an open chain (Pressman, 1968, 1969; Agtarap et al., 1967). These considerations are central to an understanding of the metabolic effects of nigericin and monensin. I n erythrocytes (Harris and Pressman, 1967 ; Pressman et al., 1967), nigericin induced electrically neutral exchange of K+ for H+. The same was observed in Strep. faecalis with nigericin (Harold and Baarda, 1968a) and monensin, albeit the latter prefers Na+ to K+ and rejects Rb+ (Estrada-0 et al., 1967b; Henderson et al., 1969; Pressman, 1969; F. M. Harold and J. R. Baarda, unpublished observations). The loss of K+, by exchange for either H+ or Na+, is a sufficient explanation for the inhibition of growth by nigericin and for its reversal by excess K+. However, unlike the other cation-conducting antibiotics, nigericin also strongly inhibits net uptake of K+, phosphate and alanine by Strep. faecalis. Since nigericin induces K+-H" exchange, we may regard it as a proton conductor of a special kind, and it is this entry of protons which accounts for the inhibition of transport (Harold and Baarda, 1968b). Whether catalysis of both cation-cation and cation-proton exchange can account for the complex effects of nigericin and monensin on mitochondria is not yet certain. Graven et al. (1966a) originally found that nigericin counteracts the effects of valinomycin and gramicidin, causing release of K+. This cation efflux was not energy-dependent, nor did it require prior exposure of the mitochondria to valinomycin (Graven et al., 1966a; Lardy et al., 1967; Pressman et al., 1967). I n addition, nigericin inhibits phosphate uptake and the oxidation of certain NADH2linked substrates and, also unmasks ATPase (Estrada-0 et al., 1967b).

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FRANKLIN M. HAROLD

The effects of rnonensin are essentially similar, except for different cation specificity (Estrada-0etal., 1967a).Pressman (l968,1969;Pressmanetal., 1967) believes that the basis for these effects is to be sought in the induction of neutral, K'-H+ exchange a t random sites on the mitochondrial membrane with release of Kf and entry of protons. A very different interpretation has been considered by Lardy and his associates (Lardy et al., 1967; Estrada-0 et al., 1967b). The ion exchanges induced by nigericin have been used as a test of the chemi-osmotic hypothesis. Conditions can be found under which nigericin and monensin induce extensive Kf-H+ exchange yet do not uncouple oxidative phosphorylation (Graven et al., 1966a; Estrada-0 et al., 1967a; Pressman et al., 1967). According to the chemi-osmotic hypothesis, ATP synthesis requires a proton gradient across the membrane and therefore nigericin, which induces H+ entry, should act as an uncoupler (Pressman P t al., 1967). The same argument has been applied bo photophosphorylation. Nigericin completely blocks proton uptake by chromatophores of Rhodospirillum rubrum, yet does not uncouple photophosphorylation (Shavit et al., 1968; Thore et al., 1968). This was held to be incompatible with the chemi-osmotic hypobhesis which calls for a proton gradient as the driving force for photosynthetic ATP formation. Jackson et al. (1968) confirmed the observations but interpret them in quite another way. Induction of K+-H+ exchange by nigericin would obliterate a pH gradient but, being electrically neutral, would not affect a membrane potential ; whether or not uncoupling is observed would then depend on whether a p H gradient or a membrane potential is the primary form of energy storage. Such a potential should, however, be collapsed by valinomycin which permits electrogenic K+ movements ; the combination of nigericin plus valinomycin did, indeed, uncouple photjophosphorylation (Jackson et al., 1968; Thore et al., 1968). I n fine, the failure of nigericin to uncouple oxidative and photophosphorylation is not necessarily incompatible with the chemi-osmotic hypothesis (see also Henderson et al., 1969).

P. OTHERIONOPHORES The list of known and potential ionophorous agents is by no means exhausted and we should mention some of the others because they may have attained prominence by the time this review appears in print. 1. Monazomycin is a basic antibiotic of unknown structure, which induces net uptake of Kt, Na+ and Li+ by mitochondria (Lardy et al., 1967; Estrada-0 et al., 1967b) and also uncouples oxidative phosphorylation. Nothing appears to be known concerning its effects on microorganisms.

ANTIMICROBTAL AGENTS AND MEMBRANE FUNCTION

81

2. Polyethers. Pedersen (1968) has synthesized a series of polyethers which exhibit varying degrees of selectivity in complexing cations (Eisenman et nl., 1968; Pedersen, 1968; Pressman, 1968; Tosteson, 1968) and which facilitate passage of ions across lipid phases and membranes. These are structurally related to the macrotetralide nactins. Some of these induce ion uptake in mitochondria ; others, inactive in themselves, inhibit the induction of K+ uptake by valinomycin (Lardy, 1968). Although nothing has been reported on the effects, if any, of such compounds on micro-organisms, they hold the promise of tailor-made ion conductors and should be kept in mind. 3. A lamethicin is a cyclic polypeptide produced by Trichoderma viride (Meyer and Reusser, 1967). It is surface active and inhibits growth of various Gram-positive bacteria albeit only a t rather high concentrations. Pressman (1968) mentions that it induces cation uptake by mitochondria. Of greatest interest is the discovery by Mueller and Rudin (1968) that alamethicin induced electrical excitability in artificial phospholipid bilayer membranes. Little is known concerning its effects on microorganisms ; experiments in this laboratory (unpublished) suggested that i t may disrupt the permeability barrier of Strep. faecnlis.

VI. Inhibitors of Energy Transfer and of the Respiratory Chain The compounds considered in the preceding sections interact primarily with the lipid moiety of membranes. Some penetrate into the membrane to distort its structure, others form lipid-soluble complexes with specific ions. The mode of action of these compounds could be reduced to physical and chemical phenomena which account for most (if not all) of their biological effects. We now turn to inhibitors which interact with individual proteins, structural or catalytic, to block specific steps in the metabolic pathways associated with membranes. These include the coupling of electron transport t o ATP generation, the utilization of metabolic energy for active transport or other functions, and the respiratory chain itself.

A. ATPase

AND

ENERGY TRANSFER

ATPases are quite universally associated with biological membranes and participate in energy transfer and transformation by these organelles. The ATPase of the inner mitochondria1 membrane, for example, requires Mg2+ and is localized in spherical particles attached to the basal membrane by a stalk. It seems to be generally accepted that this enzyme

82

FRANKLIN M. HAROLD

catalyses the terminal step in oxidative phosphorylation, the synthesis of ATP itself (see reviews by Mitchell, 1966; Racker, 1967; Pullman and Schatz, 1967). The same enzyme, but acting in reverse, mediates the utilization of exogenous ATP to energize reversed electron flow, transhydrogenase or ion transport when substrate oxidation is blocked (Section II.B, p. 48). An analogous ATPase occurs in chloroplasts (Moudrianakis, 1968). The other process in which ATPase has been definitely implicated is ion transport. ATPases which require Mg2+and are stimulated by Na+ and K+ are commonly found in cytoplasmic membranes of mammalian cells and in the microsome fraction. There is overwhelming evidence, recently reviewed by Skou (1965), Stein (1967) and Albers (1967), that these enzymes mediate the translocation of Naf and K+. Other translocations may be secondarily driven by this enzyme through gradient coupling (section II.B, p. 48). The membrane fraction isolated from bacteria also has ATPase activity; how many enzymes are invoived is not clear. I n some cases the ATPase activity is associated with stalked particles, and may be part of the machinery for generating ATP by oxidative phosphorylation (Gel’man et al., 1967; Mufioz et al., 1968, 1969), but this cannot be the function of ATPase in fermentative bacteria. In general, ATPases of bacterial membranes are only slighbly stimulated by Na+ and K+ or not stimulated at all (see Abrams, 1965; Harold et aZ.,1969a, for references). All the same there is good evidence that, in bacteria also, ATPase is involved in ion transport. This section will survey compounds which inhibit ATPase and related energy-transfer reactions. The best known of these, oligomycin, was discovered as a result of Lardy’s search for antibiotics which affect oxidative phosphorylation (Lardy et al., 1958); although of limited application to bacterial systems, oligomycin is the prototype t o which all other inhibitors must be compared. 1. Oligomycin, Rutamycin and Related Antibiotics. The oligomycin complex of antibiotics is produced by Streptomycesspeciesas isrutamycin, a related but distinct compound. Oligomycins and rutamycin inhibit the growth of many filamentous fungi and some yeasts but have no antibacterial action. Several other antibiotics have recently been added to this group. Peliomycin, ossamycin and venturicidin cIosely resemble rutamycin in their biological effects (Walter et al., 1967); peliomycin strongly inhibits growth of Micrococcus lysodeikticus (Price et al. , 1963) and may thus prove particularly useful in the study of bacterial ATPase. Like other antibiotics which act upon membranes, all are insoluble in water but soluble in organic solvents. Their structures have not yet been determined.

ANTIMICROBIAL AGENTS A N D MEMBRANE BUNCTION

83

It became clear very early that oligomycin affects respiration since only aerobic organisms are sensitive to this antibiotic. I n a recent illustration of this generalization, Parker et al. (1968) showed that oligomycin inhibits growth of yeast on ethanol, which must be respired, but is much less inhibitory to growth on glucose which can be fermented; the antibiotic also increased the frequency of mutants deficient in respiratory functions. The effects of oligomycin and related antibiotics have been studied most extensively in animal mitochondria, but yeast mitochondria respond in much the same way (Shaw, 1967a; Schatz, 1968). The summary which follows leans heavily on the comprehensive review by Shaw (1967a). I n intact mitochondria, oligomycin and rutamycin inhibit both phosphorylation and respiration but do not alter the P / O ratio; uncouplers restore respiration but not, of course, phosphorylation. I n damaged or fragmented mitochondria, oligomycin tends to block phosphorylation but not respiration. Thus these antibiotics do not inhibit electron transport as such but the energy-transfer steps which couple ATP synthesis to the redox reactions. Inhibition of respiration is a secondary effect which reflects respiratory control rather than the site of action of the inhibitor. These findings led to the discovery that oligomycin and rutamycin inhibib reactions closely related to ATP synthesis, including ATP/ADP exchange and ATP hydrolysis (Lardy et al., 1958; Shaw, l967a). The molecular basis of these inhibitions is under active investigation in many laboratories. Briefly, oligomycin and rutamycin bind to a component of the inner mitochondria1 membrane which is distinct from the ATPase itself; the enzyme is sensitive t o the antibiotic when attached to the membrane but becomes resistant when solubilized. Sensitivity to oligomycin involves one or more proteins which are part of the stalk by which the ATPase particle is attached t o the membrane. Inhibition of ATPase activity is thus a secondary phenomenon, due perhaps to a transmitted effect on the conformation of the enzyme (Bulos and Racker, 1968; Kagawa and Racker, 1966; Kopaczyk et al., 1968; MacLennan and Tzagaloff, 1968). For reviews see Pullman and Schatz (1967) and Racker (1967). The precise mechanism by which oligomycin inhibits phosphorylation and respiration is still quite controversial, and the schemes which have been proposed (reviewed by Shaw, 1967a; Lee and Ernster, 1968; Roberton et ab., 1968; Mitchell, 1966) reflect the authors’ divergent views of the basic nature of energy coupling in oxidative phosphorylation. For the purposes of the present discussion, the generalized scheme of Pig. 11 will suffice, based on the views of Lee and Ernster (1968) and of Roberton et al. (1968).

84

FRANKLIN M. HAROLD Huccinstc

Flavoprotein S Piericirliri

NAUHn

YlnV(J-

; i l)rotcin

i-1 -

enzyme

Q

Antimycin Heptylqui noline oxide

T---

5.

+Cyt. a, a3

C'yt. b w Cyt. C1

1.

A

7 I

~

A D P + P i ATP 1011 trnrisport, other functions Oliyomycin Dicyclohoxylcarbodiiniidc ~

x-P aurovertiit

Dio !J? AT1'

FIG.11. Elcctron transport and oxidative phosphorylation (in terins of tho chemical-coupling hypothesis), showing the sites of action of the inhibitors piericidin, antimycin, heptylquinoline oxide, oligomycin, dicyclohexylcarbodiimide aurovertin and Dio 9.

Certain energy-linked functions including ion transport, transhydrogenase and reversed electron flow can be energized by oxidizable substrates even in the presence of oligomycin. Since the antibiotic blocks ATP formation, this is evidence that processes such as ion transport can be supported by energy-rich intermediates or states, of unspecified nature, preceding ATP itself. When electron transport is blocked, either by lack of a suitable substrate or by an inhibitor of the respiratory chain, ion transport can be energized by ATP; under these conditions it is sensitive to oligomycin, in keeping with the scheme (Fig. 11;Lehninger et al., 1967; Pullman and Schatz, 1967; Shaw, 1967a; Roberton et al., 1968; Lee and Ernster, 1968). A most interesting development is the recognition that oligomycin inhibits ion transport, not only in mitochondria but also in tissues which depend upon the Na+- and K+-stimulated ATPase. This enzyme is inhibited by the antibiotic, albeit a t higher concentrations than are required in mitochondria (Blake et al., 1967; Inturrisi and Titus, 1968; Whittam et al., 1964). I n some cases, at least, oligomycin inhibited the K+-dependent dephosphorylation of the enzyme, but not the Na+dependent phosphorylation (Pahn et al., 1968). The unique importance of antibiotics of the oligomycin type is precisely that they block energy transductions in a variety of membranes, and thus reveal the unity of

Oa

ANTIMICROBIAL AGENTS AND MEMBRANE PUNCTION

85

structure and function which underlies the diversity. On the other hand, bacterial ATPases are generally resistant to oligomycin. Brief mention should be made of aurovertin, a fungal metabolite toxic t o animals but which has little effect on either fungi or bacteria. It is of interest in the present context because, like oligomycin, aurovertin inhibits ATP synthesis by oxidative phosphorylation. Its site of action may be the ATPase (F,) particle itself, since it can inhibit even the solubilized enzyme (see the discussions in Lee and Ernster, 1968; Shaw, 1967a; Roberton et al., 1968). However, some of the effects of aurovertin on mitochondrial function are not readily reconciled with this hypothesis.

FIG.1%.Clieniical structure of dicyclohexylcarbodiimide.

2 . Dicyclohexylcarbodiimide. Dicyclohexylcarbodiimide (DCCD), a synthetic reagent of known structure (Fig. 12), is used in organic chemistry as a dehydrating agent. Curiously, the effects of this compound on mitochondrial processes are almost identical with those of oligomycin and rutamycin : it inhibits respiration and coupled phosphorylation, as well as energy-linked functions supported by ATP. Dicyclohexylcarbodiimide inhibits mitochondrial ATPase by reaction with a protein component of the basal membrane, and thus does not inhibit the solubilized enzyme (Beechey et al., 1967; Bulos and Racker, 1968 ; Roberton et al., 1968). Dicyclohexylcarbodiimide apparently forms a covalent bond with its receptor site, which is thought to be a proteolipid and may be identical with the rutamycin receptor (Beechey et al., 1967; Bulos and Racker, 1968; Knight et al., 1968; Roberton et al., 1968). Inhibition of oxidative phosphorylation presumably accounts for the effects of dicyclohexylcarbodiimide on growth and respiration of yeast (Kovad et al., 1968) but cannot explain its effects on fltrep. faecalis which does not carry out oxidative phosphorylation. Growth of this homofermentative organism was inhibited by 0.1 mM-dicyclohexylcarbodiimide. The drug also inhibited energy-dependent accumulation of Kf by exchange for Na+ and Hfas well as the transport of alanine and phosphate ;it decreased the rate of glycolysis by inhibiting ATP degradation, but apparently did not interfere with the synthesis of ATP. These physiological effects were correlated with studies on the ATPase of membranes of Strep. faecalis. Dicyclohexylcarbodiimide irreversibly inhibited the membrane-bound enzyme, but not the solubilized enzyme (Harold et al., 1969a). Thus dicyclohexylcarbodiimide, like oligomycin,

86

FRANKLIN M. HAROLD

promises to be a valuable reagent not only in the study of oxidative phosphorylation but for energy-dependent membrane functions in general. 3. Dio 9 is a n antibiotic of unknown structure active against Grampositive bacteria. It is another compound which has, so far, been employed largely in the study of energy transfer in mitochondria and chloroplasts. Depending upon conditions, Dio 9 either inhibits or uncouples respiration and phosphorylation (Guillory, 1964), and also initiates pronounced swelling of mitochondria (Fisher and Guillory, 1968). Dio 9 also inhibits energy-conservation reactions in chromatophores of Rhodospirillum rubrum (Fisher and Guillory, 1967). The very complex effects of this antibiotic are presumably related to inhibition (and sometimes stimulation) of ATPase. It is particularly noteworthy that Dio 9 inhibits both solubilized and membrane-bound ATPase (Fisher and Guillory, 1967; Schatz et al., 1967; Schatz, 1968). Dio 9 inhibited growth of Strep. faecalis a t 5 pg./ml. Its gross physiological effects were similar to those of DCCD; it blocked cation transport and alanine uptake, and inhibited the ATPase. However, as in the other systems, it inhibited both the solubilized ATPase and the membranebound enzyme, suggesting that it may act directly on the enzyme protein itself. To what extent its antibiotic activity is explained by the inhibition of ATPase remains to be ascertained (Harold et ab., 1969b). 4. Biguanides are potent synthetic antimicrobial agents, of which chlorhexidine (Fig. 1, p. 55) is probably the most widely used as a disinfectant in industry and medicine. The biguanides are surface-active and disorganize the cytoplasmic membrane (Hugo and Longworth, 1964a, b ; Weinberg, 1968). However, chlorhexidine is bactericidal a t concentrations below those required to induce leakage of cell constituents (Hugo and Longworth, 1964a). Certain guanidine derivatives, including alkylguanidines, phenethylbiguanide and decamethylenediguanide, are inhibitors of energy transfer in mitochondria and chloroplasts (Guillory and Slater, 1965; Gross et al., 1968). Prompted by this clue, Harold et al. (1969b) explored the effects of low concentrations of chlorhexidine and of a related compound, vantocil IB, on Strep. faecalis. They proved to be good inhibitors of both cation transport and of the membrane-bound ATPase at concentrations which did not lyse the cells, and this metabolic effect may contribute to their antimicrobial action.

B. INHIBITORS OF THE RESPIRATORY CHAIN The respiratory chain of bacteria is similar in principle to that of mammalian and fungal mitochondria, though there are considerable

ANTIMICROBIAL AGENTS AND MEMBRANE FUKCTION

87

diffcrences in detail. Gel’man et al. (1967) and Smith (1968) have reviewed the information currently available ; the electron-transport chain of bacterial photosynthesis is less well established (Vernon, 1968). Like mitochondria of higher organisms, the bacterial respiratory chain contains dehydrogenases for various substrates and cytochromes of the 6, c and a types in that order ; quinones participate in electron transport and may be intimately involved in oxidative phosphorylation. Although the phosphorylation etliciency of extracts is generally low, intact bacteria have two or even three coupling sites. However, it has not yet been possible to fractionate the bacterial respiratory chain into complexes analogous to those obtained from mitochondria, and thus the location of the coupling sites is less well defined. Finally, although the mode of action of many respiratory inhibitors is similar in bacterial and mammalian systems, substantial differences exist in some cases-not only between bacterial and mammalian systems but from one bacterial species to another. For example, bacterial respiration is generally insensitive to antimycin, but particles from Bacillus subtilis are strongly inhibited and bacterial photophosphorylation is exceedingly sensitive to antimycin I (see the review by Rieske, 1967). There may thus be variation in .the I detailed structure or accessibility of the sites subject to these inhibitors. The available information on inhibitors of electron transport comes largely from studies with mammalian mitochondria (Fig. 11, p. 84). The present survey of these compounds will be a cursory one, not to minimize their importance but because the insights derived from this work bear upon the intimate details of electron transport rather than upon the structure and function of membranes in general. 1. Piericidin and Rotenone. Rotenone, a toxic substance of plant origin, has been employed for years as an inhibitor of NADH, oxidation and coupled phosphorylation in the region of the first coupling site, but is generally not an effective inhibitor of bacterial electron transport. j There is therefore much interest in the novel antibiotic, piericidin A. A product of Xtreptomyces, piericidin was first recognized as an insecticide with little antimicrobial activity (Tamura et ul., 1963). It was subsequently found to inhibit both bacterial and mitochondria1 respiration at the rotenone site. i The structure of piericidin A (Takahashi et al., 1965) is shown in Fig. ’ 13. The resemblance between the antibiotic and coenzyme Q suggested that piericidin might interfere with quinone function and this appears t o be the case. I n mammalian mitochondria, very low concentrations of piericidin inhibit oxidation of NADHB and reduction of coenzyme Q (Fig. 11, p. 84) ; higher concentrations of the antibiotic block electron transport between succinate and coenzyme Q as well (Hall et al., 1966; Jeng et al., 1968). Indeed, piericidin appears to be the most potent ~

‘

1

88

FRANKLIN M . HAROLD

inhibitor known to act in the region of the first coupling site of oxidative phosphorylation (Vallin and Low, 1968).Piericidin binds to mitochondria with very high affinity for a specific site, apparently identical with that to which rotenone binds (Horgan and Casida, 1968; Horgan et al., 1968a, b ; Coles et al., 1968; Jeng et al., 1968; Hatefi, 1968). Binding is apparently not covalent, and the antibiotic can be recovered by extraction with solvents and other treatments (Horgan et al., 1968a, b ; Coles ef al., 1968). OH

Piericidin A

~HCHO Antimycin A (R = hexyl)

Heptylquinoline oxide

R (R=heptyl)

4,

O

FIG.13. Chemical structures of some inhibitors of electron transport.

Bacterial respiration is generally, but not always, less sensitive to piericidin than is mitochondria1 respiration, but the sites of inhibition appear to be very similar (Jeng et al., 1968; Knowles et al., 1968;Kosaka and Ishikawa, 1968; Snoswell and Cox, 1968).I n several cases ubiquinone or related compounds were shown to overcome the inhibition of respiration, perhaps by displacement of the antibiotic. Respiration of intact cells is insensitive to piericidin, presumably because the antibiotic fails to reach the sensitive sites. 2 . A n t i m y c i n and A l k y l Quinoline Oxides. The antimycins are a family of closely related antibiotics produced by Streptomyces species ; the structure of antimycin A, the only member extensively used, is shown in Fig. 13. The antibiotic is highly inhibitory to many fungi and mammalian

ANTIMICROBIAL AGENTS AND MEMBRANE FUNCTION

'

89

cells but ddes not inhibit the growth of bacteria. An excellent and coniprehensive review by Rieske (1967)forms the basis of this section. Antimycin is the respiratory inhibitor par excellence, and its mode of action has been much studied. It binds specifically and stoichiometrically to a site which may form the junction between cytochromes b2 and c1 (Fig. 1 1 , ~84). . The antimycin-sensitive site, like that to which piericidin binds, is of particular interest since it is also thought to be the coupling site for phosphorylation (Rieske, 1967; Rieske et al., 1967). The growth of yeasts such as Torulopsis is inhibited by antimycin when ethanol serves as substrate, but not when glucose is provided; results of this kind first suggested that antimycin acts specifically at the level of mitochondria1 respiration (Rieske, 1967 ; Butow and Zeydel, 1968). A mutant of Torulopsis has been described in which respiration, both of intact cells and of mitochondria, is relatively resistant to antimycin. Binding of antimycin to the particles was also decreased. These mutants are reminiscent of yeasts which are naturally resistant to antimycin, such as Saccharomyces ; these lack the antimycin-sensitive site and indeed have only two coupling sites for oxidative phosphorylation. It will be of interest to see whether mutation to antimycin resistance involves loss of this coupling site. Bacterial cells as well as membrane fragments are generally resistant to antimycin, although a few sensitive systems have been found (Gel'man et al., 1967; Rieske, 1967; Smith, 1968). Photophosphorylation by particles of Rhodospirillum rubrum is particularly sensitive to antimycin (Rieske, 1967; Vernon, 1968). It is thought that the coupling site in the cytochrome b-c region of the bacterial respiratory chain has a structure slightly different from its mitochondria1 counterpart, but is not algether absent, because bacterial respiration is generally sensitive to another class of antibiotics which act at the same site as does antimycin, namely the alkyl quinoline oxides. Lightbown and Jackson (1956) originally isolated the alkyl quinoline N oxides from culture filtrates of certain Pseudomonas strains by virtue of their capacity to antagonize the action of streptomycin. Their chemical structure, determined by Cornforth and James (1956), is shown in Fig. 13. Alkyl quinoline oxides inhibit oxygen uptake by intact cells of Bacillus subtilis and Staphylococcus spp., and this presumably accounts for their incompatibility with streptomycin. Cells of Escherichia coli were resistant, but particles derived from the envelope were sensitive. Heptyl quinoline N oxide has proven to be a versatile inhibitor of respiratory and photosynthetic electron transport, both in mitochondria and bacteria. Its site of action is probably identical with that of antimycin (Lightbown and Jackson, 1956; Rieske, 1967; Rieske et al., 1967). This statement is well supported by the finding (Butow and Zeydel, 1968) that mutants

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selected for resistance to antimycin were resistant to heptyl quinoline oxide as well. Not all the cellular effects of antimycin can be ascribed to the inhibition of electron transport. I n Bacillus megaterium, one of the few bacterial species that is relatively sensitive to antimycin, the antibiotic did not markedly depress respiration but blocked active transport of a-methylglucoside and of a-amino-isobutyric acid ;higher concentrations induced lysis of protoplasts, apparently by disorganization of the cytoplasmic membrane (Marquis, 1965). 3. Other inhibitors of electron transport. Numerous other compounds inhibit electron transport in micro-organisms, but their sites of action are as yet ill defined. Among these is flavensomycin, an antifungal compound which also inhibits the growth of certain bacteria; it inhibits electron transport prior to the antimycin-sensitive step, probably between the dehydrogenases and coenzyme Q (Gottlieb, 1967 ; Gottlieb and Inoue, 1967). Other antibiotics which may prove to be of interest to students of electron transport are U19718 (Reusser, 1968) and also pyocyanine, patulin and usnic acid (reviewed in Gottlieb and Shaw, 1967).

C. INTERACTION OF HEAVY METALSWITH

THE

MEMBRANE

The antibacterial properties of mercury compounds were applied by Arab physicians in the middle ages, and until the advent of sulphonamides in the 1930s mercury compounds were the chief antiseptics available. Heavy metals other than mercury are not sufficiently toxic to micro-organisms for use as antimicrobial agents, but concentrations in the millimolar range sometimes exhibit pronounced effect on membrane transport in bacteria and fungi. The physiological effects of mercury derivatives are generally ascribed to interaction with sulphydryl groups, though the metal does bind to other groups of biological importance (Passow et al., 1961). Inhibitory effects of Hg2+ on both cells and enzymes are usually reversed by sulphydryl compounds such as cysteine. To be sure, Hg2+ blocks sulphydryl groups of intracellular enzymes but proteins exposed at the outside of the permeability barrier are the most sensitive, and many of the effects of mercury derivatives on intact microbial cells can be understood in terms of the inhibition of membrane processes. It is Mpossible to cite but a few of many known examples. Addition of HgCI, instantaneously stops glycolysis by Strep. faecalis ; the inhibition is completely reversed by cysteine and is apparently due to a block in the transport of glucose into the cells. Para-Chloromercuribenzoate inhibits transport of /3-galactosides in E. coli by interaction with a sulphydryl

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group which forms part of the active site of the galactoside-transporting M protein (Fox and Kennedy, 1965). The effects of other divalent metal ions, UOZ2+,Ni2+ and Co2+, on membrane functions have received detailed study in yeast. This work has been reviewed (Rothstein and Van Steveninck, 1966) and a very brief summary may suffice here. Uranyl ion appears to block specifically the transport of sugars into cells ; energy-linked transport is more sensitive than facilitated diffusion. Ni2+and CoZf have similar but not identical effects. At least two kinds of binding sites for UOZzfhave been detected, differing in specificity. The binding sites with the highest affinity for metal ions were identified as inorganic polyphosphate, which is thought to participate in sugar transport as a phosphate donor. Other metal ions also affect transport processes. The trivalent La3+ion stops metabolism of Strep. faecalis apparently as the result of interference with membrane functions (Wurm, 1951). It is a potent inhibitor of ion translocation in mitochondria (Mela, 1968). Zinc and cadmium, which induce active accumulation of K+ (Brierley and Settlemire, 1967) and of Mg2+ (Brierley, 1967) by mitochondria, and uncouple oxidative phosphorylation, inhibit transport in bacteria (Eagon and Asbell, 1969). This would appear to be a promising field for further research. Finally, mention should be made of the remarkable observation of Novick and Roth (1968), that genes which control the resistance of Staphylococcus to lead, zinc, cadmium and mercury are located on the penicillinase plasmid. The physialogy of resistance to metal ions is almost totally unexplored.

VII. Bacteriocins: Antibiotics which Interact with Specific Membrane Receptors Bacteriocins are macromolecular antibiotics produced by certain strains of bacteria and active only against other strains of the same species. Many appear to be simple proteins, and even in those that are structurally complex the specificity resides in proteins. It is their narrow spectrum of antimicrobial activity that sets the bacteriocins apart from other antibiotics, and this is ascribed to their interaction with highly specific cellular receptors. The bacteriocins have been the subject of several recent reviews (Holland, 1967; Nomura, 1967a, b; Reeves, 1965). This section will be confined to the interaction of bacteriocins with specific membrane receptors and the role of the membrane in the remarkable physiological consequences of this interaction ; most of the relevant reports deal with the bacteriocins of E. coli, the colicins. Colicins are classified by the pattern of receptor specificity and also of immunity. Colicins designated by the same letter, for example the E group, share the same receptor. A sensitive cell may adsorb many

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colicin molecules, and have hundreds of receptor sites per cell. Nonetheless, the killing action of most colicins follows single-hit kinetics, indicating that even a single colicin molecule has a finite probability of causing a lethal event (Nomura 1967b; Shannon and Hedges, 1967). Colicins produce diverse biochemical effects. Colicins of types E l , I and K inhibit the synthesis of DNA, RNA and protein but do not inhibit respiration. Colicins E2 inhibit DNA synthesis and induce DNA degradation, whereas E 3 colicins inhibit synthesis of protein but not of DNA and RNA (Romura, 1967a, b ; Reeves, 1968). It is curious that colicins E2 and E3, which share a common receptor, produce different metabolic effects. Nomura (1967a, b) showed that cells exposed to colicins of various types can be rescued by treatment with trypsin, which presumably digests the colicin. Thus the colicins remain at their receptor site, external to the permeability barrier, and act from there. ' The biochemical processes set in motion by the adsorption of colicins are far from clear. Colicin E2 may induce a DNAase (Holland, 1968; Holland and Threlfall, 1969). Colicin E 3 brings about the inactivation of ribosomes. Most pertinent to this survey, colicins E l , K and I appear to block energy metabolism. These colicins do not inhibit respiration but result in drastic lowering of $he cellular ATP level. The synthesis of all macromolecules (DNA, RNA, protein and polysaccharide) ceases, as do active transport and motility (Nomura, 1967a, b ; Levisohn et al., 1968; Fields and Luria, 1969a, b). The effects of colicins El and K on transport systems are particularly interesting (Fields and Luria, 1969a), Accumulation of thiomethylgalactoside, which is an energy-dependent process, was abolished. However, there was no gross breakdown of the permeability barrier and the transport system continued to equilibrate galactosides across the membrane. This is reminiscent of the effects of 2,4-dinitrophenol and consistent with an earlier suggestion that colicins E l and K uncouple oxidative phosphorylation. However, the situation appears t o be more complex. Levisohn et aH.(1968)found that the inhibition of ATP turnover by colicins is less complete than that caused by 2,4-dinitrophenol, and various non-nucleotide phosphorus compounds accumulate. Indeed, pyruvate and various phosphorylated intermediates of glycolysis are excreted by colicin-treated cells (Fields and Luria, 1969b). Moreover, the effects of colicins depend to a considerable degree upon the presence of oxygen. One consequence of colicin binding appears to be inhibition of pyruvate dehydrogenase but this is clearly not the primary lesion. Fields and Luria (1969b) suggest that colicins may promote leakage of protons through the membrane. Their results are compatible with this hypothesis but it fails to account for the role of oxygen since protonconducting compounds act anaerobically as well (PavlasovB and Harold, 1969).

~

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How can a single colicin molecule, adsorbed to a receptor located a t the outer surface of the plasma membrane (Nomura, 1967a, b ; Srnarda and Taubeneck, 1968), induce DNA breakdown or disrupt energy metabolism? Nomura proposed that each colicin has a “biochemical target”, and affects it by means of stimuli sent through a specific transmission system, presumably located in the cytoplasmic membrane. Indeed, all colicin-sensitive processes are directly or indirectly associated with the membrane. This hypothesis predicts that mutants resistant to colicins may arise in a t least two general ways. Loss of the receptor results in mutants that can no longer adsorb the colicin. Alterations in the transmission system should produce mutants which still adsorb the colicin but have become tolerant. Colicin-tolerant mutants have been isolated in several laboratories (Holland, 1968; Nomura and Witten, 1967; de Zwaig and Luria, 1967). Some of these mutant cells are fragile and hypersensitive to detergents ; others are hypersensitive to dyes such as methylene blue. It was suggested that colicin-tolerant mutants have suffered loss or modification of a membrane protein; identification of such proteins and definition of their role in membrane function is an urgent task. A physical model intended to rationalize the role of the membrane in colicin action has been proposed by Changeux and Thii?ry (1967). They regard the membrane as a structure composed of repeating subunits which interact co-operatively with each other ; binding of a single colicin molecule would change the conformation of one or a few subunits, followed by a molecular domino effect by which the entire membrane shifts into a new pattern. Proteins attachedto the inner surface of‘the membrane would thus find themselves in an altered environment, and this may lead to the arrest of functions mediated by such proteins. It should be noted that bacteriocins are a mixed lot, and not all the antibiotics so classified appear to act via specific receptors. For instance, megacjn A, the best known of the bacteriocins produced by Grampositive bacteria, disrupts the cytoplasmic membrane of many strains of B. megaterium (see Holland, 1967, Nomura, 1967b forreferences tothe original papers) ; megacin A is now thought to be a phospholipase.

VIII. Summary and Prospect

I have attempted to classify the antimicrobial agents which act upon membranes according to their primary effect at the molecular level. At least three general modes of action can be distinguished a t present. (1) Compounds which associate with membrane components and disorganize lipoprotein structures. The detergents are familiar representatives of this class, as are the surface-active antibiotics tyrocidine and polymyxin. Polyenes are not particularly surface-active but forni

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complexes with sterols. Most of these compounds do not destroy the physical integrity of membranes, but rather disorganize the permeability barrier which depends upon hydrogen bonds and hydrophobic interactions. The precise changes in molecular architecture cannot yet be specified. (2) The cation-conducting compounds have remarkable and diverse effects on the functions performed by biological membranes, but these can largely (perhaps entirely) be ascribed to the formation of lipidsoluble complexes with alkali-metal ions or protons. To these compounds we owe the recognition that ion transport is not only a central function of living membranes but an integral part of energy generation. The ionophores are both research tools and models which afford insight into membrane transport generally. All the ionophores reported thus far conduct cations. Future research may reveal anion conductors (according t o Bodanszky and Bodanszky ( 1 968) the structure of the new antibiotic stendomycin would be compatible with such a role) and indeed lipid-soluble carriers for non-ionic compounds. (3) The targets of many antimicrobial agents are, or include, specific proteins which serve structural or catalytic functions. This category includes quite non-specific reagents, such as Hg2+,but interest centres on inhibitors of the various energy transductions which membranes carry out. I n oxidative and photosynthetic phosphorylation, the free energy of redox reactions is converted into ion gradients and ATP. Membrane transport utilizes ATP to establish concentration gradients of ions and other metabolites; ATP can also be used to reverse the normal direction of the respiratory chain, and finally the potential energies of ion gradients and ATP are sometimes mutually interconvertible. Among the many inhibitors which block specific steps in energy metabolism, oligomycin and other inhibitors of “ATPase’) deserve special mention because of the central role of this enzyme in mediating the interconversion of chemical and potential energy. These few categories are not intended to be either final or exhaustive. The colicins, for example, bind to highly specific receptors a t the outer surface of the membrane and remain there, yet a single molecule may block active transport or protein synthesis. It appears that the membrane can function as a unit such that perturbations may be transmitted from one end of a cell to the other, but virtually nothing is known regarding the molecular basis of this communication process. Finally, some known antibiotics are not easily accommodated by this Procrustean bed. Further research will no doubt reveal that some of the compounds relegated to this paragraph do not primarily affect membrane processes a t all; others may be assigned to established categories. Yet,

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it is by no means unlikely that an obscure compound will afford insight into a principle of membrane function which is still unrecognized. Readers in search of a project may find the foIIowing of potential interest : albomycin and other sideromycins, flavensomycin, patulin and usnic acid (all reviewed in Gottlieb and Shaw, 1967); azalomycin (Sugawara, 1967,1968) ; pinosylvin methylether, thujaplicin and other antimicrobial constituents of wood (Lyr, 1966; Aggag and Schlegel, 1966); nisin, a polypeptide antibiotic produced by lactobacilli (see White and Hurst, 1968, for earlier references) ; and showdomycin, a novel antibiotic of known structure which may interact with sulphydryl groups at the cell surface (Hadler et al., 1968a, b). One obvious category is, so far, almost unrepresented. I am not aware of any antimicrobial agent which primarily inhibits membrane synthesis. Perhaps the best example is diphenylamine which blocks carotenoid synthesis, and inhibits the growth of certain Gram-positive bacteria (Salton and Schmitt, 1967). A major question, which can be raised but not answered, is the role of antibiotics in the organisms which produce them. Of what use or significance are valinomycin, monactin, nigericin, antimycin, piericidin, oligomycin and many other remarkable inhibitors which we have surveyed to Xtreptomyces spp. ? And why do streptomycetes produce the great majority of the membrane-active antibiotics? It has been argued (Brock, 1966) that antibiotics are agents of chemical warfare, secreted by the producing organism to suppress the growth of competing species. This view has failed to gain general acceptance. Most investigators regard the antibiotics as secondary metabolites, whose production is due to breakdown of certain control mechanisms (Woodruff, 1966; Bu’lock, 1965), but which confer no selective advantage and, indeed, serve no 1 functionin themselves. I find this view unsatisfactory (seealso Bodanszky i and Perlman, 1969) and believe that the remarkable specificity of struc- ’ ture and function displayed by a compound such as valinomycin requires . a teleonomic explanation. Finally, antimicrobial agents which act upon membranes offer an amusing instance of compartmentation in science. The torrent of papers describing the application of the ionophores to mitochondria contrasts with a bare trickle of microbiological studies in this field. This review will have accomplished its purpose to the extent that it helps disorganize such artificial barriers to the diffusion of knowledge.

IX. Acknowledgements Major J. W. Powell is quoted from “Beyond the Hundredth Meridian” by Wallace Stegner, with kind permission of the author and of the

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publisher, Houghton-Mifflin Co. Thanks are due to Dr. J. D. Dunitz for permission to reproduce Fig. 9, and to Drs P. Mitchell and B. C. Pressman for permission to read unpublished manuscripts. Ethel Goren, Rudolf Love and Nadia de Stakelburg helped generously in the preparation of this article. Original research from my laboratory was supported in part by U.S. Public Health Service Grant AI-03568 from The Institute of Allergy and Infectious Diseases. REFERENCES Abram, D. (1965). J . Bact. 89,856 Abrams, A. (1965).J . biol. Ghem. 240, 3675. Aggag, M. and Schlegel, H. G. (1967). I n “Wirkungsmechanisnien von Fungiziden und Antibiotika”, Biologische Gesellschaft der DDR, Sektion Mikrobiologie. pp. 17-24. Akademie Verlag, Berlin. Agtarap, A. and Chamberlin, J. W. (1967). Antimicrob. Agents Chemother. pp. 359-362. Agtarap, A., Chamberlin, J. W., Pinkerton, M. and Steinrauf, L. K. (1967). J . Am. chem. Soc. 89, 5737. Albers, R. W. (1967). A . Rev. Biochem. 36, 727. Andreoli, T. E. and Monahan, M. (1968). J . gen. Physiol. 52, 300. Andreoli, T. E., Tieffenberg,M. andTosteson, D. C. (1967).J . gen.PhysioZ. 50,2527. Asano, A. and Brodie, A. F. (1965). J . biol. Chem. 240, 4002. Asbell, M. A. and Eagon, R. G. (1966). J . Bact. 92,380. Bangham, A. D., Dingle, J. T. and Lucy, J. A. (1964). Biochem. J . 90, 133. Bangham, A. D., Standish, M. M. and Miller, N. (1965). Nature, Lond. 208, 1295. Beechey, R. B. (1966). Biochem. J . 98,284. Beechey, R. B., Roberton, A. M., Holloway, C. T. and Knight, I. G. (1967). Biochemistry, N . Y . 6,3867. Bergy, M. E. and Eble, T. E. (1968). Biochemistry, N.Y. 7, 663. Bielawski, J. (1968). E u r . J . Biochem. 4, 181. Bielawski, J., Thompson, T. E. and Lehninger, A. L. (1966). Biochem. biophys. Res. Commun. 24, 948. Birdsell, D. C. and Cota-Robles, E. H. (1967). J . Buct. 93, 427. Birdsell, D. C. and Cota-Robles, E. H. (1968). Biochem. biophys. Res. Commun. 31,438. Blake, A., Leader, D. P. and Whittam, R. (1967). J . Physiol., Lond. 193, 467. Bodanszky, M. and Bodanszky, A. (1968). Nature, Lond. 220, 73. Bodanszky, M. and Perlman, D. (1969). Science, N . Y . 163,352. Borowski, E. and Cybulska, B. (1967). Nature, Lond. 213, 1034. Bragg, P.D. and Hou, C. (1968). Gum. J . Biochem. 46, 631. Brierley, G. P. (1967). J . biol. Chem. 242, 1115. Brierley, G. P. and Settlemire, C. T. (1967).J . biol. Ghem. 242, 4324. Brock, T. D. (1966). “Principles of Microbial Ecology”. Prentice Hall, Englewood Cliffs, N.J. Brock, T. D. (1967). I n “Antibiotics”, (D. Gottlieb and P. D. Shaw, eds.), Vol. I, pp. 651-665. Springer Verlag, New York. Brown, M. R. W. and Richards, R. M. E. (1963). Nature, Lond. 207, 1391. Browning, C. H. (1964). In “Experimental Chemotherapy”, (R. J. Schnitzer and F. Hawking, eds.), Vol. 11, pp. 1-36. Academic Press, New York.

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Tostcson, D. C., Cook, P., Andreoli, T. E. aridTieffenberg, M. (1967).J.gen.PhysioZ. 50, 2513. Tostcson, 11. (j., Andreoli, T. E., Tieffenberg, M. and Cook, P. (1968). J. gera. Physiol. 51, 3738. Vallin, I. arid Liiw, H. (1968).E u r . J . Biochem. 5 , 402. Varicchio, F., Doorcnbos, N. J . and Stevens, A. (1967).J . Bact. 93, 627. Vernon, L. 1’. (1968). Hact. Rev.32, 243. Voss, J . (2. (1967). J . gen. Microbiol. 48, 391. Wahri, K., Lutsch, G., Rockstrob, T. and Zapf, K. (1968).A r c h . Mikrobiol. 63, 103. Wallsch, D. F. H. and Gordon, A. (1968). li’edn Proc. Fedn. Am. Socs exp. Biol. 27, 1263. Waltjcr, P., Lnrdy, H. A. and Johnson, D. (1967).J . biol. Chem. 242, 5014. Wcber, M. M. arid Kinsky, S. C. (1965). J . Bact. 89, 306. W c i i i l ~ x h E. , C. (1957).Proc. n!atn. A c a d . S c i . U . S . A . 43, 393. Woiribach, E. (J. and Garbus, J. (1968a). Biochem. J . 106, 711. Wc:iribach, E. C. arid Garbus, J . (1968b).Biochim. biophys. A c t a 162, 500. Wciriberg, E. U. (1967). I n “Antibiotics”, (D. Gottlieb and P. D. Shaw, eds.). Vol. I, pp. 90-101. Springer Verlag, New Yorlr. Wciriberg, E. D. (1968).Ann. N. Y . A c a d . S c i . 148, 587. Wcisor, It.,Asscher, A. W. and Wimpenny, J. (1968). N a t u r e , Lond. 219, 1365. Woissmanir, Q. arid Sessa, GI. (1967).J. biol. Chem. 242, 616. Wkiit>e,R . J. arid Hurst, A . (1968).J. gem. 3ZicrobioZ. 53, 171. Wkiitchousr:, M. W. (1964).Riochem. Pharmac. 13, 319. Whittam, R.,Wheclor, K. P. and Blake, A. (1964). N a t u r e , L o n d . 203, 720. Wilkiiisorr, R. G . (1967). J . gen. M%crobiol.47, 67. Williamsori, 12. L. arid Metcalf, R. L. (1967).Xcience, N.P. 158, 1694. Windisch, F.and Hcumann, W. (1960). Naturwissenschuften 47, 209. Winkler, H . H. arid Wilson, T. H. (1966).J . biol. Chem. 241, 2200. Wipf, H. K., Pioda, L. A. K., Stofanac, Z. arid Simon, W. (1968).Helv. chim. A c t a 51, 377. U’oodroff’e, It. C . S. and Wilkinson, B. E. (1966a).J . gen. Microbiol. 44, 343. Woodrofle, 1%. C . S. arid Wilkirison, B. E. (1966b). J . gen. Microbiol. 44, 353. Wootlriiff, H. B. (1966). I n “Biochemical Studies of Antimicrobial Drugs”, iYym,p. ~’ioc.gen. iWicrobio1. 16, 22. Wurm, M. (1951). ,7. b%ol.Chem. 192, 707. Ypl-innt,is,.I). A., Dainlro, J. L. and Schlenk, F. (1967).J . Bact. 94, 1509. do Z ~ r a i g It. , I?. a r i d Luria, S. E. (1967).J . Bact. 94, 1112.

Encyst ment in Amoebae A. J. GRIFFITHS Department of Microbiology, University College of Xouth Wales and Monmouthshire, Cathays Park, Cardiff, Wules .

106

II. ICxp(~rimorita1Approaches Employcd in the Study of Encystment

107 107 107 107 108

1. Irrtrotliiction. A . Mixed Cultures . B. Axmic Cultures . ( ’ . ltclplacement Technique. D. Measurement of Encystmt-nt

.

. . .

.

.

111. Structiiral Changes lluring Kncystmcnt . A. General B. The Cyst Wall . C. The GolgiBody . D. Aiitolysosomes . E. Mitochondria . F. Othcr Cytoplasmic OrganclI(~s C:. The Nucleus and Nucleolus . H. FoodReserves . I. Time-Course of Structural Changes

. . . . . . . . .

109 109 109 113 114 115 115 115 116 116

LV. Physiology of Encystment . ,4.Encystment in Mixed Cultures

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117 117 118 119

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123 123 124 124 125

.

126

.

126

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B. Encystment i n Axenic Cultures C. Induced Encystment .

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V. I3iocbcmical Aspects of Encystment .

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A. Respiratory Metabolism . B. Fate of Major Cell Componerits . C. Ermyme Synthesis. . D. Control of Ericystmerlt, by Metabolites VL. Kxcyst>ment.

VI I . Kesistilrice arid I’wictiori of C y sts

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W I T . Concluding Remarks

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IX. Acknowlcdgemcnts

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127 128

Heferences

. 105

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A . J GRIFFITHS

I. Introduction Many of the eucaryotic protista appear to be capable of encystment. Tn some cases, the formation of a cyst is intimately related to the processes of sexual or asexual reproduction, but it is generally considered t o be a response to environmental conditions which are suboptimum for growth of the organism. Encystment usually involves a drastic reorganization of the subcellular structure of the vegetative cell in which cilia, flagella, vacuoles and other inclusions disappear. The process is not wholly dcgradative however, as these changes are usually accompanied by the synthesis of the various elements of the cyst wall. The resulting cyst, which is usually a spherical, refractile cell with a wrinkled cyst wall, apparently represents a cryptobiotic stage in the life-cycle of the organism and is thought to be refractory to adverse environmental conditions. Much of our knowledge of encystment is based on data which are included in reports of a more general nature. For this reason it has not been possible to review the literature exhaustively. An earlier review (van Wagtendonk, 1955) has emphasized the contradictory nature of much of the information relating to encystment. This is undoubtedly due to the unsatisfactory agnotobiotic culture methods used for many protozoa and algae, and the resulting lack of control of experimental conditions. The soil-inhabiting hartmannellid amoebae may now be grown axenically in complex and defined media (Adam, 1959, 1964; Band, 1959, 1962) and this review will centre mainly on recent studies of the encystment of these organisms. Although the present state of knowledge of the hartmannellids is still only in a rudimentary stage, these organisms nevertheless present an opportunity for constructing models of encystment which will further our understanding of encystment in thosc organisms which are difficult to culture under axenic conditions. For example, the cyst is important in the transmission of the parasitic amoebae, and encystment is believed to be important in preserving the invasiveness of Entamoeba histozytica (Neal, 1965) but a recent review (Mcconnachie, 1969) has shown the extent to which inadequate culture techniques are hampering investigations of encystment in members of this genus. Encystment can also be considered as an example of cellular differentiation (‘Frager, 1963; Neff et al., 196410; Tomlinson and Jones, 1062). Similar studies of the sporulation of cellular slime-moulds (Sussman and Sussman, 1969) and bacterial sporulation (Murrell, 1967 ; Mandelstam, 1969) show that simple microbial systems provide useful models for cellular differentiation. Their attraction lies in their amenability to investigation under controlled experimental conditions

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and, unlike cellular differentiation in Metazoa and Metaphyta, differentiation of microbial cells is usually uncomplicated by simultaneous growth.

11. Experimental Approaches Employed in the Study of Encystment A. MIXED CULTURES Many of the earlier investigations of encystment were carried out in mixed cultures, which ranged from the completely agnotobiotic to monoxenic cultures in which the protozoan was grown with a known microbial associate, usually a bacterium. Much of the work carried out under these conditions has been only observational but there have also been a number of experimental investigations (van Wagtendonk, 1955). The use of these types of cultures imposes considerable practical difficulties even when the biotic composition is known, and it makes the establishment of satisfactory controls and interpretation difficult. It is not surprising that the earlier work on encystment has yielded a body of largely contradictory information (see Section IV.A, p. 117). Studies are now beginning of the nature and extent of the interactions which occur in mixed microbial cultures (see, for example, Hobson, 1969) but it is quite clear that our knowledge is so rudimentary that i t is doubtful that the use of mixed cultures is of any value a t present in the study of encystment.

B. AXENICCULTURES Although axenic cultures, in which the protozoan is maintained in the absence of any other microbial associate, are far more controllable than mixed cultures they are not necessarily completely specified. For example, the complex media usually contain highly variable components such as protein digests, tissue extracts, and skimmed milk. Despite the fact that these complex media often give reproducible growth yields they are, in many ways, unsuitable for developmental studies. This is particularly true ofencystment whichis quiteincompatible with growth and usually occurs in cultures in which growth has ceased presumably as a result of nutrient depletion or accumulation of waste materials. Even completely defined media can only be considered to be specified a t the time of inoculation (see XectionIV.B, p. 118) and lose their definition as growth proceeds.

C. REPLACEMENT TECHNIQUE The existence of axenic cultures of amoebae has made possible another approach to the investigation of encystment which consists of replacing the growth medium by a non-nutrient saline (Band, 1963; Neff et al.,

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l964a). The method is essentially similar t o that which has been used in studies of cndotrophic sporulation in bacteria and has the advantage that enczystment can be separated completely under conditions which do not allow growth. By employing this technique, Neff et al. (1964a) found that Acantharnoeba sp. gave 90% encystment in the medium shown in Table I(a) and that the process showed a good degree of synchrony. Band (1963) has obtained an average encystment of 55% (calculated from Band’s results) with Hartrnannella rhysodes in an unbuffered replacement medium (Table I(b)). Tomlinson (1967) used a modification of the Neff group’s medium in which the amine buffer was replaced by 2 mM-phosphate buffer (pH 6.8) and bicarbonate was eliminated. Tomlinsoii (1967) does not give details of the degree of enc.ystment obtained with Acantharnoeba in this medium. The simplest encystment medium is that of Griffiths and Hughes (1968) which consists of‘ unbuffered 50 mM-magnesium chloride and in which 90-92yo rncystment was obtained with Hartmannella castellanii, H . astronyxis and Mayorella palestin ercsis. T A m E

1 . Cornposjtiori of Rcplaccmcnt M d i a for Encystment of Amocbao

for Acrxnthamoeba sp. (Ncff et al., 1 9 6 4 ~ ) Potassium chloride 100 mM Amirie buffer 20 m M Magnesiirm sidphate 8 mM Cttlciiim chloride 4 mM Sotliiim bicarbonate 40-1.0 mM

(a,) Mlctlium

( b ) Mcdiurn for Hartmannellu rhysodes (Band, 1963) Sodium chloridc 0.25 M (0.5 osmolar) Magnesium chloride 5.16 mM Calcium chloride 0.36 mM

U. MEASUREMENT OF ENCYSTMENT Most studies of encystment have relied on microscopic examination of samples and direct counts of organisms using conventional haemocytometer counts. Apart from the inaccuracies inherent in these procedures, there is also a coiisiderable subjective element when attempts are made to distinguish between two cell types. This is especially true of encystment. For example, Band (1963) refers t o the occurrence of “round-forms” in encysted cultures, and Griffiths and Hughes (1969) wcrc able to produce refractile amoebae by adding magnesium chloride to growing cultures of H . castellanii. Neither of these cell types is a true cyst but, by lacking pseudopodia and in the latter case possessing refravtility, could be mistaken as such in a cell count. Quite clearly the

ENCYSTMENT IN AMOEBAE

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existence of aberrant cell types such as these casts doubt on the validity of microscopic methods in quantitative studies of encystment. Another technique has been used by Griffiths and Hughes (1969) which involves measurement of the cellulose produced by encysted amoebae. The cellulosic inner walls of the cysts are easily extracted by hot alkali and are left as intact “ghosts” which can be measured turbidimetrically or gravimetrically. This method, involving the measurement of’ a component characteristic only of cysts, was found to be particularly valuable in studies involving the use of inhibitors and promotors of encystment in which interference by necrotic, non-encysting amoebae which do not contain cellulose was eliminated.

111. Structural Changes During Encystment A. GENERAL The degree of structural re-organization in an encysting organism will depend on the morphological complexity of the vegetative form. The general protozoological texts (Hyman, 1940 ; Kudo, 1954 ; McKinnon and Hawes, 1961) give good accounts of the gross structural changes which accompany encystment. I n the majority of Protozoans which exhibit encystment, the process usually involves the degradation of the organelle systems, such as the resorption of cilia, flagella and myonemes, and the disappearance of the food vacuoles and eventual loss of the contractile vacuoles. These changes are usually accompanied by a decrease in the size of the organism and an increase in its surface area: volume ratio by the adoption of a spherical form. The cyst is frequently more refractile than the vegetative organism and this is usually attributed to dehydration during encystment; but the work of Klein (1959), which suggested that cysts of Acanthamoeba had a higher water content than the vegetative amoebae, must be set against these observations.

B. THECYSTWALL The cyst wall may consist of from one t o three layers, and there appears t o be a great degree of variation in the chemical composition and morphology of these structures from species to species. Kudo (1954) cites chitin and cellulose as being the comnionest components of cyst walls. The cyst walls of parasitic amoebae contain a proteincarbohydrate complex (see McConnachie, 1969). Incrustation of the outer layer with minerals is not uncommoii (Hyman, 1940). Much of the information regarding the chemical composition of cyst walls has been obtained by the application of cytochemical techniques. The cyst wall of Acantharnoeba sp. has, however, been the subject of chemical analysis by Tomlinson and Jones (1962) who found that cellulose was a major component comprising about 30% of the wall dry

110

A . J . GRIFFITHS

weight, and was synthesized de novo by the encysting amoebae. Neff et al. (1964a) have shown that the mature cyst wall of Acanthamoeba contains, as well as cellulose, up to 6.5% of its dry weight as lipid, 33% protein, and 7-8% ash. These authors also detected the presence of nucleotides and organic acids. The cysts of Acanthamoeba sp. require lipid extraction before giving a positive reaction with Schulze’s solution, which is a specific reagent for the demonstration of cellulose (Neff and Benton, 1962). Cysts of Hartmannella custellanii have also been shown to contain cellulose after lipid extraction or heat-fixation (Griffiths and Hughes, 1968) and, in this organism, cellulose constitutes up to 27% of

FIG.1. Cyst of Acanthamoeba sp. This is an early cyst in which the lipid droplcts ( 1 ) have not yet moved t o the periphery of the cell. Autolysosomal material (a) is seen omlncdded in the exocyst (ex) and the Golgi vesicles (g) are discernible below thc endocyst (en). The water-expulsion vesicle (wev), ostioles (0) and mitochondria (m) are also t o bc seen. Magnification x 7620. From Bowers and Korn (1969).

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the dry weight of the whole cyst (Griffiths and Hughes, 1969). Cellulose also appears to form a large proportion of the walls of slime-mould cysts (Blaskovics and Raper, 1957) and is also one of the principal products synthesized during the complex morphogenesis shown by the Acrasiales. The cyst walls of the free living amoebae Acanthamoeba spp. (Vickerman, 1962; Bowers and Korn, 1969) and Naegleria sp. (Schuster, 1963) bear some resemblance to that of Entamoeba invadens (Deutsch and

FIG.2 . Dptail of the cyst wall of Hurtrraannella castellanii. The ostiole ( 0 ) is formed by the closely apposed exocyst (ex) and endocyst (on). Magnification x 30,000.

Zaman, 1959) in being composed of two electron-dense layers separated by an electron-transparent layer (Figs. 1, 2 and 3). I n E . inwadens, the electron-transparent layer appears stratified in sections which were treated with formalin (Deutsch and Zaman, 1959). The NaegZeria cysts have definite pores which are closed by an amorphous electron-transparent material (Schuster, 1963). Bowers and Korn (1969) have also demonstrated pores in Acnnthnmoeba but these have an operculum which retains the structure of the wall in that it is composed of two layers which are more closely apposed than in the wall proper. The outer layer of the Acai.zthamoebawall (the exocyst) is itself doublelayered with an outer amorphous layer overlying an essentially fibrillar layer (Bowers and Korn, 1969). The fibrillar exocyst may also contain

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A . J . GRIFPITHS

FIG.3. A mature cyst of' Acanthamoeba sp. with a prominent water-expulsion vcsicle (wrv) arid i n which thc lipid droplets (1) have moved t o the pcriphcry of tho ccll. Magnification x 18,600. From Bowers and Korn (1969).

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amorphous material or even cell debris, including glycogen granules, embedded within it. The inner component (the endocyst) of the walls of Acnnfhamoebn sp. and H . castellanii has been shown, by cytochemistry, to be the celluloselayer (Neff and Benton, 1962; GriffithsandHughes, 1969). In the electron micrographs of Bowers and Korn (1969), the endocyst is seen to consist of fibrils which have a diameter of less than 50 A, but Vickerman (1962) estimates their diameter as about 100 A. Bowers and Korn (1969) have followed wall synthesis in amoebae induced t o encyst by the replacement technique. It appears that the exocyst is secreted first and that this process is initiated a t the time when the amoebae assume a spherical form during the first few hours immediately following suspension of the amoebae in the encystment medium (see Section IV.C, 1’. 122). C. THEGOLGIBODY Certain changes in the appearance of the Golgi apparatus also seem t o coincide with exocyst synthesis. In encysting amoebae, the volume fraction of the cell occupied by the Golgi body increases and the complex becomes more dispersed. Vesicles, about 70 nm. in diameter, are pinched off the Golgi body and these seem to migxate t o the periphery of the

FIG.4. Cyst wall of Hartwbannella castellanii containing autolysosomal material (a). The membrane-bound elements below the endocyst (g) are suggestive of the vcsicles produced by the Golgi bodies which have been implicatcd in wall synthesis. Magnification x 60,000.

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amoebae where they discharge their contents on to the surface of the cell (Bowers and Korn, 1969; Fig. 4). Cytochemical tests (Neff et al., 1964a) and incorporation studies with labelled hydroxyproline (Bauer, 1967) indicate that the exocyst is proteinaceous.

D. AUTOLYSOSOMES When the cell has become spherical in the initial stages of encystment, autolysosomes (de Duve and Wattiaux, 1966) appear and persist throughout encystment. These vacuoles contain mitochondria, lipid droplets, glycogen and other cytoplasmic material and have also been shown to contain acid phosphatase (Bauer, 1967). The autolysosomes migrate to the surface where they discharge their contents which become trapped in the exocyst (Figs. 1 , 4 , 5 ) . The presence of cell debris in this layer may account for the detection of nucleotides and organic acids in isolated cyst walls reported by Neff et al. (1964a).

FIG.5. An autolysosome (A) in encysting Hartmannella castellanii. Magnification x 120.000

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E. MITOCHONDRIA The mitochondria of Acanthamoeba, Schixopyrenus and Naegleria spp. show interesting structural features during encystment (Vickerman, 1960, 1862; Bowers and Korn, 1969; Schuster, 1963). I n Naegleria sp. the mitochondria become elongated in a manner resembling division stages but, in the cysts of both Xchixopyrenus and Acanthumoeba sp., they are spherical and of a smaller overall diameter than those of the vegetative amoebae although occupying a fairly constant fraction of the cell (Schuster, 1963; Vickerman, 1962; Bowers and Korn, 1969). Bowers and Korn (1969) observed coiled lamellated structures within the mitochondria in Acanthamoeba sp. These appeared to be extruded by the mitochondria as they were also seen free in the cytoplasm but are not present, in any situation, in the mature cysts. Mitochondria from Acantharnoeba sp. also contain granules which are probably of some inorganic material. I n the cyst these granules become swollen and occupy a considerable proportion of the lumen of the mitochondria in which they appear as vesicles 3,000-5,000 A in diameter. Although they are still recognizable, the cristae of cyst mitochondria are less well defined than those of the vegetative cell and have presumably undergone some degeneration (see Section V.A, p. 123).

B. OTHERCYTOPLASMIC ORGANELLES During the secretion of the exocyst, a definite cortical layer is distinguishable at the periphery of the cell. This is a quite hyaline region which is devoid of organelles or other inclusions except for small amounts of rough endoplasmic reticulum. Smooth endoplasmic reticulum has not been observed in cysts (Bowers and Korn, 1969). The encystment of amoebae is accompanied by a period of considerable vacuolar activity and, in Acanthamoeba sp., by intense activity of the water-expulsion vesicle (Fig. 3, p. 112) which, it is claimed, results in dehydration of the encysting amoebae. The activity of the waterexpulsion vesicle is believed to be particularly responsible for the wrinkled appearance of the cyst which occurs as a result of the cytoplasm drawing away from the exocyst. This occurs prior to cellulose synthesis and explains the separation of the endocyst and exocyst in mature cysts (Bowers and Korn, 1969).

G. THE NUCLEUS AND NUCLEOLUS Encystment is also a period of visible nuclear and nucleolar activity. Both the nucleus and nucleolus decrease in volume during cyst formation.

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The nucleus of Acanthamoeba sp. has been seen to form buds which do not appear t o contain nucleolar material, and many of these buds are incorporated in the autolysosomes. Schuster (1963) reported the existence of localized concentrations of RNA in Naegleria sp. which occur as particles packed within vacuoles. These concentrations probably occurred at the expense of nucleolar RNA. A similar dissimilation of the nucleolus has also been reported for Acanthamoeba sp. (Bowers and Korn, 1969), H . castellanii (Volkonsky, 1931), H . astronyxis (Ray and Hayes, 1954), and Entamoeba sp. (Barker and Svihla, 1964). The chromatoids of Entamoeba spp. are extreme examples of extra-nucleolar concentrations of RNA (McConnachie, 1969). These have been shown to be aggregates of ribosomes which, in some cases, degenerate during maturation of the cyst (Morgan et al., 1968).

H. FOOD RESERVES I n free-living amoebae, the glycogen reserves of the vegetative amoebae become depleted during encystment but may become incorporated in the exocyst. The glycogen of parasitic amoebae, however, forms into one or more masses which occupy substantial volumes in the cyst (McConnachie, 1969). In Acanthamoeba sp. lipid droplets occupy an increased volume fraction of the cyst which may be explained by the decrease which occurs in the general cytoplasmic volume (Bowers and Korn, 1969; Fig. 3, p. 112).

I. TIME-COURSE OF STRUCTURAL CHANCES The use of the replacement technique has made it possible to follow the time-course of encystment. Using visual criteria, Neff et al. (1964b) suggest that cyst formation in Acanthamoeba sp. proceeds by three stages-pre-encystment, cyst initiation and, finally, the phase of cyst wall synthesis. During the initial pre-encystment stage, the amoeboid character of the cells is still apparent in that pseudopodia are still formed. The initiation of the cyst occurs 5-6 hr. after suspending in the encystment medium when the amoebae lose their pseudopodia and become rounded. One feature of this stage is that the cells become “sticky” and aggregate into clumps. The cyst wall is not yet visible and does not appear until about 12 hr. after removal from the growth medium. The rest of encystment appears to be concentrated on the thickening of the wall; this is completed in about a further 12 hr. Thus, according to this report, encystment is completed after about 24 hr. which is considerably shorter than the 32-36 hr. reported by Band (1963) to

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be necessary for the complete encystment of H . rhysodes. By measuring cellulose synthesis in encysting H . castellanii, Griffiths and Hughes (1969) found that measurable quantities first appeared a t about 14 hr. and increased up to about 30 hr. I n the first 8-10 hr. incubation in the encystment medium, the amoebae are highly vacuolated and also have pseudopodia. At 10-15 hr., they adhere to form clumps and become rounded ; the vacuoles and pseudopodia disappear a t this stage. At 15 hr., the cyst wall is discernable and 7 0 4 0 % of the cells give a positive reaction with the zinc chloro-iodine reagent. These authors concluded that, after its first appearance, much of the increase in the quantity of cellulose was attributable to an increase in its quantity per cell rather than being a measure of an asynchronous encystment response. It is quite clear that encystment is a considerably longer process than growth. The fastest generation time for Acanthamoeba sp. appears to be 11-12 hr. at 30”, which is also the optimum temperature for encystment (Neff et al., 1964b), and H . castellanii has a mean generation time of 8-75 hr. (A. J. Griffiths, unpublished observations).

0

IV. Physiology of Encystment A. ENCYSTMENT IN MIXED CULTURES

Much of the earlier work relating to the elucidation of the environmental factors which act as stimuli for the encystment response was carried out in monoxenic cultures. This literature has been reviewed by van Wagtendonk ( 1955) who emphasizes the contradictory nature of the information obtained from these sources. Among the causes listed by van Wagtendonk (1955) are (i) deficiency of food, (ii) excess of food, (iii) accumulation of products of both protozoan and bacterial excretion, (iv) change in the pH value of the culture, (v) desiccation of the culture, (vi) lack of oxygen, and (vii) crowding. There have also been reports of “encystment-inducing factors” in crowded cuItures (see, for example, Strickland, 1940). Cyst formation by Entamoeba sp. has also been largely investigated in mixed cultures in which the amoebae are generally associated with bacteria. I n many cultures only a few amoebae in the population encyst. In others, however, a mass synchronous encystment may occur (McConnachie, 1969). Encystment in these amoebae generally follows periods of vigorous growth which culminate in conditions unfavourable for growth. Crowding, depletion of starch in the medium, and exposure to hypotonic conditions are also reported to be effective stimuli to encystment. Cysts have not been found in cultures of E . histolytica grown with Trypanosoma cruzi.

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B. ENCYSTMENT IN AXENICCULTURES Neff et ul. (1964b) found that amoebae of Acantlmmoeba sp. undergo asynchronous encystment in growth medium 5-10 days after growth had stopped. I n these experiments, in which the amoebae were grown in shallow liquid medium, only 50-60% of the cells present a t the end of the growth phase encysted and the process extended over a month or so. I n aerated cultures of the organism in the same medium, 60-70% of the amoebae formed mature cysts within about 1-2 days of the cessation of growth. Griffiths and Hughes (1969) found that, by adding magnesium chloride to a culture of H . castellanii in the logarithmic phase, growth Time (hours)

Arsenite and azide Inhibit

j

I

Dry weight decreases : Q ~ ~ i n c r e a s e: s actinomycin -D inhibits -

Tetracycline inhibits

0

+

-

Sontents of pentoses and hexoses increase

o n E C

Sontents of protein and amino acids decrease

50 xLY O W

Exocyst initiated autolysosomes appear

t c c o 3 w

Glyoxylote cycle operates

;T)

a w 01,. Glutamate and histidine inhibit

€$

0"s

FIG.6. A diagram of some of the events occurring during the encystment of hartmannellid amoebae in replacement media. The data are based on those of Neff et al. (1964b), Griffilhs and Hughes (1969), Bowers and Korn (1969) and unpublished observations of S. M. Bowen.

ceased and the amoebae became spherical and refractile. Cellulose was not detectable in these forms which therefore did not satisfy the biochemical criterion of encystment adopted by these authors. Neff and Neff (1966) were able to induce encystment in Acanthamoebu sp. by adding various inhibitors to shallow cultures of the organism, . but the amoebae were only susceptible to induction by these agents

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during a restricted portion of the “growth division cycle”. The most effective inducers of encystment in this system were inhibitors of DNA function or synthesis and inhibitors of protein synthesis (cf. Band, 1962; Griffiths and Hughes, 1969; see also Section V, p. 124 of this review). It was concluded that encystment occurs when the cells are arrested during the phase of DNA synthesis. Cysts have not been found in axenic cultures of Entamoeba histolytim but Richards et al. (1966) were able to induce cyst formation by E. invadens in hypotonic media. McConnachie (1969) confirmed this result and increased the yield of cysts to 60-70% of the population from the low values of l - l O % which are usual in normal undiluted growth media. Band (1962) examined the encystment of H . rhysodes on solidified complex media. I n these experiments, treatments which maintained the humidity of the culture delayed or even prevented encystment. Another approach to the study of encystment of amoebae in growth medium is promised by the use of defined media (S. M. Bowen, personal communication). This involves the elimination (or addition) of metabolites from the basic medium in an attempt to obtain information about the balance which undoubtedly exists between growth and encystment. C. INDUCED ENCYSTMENT I . Osmotic and Ionic Requirements f o r Encystment

Reports of the osmotic requirement for encystment of soil amoebae give conflicting results. According to Band (1 962) the optimum tonicity for cyst formation in H . rhysodes in carbon- and nitrogen-deficient media is given by unbuffered 0-2-0.3 M-sodium chloride. Sucrose or potassium chloride a t iso-osmotic concentrations were equally effective in supporting encystment. Neff et al. (1964b) obtained maximum encystment of Acanthamoeba sp. in 0.1-0.15 M-potassium chloride buffered with 0.02 M-amine buffer. I n this medium also the potassium chloride could be replaced by sodium chloride. None of these media was found to be suitable for the encystment of H . castellanii (Griffithsand Hughes, 1968) which encysted in 0.02 M-0.05 M-magnesium chloride alone. This medium was also found to be effective for H . astronyxis and Ma yorella palestinensis. As well as the osmotic requirement, both Acanthamoeba sp. and H . rhysodes also required magnesium and calcium ions. When present together these ions appeared to accelerate the wrinkling of H . rhysodes cyst wall. Band (1963) found that the chelating agent ethylenediaminetetraacetic acid (EDTA) inhibited encystment of H . rhysodes, and Neff et al. (1964b) attributed the inhibitory effect of phosphate

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buffer on cyst formation by Acanthamoeba sp. to its likely removal of Mgz+ and Ca2f a t the higher pH values used in their system. Griffiths and Hughes (1968,1969)investigated some of the effects of the magnesium ion on H . castellanii in nutrient-deficient media. I n short, they found that magnesium chloride inhibited the efflux of ultraviolet-absorbing materials (nucleotides, amino acids and proteins) from the amoebae, decreased cell loss by lysis, and promoted cellulose synthesis, The requirement for magnesium was not a result of its deficiency during growth. These authors did not find any calcium requirement in this organism. Similar effects exerted by magnesium ions have also been noted for amoebae of the cellular slime-mould Dictyostelium discoideum (Krichevsky and Love, 1965; Krichevsky and Wright, 1963). It was suggested by Krichevsky and Love (1965) that the magnesium ion has its effect a t the surface of the amoeba thereby altering the permeability of the membrane and preventing the efflux of macromolecules into the suspending medium. However, magnesium ions are known to activate many enzyme systems (Dixon and Webb, 1964), stabilize ribosomes (Dagley and Sykes, 1957) and increase the availability of hydrolases in isolated lysosomes (Sawaiit et ak., 1964) and it is possible that they may exert some effect a t these levels in encysting amoebae. Encystment is a period of demonstrably intense metabolic activity (see Section V, p. 123) and is sensitive to inhibitors of protein synthesis and RNA synthesis (Band, 1963; Griffiths and Hughes, 1969). Furthermore the identification of autolysosomes in encysting Acanthamoeba sp. makes this another possible site of action for magnesium, and there is now some biochemical evidence for this possibility (A. J. Griffiths and S. M. Bowen, unpublished observations; see also Section V, p. 124). I n a “closed system” such as encystment (Wright, 1967), inhibition of cell leakage should not be minimized however as the amoebae must be entirely dependent on endogenous materials for all metabolic activities. 2. Age of Amoebae and Composition of the Growth Hedium

It is not surprising that the physiological state of the amoebae is an important determinant of their encystment response in non-nutrient conditions. Acanthamoeba sp. was judged to give the most rapid and synchronous encystment when removed to the replacement medium in the stationary phase after growth in a medium which produced the shortest generation time (Neff et al., 1964b). The ability of cultures of H . castellanii t o undergo encystment, as measured by celluloseproduction, appeared t o decline throughout the growth phase (Griffiths and Hughes, 1969) although it is possible that the proportion of amoebae which eventually formed cysts did not decline in the same manner. This organism was also sensitive to the composition of the growth medium. The growth

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rnediuni normally used in the studies with H . castellanii was a liver digest, but it was found that an essentially equivalent response was obtained with amoebae grown in a peptone-glucose-yeast extract medium. Elimination of glucose from this medium, however, considerably impaired the ability of the amoebae to undergo encystment. Addition of glucose to the liver-digest medium did not greatly affect subsequent encystment but the dry-weight loss by the amoebae, which is usually considerable during encystment (about 40%)) was decreased quite dramatically (lowest value, 13.2%). Band (1963) reported that the encystment of H . rhysodes was equally efficient in amoebae grown in defined media as in organisms harvested from a richer peptoneglucose medium. He did find, however, that amoebae grown in static cultures gave higher yields of cysts than thosegrownin ashakerincubator. Some populations of Acunthamoeba sp. gave a higher degree of synchrony following a period of “training” which entailed the serial culture of the organism in aerated cultures (Neff and Benton, 1964). Some of these lines required only about one month of “training” and these were characterized by a generation time which decreased. Other populations in which the generation time remained low even after three months’ “training” gave a low degree of encystment synchrony. It appears that, in this laboratory prior to these studies, the organism was routinely maintained in shallow, static cultures. I n the studies of Griffiths and Hughes (1968, 1969) the amoebae of H . castellanii were maintained in exactly the same manner as that used for growing them for encystment experiments which, in principle, is the same as that adopted in the “training” procedure. Under these conditions, the generation time of H . custellanii has not been observed to change over four years. 3. The Effect of p H value on Encystment

The importance of the buffering capacity of the medium in the encystment of Acanthamoeba sp. has been thoroughly investigated by Neff et al. (1964b). It appears that different p H values are required at the two stages during the pre-encystment phase and cyst-wall synthesis. The highest degree of synchrony was obtained by maintaining the pH value a t neutrality in the early stages of encystment followed by the addition of alkali during wall formation which is optimum a t pH 8.6-9-0. Both H . castellunii and H . rhysodes encysted in unbuffered media Griffiths and Hughes, 1969; Band, 1963), but in these studies synchrony was not measured as was the case in the Acanthamoeba investigation. Griffiths and Hughes (1969) found that, in their unbuffered media, encystment of H . castellanii occurred in media in which the initial p H value had been adjusted within the wide range pH 3-8.5. At

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the end of encystment, however, the pH value of the medium had risen in these cultures, initially a t pH 3-7, to a value of p H 8.5. Addition of either tris- or phosphate buffer to the replacement medium impaired encystment in this organism. 4. Temperature and Aeration

Encystment of the hartmannellid amoebae is maximal a t temperatures in the range 25"-30" (Band, 1963; Neff et al., 1964; Griffiths and Hughes, 1969). Encystment does not occur a t 37"-40", but short exposures of H . rhysodes t o 10" during the washing and harvesting stages does not affect the encystment response. Neff et al. (1964b) obtained evidence that cyst initiation was more temperature-sensitive than the preencystment phase. Encystment will not proceed under oxygen-deficient conditions. Incubation of H . castelhnii in replacement medium without shaking inhibited encystment by SO-SO% (Griffiths and Hughes, 1969) and aeration of encysting Amnthumoeba sp. below 1.5-2.0 cubic feet per hour per litre of culture lengthened all stages of encystment. Using cell concentrations of 105-2 x loGcells per ml., Neff et al. (196413) found that optimum synchrony of Acanthumoeba sp. was obtained with an aeration rate of 4 cubic feet per hour per litre, but cell breakage occurs a t rates above 6 cubic feet per hour per litre. Hartmnnella rhysodes did encyst in static cultures but it may be of significance that the amoebae were also grown under these conditions (Band, 1963). 5. Phagocytic Behuviour

I n an investigation of the phagocytic activity of encysting Amnthamoeba, Weisman and Moore ( 1969) demonstrated a remarkable decline in the ability of the amoebae to engulf polystyrene and polyvinyltoluene beads. This decline appears to begin almost immediately following the suspension of the amoebae in the encystment medium. At zero time encystment, uptake of the beads was about 30% of that of amoebae suspended in growth medium. After encystment for three hours, this had fallen to about 25% and progressed further until, after 24 hr. encystment when 87% of the amoebae had formed mature cysts, bead uptake was immeasurable. Another aspect of this behaviour was that amoebae which contained engulfed beads a t the start of encystment lost them by about 20 hr. incubation in the encystment medium. These beads which were lost from the cells could be observed in the encystment medium. The time-course of these changes suggests that changes in the phagocytic behaviour of the amoebae occur when the cells are assuming the spherical appearance which is prior to and during primary wall (exocyst) synthesis.

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V. Biochemical Aspects of Encystment A. RESPIRATORY METABOLISM The first ten hours of encystment in non-nutrient replacement media is marked, in H . castellanii, by a dramatic increase in the rate of oxygen uptake of the amoebae. After this initial increase, the rate of oxygen consumption decreases gradually to an immeasurable value by the time cyst formation is complete (Griffiths and Hughes, 1969). The amoebae also show changes in the utilization of exogenous substrates during the first ten hours or so of encystment. Encystment of H . castellanii is inhibited by iodoacetate, arsenate and arsenite. Malonate and 2,kdinitrophenol were not inhibitory, but amoebae that encysted in the presence of these compounds lost about 20% or more dry weight than the controls, and they were also effective in decreasing the rate of oxygen consumption by the cells. Sodium fluoride actually stimulated cellulose production by 50%. Band (1963) found that both malonate and 2,4-dinitrophenol inhibited encystment in H . rhysodes, but that the inhibition could be reversed by addition of sodium acetate or glucose. Mitochondria isolated from encysting amoebae showed considerable variations from those isolated from vegetative amoebae (Griffiths, 1967; Griffiths et al., 1967; Lloyd and Griffiths, 1968). Respiration rates with a number of substrates were higher in mitochondria isolated from amoebae after 4 hr. encystment but, a t later stages of encystment, respiration with the same substrates had decreased. These changes were accompanied by impairment of the phosphorylating ability of mitochondria and an apparently progressive loss of cristal organization. In their investigation of the osmotic properties of mitochondria isolated from Acanthamoeba, Klein and Neff (1960) found that hypotonicity of the medium led to an increase in mitochondria1 size and respiration rates. It is interesting to note that magnesium had a similar effect. The suggestion of these authors that dilution of the environment could result in an increase in the rate of respiration of whole amoebae is certainly supported by the measurements made with H . castellunii. This may indicate an extremely intimate relationship between the environment and the intracellular organelles of these amoebae. Although of an essentially preliminary nature, these results reveal encystment under these conditions as requiring initially intense respiratory activity. The experiments with some of the inhibitors may have been complicated by permeability factors, but it is not possible, a t this stage, to draw any conclusions regarding the pathways involved in energy production.

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B. FATEOF MAJORCELL COMPONENTS Soil amoebae show considerable similarities in the pattern and extent of their endogenous metabolism during encystment to that of sporulating slime-moulds. One of the features of cyst formation is the use of endogenous materials in a controlled fashion. Hartmannella castella?aii loses 40% of its dry weight, mostly in the first ten hours of encystment, and the same proportion of cellular protein is degraded or excreted over a period of thirty hours (Griffiths and Hughes, 1969). There is also an increase in the amount of carbohydrates and ribose per cell during the initial 20 hr. and 10 hr. of encystment respectively. The amount o f free amino acids per cell does not change measurably. I n other experiments in which 14C-labelledamoebae were encysted, 27.5% of the label appeared in the supernatant after 72 hr. encystment and 14% was collected as 1 4 C 0 2 . O f the label remaining in the amoebae, 32% was incorporated into the cellulose fraction. I n contrast to this, Neff et al. (1964a) reported that 80% of the nitrogen lost by Acantharnoeba sp. during cyst formation was recovered from the supernatant encystment medium. More than half of this could be accounted for as identifiable amino acids. These changes in the chemical composition o f encysting amoebae are in accord with the report of the presence of autolysosomes in Acanthamoebu sp. (Bowers and Korn, 1969; see Section 111, p. 114). Recent studies in which the distribution and behaviour o f acid phosphatase (a convenient lysosomal marker enzyme) were examined in encysting H . castellanii have shown that magnesium ions, glutamate and glucose, which are capable of initiating or modifying the encystment response, also affect this enzyme (Bowen and Griffiths, 1969).

C. ENZYME SYNTHESIS Encystment of both H . castellanii and H . rhysodes is sensitive to inhibitors of protein synthesis and RNA synthesis (Griffiths and Hughes, 1969 ; Band, 1963). Hartmannella castellanii is completely inhibited by tetracycline and actinomycin, and chloramphenicol inhibits encystment of H . rhysodes by 70-80%. This suggests that enzyme synthesis is necessary for encystment in replacement media but, as yet, there have been no published reports of newly synthesized enzymes in soil amoebae during encystment. Tomlinson (1967) found that the levels of two key enzymes of the glyoxylate shunt, isocitrate lyase and malate synthase, both increase in Acanthamoeba sp., and he proposes that this pathway is involved in the synthesis of cellulose a t the expense of cellular lipids. The hartmannellid amoebae have been shown to possess peroxisomes, which are usually the locales for the enzymes of the

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glyoxylate pathway (Muller and Mdler, 1967 ; Bowen and Griffiths, 1969) but this work is still only in its preliminary stages. I n the slime moulds, the newly synthesized enzymes appear to be largely involved in the synthesis of the mucopolysaccharides and polysaccharides which are characteristic of the sporulating amoebae and it would be surprising if this were not the case in encystment. I n H . castellanii, actinomycin begins t o exert its effect up to about 10 hr. encystment, and tetracycline up to 17 hr. (S. M. Bowen, unpublished results), which coincides with the phase of encystment a t which cellulose synthesis begins in this organism. Furthermore, glucose was effective in reversing inhibition by actinomycin D. Examination of more enzyme activities measured in cell homogenates without reference to the possible compartmentalization of the enzymes in question, and the concentrations of their substrates or the products of their activity, are clearly of limited value in studies of this nature. These points have been discussed a t length elsewhere (Wright, 1964; Sussman and Sussman, 1969) but have been frequently overlooked by previous workers. It is also important that care should be exercised in choosing the unit of activity. The use of specific enzyme activities based on protein or dry weight can frequently lead to misinterpretation when applied to encysting or sporulating amoebae in which protein degradation or dry-weight loss are necessary features of the differentiation process (Wright, 1964).

D. CONTROLOF ENCYSTMENT BY METABOLITES Until further progress is made using defined media in studies on encystment, another fruitful approach is likely to be provided by the addition of metabolites to the replacement medium. The presence of glucose during encystment stimulates cellulose synthesis by H . castellanii and also exerts a sparing effect on the dry-weight loss by the amoebae (Griffiths and Hughes, 1969).This suggests that much of the degradation of cellular components of the vegetative amoebae which occurs in cyst formation is directed towards the provision of the hexose monomers required for cellulose synthesis. The intermediate, a-ketoglutarate has, to a lesser extent, the same effect as glucose which indicates that the source of the cellulose precursors may be protein. Glutamate and histidine are both inhibitory to the encystment of H . castellanii. Other amino acids have no effect, however, and encystment will occur in the defined medium under certain conditions. It isinteresting, in this context, that glutamate is the most abundant of the free amino acids found in the peptone growth-medium used for this organism, and that even 1% (w/v)peptone is, itself, an effective inhibitor of encystment although it does not support growth.

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I n contrast to these results, Band (1963) found that glucose inhibited the encystment of H . rhysodes as did acetate and citrate. This is rather surprising in view of the reported reversal of malonate inhibition which was obtained with glucose and acetate in this organism (see Section V.A, p. 123).

VI. Excystment The process of cyst germination, that is excystment, has received even less attention than encystment. I n some ways, it holds even more interest than cyst formation in that it involves the activation of the quiescent cyst and a re-establishment of the vegetative metabolism. The reversal of the degenerative changes in the mitochondria should, for example, throw some light on the mechanism, and its control, of the biogenesis of these structures which is one of the central problems of contemporary cell biology. The small amount of evidence available shows that, in their germination behaviour, the cysts of H . castellanii bear some resemblance to slime-mould spores. Using essentially bacteria-free conditions, Cotter and Raper (1966, 1968) have shown that Dictyostelium spores germinate fully within 5 hr. in a variety of media after a short (30 min.) heat-shock treatment a t 45". Germination, which was measured as the actual emergence of myxamoebae, exhibited the same temperature optimum as growth (25") and was most easily induced in young spores (one day old). Excystment of H . castellanii cysts is a considerably longer process, 1-3 days being required for emergence of amoebae (Griffiths, 1967). The two systems have the same p H optimum (pH 6-0-7.0) and young cysts emerge more rapidly than old ones but did not require, and were unaffected by, heat-shock treatments. As yet, excystment of H . castellanii has been obtained only in peptone growth medium. Higher concentrations of peptone than that used in the normal growth medium (in the range S-lO%, w/v) were inhibitory. Although emergence of the amoebae occurs over a period of days, addition of growth medium to cysts can initiate oxygen uptake over a period of three hours or so.

VII. Resistance and Function of Cysts There is some evidence for the resistant nature of the cyst (Goodey, 1915; Hyman, 1940; Bridgeman, 1957; Stout and Heal, 1967).Various flagellates, ciliates and amoebae have been recovered from cysts which had been eontained in dried samples of soils after as much as 49 years storage (Hyman, 1940). Taylor and Strickland (1936) have shown that dried cysts of the ciliate C'olpoda are not injured by high vacuum if this is applied gradually and will survive-180" for 13-5hr., 70" for 26 hr.

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and 106" for one hr. More recently, Band (1963) found that cysts of H . rhysodes which had been produced by the replacement method retained their viability after drying over calcium chloride. Cysts of the obligately anaerobic Entamoeba sp. are insensitive to oxygen (McConnachie, 1969). However, resistance to lesser enviroiimental extremes, such as the ability to withstand dilution of the medium, fluctuations in temperature, and the presence of antimetabolites, is undoubtedly just as important to the survival of the organism. Resistance must be considered as a secondary attribute of the cyst or any other cryptobiotic structure (Foster, 1956). The most important property of the encysted state must be its depressed metabolism which renders the cell insensitive to nutritionally poor environments. This is supported by the few quantitative studies of resistance (see for example, Beers, 1937) which show that, although subjected to sometimes spectacular environmental extremes over long periods, only a small proportion of cysts remain capable of germination.

VIII. Concluding Remarks Despite the growing use of axenic cultures and the replacement technique, some contradictions remain in our knowledge of encystment. These may be attributable to species differences even within a small group of organisms such as the hartmannellid soil amoebae, but for the moment this must remain unanswered (see Page, 1967a, b). Investigations of processes such as encystment must eventually involve the study of switches in the metabolism of the differentiating organism. It therefore becomes necessary to obtain a greater knowledge of the modulations which can occur in the metabolism of the organism at other stages in its life cycle. To this end, it is either necessary to standardize the conditions of growth or obtain more information about the effects of such variables as medium composition, aeration rates, and inoculation regimes on metabolism. Batch-culture methods as opposed to continuous-culture methods should also be re-evaluated with regard to their usefulness in studies which involve the measurement of levels of enzymes and cofactors, the chemical composition of the cells and other parameters on which models of differentiation are frequently based and which may vary independently of differentiation in uncontrolled environments.

IX. Acknowledgements I am grateful to Mrs. S. M. Bowen for allowing me to include some of her unpublished results and to Mr. M. P . Stratford for Figures 2 , 4 and 5. My thanks are also given to Professor D. E. Hughes and Dr. D. Lloyd for their helpful discussions and collaboration in some of this work.

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The manuscript was prepared while the author was a holder of a Medical Research Council Fellowship.

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Serotype Expression in Paramecium JOHNSOMMERVILLE Protozoan Genetics Unit, Institute of Animal Genetics, Edinburgh University, Edinburgh 9 , Xcotland I. Introduction . A. Theorganism R. Serotypes

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11. Structure of i-Antigen Molecules . A. General Properties . B. Subunits . C. Relationship between Different i-Antigens D. Hybrid Molecules . E. Secondary Antigens

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111. Cellular Location of i-Antigens A. Nature of Surface Association B. Internal Sites . IV. Genetics of Serotype Expression A. Nuclei and Chromosomes . H. i-Antigen-Determining Genes C. Regulation of Gene Expression

V. Function of i-Antigens VI. Formation of i-Antigens A. Synthesis in vivo B. Synthesis in vitro C. Transportation VII. Serotype Transformation A. Induction Kinetics H. Nuclear Activity C. Regulation . VIII. Conclusions

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Introduction The central theme of cell biology is the control of phenotype expression : how cells with an apparently fixed genetic constitution develop variations in a large number of characters. Our current views on the mechanisms involved in the regulation of genic activity are derived mainly from bacterial studies where the emphasis has been on the action of small molecules in influencing the rate of synthesis of those enzymes concerned with their metabolism. This action is believed to be mediated through the reversible activation/inactivation of cistron-specific repressors of DNA to RNA transcription (see Epstein and Beckwith, 1968). I n this case the problems of cell “differentiation” resolve, fairly simply, in terms of the functional economy of the cell, each cell regulating its macromolecular syntheses in response to its particular environment. I n the realm of biological systems, inducible/repressible enzyme synthesis in bacteria may represent a uniquely simple and experimentally accessible control phenomenon. Apart from the probability of control at the level of translation of pre-existing RNA templates (see Harris, 1968), highly organized cells, due to the required spacial and temporal integration of many components, may exhibit other forms of control. For instance, the ordering of cell structure as a process may be under the influence of pre-existing cell structure rather than a consequence of random assembly of cell products (see Sonneborn, 1964). Whether or not another principle is involved in its genesis, cell structure influences the distribution and activity of the products of genic expression. I n this article I wish to discuss the ciliated protozoan Paramecium aurelia, in particular the differentiation of its serotypes or surface immobilization antigens (i-antigens). This system has several features particularly advantageous in the study of gene expression. For example, paramecia exhibit a range of readily distinguishable, alternative celltypes, expressing a character which is essential for the existence of the cell but is nevertheless neutral in the selective sense. These serotypes are generally mutually exclusive and can often be made to change reversibly, one to another, in response to standard changes in their environment. Since all cells have antigenic substances on their surfaces, I hope that some of the points discussed may have a more general relevance. As well as considering the control mechanisms which select the expression of only one i-antigen, part of the discussion will be concerned with the particular problems involved in the formation of a specific cell-surface protein which may demonstrate control phenomena of another type. I have tried to give prominence to the latest work in this field which, although largely incomplete, has already done much to supplement the

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original descriptive work with a more detailed biochemical basis. Indeed, in the study of i-antigen formation and transformation, the data from genetical, chemical and cytological analyses have provided the beginnings of a synthesis.

A. THE ORGANISM Paramecium aurelia is routinely considered as a large micro-organism or a small animal and even has features normally associated with plants. Whether Paramecium can be regarded as a useful point for reference to both procaryotes and eucaryotes or whether it represents an evolutionary backwater remains to be seen, but the important point is that it can be studied using the methodology of both. The following points should be noted : (a) The cells are almost visible to the naked eye; these cells can be handIed singly and induced to mate ; exconjugants can be isolated and grown up as clones; heterozygotes can be induced to undergo a nuclear re-organization (autogamy) which selects for survival one haploid division nucleus resulting in cells homozygous a t all loci. During conjugation haploid nuclei are exchanged, generally without exchange of cytoplasm, enabling the separate effects of nuclei and cytoplasm on the expression of cell characters to be distinguished. (b) There exists a large number of easily observable differentiated structures, including those organelles normally encountered in eucaryote cells. Of particular significance to a consideration of gene action is the diversification of nuclei. The dual nuclear function is here separated into one unit primarily concerned with germinal replication (micronucleus) and one unit governing somatic activity (macronucleus). (c) These cells are small enough to be cultured in large numbers (as many as lo4 per ml.) and may undergo binary fission as frequently as every 4-5 hr. I n this account, reference will be made t o stocks and syngens of paramecia. I n general, a stock is a culture of cells derived from an individual collected in the wild and may differ from another stock by one or more alleles; a syngen is a group of stocks capable of mating together and yielding viable offspring.

B. SEROTYPES When paramecia are placed in a dilute solution of anti-serumprepared from a rabbit after injecting a homogenate of homologous cells-the cilia are seen to clump together and the normal swimming motion is inhibited. This process is called immobilization and is due to the presence of antigenic substances on the surface of the cilia. However, not

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all paramecia of the same stock are immobilized by the same antiserum ; resistant lines can be used to elicit antibodies specifically directed against their own ciliary antigens. Such variation does not arise by mutation, or by a conformational change of the antigen; rather it is due to a physiological “switch” involving the expression of an alternative gene. There exists in all isogenic stocks of Paramecium a spectrum of possible serotypes (up to 12) directed by genes a t different unlinked loci. This source of serotype variation is called non-allelic. Normally one locus is expressed to the exclusion of all others. Another source of serotype variation is that encountered in different stocks which are usually isolated from different geographical locations. Here allelic differences occur and antisera tend to cross-react with the serotypes determined by these alleles. Cells heterozygous a t the expressed locus may react strongly with the two antisera. The early work on serotype inheritance has been reviewed by Beale (1957) and more recently by Preer (1968). A proper understanding of the mechanisms involved in the control of serotype expression depends upon a careful analysis of the components involved. I n the next section the chemical nature of the serotype substance (i-antigen) will be considered.

11. Structure of i-Antigen Molecules A. GENERAL PROPERTIES Although each serotype of Paramecium has a specific serotypecorrelated i-antigen, these various substances have been found to share a number of similar chemical properties. Fortunately, the i-antigens are water-soluble and retain their specific immunological form in solution, so that antigenic extracts can be readily characterized. For instance, cell homogenates generally form specific single-band precipitates with homologous antiserum in agar diffusion tests (Finger, 1956). As well as differentiating i-antigens, this double diffusion method can be used as a quantitative assay, as can the indirect technique of blocking the immobilization reaction specifically by absorbing antibodies from the serum with i-antigen extracts (Preer, 1959a ; Bishop, 1963). On assaying fractionated homogenates, Preer and Preer (1959) were able to show that the i-antigen was associated mainly with cell-surface structures, i.e. the cilia and cell walls. The various properties mentioned above enable good yields of material t o be extracted from the cell surface. This is done by suspending the cells in a salt-alcohol solution, a procedure which does not lyse the cells but nevertheless breaks the cilia and releases the i-antigen. Extracts can be fairly easily purified by a number of techniques : fractional precipitation

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with ammonium sulphate (Preer, 1959b), starch-gel electrophoresis (Bishop 1961) and by column chromatography using hydroxyapatite (Bishop, 1961)or cation-exchange resins (Jones, 1965a).Such preparations were shown to be relatively pure by electrophoretic, rate-sedimentation and immunological criteria. Preer (1959~) working with syngen 4,and Bishop (1961)working with syngen 1, showed that the i-antigen preparations were largely or wholly protein. Molecular weight determinations were reported from a number of different serotypes to be in the range of 240,000-260,000 (Preer, 1959c ; Bishop, 1961 ; Jones, 1965a) but a more accurate determination, with extrapolation of all parameters to zero protein concentration, is 310,000320,000 (Steers, 1965). As to the general form of the i-antigen proteins, Preer (1959~)has suggested that the marked dependence of the sedimentation constant on concentration indicates an asymmetrical molecule. This view is supported by his estimates of intrinsic viscosity (0.11 g./100 ml.) and frictional ratio (1.8). The likely conclusion is that these proteins are fibrous in shape, although electron micrographs of negatively-stained extracted i-antigen have revealed hexagonal shaped units 200 I% in diameter (Mott, 1964). Different means of preparation and storage (e.g. in frozen solution or as lyophilized solid) may affect the general structure or state of aggregation.

B. SUBUNITS In the presence of dissociating agents, such as 8 M-urea and 6 M guanidine hydrochloride, the i-antigen molecule retains its native molecular-weight value. However, under conditions of chemical reduction, e.g. in the presence of 0-1 M-mercaptoethanol, smaller derivative molecules are obtained. The molecular-weight values of such reduced molecules were determined by equilibrium centrifugation and found to be 35,000 in syngen 4 (Steers, 1965) but more variable from preparation to preparation (16,000-80,000) in syngen 1 (Jones, 1965a). On gel-filtration, reduced syngen 1 i-antigens elute as single peaks estimated to contain material with molecular weights of approximately 40,000 (Fig. la). Assuming a native molecular weight of 310,000, these results show that the i-antigen molecule is composed of a number of similar-sized polypeptide chains, the actual number being, most likely, nine. Prom the reduction and dissociation data, it can be seen that the subunit polypeptides are held together by covalent linkages in the form of disulphide bonds. Normally the -SH groups of the reduced components are carboxymethylated to prevent S-S bonds reforming, thereby allowing the i-antigen to be isolated in its subunit form.

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Amino-acid composition studies have shown the cysteine content of various i-antigens to be remarkably high; 272 residues per molecule in stock 51A, syngen 4 (Steers, 1965), and 244-253 residues per molecule in several stocks of syngen 1 (Jones, 1965a). These figures represent 10%

Reduced 168 G i-antigen

I68 G i-antigen

. . . .

:.

. . .

. .

Reduced

. . .

.

. . . .

.

t

Anti-168G-serum

FIG.1. Analysis of reduced 168G i-antigen. (a) Gel filtration profiles of native i-antigen (in 4 M-urea, 0.05 M-tris-HC1; pH 7 . 5 ) and reduced i-antigen (in 4 M urea, 0.05 M-tris-HC1; pH 7 . 5 ; 0.1 M-P-mercaptoethanol) on Sephadex G-200. Molecules of known molecular weight were used to calibrate the column. (b)Acrylamide gel disc electrophoresis of native i-antigen and reduced i-antigen (in the presence of 0.1 M-P-mercaptoethanol).Usual protein bands are shown solid black; variable additional bands are shown dotted. The positions of precipitin arcs formed by antigenic material diffusing from the acrylamide gels through agar t o meet diffusing homologous antiserum are also indicated.

(w/w) of the molecule or one cystinyl residue in every 20 amino acids. Since no free sulphydryl groups appear to be present in the native molecule, 122-136 disulphide bridges are available for linking together the polypeptide chains, although some, or even most, of these bonds may be of an intrachain nature. The presence of many disulphide bonds appears to be a general feature and has two likely consequences for the properties

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of the i-antigens : (a) they are very stable proteins, being held in conformation by a large number of covalent bonds. This is borne out by the observed retention of antigenic activity under a wide variety of physical conditions; (b) the polypeptide chains are subject to a high degree of folding. The facility of i-antigens in stimulating the formation of various specific antibodies may be partly derived from such a property. It has been found that some i-antigens have a t least 11 antigen determinant sites per molecule (Finger, 1964). Further information on subunit structure, particularly on the question of any of the polypeptide chains having identical primary sequence, may be derived from a comparison of the amino-acid composition and the peptide maps of tryptic digests. Since trypsin breaks only those bonds involving the carboxyl groups of arginine and lysine, the expected number of peptides can be compared with the observed number (see Table 1). Steers (1965) found that the number of observed peptides spots was one-third of the number expected. He concluded that there exist three identical subunits within the molecule, i.e. 103,000 molecular weight of unique primary sequence. Also, fingerprints, specifically stained for arginine, revealed one-third of the expected number of arginine-containing peptides. A similar study on syngen 1 i-antigens (Jones, 1965a) showed that the observed numbers of tryptic peptides and peptide spots specifically staining for arginine and tryptophan were slightly less than one-half of the number expected. Jones concluded that the i-antigens probably contain two identical subunits. However, if the molecular weight of the syngen 1 i-antigens is 310,000, rather than the value of 250,000 estimated by Jones, this correction would make the data fit a three-common subunit model. These results are summarized in Table 1. Now if, as already discussed, there exist nine polypeptides per molecule, the common subunits would each contain three polypeptides. This arrangement of the i-antigen molecule is shown in Fig. 2 . The correctness of this model could be tested by analysing the products of completely reduced i-antigens. Assuming they could be separated, equal amounts of three types of polypeptide would be expected. The analysis of reduced syngen 1 i-antigens by acrylamide disc electrophoresis has shown several protein-staining bands which migrate from four to seven times faster than the native i-antigen (J. Sommerville, unpublished observations). Although the actual number of bands is sometimes variable, patterns of three bands are commonly observed (Fig. l b , p. 136). Variability in band number is probably due to incomplete reduction of the protein. An interesting property of these separated subunits was demonstrated by setting the acrylamide gels in agar and allowing the contents of the gels t o diffuse through the non-reducing conditions of the agar to meet a diffusing band of antiserum. After incubation, a precipitin band was

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TABLI.:1. Comparison of Expected and Observed Peptide Number in Tryptic Digests of I-Antigensa i-Antigen

51A

90D

~~

9OG ~

Tryptophan Calculated number of residues Observed number of specifically staining spots

-

20 7

32 14

Argiriirrr Calculated number of residues Observeti number of specifically staining spots

38 13

43 17

43 17

148

257

171

187

301

215

Lysine Calculated number of residues Pcptides Calciilated number of trypsin-sensitive bonds (arg + lys residues) + 1 Observed number of spots

66 90-100 65-70

a Data from Jones (1965a) and Steers (1965) assuming the molecular weight of each i-antigent o be 310,000. -- No data available.

formed between one subunit position and the homologous antiserum trough (Fig. l b , p. 136). Apparently one type of subunit had refolded on diffusion to form its specific molecular configuration.

Bic:. 2 . Model of' i-aritigen molecule. a , /?and y are three non-identical polypeptide chains of' approximately 34,000 molecular weight.

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C. RELATIONSHIP BETWEEN DIFFERENT I-ANTIGENS Previously this review has been concerned with the general and common properties of i-antigens, but what is the chemical basis of their immunological differences? Two types of relationship will be considered ; those between alternative non-allelic i-antigens within a stock and those between i-antigens determined by allelic genes in different stocks. Preer (1959d) showed that the non-allelic i-antigens in both syngen 2 stocks and syngen 4 stocks differ in respect to solubility in ammonium sulphate and that the pattern of these differences reflects the known degree of immunological difference. Differences have also been found between i-antigens in syngen 1 stocks on electrophoresis and column chromatography. For instance, the G, D and T i-antigens of stock 60 migrate a t different rates on starch-gel electrophoresis and separate by chromatography on hydroxyapatite (Bishop and Beale, 1960). The G and D i-antigens of stock 90 have also been separated by cationexchange chromatography ( J . Sommerville, unpublished observations). However separation by charge difference is generally poor, even a t pH 9.6 which is well above the isoeIectric point (pH 3.8-4-2) of the proteins. This is somewhat surprising in view of the fact that the aminoacid compositions, particularly with respect to the number of charged amino acids, of G and D i-antigens are quite distinct. Jones (1965a) suggests that the poor migration of these i-antigens may be due to many of the charged groups being buried within the molecule. More conveniently studied are peptide pattern differences. The serotypes A, B and D of syngen 4 (Steers, 1962) and G and D of syngen 1 (Jones, 1965a) can show as many as 80-90% of the peptide spots as being different. Therefore non-allelic i-antigens are quite distinct on the basis of peptide analysis. These differences are many more than are generally encountered between i-antigens controlled by alleles in different stocks. Steers (1962) found no difference between the peptide pattern of two A types and a t most 15% of the peptide spots were found to be different between two D types and two G types (Jones, 1965a). A survey of the allelic serotypes of syngen 1 (Beale, 1954; Jones and Beale, 1963) shows that they can be arranged as a number of immunologically similar, or identical, groups. But even immunologically identical alleles can be distinguished by the more sensitive technique of peptide analysis. Of nine allelic D types, no two were shown to be identical in peptide pattern (1-12% of the peptides were different) although they all showed immunological cross-reaction and five were indistinguishable by immunological criteria (Jones and Beale, 1963). I n general, there is a good correlation between the degree of serological cross-reaction and similarity of peptide

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pattern, indicating that the immunological properties of a given i-antigen are dependent upon the overall chemical structure of the molecule. The conclusion that must be drawn from these results is that different i-antigens have different primary sequences although allelic types may differ in the substitution of only a few amino acids. Another approach to the analysis of molecular differences involves a consideration of the surface structure of the i-antigen molecule (antigenic sites) rather than the amino-acid sequence (Finger, 1964). Here gel diffusion tests are used t o compare various i-antigens in their interaction with different cross-reacting antisera. I n principle, the lines of antigenantibody precipitate, formed by the diffusion of two adjacently placed antigens to meet a cross-reacting antiserum, may interact in three possible ways : (a) They may fuse completely, i.e. the i-antigens are apparently identical since no antibodies present can distinguish them. (b) They may cross completely, i.e. the i-antigens are unrelated since neither precipitate interferes with the diffusion of the unrelated antigen and antibody molecules. (c) They may fuse partially and form a spur, i.e. the antigens are related but not identical, spurs being formed by one antigen reacting exclusively with a t least one typeof antibody. Onthe basis of comparisons of different i-antigens with their cross-reacting antisera, antigenic determinants have been assigned to the allelic series of five i-antigens a t the c locus in syngen 2 (Finger, 1964; Finger et al., 1963, 1966). The determinants can be arranged in matrix form to show the degree of relatedness between antigenic types (see Table 2 ) . Of eleven detected C determinants five or more are found in each i-antigen with as many as nine differences between two i-antigens. Although the fingerprint data indicated few differences in peptide pattern between allelic i-antigens of syngen 1, and no difference between alleles of syngen 4,the number of actual amino-acid substitutions may be much greater since only charge differences would be detected. The data on antigenic determinants of alleles in syngen 2 a t first sight seem to show quite large differences, but this may be due to relatively few aminoacid substitutions influencing substantial areas of the general configuration of the molecule, and so affecting the immunological specificity. I n fact these determinant differences appear somewhat less when arranged as substitutions and omissions within linked groups corresponding to i-antigen subunits (see Section II.D, p. 141). Any conclusion about the significance of i-antigen differences must reconcile two sets of results ; the relatively few (permitted?) chemical differences found between allelic i-antigens and the surprising amount of

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variation found between non-allelic i-antigens. Needless to say, this ties up with questions about the function and evolution of these antigens, a topic which will be discussed later. TABLE2 . Matrix of Antigenic Determinants for C Serotypos as Determined by Gel Diffusiona Sorotype 30C 7C 72C 83C 197C ~~~

a

Determinants

C

F G F G F

F

H

I

J

L

I J H H H

J

I I

M

0

111 K

L

K

L

M M

N

N

0 O

~

From Finger (1964).

D. HYBRID MOLECULES An important question, pertaining t o subunit structure and the control of subunit assembly, is what type and number of types of i-antigen are formed in cells heterozygous a t the active locus? It has long been known that such heterozygous clones react with both parental-type antisera. However, the general observation is that cells may react more strongly with one parental type than with the other, i.e. the two alleles are not equally expressed. Are these reactions due to parental i-antigens being differentially expressed or to hybrid molecules which may be predominantly one homozygous type in their determinant constitution? On the basis of serological screening tests, it has been shown that cells heterozygous a t the c locus and the e locus in syngen 2 form a number of hybrid i-antigens. These molecules appear to be variable in the proportion of antigenic determinants contributed by each parent, ranging from equivalence to proponderanee of one type. However, the unusual feature of this system is that only a single species of hybrid molecule is present in any one clone, although different isogenic clones may synthesise different types of hybrid molecule. This phenomenon may be related to the peculiar effect whereby, under certain conditions, only one of the e alleles is selected for expression (see Section IV.C, p. 154). Another observation is that antigenic determinants do not combine randomly to form hybrid molecules ; rather there is a restricted spectrum of types. This may be due to some antigenic specificities being linked on the same polypeptide chain. From a consideration of the number of types of hybrid molecule formed, Finger suggests that the i-antigen model most compatible with his results consists of three dissimilar polypeptide chains a , /3 and y , each represented a t least twice. This agrees with the chemical

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evidence already discussed. The proposed model for distribution of determinants among the subunits of the C antigens is shown in Table 3. Hybrid i-antigen molecules are also formed by cells heterozygous a t the d locus in syngen 1 (Jones, 1965b). However, the situation is different from that already described since a mixture of molecular species was found. It is not certain that the large number of cells used in this experiment belonged to a single clone, and the result may represent the cumulative serotype expression of a number of differentiated clones. The TABLE3. Matrix of Antigenic Determinants for C Serotypes in Proposed Subunit Arrangementa Subunits

P

CL

Serotype 30C 7C 72C 83C 197C a

Y

Determinants

M G M G M K M K

H

O

J C J H N H N H

0 O

J

L

F I F I

L

F

L

F

I

From Finger rt nl. (1966).

results were obtained by fractionating the antigenic material derived from d60/dg0heterozygotes on a column of hydroxyapatite. Of the total effluent material, 50% was recovered in one peak corresponding to one parental antigenic type. The remainder of the material formed several minor peaks and reacted, to different degrees, with both parental antisera. Jones concluded that there were at least two species of hybrid iiiolecule and he proposed a scheme for random assortment of subunits. Although both alleles at the d locus are expressed there is a quantitative difference in the activity of each, 70-80y0 of the serological reactivity being of the 6OD type. The seemingly random assortment of i-antigen subunits implies that the polypeptides are synthesized separately and are derived from distinct cistrons. However, on top of this random type of event are superimposed two “control” principles : (a) I n a given heterozygote, one allele may be more active than the other, or else the products of one allele may be preferentially utilized. (b) There may be a restriction of the i-antigen formed to only one type. This is equivalent to a fixation of differential activity both of alleles and cistrons within alleles. These genetic control mechanisms will be discussed in more detail later. Apart from this type of differentiation,

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there may be restriction of the number of observed hybrid types due to : (a) the presence of common or indistinguishable polypeptides; (b) an order difference or preference in subunit assembly.

E. SECONDARY ANTIGENS Although allelic i-antigens tend to be serologically related, alternative non-allelic types are normally serologically distinct, especially in syngen 1. However, instances have been cited of strong interaction between non-allelic serotypes ; antiserum against one serotype may immobilize cells expressing another type, or precipitate its extracted i-antigen. Such interaction may be due to either structural relatedness between antigenic determinants (cross-reaction)or to the presence of more than one i-antigen within a single cell (secondary i-antigens). These effects can be detected and distinguished by gel-diffusion tests but neither need involve immobilization by the heterologous antiserum (Baldinder and Preer, 1959; Finger et al., 1962). I n fact, a variety of different observations has been reported, which may reflect as many different types of effect. In stock 172, variety 4, Margolin (1956) reported the simultaneous expression of two unrelated serotypes, D and M, both of which could be detected by immobilization with specific anti-D and anti-M sera. The results suggest that both i-antigens have equal status as surface antigens, although variations occur in the proportion of each. Where serotypes react with heterologous antiserum in gel-diffusion tests, but fail to react in immobilization tests, both secondary i-antigen and cross-reaction have been suggested as explanations. For instance, in serotype E of stock 197, syngen 2, the related G i-antigen may also be present; similarly, type E may be present as a secondary i-antigen in cells of serotype G (Finger et al., 1962). Here appearance of secondary i-antigen is infrequent and its relative amount variable. On the other hand, Balbinder and Preer (1959) were unable to detect any secondary i-antigen of the closely related E type in G serotype cultures of stock 28, syngen 2. Secondary i-antigens have also been found in immunologically unrelated serotypes. I n various stocks of syngen 2, G type has been found as a secondary antigen in cells expressing the C serotype (Seed et al., 1964). Again the appearance and amount of secondary antigen is variable. These secondary G antigens are indistinguishable from primary G antigens in their immunological specificities, electrophoretic mobilities, sedimentation rates and solubilities in ammonium sulphate. It is surprising that no secondary i-antigens have been detected in a large number of types, especially those of syngens 1. and 4 whose chemical

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structure has been extensively studied (Preer, 1959b-d; Steers, 1962 ; Jones, 1065).I n fact, secondary i-antigens may be a peculiarity of certain clones of paramecia which tend to be unstable in the expression of their serotype. The significance of these results as exceptions to the usual condition of mutual exclusion among i-antigens will be discussed later.

111. Cellular Location of i-Antigens Preer and Preer (1959) separated cell homogenates by differential centrifugation and showed that extractable i-antigen was associated mainly with cilia and pellicle fractions. This is what we would expect of a substance diagnosed by the immobilization of cilia. However, it is important to know, from the point of view of i-antigen formation and function, what sort of association it forms with the surface structures and whether any i-antigen, perhaps of a different type, exists intracellularly. Information on these issues has been provided by refinements in cytological studies utilizing antibody conjugates.

A. NATUREOF SURFACE ASSOCIATION Fluorescein-conjugated antibodies were used to label whole cells (Beale and Kacser, 1957) and cell sections (Beale and Mott,, 1962). Such studies showed that osmium-fixed preparations specifically absorbed antibodies as a thin layer around the entire surface of the organism (Fig. 3). However, in unfixed cells, the complexing of antigen with antibodies had the effect of stripping the antigen from the stems of the

F r G . 3. Location of i-antigen using fluorescein-conjugated antibody; (a)cell section treated directly ; (b) cell section pretreated with heterologous antibody. Unpublished data of R. E. Sinden.

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cilia, resulting in an accumulation of “sticky” material a t their tips. Such cilia no longer reacted with the antibodies, yet their structure was not visibly altered. It would appear that the i-antigen is not an integral part of the surface structure ; rather it is considered to be a “fluid” substance secreted onto the cell surface where it is in some way secured. Further, the i-antjigens are readily solubilized from the cell surface by a mild extraction procedure (Preer, 1959b). The chemical data suggest that extracted i-antigen is more like a globular protein than a fibrous one. Although the extracted material has identical immuiiological properties t o the i-antigen i n si tu , attachment to the surface membranes may modify the general configuration or state of aggregation. Mott (1963, 1965) treated whole cells with ferritin-conjugated antibodies prior t o sectioning and located the electron-dense molecules by electron microscopy. By this method the i-antigen is shown to be distributed as a layer over the entire outermost pellicular membrane, a membrane which is continuous over the surface of the pellicle and cilia. I n cells transforming from one serotype t o another, Mott (1963, 1965) found that new-type i-antigen appeared initially a t isolated sites on the pellicle, later increasing to cover the pellicle and subsequently the cilia. However, there was no evidence for the spread of new-type i-antigen along the surface from any particular point of emergence and no pores in the membrane were observed. Nevertheless, newly formed i-antigen molecules may be transported through the general structure of the surface membranes, the points of appearance perhaps being sites made available by membrane growth or detachment of pre-existing i-antigen.

B. INTERNAL SITES If the i-antigen molecules continually appear on the surface membrane, we might expect to locate them as cytoplasmic precursors, unless the i-antigens are synthesized a t their final site. However, this latter suggestion is highly unlikely in view of the number of components which would be required to be situated on these membranes. For instance, there are no ribosomes seen to be on or near the pellicle. Although fluorescence was observed in the cytoplasm of sectioned cells after treating with conjugated antibody (Beale and Mott, 1962), this was said t o be due to the presence of antibodies against antigenic material other than i-antigens. I n this case antiserum was derived against whole cell homogenates. The evidence was that the cytoplasmic reaction could be effectively blocked by pretreatment of the sections with unconjugated heterologous antiserum. However, it has been shown recently that fluorescein-conjugated globulin from rabbits injected with purified

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i-antigen labels the cytoplasm to a certain extent (R. E. Sinden, unpublished observations). Also, Sinden has shown that blockage by pretreatment with heterologous globulin is affected by a large amount of globulin-globulin interaction, resulting in a thick coating of the material which later treatment with homologous globulin would not penetrate. Nevertheless, it is still possible that cytoplasmic fluorescence is due mainly, if not entirely, to cross-reacting material. Ferritin-conjugated globulin, although allowing an exact localization of reacting material, is unsuitable for detecting internal antigens because it is difficult to distinguish the electron-dense ferritin granules from the generally granular appearance of the cytoplasm in electron micrographs. A promising approach is provided by the use of lZ5I-labelledglobulin (R. E. Sinden, unpublished observations). Here, by means of high resolution autoradiography, the labelled globulin complexing with cell structures can be localized in electron micrographs. However, so far Sinden has been unable to show serotype-specific labelling of cytoplasmic structures. Thus the conclusion must be that, although i-antigenic material has been shown to exist in association with ribosome preparations (Preer and Preer, 1959; Seed et al., 1964; Macindoe and Reisner, 1967) and to be synthesized in such preparations (Sommerville, 1967), intracellular i-antigen has not been detected directly by cytological means, There are a number of possible explanations for this seeming paradox (a) Internal i-antigen exists in such small amounts that it is not easily detected. The amount associated with isolated ribosomes may be exaggerated by contamination after homogenization. (b) It exists internally in a form lacking complete antigenic specificity, perhaps as subunits. The i-antigenic material may assume a more specific form when extracted from homogenates than i t possesses in situ a t its internal sites. (c) Its site of synthesis is very near the pellicle membranes and may be confused with the surface reaction. A candidate for internal localization is the secondary i-antigen. This is a minor component of a different serotype which is occasionally found in cell homogenates but is not normally detected by immobilization (see Section II.E, p. 143). The distribution of secondary G-type antigen in syngen 2 cells of C serotype was studied by Seed et al. (1964) and compared with the cellular distribution of the primary i-antigen. There are a number of difficulties inherent in this type of study, for instance, the variable amounts of secondary i-antigen in different cultures and even the variable distribution among cell fractions of primary i-antigen in different preparations, perhaps due to differential leachiqg of i-antigen

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from cell structures. I n spite of these difficulties, a number of conclusions were reached. As with the primary i-antigen, the major secondary i-antigen-containing fraction is the ciliary fraction. Although both primary and secondary i-antigens are found in association with the same organelles, differences may exist in their relative distribution. For instance there appears to be more secondary than primary i-antigen in the soluble supernatant fraction. Thus much of the secondary i-antigen may be internal, not attached to any particular structure.

IV. Genetics of Serotype Expression I have so far discussed the nature of the i-antigen molecule and its cellular location, the chemistry and cytology of the system. However, the earliest studies on i-antigens were primarily concerned with a third approach-that of genetic analysis.

A. NUCLEI AND CHROMOSOMES Although there have been few direct observations on nuclear activity in relation t o serotype expression, it is perhaps worth while considering the general aspects of the nuclei and chromosomes of Paramecium as a background to what we know of the genetics of the system. We might even consider any peculiar features of phenotype expression in Paramecium to be related in some way to the organization of its nuclear material. As with other ciliated protozoa, the organization of nuclear material appears to involve two levels of complexity which probably had separate phylogenetic origins (Raikov, 1963)-nuclear diversification and polyploidy. Paramecium possesses two types of nuclei which are morphologically and functionally distinct. The micronuclei (usually two), which closely resemble the nuclei of eucaryotic cells, are the germinal nuclei. They are generally considered to be genetically inactive, preserving an intact diploid complement of genetic information which is available for fertilization (after reduction division) and the generation of both micronuclei and new macronuclei. On the other hand, the single macronucleus is very large and is the physiologically active component, controlling the phenotypic features of the cell. Chromosomes are observable in preparations of micronuclei undergoing meiosis. There are, however, considerable differences in chromosome number between different stocks, the variation within syngens being as great as that between syngens. For instance, the diploid number of six syngen 1stocksrangesfrom 86to 126 (Koiciuszko, 1965)while the diploid number of five syngen 4 stocks ranges from 66 to 102 (Dippell, 1954). A consequence o f this large chromosome number is the scarcity of linked

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loci; a consequence of the variable complement is the mortality of F, generations in crosses between certain stocks. It is the structure and organization of the macronucleus, however, which arouses a greater amount of interest. Here are found some special features befitting the metabolism of a large and active cell. For instance, photometric measurements have shown that the macronucleus, compared to the micronucleus, has vastly increased amounts of both DNA and RNA. The ratio of macronuclear t o micronuclear DNA is 430 to 1 (Woodard et al., 1961) but how this degree of polyploidy is organized into macronuclear structure remains uncertain. It has long been postulated, on the basis of the ability of macronuclear fragments to regenerate a complete new macronucleus, that the macronucleus contains a t least 40 sets of chromosomes possibly arranged as separate sub-nuclei (Sonneborn, 1947). However, electron-microscope studies have failed to reveal structures which can be positively identified as sub-nuclei (Jurand et al., 1962, 1964). Of the structures found, it is generally considered that the many large RNA-containing bodies are analogous to nucleoli while the DNA-rich matrix containing small bodies and filaments represents the chromosomal material in a dispersed state (Dippell and Sinton, 1963). Although there is no cytological evidence for a polygenomic arrangement of chromosomes there is evidence for another type of organization of the polyploid material. Recent work has shown the developing macronuclei of Paramecium and other ciliates to contain polytene-type chromosomes (Alonso and Perez-Silva, 1966) which may be composed of the bundles of numerous extensible DNA-histone filaments found in the macronucle of other related ciliates (Seshacher, 1964). However, there may still be polygenomic arrangements of these polytene chromosomes. Thus the macronucleus may be considered to have attained a complexity comparable with the salivary gland nucleus of Diptera and the oocyte nucleus of amphibia. How this complexity affects the control of gene expression a t the molecular level is not known. The differentiation of certain phenotype characters, particularly the mating types of Paramecium and other ciliates (see Preer, 1968) and various enzymes and serotypes of Tetrahymena (see Nanney, 1963), can be related to stages of macronuclear development. However, the relationship between nuclear differentiation and serotype expression in Paramecium is more tenuous.

B. I-ANTIGEN-DETERMINING GENES 1. Xpecificity Loci A large number of different serotypes (as many as twelve) can be expressed by a totally homozygous strain of paramecium when cultured under different conditions. It is now evident, from the chemical data already discussed, that the differences between alternative i-antigens are

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due t o differences in amino-acid sequence rather than to reversible structural modifications of a common protein molecule. The earlier genetical studies of Sonneborn with syngen 4 and Beale with syngen 1 (see Beale, 1954) clearly showed that these alternative forms were in some way due to the differential expression of a series of genes a t different loci. Where tested, these genes were shown to be unlinked. The structure of any given i-antigen is completely specified by genetic information a t one locus. This was demonstrated by comparing two preparations of purified i-antigen, one derived from the normal stock, the other from a strain expressing this i-antigen in a completely different genetic background (Jones and Beale, 1963). The “artificial” strain was obtained by repeatedly backcrossing the normal stock with an unrelated stock and selecting for the required serotype. The two preparations of i-antigen had indistinguishable peptide patterns. It has been suggested (Allen, 1966)that different i-antigens are derived from either the substitution of a specificity-conferring polypeptide in an otherwise common i-antigen molecule or the recombination in different ways of protein subunits specified by a small number of genes. However, the chemical evidence already presented has shown that, at least in peptide composition, alternative i-antigens are completely different, i.e. it is unlikely that there are subunits common t o two ormorei-antigens. Further, the genetic evidence is a t variance with any scheme proposing that more than one locus contributes towards the specificity of one i-antigen. This is made even more unlikely by the finding that the antigenic determinants are distributed throughout the structure of the molecule (Finger et al., 1966). Any structural homology between i-antigens should be considered as a function of common genetical descent rather than a sharing of gene products. Thus we can now say that the 12 alternative serotypes expressed by stock 51, syngen 4, probably eachhavetheirprimarysequencedetermined by one of 12 distinct genes. The model already proposed for the i-antigen molecule (Fig. 2 , p. 138) requires the specification of approximately 103,000 molecular weight of unique primary structure. However, since each identical subunit in turn probably consists of three different polypeptides, each i-antigen specificity locus may be considered to be a polycistron composed of three closely-linked units. 2. Alleles and Polymorphism

The number of possible alleles a t a given locus is unknown. Stocks of independent geographical origin express allelic i-antigens which appear to fall into a limited number of immunologically related categories. Certain indistinguishable serotypes are found in completely different regions of the world and in addition every region contains an assortment

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of the limited allelic types. It has also been shown that polymorphism may be extended to small populations. For instance, three alleles a t the g locus and six alleles a t the x locus, sometimes combined as heterozygotes, have been found in syngen 9 paramecia from a small localized habitat (Pringle, 1956; Beale and Pringle, 1960). However, the amount of allelic variation is greater than originally thought. With the advent of the more discriminating fingerprint analysis, even seemingly identical serotypes have been distinguished. It has been shown that, at the d locus in syngen 1, no two stocks of the nine tested have identical i-antigen peptide patterns (Jones and Beale, 1963). Thus there exists a large amount of natural genetic variation, although the degree of structural difference between i-antigens may, in some instances, be small. 3. Control Genes

I n addition to structure-determining (structural) genes, there may exist genes concerned with the control aspects of i-antigen formation. However, there is as yet no direct genetic evidence for the existence of such elements although phenomena best explained in terms of controlgene activity will be discussed in the next section. All serotypes have a, characteristic range of conditions favouring their expression. I n general, the stability of i-antigen expression is inherited with the structural determinants and no recombination of these properties has been observed. Also, samples of one allelic type (g60) collected from widely separate locations behave identically in respect of the conditions under which they are expressed (Beale, 1957). Beale suggested that the stability of expression may be a function of the structural genes themselves, but the existence of closely-linked structural and control genes has not as yet been ruled out. Indeed, the ability to form the conditions necessary for the expression of a given locus may be missing, although the structural components can be shown to be present (see Section III.C, p. 156). It should be added that, in syngen 4, it has been shown that loci other than the active i-antigen locus influence the stability of its expression (Sonneborn et al., 1953). However, these were identified as the specificitydetermining loci of other unexpressed i-antigens, in fact those that would be expressed under slightly altered environmental conditions. This finding may relate to the mechanism ensuring co-ordinated control of serotype transformation.

C. REGULATION OF GENEEXPRESSION It has long been known that serotype expression is influenced by extranuclear components which collectively constitute the “cytoplasmic state” of the cell (Sonneborn, 1948; Beale, 1952). The composition of the

SEROTYPE EXPRESSION IN Paramecium 151 “cytoplasmic state” is in turn determined by a number of factors, not only the genes present and the recent history of the cytoplasm (earlier physiological condition) but various environmental conditions such as temperature, p H value, ionic strength and availability of nutrients. The “cytoplasmic state” may also be altered by conditions not normally encountered by the cells, such as treatment with homologous antiserum, radiation and antibiotics. Both “natural” and “artificial” variables provide means for studying the stability of serotype expression (see Section VII, p. 165). The elegant series of genetic experiments leading t o the postulation and description of “cytoplasmic states” has been extensively reviewed (Beale, 1957 ; Sonneborn, 1960; Preer, 1968). The concept of control of gene activity by cytoplasmic factors has more recently gained general acceptance and has been precisely defined in some bacterial systems (Jacob and Monod, 1961). Although modifications of the general theory have been suggested to account for phenomena observed in other systems, the present position in Parameoium appears to differ, albeit in respect to the complexity of the system. The complexities, some of which I shall discuss in more detail in this section, are: (i) the exclusion of expression of all but one of a dozen or so potential serotypes ; (ii)the refinement of genic control involving mutual exclusion between alleles a t an active locus and even between cistronic regions within the locus; (iii) the specific and predictable response (in selection of serotype) to a variety of conditions) a property which may reflect the general function of the i-antigens (see Section V, p. 157); (iv) the coordinated shift in activity of all the elements involved in serotype transformation (see Section VII, p. 165). Nanney (1963), in a description of mutual exclusion in the related ciliate Tetrahymena, has used the teminology inter-locus repression, allelic repression and intra-locus repression to denote the three types of genetic control mentioned above, I n this section, I have considered these various control phenomena to be exerted a t the level of the genes. It is not certain that this is the case ; control a t the level of gene products is a possibility. However, I shall tentatively refer to the regulationof serotype expression in terms of the control of loci, alleles and sub-loci (cistrons).

1. Control of Expression of Loci

The most general action of cytoplasmic regulatory factors in the i-antigenic system is to select for expression the information content of one locus to the exclusion of all other equivalent loci. Considering a cross between two stocks manifesting different serotypes, e.g. 90G and 60D (Fig. 4), the main observation is that, although both I?, clones contain the same genes, they express different loci, the particular locus expressed being a property of the cytoplasmic parent. Furthermore, both alleles a t

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the selected locus are expressed. Subsequent environmental conditions may generate a new cytoplasmic state causing a switch to the expression of an alternative locus. Since the features of this process have been described in detail by Beale (1954, 1957) only a few relevant points will be mentioned here. Some of the conceptual difficulties originally inherent in this system now seem less significant due to the modification of two points in line with current theory, First, the condition of mutual exclusion is simplified by shifting the emphasis t o the activation of one locus rather than the simultaneous inactivation of all but one locus. Secondly, the seeming paradox of the gene-cytoplasm interaction, whereby the i-antigen locus “conditions” the cytoplasm which in turn stimulates (in terms of the Parental serotypes

9OG

E’1-serotypes after five fissions

FI-serotypes after many fissions

BOD

9OQ at 270 9 -

600;

BOD

‘OD

90G 60G

SOD

Pig. 4. Inhoritancc of sorotypes on crossing types 90G and BOD. From Beale (1954).

first point) the phenotypic expression of that same locus, is to some extent resolved by considering the separate functions of control and structural genes. I n these terms, locus selection could operate as follows. Specific products of a control gene “condition” the cytoplasm/cell to favour the synthesis of the i-antigen product ofa closely-linked structural gene. The environment and general physiological conditions act indirectly either by activating the controlling factors themselves or by stimulating their synthesis. However, an additional mechanism probably operates in this control system-pcsitive feedback. It has been suggested (Kimball, 1964) that the i-antigen loci may be self-induced by their own products. This view is supported tosomeextent by thedemonstration that added end-product (i-antigen) tends to stabilize its own synthesis (Finger, 1967). Thus it is generally considered that there are a t least two stages in the control of i-antigen synthesis ; the initiation of expression of a locus in favourable physiological conditions by derepression and the continued stability of expression of that locus by self-induction. These

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two processes could be linked together if, for instance, the i-antigen, or a precursor, bound its own “repressor)’. Several additional features concerning the relative stabilities of the cytoplasmic states deserve consideration. For instance, the time taken for a new effective cytoplasmic condition to be established, a function of the relative stabilities of expression of the old and new serotypes, is highly variable. A standard change in external factors may bring about a serotype change in a matter of a few fissions or only after asmany as fifty or more fissions. I n either case, the actual transformation period, i.e. when two i-antigens can be detected simultaneously in one cell, is a short 2-3 fissions. This suggests a variable lag ti11 a threshold effect is achieved. Ranges of stability can be assigned to the cytoplasmic state for each 10” I

15O

20”

25’

30’

40’

35O

-G I

D

FIG. 5. Approximate temperature ranges for stability of cytoplasmic states in stocks 60 arid 90, syngen 1. From Beale (1954).

serotype, for example the serotype-characteristic temperature ranges shown in Fig. 5. In general, the wider the range of stability, the longer the time or more drastic the stimulus required to transform that serotype. Each alIeIe has its own particular range of stability, e.g. 90G is more stable than GOG, GOD is more stable than 90D. As already mentioned, this stability is inherited with the serotype itself. Of particular interest is the effect in heterozygotes whereby a cytoplasmic state is formed which in stability is intermediate between that of the two homozygous parents, i.e. such heterozygotes transform from the expression of the two alleles a t one locus to the expression of the two alleles a t another locus in a single step irrespective of the differences in stability of expression of each gene. Thus, although the cytoplasmic components are allele-specific in specification they appear to be locusspecific in action, acting in a “trans” position analogous to the “repressors” postulated for other systems.

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Unfortunately, we know nothing of the biochemical identity of the cytoplasmic control elements. What we do have is information on the physiological conditions under which they are activated (see Section VII.A, p. 166) and information which suggests that they exist externally of the cells in the culture medium (see Section VII.C, p. 175). 2 . Control of Expression of Alleles The main conclusion of the preceding section is that all alleles a t a given ocus can be expressed through the same cytoplasmic state. This, no doubt, is generally true but an additional degree of discrimination in the control of phenotypic expression appears to be operative in certain instances. A study of serotype inheritance in syngen 2 has shown that there may be preferential expression of but one allele in a heterozygote (Finger and Heller, 1964; Finger, 1967). The circumstances in which this phenomenon was demonstrated are as follows. Parental serotypes 197E

197E

34

72G

197E

197E

197E 72E

72E

197G

__ 197E 72E

72E

x

72E

72E

F,-serotypes at a temperature favouring the expression of the e locus.

FIG.6. Inheritance of serotypes on crossing stocks 197 and 7 2 when both parental cells are expressing the E-type and when only one parent is expressing the E-type. From Finger arid Heller (1964).

Crosses between cells of different stocks expressing the same locus (elg7x e72;Fig. 6) yield F, clones manifesting the hybrid E phenotype, as expected on the basis of locus selection. However, when parental cells express separate loci (elg7x g72,glg7x e72;Fig. 6) and F1 clones are grown in conditions favouring one locus (the e locus), there is a strong tendency for the manifestation of only one serotype, that derived from the previously active allele. Furthermore, this same single allele is expressed in B , clones irrespective of the cytoplasmic parentage. If, a t conjugation, there is only transfer in either direction of a haploid macronucleus without cytoplasmic exchange, we must conclude that these migratory micronuclei are predetermined for serotype in accordance with the phenotype from which they came. As pointed out by Finger (1967) this is somewhat surprising in view of the fact that, even in an unfavourable cytoplasm, the micronuclear genes (considered to have been inactive in the parental cells) determine an allele-specific cytoplasmic condition which in turn differentiates the developing macronucleus

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and the phenotype of the cell. The implication of these results is that some control factor (repressor?)can discriminate between alleles. Preer (1968) suggests that these results favour a primarily nuclear localization of serotype-controlling factors, i.e. a “nuclear state” rather than a “cytoplasmic state”, the only exception being at conjugation when the pattern of serotype inheritance undoubtedly shows a cytoplasmic effect. Other lines of evidence supporting this view are that cells tend to change their serotype more readily a t nuclear re-organization (conjugation and autogamy) than a t binary fission (Dryl, 1959) and that serotype transformation occurs much more frequently in clones forming new macronuclei than in clones whose macronuclei are descended from fragments of the old macronucleus by regeneration (Preer et al., 1963). Presumably a t such nuclear re-organization the control factors are liberated from the fragmenting macronucleus into the cytoplasm where they may be more susceptible to environmental influences and may partly determine the micronuclei. Whatever the localization of the controlling factors the fact remains that one allele cam be preferentially expressed in a heterozygote. Allele selection is a more general phenomenon in Tetruhymena. A number of characters-mating type, esterases and phosphatases as well as serotypes-have been shown to differentiate pure phenotypes in heterozygous cells (Nanney, 1963 ; Allen, 1967). However, this process (inter-allelic repression) is slightly different from that in Paramecium, for here the differentiation is stable and irreversible and confined to nuclear events whereas with Paramecium there is a cytoplasmic influence and the suppressed allele can come t o expression after suitable treatment. It can be argued that these special phenotypic effects are due to the complex structure and mode of replication of the ciliate macronucleus but analogies can be found in the differentiation of other cell types. For instance, Finger (1967) compares these effects in ciliates with other cases of allele preference such as X-chromosome inactivation in mice and immunoglobulin determination in heterozygous mammalian cells. 3. Control of Expression of Sub-Loci (Cistrons)

Some of the characteristics of mating-type differentiation in Tetrahymena have been interpreted in terms of the activation of only one of seven specificity-conferring cistrons at the mt locus. The remaining six cistrons are subjected to intra-locus repression (Nanney, 1963). The factors influencing this differentiation are various and include temperature, structure of the mt locus and genetic background. Apparently a somewhat similar phenomenon can influence serotype expression in Paramecium. This concerns the assortment of i-antigen

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subunits in heterozygotes of syngen 2 (Finger et al., 1966). The observation is that only one type of hybrid i-antigen molecule is formed in each heterozygous clone, yet different clones form different hybrid molecules. Furthermore, the overall proportion of different types expressed by a large number of clones seemingly corresponds to that expected from the random assortment of different subunits. It would appear that, after an initial random event, one particular arrangement of subunits is determined for that cell line. This is operationally equivalent to a fixation of the differential activity of each pair of allelic cistrons, assuming the three types of polypeptide to be determined by a polycistronic locus. However, there is no evidence that this mechanism operates a t the genelevel, indeed its significance as a process will remain obscure until more information is available. 4. Exceptional Types The usual approach in studying genic function and regulation is the detailed analysis of a number of precisely mapped mutations which give rise to readily assayed altered biochemical activities. Such fine-structure analysis of the i-antigen locus has been impracticable for a number of reasons, for instance, the lack of genetically linked markers and the readiness of treated cells to switch to the expression of another i-antigen locus. However, those few mutations that have been obtained in the laboratory have been adequately supplemented by a wealth of natural variation. As already mentioned, i-antigen alleles vary greatly in the stability of their expression; indeed some are never, or only rarely, expressed. For example, when heterozygotes a t the D locus in syngen 1 (containing the allele d 6 0 along with P ,d103,or d145)are passed through autogamy, they give rise a t low frequencies to cells which produce no D serotype, instead switching to the expression of another locus (Beale, 1957). Subsequent genetic analysis of such types showed that the ability to form an i-antigen specified by this locus was completely missing. These so-called “nulalleles” (do)appear to contribute no antigenic specificity in heterozygous cells, i.e. the serotype is totally determined by the normal allele. Therefore, they are operationally equivalent to gene deletions in which the properties of structure and control cannot be differentiated. As well as arising through an error a t recombination and segregation, “nul-alleles” have been obtained following irradiation of syngen 4 cells with X-rays (Reisner, 1955) and occasionally occur in natural populations. A serotype missing from several stocks of syngen 2 is type E. This deficiency has been shown to be due to a single gene effect (Finger, 1957). Further analysis has revealed that heterozygotes containing a deficient and normal E-forming allele make a complete i-antigen which differs in

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specificity from the normal parental serotype (Finger and Heller, 1963). The deficient allele appears to contribute specific determinants to the E-type antigen molecule. It is not known whether E-deficiency is due to a defect a t the structural locus (incomplete protein formed) or at some control locus (structural locus only active in heterozygotes). Presumably the former possibility would be favoured if only a fragment of the i-antigen molecule could be shown to be produced in the deficient strain, the latter if all the specificity determinants could be identified in the hybrid molecules of heterozygotes. An example, more informative on this issue, has been reported by Beale (1957). Stock 192 of syngen 1 is incapable of forming the G serotype. However, the specificity locus was present and apparently complete, for in a hybrid 192190strain, both i-antigen specifities were expressed. But, on segregating the alleles by making this hybrid strain undergo autogamy, the 192G serotype became unstable and soon disappeared, being replaced by an i-antigen determined by another locus. It would appear that the genetic information for the specification of the 192G-type antigen is complete but that the factors ensuring expression are missing. These examples lend further support to the view that there exist closely-linked control and structural genes a t the i-antigen locus. On the basis of mutual exclusion between loci being the norm, the secondary i-antigen formation described in Section 1I.E (p. 143) may be considered as an exceptional breakdown of the control mechanism coordinating the action of the various i-antigen loci. Two possible explanations are : the inability of control factors to discriminate between two or more loci, or the stabilization of expression of two or more loci due to environmental conditions intermediate between their stability ranges.

V. Function of i-Antigens It is remarkable that so much is known about paramecium i-antigens yet their biological function remains obscure. Let us first examine some of the circumstantial evidence already mentioned in the context of the genetics, location and chemistry of these proteins. Whatever their function, i-antigens appear to be indispensible. Paramecia lacking these surface proteins have never been found; indeed a complex genetic system has been selected for, to ensure that one i-antigen is always formed. When one serotype-conferring locus is inactivated, a n alternative active locus is immediately substituted. Their essential function is also suggested from the behaviour of paramecia treated with specific antiserum. Homologous antiserum not only immobilizes the cells but subsequently kills them. Also, dilute antiserum, not strong enough to immobilize, kills cells which have had their protein-synthesizing

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systems inhibited (Finger and Heller, 1964). The complexing of surface antigen with specific antibodies presumably destroys some vital function. Whether this lethal effect results directly from i-antigen inhibition or from more general blockage of a cell-surface property is not known. The chemical data describe a variety of i-antigen forms differing only in amino-acid composition and sequence. The similarity of allelic forms suggests that the level of variability is restricted by some common functional requirement. On the other hand, the large differences between non-allelic i-antigens suggest that the equivalent function is performed by an alternative molecular form. These alternative forms may, nevertheless, have had a common ancestry, diverging after gene duplication. Concomitant with this view is that different i-antigens function more efficiently under different sets of conditions. If this is so we might expect one serotype to have a selective advantage over another in a particular environment. I n general this is difficult to demonstrate for two serotypes cannot be maintained together for long enough to estimate survival meaningfully without the transformation of one type to the other. At truly intermediate conditions, both serotypes would be equally favoured. Of course, in a case like antiserum-induced change, serotype transformation has an obvious adaptive value, but here any other rather than a particular serotype confers the advantage. However, a true advantage can be claimed in the case of patulin-induced serotype change from 51D to 51B in syngen 4 ; here the B type had a better rate of survival than the untransformed D type (Austin et al., 1956). Since the i-antigen forms a buffer between the cell and its environment, a likely explanation is that it is an enzyme concerned with some aspect of membrane transport. However, with specialized organelles concerned with the intake (oral apparatus) and output (contractile vacuoles) of ions and molecules, a function of the surface in general may be difficult to demonstrate. The only evidence for enzyme activity is that cell extracts with i-antigen specificity have an associated ATPase activity (Van Wagtendonk and Vloedman, 1951). Perhaps a more thorough investigation of enzymic activity of purified i-antigen will reveal its true function.

VI. Formation of i-Antigens Much of what has already been said about serotype expression concerns the different ways of analysing the properties of the system but tells little of the actual processes involved. Mostly these processes are implied from the sets of relationship found to exist between environment, gene and cell-surface structure, and are described in terms of the mechanisms known to operate in other systems. Recently attempts have been made to fill in some of the details with a biochemical description of 6wo

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intermediate processes ; between the genes and the phenotype-i-antigen formation, and between environment and gene activity (serotype transformation; see Section VII, p. 165). The formation and transport of i-antigen have been studied either by following the appearance of the “new-type” i-antigen during serotype transformation, or by radioactively labelling protein immunologically identified as i-antigen. Both cell cultures and cell-free systems have been examined for their synthetic potential. A. SYNTHESIS I n Vivo Since a completeIy defined medium is as yet unavaiIable for the culture of Paramecium, the cells are fed on the bacterium Klebsiella aerogenes. Bacteria are ingested into food vacuoles where digestion takes place and metabolites are released into the cytoplasm. As a result, radioactiye labelling studies have utilized this normal feeding route by supplying the paramecia with bacteria labelled with the required isotope. Indeed this method has been shown to be more efficient than supplying the label as an exogenous source, in giving specific incorporation into Paramecium nuclear DNA (Berger and Kimball, 1964) and protein (Sommerville, 1967a). The passage of radioactivity from bacteria labelled with 14C-leucineor 35S-magnesiumsulphate has been followed by high-resolution autoradiography (R. E. Sinden, unpublished observations; Fig. 7) and by analysis of the distribution of radioactive protein in subcellular fractions (Sommervilleand Sinden, 1968).After a 15min. feeding pulse, radioactive material has already diffused from the labelled food vacuoles into the cytoplasm where it appears to become associated first with cell particulates (fractions containing membranous vesicles and ribosomes) then in increasing amounts with soluble material. From about 30 min., cellsurface structures become labelled. The timing of these events has been correlated with the appearance of labelled i-antigen (Fig. 8) which can be separated from the other labelled proteins by the techniques of column chromatography, immunoelectrophoresis and direct precipitation by specific antiserum. The radioactivity in these preparations is assayed by either liquid scintillation counting or autoradiography (Sommerville,1968).During the early stages of labelling (30-45 min.) before there is much detectable antigen-specific activity on the cell surface, most labelled i-antigen is found in a “membrane fraction” which sediments after mitochondria but before the bulk of the ribosomes on sucrose-density centrifugation. This fraction, which has been shown to containmembrane-boundribosomes as the components active in protein synthesis (Sommerville and Sinden, 1968), represents

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FIG.7 . Electron microscope autoradiographs of Paramecium after feeding with 3%-labelled bacteria. (a) Several minutes after feeding, the autoradiographic grains are contained in the area of a food vacuole (fv) near the gullet which shows many cilia ( c )in section. (b) After one hour, autoradiographic grains are found in the cytoplasm and on the surface structures, e.g. the cilium (c) shown. TJnpublished data of R. E. Sinden.

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the most likely site of i-antigen synthesis. Also examination of this fraction after detergent treatment (Figure 9) suggests that the radioactive i-antigen is at least partly associated with membrane-released

FIG.8. Electron micrograph showink simultaneous localization of transformed type i-antigen using ferritin-conjugated antibody (arrowed) and biosynthetically incorporated 35s (autoradiographic grain). The conditions of labelling are similar t o those of Fig. 7b. Unpublished data of R. E. Sinden.

ribosomes, particularly those that are arranged as ribosomal aggregates (polyribosomes). There is also evidence for i-antigen activity in association with unbound ribosomes. Such ribosomes tend to adsorb to unlabelled washed “membrane fraction” whereas labelled soluble i-antigen does not. Unbound ribosomes with associated i-antigen activity may represent either breakdown products of the isolation procedure or also some precursor form of the natural process. The significance of the membraneribosome-antigen association is not altogether clear. 6

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The distribution of i-antigen associated with subcellular fractions has been assayed by the immunologicaltechniques of gel-diffusion (Preer and

Fraction number

FIG.9. Sucrose density-gradient analysis of labelled membrane fraction. Cells were labelled with 1%-labelled bacteria for 15 min. and incubated for a further 30 min. After homogenization, the fraction, pelleted between 10,000 g x 10 min. and 20,000 g x 20 min., was centrifuged through a 10-25y0 sucrose gradient with a 50% sucrose cushion. The gradient fractions were analysed for absorbance a t -), total protein radioactivity (-- o --) and specificallyprecipitated 260nm (radioactivity (counts/min. precipitated by homologous antiserum minus counts/ min. precipitated by heterologous antiserum; indicated as histograms). (a) The membrane fraction pelleted to the dense sucrose cushion. (b)After treatment of the same material with 0.5% sodium deoxycholate. The peak between fractions 12 and 13 has an approximate sedimentation coefficient of 80s. The final fraction had a radioactivity of 12,000 counts per min.

Preer, 1959; Seed et al.) 1964) and adsorption titration (Seed et al., 1964; Macindoe and Reisner, 1967). These studies implicate ribosomes as the sites of i-antigen synthesis but have not distinguished their morphological form.

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B. SYNTHESIS In Pitro Paramecium cell-free extracts are capable of incorporating amino acids into protein (Reisner and Macindoe, 1967;Sommerville and Sinden, 1968)and such a system has been used to study the synthesis of i-antigen (Sommerville, 1967a, b). The particular advantage of in vitro-synthesizing systems is that they often provide a meansof studying macromolecular syntheses in simplified conditions. Here the specific requirements of the process can be identified and related back to the generally more efficientand controlled in vivo process. Apparently the informational requirements for the synthesis of i-antigens are present in supernatants derived from cell-homogenates. There are two points worth considering; the presence of the “membrane fraction” is essential for the production of significant amounts of labelled material with i-antigen specificity, and this specificity is the same as that of the i-antigen expressed by the cells prior to homogenization. The first point agrees with the finding of the in vivo study that membrane-bound ribosomes are the major sites of i-antigen synthesis. Supernatants including only unbound ribosomes and soluble material formed little or no labelled i-antigens. The second point implies that only one type of i-antigen is being manufactured by each cell type and so the process of mutual exclusion between different i-antigens is probably exerted a t some stage before the synthesis of the protein. However, it is possible that both of these points can be re-interpreted by considering that the detection of i-antigen activity relates to the genesis of specific moIecular configuration rather than to the completion of synthesis, i.e. both properties may not appear co-incidentally, antigenic specificitymay arise at some later stage, perhaps by aggregation of polypeptides. It would be of some interest to know if the different polypeptides constituting the complete i-antigen molecule are synthesized separately, aggregating after the completion of synthesis, or whether they are synthesized in close proximity on some compound (polycistronic) messenger. Certainly the findings that some heterozygotes form only one hybrid molecular species (Finger et al., 1966) would tend to rule out a random association of subunits and favour the second proposal. A study of the sedimentation values of those ribosomal aggregates giving an i-antigen reaction has shown that quite large aggregates (each consisting of up to 20-30 ribosomes) may be involved in i-antigen synthesis (Sommerville, 1967a). If there is a direct relationship between polyribosome size and length of polypeptide being synthesized,this result would suggest that the aggregates contain enough information for the synthesis of more than one polypeptide but less than the complete protein. However, the nature of the aggregation is critical since the ribosomes may be linked by

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virtue of their nascent polypeptide chains rather than a messenger RNA. I n fact, breakdown or aggregation of ribosomal clusters may occur during the isolation procedure. The main drawback in studying i-antigen synthesis in vitro is that there is little natural release of soluble labelled i-antigen during the period in which the system is active (up to 30 min. a t 30”). However, labelled protein with i-antigen specificity is released from the particulate structures by treating them with puromycin. This “artificially” released material is then available for further analysis (Sommerville, 1967b). Labelled antigenic material is also released on treating the membrane fraction with detergent but this may be a n indirect effect due to the solubilization of degradative enzymes contained in the preparation (Sommerville and Sinden, 1968). It is somewhat surprising that protein labelled in vitro, particularly that “artificially” released from ribosomes, reacts in an immunologically specific manner. The released antigenic material is similar to the native protein in its sedimentation and electrophoretic properties (Sommerville, 1967b) suggesting that it is composed of an aggregation of polypeptides like the complete i-antigen. However, it is unlikely that complete synthesis of the molecule has occurred in the cell-free system, a more likely explanation being that the released labelled fragments have associated in vitro with pre-existing complete subunits. This view is substantiated by the analysis of such labelled material after chemical reduction. Disc electrophoresis of immunologically-precipitated labelled material has shown that the radioactivity migrates on average slightly faster than marker i-antigen subunits, indicating that the polypeptides labelled in the cell-free system were partially incomplete (J. Sommerville, unpublished observations). Further, separated subunits appear to be capable of refolding in vitro to give an immunologically specific form. When reduced i-antigen was separated into discrete subunit bands by disc electrophoresis and the acrylamide gels were set in agar, one type of subunit diffused through the agar and formed a precipitate with diffusing homologous antiserum (Figure lb). It would appear, therefore, that to a large extent the formation of i-antigen can occur in vitro. Apart from the construction of polypeptides, the formation of this large and complex protein may proceed without special control influences from the living cell.

C. TRANSPORTATION Accepting that i-antigen synthesis occurs on the endoplasmic reticulum which is distributed throughout the cytoplasm of the cells, there must be a two-stage transport of macromolecules, in the form of information from

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the nucleus to the site of synthesis, and in the form of the newly synthesized protein or its subunits from here to the cell surface. We know little about the first process. It may occur continuously or infrequently depending upon the natural stability of the m-RNA for i-antigen synthesis. Since i-antigen-specific m-RNA formation can only be conveniently detected in cells undergoing serotype transformation this aspect will be discussed in more detail in Section VII (p. 170). Attempts to study the second process are mentioned here. Since i-antigen other than that on the cell surface has not been detected by cytological means, it must be concluded that there exists no significant internal pool of the completely formed protein. One possible explanation (see Section 111,p. 146) is that the newly synthesized i-antigen is rapidly removed from the ribosomes to the cell surface. Information from cell fractionation studies are in general agreement that there is little soluble cytoplasmic i-antigen (Finger et al., 1960; Seed et al., 1964; Macindoe and Reisner, 1966; Sommerville, 1967a). I n other cell types, generally those forming “secretory” proteins such as collagen (Ross, 1968)andpancreatic proteins (Jamieson and Palade, 1967), the synthesized material appears to pass into the cisternae of the endoplasmic reticulum to be transported to the cell exterior via membrane-enclosed spaces. I n Paramecium there is no obvious connection of the endoplasmic reticulum with the cell surface although many microtubules of unknown function pass through the cytoplasm. Considering i-antigen utilization by the cell, it would be interesting to know if the growth of the surface membranes in any way regulates the synthesis of i-antigen. The fact that there appears to be little internal i-antigen indicates some sort of regulation of synthesis. Also serotype transformation normally requires cell growth and division (see p. 167). However, since i-antigen synthesis represents only a small percentage of the protein synthesized by the cell, experimental approaches to this problem are difficult.Recently the relationship bet~een~~c-lipid-labelled membranes and ferritin-labelled new-type i-antigen has been studied by doubly labelling transforming cells in order to see if there was any correlation in cellular incorporation and distribution of the two labels (R. E. Sinden and J . Sommerville, unpublished observations). However, the results are, as yet, inconclusive.

VII. Serotype Transformation I n studying the process of serotype transformation, two types of treatment have been employed. The first involves the manipulation of one of a wide variety of environmental factors which induce the cells to form an alternative i-antigen. Effects of this type are normally achieved

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by means of temperature change, addition of antiserum, treatment with the antibiotic patulin, addition of i-antigen and addition of culture fluid. Secondly, agents can be used to change the rate of the physiological response after transformation has been induced. These agents are generally antibiotics believed to act in a specific manner on certain biosynthetic processes such as actinomycin D, puromycin and chloramphenicol. Sometimes, when cells induced to change serotype are treated with antibiotics, the interaction of effects may result in either inhibition or stimulation of the transformation process, depending upon the relative intensities and timing of the treatments. Alternatively, the effects of antibiotics may not be as specific as often assumed. This description of serotype-transformation experiments will be divided into two sections ;one concerned with the kinetics of the general physiological response, the other with the effects produced by interfering with the normal regulatory mechanism. Since this phenomenon is often considered to result from differential gene activity, some points relating to nuclear activity during the transformation process will be mentioned separately. A. INDUCTION KINETICS

It has been noted (Beale, 1957) that an important factor in inducing serotype transformation is the nature of the change itself; a rapid change in environmental conditionsismuch more effective than a gradual change. Of course, at a certain level, the shock-effect is superceded by a pathological effect. Yet in some cases, transformation may involve a certain degree of catabolism (degradation of the pre-existing synthetic mechanism?) before the physiological adaptation eventually occurs. However, the shock element is not essential. I n all probability most naturally occurring environmental changes are relatively slow and initiate a smooth physiological switch. Mild stimuli when applied to laboratory cultures result in a delay of many fissions before transformation takes place but, once initiated, the process is completed within a standard 2-3 fissions. Since most laboratory stimuli are fairly drastic, it should be remembered that the effect may be slightly different from the natural phenomenon. Some special points appropriate to different types of stimulus are as follows. 1. Temperature

Serotype transformation in response to temperature change has been studied primarily with syngen 1 stocks where there tends to be a direct and regular relationship between temperature and the serotype formed (Beale, 1957). This regularity of response can be measured in a number of

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ways. For instance there is a specific temperature range for the stable expression of each serotype allele (see Fig. 5, p. 153) and with a standard stimulus the time required for the change of one specified serotype to another is constant. These are properties of the "cytoplasmic state" of the cells and have been discussed (Section 1V.C)p. 150). However, the time required for the completion of serotype transformation can be varied with two properties of the temperature change. First, the extent of the temperature difference is important. Cells of type 41G previously grown at 18") when transferred to 34", 30" or 28", completed transformation to 41D after 16 hr. growth (4 fissions), 30 hr. growth (7-8 fissions) and 50 hr. growth (10-11 fissions), respectively (Beale, 1957). I n general, the smaller the change, the longer the period of growth required for complete serotype change. Yet, as already mentioned, the actual process of transformation (when both G and D types are detected simultaneously) is a relatively short period of 2-3 fissions. Thus there appears to be a variable lag in the time required to establish the synthesis of the new type i-antigen. The other important factor is the length of stimulus.For transformation to occur the cells need not be grown continuously at the changed temperature; they can be transferred back to the original temperature after a critical period which again is dependent upon the extent of the change. For instance, 2.5 hr. at 36", 4 hr. at 34" and 5 hr. a t 32" each produced an eventual change of 50% of 41G cells to the 41D type (Beale, 1957). Hence a drastic change in temperature for a short period is sufficient to bring about an internal event which determines the eventual phenotypic change. No doubt, these effects stem from the properties of the cytoplasmic control elements. Normally, serotype transformation only becomes apparent after a certain amount of metabolism and growth. Since de novo synthesis of protein is required, the cells must be metabolically active, but is an increase in cell surface also a necessary factor;! Apparently not, for in the case of patulin-induced transformation from 51D to 51B in syngen 4 (seelater) the change was shown to occur without cell growth and division (Austin et al., 1956). Also the rate of transformation normally proceeds faster than the dilution of pre-existing surface area expected on the basis of the known growth rate. Therefore the transformation process may be aided by the loss of old-type i-antigen from the cell surface, a factor which is particularly relevant to the effect of antiserum treatment which does in fact give a faster rate of transformation than temperature change. The measure of transfoimation in these early experiments was based on the immobilization test which at best gives a reaction difference only after several hours. Recently some of the early events in the process have been studied with a view to following the initial biochemical changes.

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The synthesis of i-antigen during the first few hours of transformation has been followed by radioactively labelling the cells and assaying the amounts of old and new-type radioactive i-antigen by autoradiography of immuno-electrophoresis gels (J. Sommerville, unpublished observations). The cells used were type 168Gwhich transform within 2-3 fissions. Two important points have emerged: (a) the old-type (G) i-antigen continues to be synthesized for at least 4 hr. (the maximum length ofthis type of experiment) ; (b)the new-type (D) i-antigen appears at an early stage (15 min.) but its synthesis increases only slowly: even after 4 hr. the cells have synthesized far less D-type than G-type. TABLE4. First Detection of Transformation 168G to 168D as Measured by Different Techniques Total Fluorescein- FerritinSurface radiolabelled labelled radioactive antiseruma antiseruma active i-antigene i-antigenb AutoradioImmobilization Fluorescence Electron High graphy of micromicroresolution SCOPY SCOPY autoradio- precipitin bands graphy 15 min. 3-5 hr. 1-2 hr. 30 min. 7-8hr.

Marker Antiseruma

Assay

Time

From Mott (1965). From R. E. Sinden (unpublished observations). c From J. Sommerville (unpublished observations). a

b

I n order to study the continuation of this process the experiment was altered slightly by labelling the cells for 2 hr.-periods at various stages throughout transformation. Here the results were : (a) the old-type (G) i-antigen continues to be synthesized for a considerable time, at least 20 hr. (two fissions),after the stimulus was applied; (b)the new-type (D) synthesis only becomes equal to the slowly decaying old-type (G) synthesis after approximately 10-20 hr. (1-2 fissions), eventually replacing it entirely after 30 hr. (three fissions). It would appear that in this instance at least serotype transformation involves a gradual changeover in the synthesis of one i-antigen to the synthesis of another (see also p. 173). The early appearance of new-type i-antigen on the cell surface has been followed by the use of fluorescein-labelled and ferritin-labelled antisera (Beale and Mott, 1962; Mott, 1965). Again the transformation studied was from l68G to 168D. As for the first detection of new-type (D) i-antigen, what emerges from the various experiments is that the timing of this event is ent,irely dependent upon the technique used to

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measure it (Table 4).R. E. Sinden (unpublished observations) found that treatment of transforming cells with ferritin-labelled antibody can give a quantitative measure of the amount of surface i-antigen. Apparently the amount of new-type (168D) i-antigen per unit surface area increases exponentially from about the time of the temperature stimulus. So, in addition to there being little delay in the initiation of its synthesis, there is little delay in the surface appearance of the new i-antigen. The temperature-stimulated transformation of stock 28 syngen 2 from type G to type E was followed by gel-diffusion assay over the length of the process (Balbinder and Preer, 1959). Here again, the increase in new-type i-antigen per cell was exponential, increasing at a constant rate till near the completion of the process and extrapolating back to zero time. I n summary, there may be three steps in the process of serotype transformation : (a) The nature of the environmental stimulus predetermines which allele is to be expressed by activating the appropriate cytoplasmic control factors. (b) There is a variable, allele-specific lag period till the cytoplasmic conditions are established to allow the expression of the selected i-antigen locus. For some alleles, e.g. l68G syngen 1,28G syngen2, there is virtually no lag, for others the lag may be 50 fissions or more. (c) Synthesis of the new-type i-antigen and decay in synthetic rate of the old-type i-antigen proceed to the completion of the “switch” at 2-3 fissions. 2. Antiserum

Since syngen 4 cells treated with dilute homologous antiserum sometimes give rise to transformed types, it was suspected that the antibodies interfere in some way with the production of i-antigen (Sonneborn and Le Suer, 1948). However, it was demonstrated by Skaar (1956) that serum treatment can also stabilize the expression of the homologous i-antigen. The consensus of opinion is that serum treatment does not act in a specific manner but acts indirectly by causing a general disturbance in cell function (Beale, 1957). Generally antiserum treatment is used in conjunction with other controlled factors such as temperature and food-supply (growth rate). Under these special conditions serotype change can be directed in a regular fashion. Some points should be mentioned in respect of the transformation of 51D to 51B at different temperatures and in different concentrations of antiserum (Austin et al., 1967a). The two variables seem to affect the transformation in different ways; an increase in temperature tends to

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shorten the time to the onset of the process (in this case when the first cells are immobilized by anti-51B serum) whereas an increase in serum concentration tends to bring the process to completion (when all cells are immobilized by anti-51B serum) more quickly. For instance, the approximate times of onset and 95% transformation using 1:2000 strength anti-51D serum are 7 and 20 hr. at 19", 5 and 48 hr. at 27", 4 and 18 hr. at 31"; and using 1:SO0 strength antiserum are 7 and 10 hr. at 19")4 and 9 hr. at 27') 3 and 6 hr. at 31". Thus antiserum treatment may be regarded as a useful cofactor ininfluencing the rate of transformation. The times quoted above are less than thosenormally obtained with temperature-induced serotype change. The special effect of antiserum may be due to the fact that antibody removes i-antigen from the cell surface allowing more new-type i-antigen to occupy the surface sites. This would suggest some sort of control on i-antigen synthesis influenced by the condition of cell surface. 3. Chemicals The chemical agents patulin and acetamide have been shown to induce serotype transformation in a specific and predictable manner. Patulin changes the serotypes C, D, E, H and N of stock 51, syngen 4,almost entirely to serotype B, occasionally to a small percentage of serotype A, whereas serotypes AandB are unaffected bythisantibiotic (Austin, 1957; Austin et al., 1956, 1967b). The transformation of D to B induced by 1 pg. patulin per ml. is first evident at about 18 hr. and completed at about 48 hr. Acetamide changes cells of serotype D or B to serotype E, 0.25 M-acetamide producing transformation kinetics similar to those of 1pg. patulin per ml. (Austin et al., 1967b). These effects are most interesting for they suggest that patulin and acetamide act specifically on certain i-antigen molecular configurations or else the conditions favouring the synthesis of these molecules. An understanding of their action would be of great help in analysing the control of i-antigen expression. 4. Other Factors Other factors known to change serotype expression are salinity of the medium, food supply, X-rays and ultraviolet rays, and enzymes such as trypsin and chymotrypsin (see Beale, 1954).

B. NUCLEAR ACTIVITY One of the most important questions concerning serotype transformation is at what level the response is initiated. Let us examine the implications. If transformation is initiated at the level of the genes there would have to be a switch in the type of RNA synthesized; if at the level of mRNA an alternative type of preformed RNA would be selected for

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translation ; if a t the level of proteins different pre-existing protein specificities would be expressed by the cell. We already know that during transformation there is a de novo synthesis of the protein i-antigen (Balbinder and Preer, 1959 ;J.Sommerville, unpublished observations) but is there necessarily a de novo synthesis of m-RNA coding for the new-type i-antigen? Preformed RNA molecules of this type could presumably cope with the change in synthesis for a certain time depending upon the functional stability of these molecules. But for the change in serotype expression to last indefinitely, a change in the activity of the genes will eventually be required. Autoradiographic studies have shown that RNA is synthesized more or less continuously during the cell cycle and passes from the macronucleus into the cytoplasm (Kimball and Perdue, 1962). There appears to be no evidence for cytoplasmic synthesis of RNA (Prescott, 1961). If a significant part of the RNA synthesized under these conditions is informational, the information passes to the cytoplasm continuously during the cell cycle. The micronucleus is generally considered to be genetically inactive, a point of view substantiated by Kimball (1964) who found little incorporation of RNA precursors into the micronucleus compared with the substantial macronuclear incorporation. The foregoing observations concern RNA synthesis in general. Recently, the activity of both macronucleus and micronucleus has been studied in relation to serotype transformation (Pasteniak, 1967). The findings are that, after the induction of transformation by either antiserum or patulin, there is an enhanced rate of RNA synthesis as judged by autoradiographic analysis, in both macronucleus and micronucleus. The increase in RNA synthesis reaches a peak about the time when transformation is first detected by the immobilization test, i.e. at 5 and 8 hr . for antiserum- and patulin-induced transformation respectively. However, it is somewhat surprising that for both types of nuclei the maximum increase in RNA synthesis is more than double the basal (untransformed) level. The significance of this large effect is not clear, especially in the case of patulin which appears to be quite specific in its effect of inducing serotype transformation. Large amounts of newly synthesized RNA at the time of serotype transformation are also detected in DNA-RNA hybridization experiments (Gibson, 1969). This was demonstrated by the hybridization of pulse-labelled RNA recovered from cells at various stages during the transformation process with the DNA of the stable original cell type and the stable transformed cell type. Gibson (1969) suggests that there is a general synthesis of stable m-RNA species for all i-antigens after the induction stimulus, but only one of these is selected for continued activity.

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As already mentioned, the stimuli applied to promote serotype transformation may have a deleterious effect on the cells. It is perhaps worthwhile noting that with Tetrahymena relatively slight changes in environmental conditions bring about a large efflux of orthophosphate, purines and pyrimidines from the cells (Cline, 1966; Cline and Conner, 1966; Pruett et al., 1967). This excretion has been shown to result from RNA catabolism, 30% of the total cellular RNA being degraded over a period of 3 hr. It appears that ion and tonicity changes alter both the stability of cellular RNA and the properties of the membrane system. It is not known if Paramecium is influenced in the same way, but Pasternak (1967) did note that there was an initial reduction of labelled RNA to almost zero during the first few hours after antiserum treatment. Patulin treatment, on the other hand, did not result in a decrease in RNA synthesis from the basal rate. Therefore relatively strong stimuli may result in a certain amount of RNA catabolism, but whether RNA catabolism is required for serotype transformation is not known. The main question left unanswered is the meaning of micronuclear activity during serotype transformation. Does it play some special role in the regulation process? C. REGULATION 1. Effect of Antibiotics

Austin et al. (1967a, b) have studied the effects of actinomycin D, puromycin and chloramphenicol on a number of different serotype transformations in syngen 4,induced by homologous antiserum, patulin and acetamide. It is found that the transformation process can both be inhibited and stimulated by antibiotic treatment, depending upon the relationship between this treatment and the induction stimulus. That is, the relative concentrations and timing of stimulant and antibiotic are shown to be important factors. Considering the effect of actinomycin D the main results were as follows : (a) Simultaneous treatment with high concentrations of both inducer and antibiotic (6-25pg. actinomycin D per ml.) inhibits transformation. (b) As the concentration of the antibiotic is decreased a point is reached (less than 3 pg. actinomycin D per ml.) where further decrease stimulates transformation. High concentrations of actinomycin D (10-12.5 pg. per ml.) used in conjunction with a weak induction also give rise to stimulation. (c) The same low concentration of antibiotic (1-3 pg. actinomycin D per ml.) may either inhibit the transformation if added after the inducer, or stimulate the transformation if added before the inducer.

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Puromycin and chloramphenicol act in a similar manner a t their own appropriate concentrations. I n fact the results show that all three antibiotics, actinomycin D, puromycin and chloramphenicol, are effective in influencing the rate of serotype transformation. Austin et al. (1967a, b) conclude that both RNA synthesis and protein synthesis are essential for the transformation process and that interference with either level of synthesis is sufficient to initiate a switch on gene activity. Further, the apparently heterogenous inhibition/stimulation effects produced by the antibiotics can be resolved by considering that they always act by inhibiting i-antigen synthesis. The variable effect is then due to whichever i-antigen, the old (pre-existing) type or the new (transformed) type, is being actively synthesized, and hence more susceptible, at the time of antibiotic treatment. However, what remains unexplained is whether the antibiotics are acting on the synthesis of the proteins, control factors or both. The experiments described above involve a delay (phenotypic lag) before the effect of antibiotic treatment can be assessed (by the immobilization test). The actual synthetic capacity of the cells during the process of serotype transformation has been studied by assaying radioactively labelled i-antigen (J.Sommerville, unpublished observations). Cells of type lS8G have been subjected to a temperature shock (20' to 34"))some cells being suspended from this time in actinomycin D. The progress of transformation in the presence and absence of actinomycin D has been followed by measuring the relative amounts of old-type (G) and new-type (D) i-antigen synthesized during 2 hr. periods throughout the transformation process. The main findings were as follows (see Fig. 10):

(a) There is a gradual decline in total i-antigen synthesis over a period of three fissions (about 30 hr.) when the cells are suspended in 5-20 pg. actinomycin D per ml. Under these conditions there is no cell growth or division and death occurs towards the end of the experimental period. (b) The relative amounts of synthesis of old-type (G) and new-type (D)i-antigens in the presence of 5 yg. and 20 pg. actinomycin D per ml. are no different from the relative amounts of synthesis of the two types of i-antigen in untreated transforming cells. Thus it appears that actinomycin D does not specifically affect serotype transformation, but has a general detrimental effect on the cells. Continued serotype transformation in the presence of high concentrations of actinomycin D suggests two possibilities ; that this antibiotic inhibits RNA synthesis but the cells contain pre-exising stable RNA coding for alternative i-antigens, or that the antibiotic does not inhibit synthesis of RNA coding for the new-type i-antigen any more than it

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inhibits the continued activity of the RNA coding for the old-type i-antigen. The only measure of the effectiveness of actinomycin D as an inhibitor of RNA synthesis in Paramecium is that more than 60% of the

FIG.10. Autoradiograph of immunoelectrophoresis gel (see Sommerville, 1968) showing synthesis of i-antigen by transforming cells cultured in presence and absence of 20 pg. actinomycin D per ml. (indicated by A). Cells transforming from 168G to 168D were radioactively labelled for 2 hr. periods a t 0, 8, 16 and 32 hr. after the temperature change (20' to 34"). The positions where anti-168G and anti-168D sera were applied are indicated by G and D respectively.

cellular RNA synthesis is inhibited after 1 hr. in the presence of 12.5 pg. of the antibiotic per ml. (Pasternak, 1967). Although, the particular biochemical effects of actinomycin D on the i-antigen system are still unknown, the evidence suggests that this antibiotic does not act in the generally accepted way, that is by blocking synthesis of unstable informational RNA (cf. Harris, 1968). 2. Addition of End-Product It has been found that clones unstable in their serotype expression can be influenced by treatment with purified i-antigen (Finger, 1967). For instance, in syngen 2, cells of C serotype are unaffected by C antigen but tend to be induced to transform to G serotype by G antigen. This effect is enhanced by pretreating the cells with puromycin which presumably makes the existing synthesis of i-antigen more unstable. Thus, added end-product (i-antigen) may influence its own synthesis by means of a positive feed-back mechanism. However, the i-antigen may not initiate its own synthesis but act indirectly via the cytoplasmic control factors which determine the i-antigen locus to he expressed.

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3. Addition of Culture Medium

Another naturally occurring phenomenon which influences the stability of serotype expression has recently been described (Finger, 1967; Finger et al., 1967). Serotype transformation can be induced by the cellfree culture fluid in which a heterologous cell type has been growing. Unlike induction by end-product i-antigen, this effect is not directional and can best be explained by considering that the medium of a given clone of cells contains a collection of “repressor substances” which tend to inhibit the formation of all i-antigens except the one being expressed by that clone, i.e. the medium from stable C-type cells may transform unstable cells of G and X serotypes in an unpredictable fashion. Variable effects may be due to quantitative differences in the concentrations of various “repressors” in the culture media which in turn are due to the stability characteristics of donor and recipient cells. Antiserum prepared against cell-free culture medium has the effect of inducing the expression of specific i-antigen loci (Finger, 1967). As expected from the postulated action of the medium (“repressors”), the particular locus stimulated in recipient clones is a property of the antimedium serum and any one clone may be induced to form a mixture of serotypes. The action of anti-medium serum is enhanced by treating the recipient cells with antiserum against the homologous serotype. Thus the genetic controlling factors composing the “cytoplasmic state” of Paramecium (Section IV.C, p. 150) may be extended in their range to include intercellular effects. This is of interest as a mechanism for making possible communication between cells to determine a collective differentiation. The next step in this investigation is likely to be the chemical identification and characterization of these controlling factors.

VIII. ConcIusions

I have tried to discuss the significance and implications of the experimental results as they have appeared in the main text of this article. Some points omitted here have been covered by other reviews, particularly the relationship between paramecium i-antigens and bacterial antigen systems (Beale and Wilkinson, 1961) and between i-antigens and various other genetically controlled cell-surface phenomena (Beale, 1964). The main area of interest at the moment is the nature of the control of serotype expression. It is too early yet to tie together all the results mentioned into a comprehensive scheme ; some results are conflicting and others may not be generally applicable. Nevertheless, the encouraging aspect of this work is that several unusual and interesting phenomena have been discovered in the serotype system :

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(a) The reversible differentiation of one of many phenotypes involving a highly sophisticated degree of genetic control, i.e. inter-allelic and intra-locus effects as well as the co-ordinated control of a large number of loci. (b) The presence of genetic control factors in the cytoplasm and culture medium which are influenced in a highly specific manner by environmental conditions. (c) The possibility of the informational RNA for i-antigens being relatively stable and subject to translation control.

It is hoped that a description of the molecular nature of these various elements of the serotype system in Paramecium and an understanding of their integration into an ordered mechanism will soon be forthcoming.

IX. Acknowledgements I should like to thank Mr R. E. Sinden for supplying unpublished results and photographs, and Professor G. H. Beale for reading the manuscript and giving advice on many matters. REFERENCES Allen, S. L. (1966). I n “Chemical Zoology”, (M. Florkin and B. Scheer, eds.). Vol. 1, pp. 617-694. Academic Press, New York. Austin, M. L. (1957). Microbial Genet. Bull. 15, 5. Austin, M. L., Widmayer, D. and Walker, L. M. (1956). Physiol. 2001.29, 261. Austin, M. L., Pasternak, J. and Rudman, B. M. (1967a). ExpZ Cell Res. 45, 289. Austin, M. L., Pasternak, J. and Rudman, B. M. (1967b). Expl Cell Res. 45, 306. Balbinder, E. and Preer, J. R. (1959).J . gen. Microbiol. 21, 156. Beale, G. H. (1952).Genetics, Princeton, 37, 62. Beale, G. H. (1954). “The Genetics of Paramecium uureliu”. Cambridge University Press, London. Beale, G. H. (1957).I n t . Rev. Cytol. 6, 1. Beale, G. H. (1964). In “Recent Progress in Surface Science”, (J. F. Danielli, K. G. A. Pankhurst and A. C. Riddiford, eds.). Vol. 2, pp. 261-351. Academic Press, New York. Beale, G. H. and Kacser, H. (1957).J . gen. Microbiol. 17,68. Beale, G. H. and Mott, M. R. (1962).J . gen. Microbiol. 28, 617 Beale, G. H. and Wilkinson, J. F. (1961). A . Rev. Microbiol. 15, 263. Berger, J. D. and Kimball, R. F. (1964).J . Protozool. 11, 534. Bishop, J. 0. (1961).Biochim. biophys. Acta 50,471. Bishop, J. 0. (1963).J.gen. Microbiol. 30,271. Bishop, J . 0. and Beale, G. H. (1960).Nature, Lond. 186, 734. Cline, S. G. (1966).J . cell. cornp. Physiol. 68, 157. Cline, S. G. and Conner, R. L. (1966).J . cell. c m p . Physiol. 68, 149. Dippell, R . V. (1954).Caryologia 6 (Suppl.), 1109. Dippell, R. V. and Sinton, S. E. (1963).J . Protozool. 10, (Suppl.), 22. Dryl, S. (1959).J . Protozool. 6 (Suppl.), 25.

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Epstein, W. and Beckwith, J. R. (1968).A. Rev. Biochem. 37,411. Finger, I. (1956).Biol. Bull. mar. biol. Lab., WoodsHole 111,358. Finger, I. (1957).J. Genet. 55, 361. Finger, I. (1964).Nature, Lond. 203, 1035. Finger, I. (1967). In “The Control of Nuclear Activity”, (L. Goldstein, ed.). pp. 377-411. Prentice-Hall, Inc., Englewood Cliffs, N.J. Finger, I. and Heller, C. (1962). Genetics, Princeton 47, 223, Finger, I. and Heller, C. (1963).J . molec. Biol. 6, 190. Finger, I. and Heller, C. (1964a). Genetics, Princeton 49,485. Finger, I. and Heller, C. (1964b). Genet. Res. 5, 127. Finger, I.,Heller, C. and Green, A. (1962). Genetics, Princeton 47, 241. Finger, I.,Heller, C. and Smith, J. P. (1963).J . molec. Biol. 6, 182. Finger, I., Onorato, F., Heller, C. and Wilcox, H. B. (1966).J. molec. Biol. 17, 86. Finger, I., Heller, C. and Larkin, D. (1967). Genetics, Princeton 56, 793. Gibson, I. (1969).Adv. Morphogen. in press. Harris, H. (1968). “Nucleus and Cytoplasm”. Clarendon Press, Oxford. Jacob, F. andMonod, J. (1961).J. molec. Biol. 3, 318. Jamieson, J. D. and Palade, G. E. (1968).J.Cell Biol. 34,577. Jones, I. G. (1965a). Biochem. J. 96,17. Jones, I. G. (1965b). Nature, Lond. 207, 769. Jones, I. G. and Beale, G. H. (1963).Nature, Lond. 197, 205. Jurand, A., Beale, G. H. and Young, M. R. (1962).J. Protozool. 9,122. Jurand, A., Beale, G. H. and Young, M. R. (1964).J.Protozool. 11, 491. Kimball, R. F. (1964). In “Biochemistry and Physiology of Protozoa”, (S. H. Hutner, ed.). Vol. 3, pp. 243-275. Academic Press, New York. Kimball, R. F. and Perdue, S. W. (1962a).Expl Cell Res. 27,405. Kimball, R. F., Vogt-Kohne, L. and Caspersson, T. 0. (1960). ExpZ Cell Res. 20, 368 KoBciuszko, H. (1965). Polio biol., Praha 13, 239. Macindoe, H. and Reisner, A. H. (1967).Aust. J. biol. Sci. 20, 141. Margolin, P. (1956). Genetics, Princeton 41, 685. Mott, M. R. (1963).Jl. R. microsc.80~.81, 159. Mott, M. R. (1964).Ph.D. Thesis: Univ. of Edinburgh, Edinburgh. Mott, M. R. (1965).J. gen. Microbiol. 41, 251. Nanney, D. L. (1963).I n “Biological Organisation a t the Cellular and Supercellular Level”, (R. J. C. Harris, ed.). pp. 91-109, Academic Press, New York. Pasternak, J. (1967).J. exp. 2001. 165, 395. PBrez-Silva, J. and Alonso, P. (1966). Arch. Protistenk. 109, 65. Preer, J. R. (1959a).J. Immun. 83,276. Preer, J. R. (195913).J. Immun. 83, 378. Preer, J. R. (1959c).J. Immun. 83,385. Preer, J. R. (1959d). Genetics, Princeton 44, 803. Preer, J. R. (1968). I n “Research in Protozoology”, (T. T. Chen, ed.). Vol. 3, p. 129. Pergamon Press, Oxford. Preer, J. R. and Preer, L. B. (1959).J. Protozool. 6, 88. Preer, J. R., Bray, M. and Koizumi, S. (1963). Proc. X I Intern. Congr. Gen., The Hague, Vol. 1, 189. Prescott, D. M. (1961).I n t . Rev. Cytol. 11, 255. Pringle, C. R. (1956). 2. indukt. Abstamm. -u. VererbLehre 87, 421. Pringle, C. R. and Beale, 0. H. (1960).Genet. Res. 1,62. Pruett, P. O., Conner, R. L. and Pruett, J. R. (1967).J.cell. comp. Physiol. 70,217. Raikov, I . B. (1963).Proc. I I n t . Congr. Protozool., Prague, 253.

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Reisner, A. H. (1955). Genetics, Princeton 40, 591. Reisner, A. H. and Macindoe, H. (1967). J. gen. Microbiol. 47, 1. Ross, R. (1968). Biol. Rev. 43, 51. Seed, J. R., Shafer, S., Finger, I. andHeller, C. (1964). Genet. Res. 5, 137. Seshachar, B. R. (1964). J. Protozool. 11, 402. Skaar, P. D. (1955). Expl Cell Res. 10,646. Sommerville, J. (1967a). Ph.D. Thesis :Univ. of Edinburgh, Edinburgh. Sommerville, J. (196713). Biochim. biophys. Acta 149,625. Sommerville, J. (1968). E z p l Cell Res. 50, 660. Sommerville, J. and Sinden, R. E. (1968). J. Protozool. 15, 644. Sonneborn, T. M. (1947). Growth 11, 291. Sonneborn, T. M. (1948). Proc.natn. Acad.Sci. U.S.A. 34,413. Sonneborn, T. M. (1960). Proc. natn. Acad.Sci. U.S.A. 46, 149. Sonneborn, T. M. (1964). Proc. natn. Acad.Sci. U.S.A. 51,915. Sonneborn, T. M. and Le Suer, A. (1948). Am. Nat. 82, 69. Sonneborn, T. M., Ogasawara, F. and Balbinder, E. (1953). Microbial Genet. Bull. 7,27. Steers, E. (1962). Proc. natn. Acad. Sci. U.S.A. 48, 867. Steers, E. (1965). Biochemistry, N.Y. 4, 1896. Van Wagtendonk, W. J. andVloedman, D. A. (1951). Biochim. biophys, Acta 7, 335. Woodard, J., Gebler, B. and Swift, H. (1961). Expl Cell Res. 23, 258.

The Aliphatic Amidases of Pseudomonas aeruginosa PATRICIA H. CLARKE Department of Biochemistry, University College, Cower Street, London, England T. Microbial Amidases A. B. C. D.

.

Enzymes Hydrolysing Amide Bonds Aryl and Aliphatic Amidases Amide Hydrolases and Transferases Pseudomonad Amidases

.

.

. . . . .

.

. .

.

111. Amidase Mutants , A. Regulator Mutants . B. Mutants Producing Altered Enzyme Proteins

. .

11. The Amidase of Pseudomonas aeruginosa 8602 A. General Properties of the Amidase System B. Regulation of Synthesis C. Enzyme Characteristics

. .

IV. Genetic Analysis

.

. . . .

.

.

V. Genetic Homology among Pseudomonas spp. VI. Acknowledgements References

.

. . . .

.

179 179 180 182 183 183 183 184 192 196 196 206 217 218 221 221

I. Microbial Amidases A. ENZYMES HYDROLYSING AMIDEBONDS There are very few references in the literature to enzymes which are specific for the hydrolysis of simple aliphatic amides. However, several different classes of enzymes are known to hydrolyse some amide bonds. Many proteolytic enzymes can hydrolyse peptide or amino-acid amides, e.g. trypsin and papain hydrolyse benzoyl argininamide and leucine aminopeptidase hydrolyses leucinamide, aminobutyramide and glycinamide (see Bergman, 1942; Smith and Slonim, 1948; Zittle, 1951, for a general discussion of specificity of proteolytic enzymes). Bacterial proteinases range from those attacking only a few bonds of complex protein substrates to those with very wide specificity. The proteolytic 179

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enzyme produced by Streptomyces griseus hydrolyses 80% of the peptide bonds of proteins, many amino-acid amides and a few simple amides and esters including ethyl acetate (Hagihara, 1960). The action of proteolytic enzymes on simple amides may be regarded as incidental to their main physiological role of splitting peptide bonds. However, these reactions have been of great value in investigations of the mechanism of enzyme catalysis, and a comparison of specificitiesand relative catalytic efficiencies may be illuminating in attempting to trace the evolution of enzymes attacking C-N bonds. Penicillin amidases are very specific enzymes produced by various fungal and bacterial species some of which also produce penicillin /3-lactamase (Holt and Stewart, 1964). The fungal enzymes attack such substituted penicillins as phenoxymethylpenicillin more readily than benzylpenicillin (Batchelor et al., 1961) whereas the bacterial penicillin amidases are most active on benzylpenicillin. Chiang and Bennett ( 1967) found that the penicillin amidase purified fromBacillus megaterium was able to attack, although more slowly than benzylpenicillin, several natural and synthetic penicillins and a few related amide derivatives. Phenylacetamide was hydrolysed at 25% of the rate for benzylpenicillin. Urease is a highly specific enzyme (Sumner, 1951) and the ability to hydrolyse urea has been widely used as a diagnostic test in bacterial classification (Christensen, 1946). The specificity of the enzyme responsible has seldom been checked and it is possible that a number of different enzymes may carry out this reaction. Gorr and Wagner (1933) showed that the yeast Candida (Torula) utilis hydrolysed urea if it had been grown in media containing acetamide, asparagine or urea but not if the only nitrogen source was ammonium sulphate. Lamaire and Brunel (195 1) reported that Steiromagmatocystis nigra produced an inducible cyanamidase hydrolysing cyanamide to urea which was then attacked by urease. B. ARYLAND ALIPHATIC AMIDASES There are many reports of enzymes hydrolysing nicotinamide. Hughes and Williamson (1953) showed that Lactobacillus arabinosus produced a constitutive nicotinamidase. Kimura (1959a) partially purified a nicotinamidase from Mycobacterium avium which appeared to be absolutely specific for nicotinamide. A similarly highly specific nicotinamidase was purified from Candida pseudotropicalis ( Torula cremoris) by Joshi and Handler (1962) who suggested that it was involved in a salvage pathway to produce nicotinic acid for the resynthesis of the pyridine nucleotide coenzymes. The enzyme was inhibited non-competitively by NAD and was also repressed by the presence of NAD in the growth

THE ALIPHATIC AMIDASES OB Pseudomonas aeruginosa

181

medium. The nicotinamidase purified from rat and rabbit liver by Petrack et al. (1965) was also thought to be concerned with the biosynthesis of NAD. Bray and coworkers (1949, 1950) made enzyme preparations from animal tissues which were able to hydrolyse aliphatic amides; rat liver extracts hydrolysed acetamide and propionamide but higher activities were obtained with hexamides and heptamides and the preparations also hydrolysed some aromatic amides. These authors concluded that they were dealing with a single enzyme which had a very broad range of substrate specificity. Gorr and Wagner (1933) described the hydrolysis of several aliphatic amides, as well as asparagine and urea, by suspensions and dried preparations of Candida utilis but did not speculate on the number of enzymes involved in these reactions. It is interesting to note that growth on acetamide produced the highest acetamidase activity whichisthe first recordof the microbial production of an aliphatic amidase in response to the presence of the corresponding amide in the growth medium. The specificity of the Candida utilis amidases was re-examined by B. Brady (1969, personal communication). Preparations of intact organisms, grown in a medium containing a complex nitrogen source, were found to hydrolyse a wide range of amides. Asparaginase activity was lost during a 60-fold purification but the preparation was still able to hydrolyse nicotinamide as well as a number of aliphatic amides. It is likely that the nicotinamidase activity was due to contamination with a separate enzyme since another fraction also contained nicotinamidase and this second fraction had a higher affinity for nicotinamide. The substrate specificity of the aliphatic amidase was very broad and extended from acetamide to hexanoamide with very little difference in the relative rates of hydrolysis of these amides. Mycobacterium phlei was shown by Halpern and Grossowicz (1957) to hydrolyse glycinamide, formamide and acetamide while Kimura ( 1959c) observed acetamidase activity with a strain of Mycobacterium smegmatis. The characteristic patterns of hydrolysis of various amides have been used by several workers for the classification of mycobacteria (Bonicke, 1960; Juhlin, 1960; Schneidau, 1963; Urabe et al., 1965). Draper (1967), in a detailed study of amide hydrolysis by M . smegmtis, showed that amidase synthesis was induced by acetamide. With intact bacteria, and also with partially purified preparations, the highest activities were obtained with formamide which was hydrolysed about one hundred times more rapidly than any of the other aliphatic amides. Apart from formamide, the optimum substrate appeared to be butyramide since the specific activities fell off both with increased and with decreased chain length; i.e. the rates of hydrolysis were acetamide < propionamide valeramide > hexanoamide. The simplest

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interpretation of these results is that there were two enzymes involved, one specific for formamide and the other with a broader substrate range optimal for butyramide. This possibility cannot be completely ruled out although Draper (1967)found that both formamidase and butyramidase activities were induced by growth on acetamide and that the two activities were not separated by a partial purification (2.2fold). The ratio of the rate of hydrolysis of formamide to that of butyramide for extracts of acetamide-grown cultures was 77:l so that, if there is only a single enzyme involved, it is clear that formamide is much better than butyramide as a substrate for it. The nicotinamidase and benzamidase activities of M . smegmatis were not induced by acetamide, confirming other evidence that the arylamidases are not identical with the aliphatic amidases.

C. AMIDEHYDROLASES AND TRANSFERASES Amide-hydrolysing enzymes are often able to transfer the acyl moiety of the amide to hydroxylamine to form hydroxamates. Meister et al. ( 1955) showed that glutaminase preparations from Escherichia coli could also catalyse formation of the corresponding hydroxamate. Mycek and Waelsch (1960)and Folk and Cole (1966)established that the hydrolase and transferase activities of a purified glutaminase from guinea pig heart were due to the same enzyme protein. Some of the proteolytic enzymes, e.g. ficin (Johnson et al., 1950)and papain (Durrell and Fruton, 1954), can also catalyse hydroxamate formation from their amino-acid amide substrates. There are conflicting reports on the hydrolase and transferase activities of the microbial enzymes. Kimura (1959b, c, d) showed that M . avium could form hydroxamates from nicotinamide and hydroxylamine but concluded that this was not due to the enzyme which hydrolysed nicotinamide. He examined hydrolase and transferase activity for nicotinamide, benzamide, glutamine, asparagine and acetamide in several species of mycobacteria, and found that transferase activity for these amides was not always accompanied by hydrolase activity. Draper (1967) concluded that the transferase and hydrolase activities of crude extracts of M . smegmatis were not due to a single enzyme since transferase activity had disappeared from the ammonium sulphate fraction which contained the bulk of the amide hydrolase activity. Grossowicz and Halpern (1957) had previously identified separate enzymes in extracts of M . phlei for aspartyl transfer to hydroxamate and for the hydrolysis of asparagine. An enzyme preparation purified 19-fold from M . avium by Kimura (1959d) catalysed hydroxamate formation from butyric or valeric acids and hydroxylamine, and Draper (1967) found that crude extracts of M . smegmatis were also able

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Pseudomonas aeruginosa

183

to form hydroxamates from propionic and some of the other aliphatic acids although the rates were very low. The amidase purified from Candida utilis by B. Brady (1969, personal communication) appeared to have both hydrolase and transferase activities for acetamide but no other amides were tested for transferase activity.

D. PSEUDOMONAD AMIDASES The first indications that aliphatic amidases were produced by pseudomonads were the observations of den Dooren de Jong (1926) that several species could utilize amides as carbon or nitrogen sources for growth. Kelly and Clarke (1960, 1962) found that Pseudomonas aeruginosa 8602 could grow in a minimal salts medium with acetamide as the carbon and nitrogen source and that it produced an inducible aliphatic amidase which was nost active on 2- and 3-carbon amides. Buhlman et al. (1961) examined a number of isolates of fluorescent pseudomomds and found that only strains of Pseudomonas aeruginosa produced an alkaline reaction in a glucose + acetamide medium, presumably due to the ammonia produced by the hydrolysis of acetamide. This observation was confirmed by Stanier et al. (1966) who found that all their strains of P.aeruginosagrew on acetamide-agar plates. The only other strains of the fluorescent group which grew on this medium were those which they had assigned by other criteria to Pseudomonas putida biotype A. Jacoby and Fredericks (1964) isolated from soil a fluorescent pseudomonad which grew in a minimal salts medium containing acetamide and produced an aliphatic amidase. The organism was not identified further but the enzyme had a similar limited range of substrate specificity to the amidase produced by P. aeruginosa 8602 (see next section).

11. The Amidase of Pseudomonas aeruginosa 8602 Most of this review will be concerned with the amidase produced by

P. aeruginosa 8602 (see Brammar et al., 1967). This is a fairly typical strain but does not produce pyocyanin or a haemolysin. It grows well in a minimal salts medium a t 30" or 37" and can utilize a wide range of carbon compounds as growth substrates.

A. GENERALPROPERTIES OF THE AMIDASE SYSTEM Acetamide and propionamide are good substrates and inducers and can provide both carbon and nitrogen for growth. A few other amides, such as glycollamide and acrylamide, are also good substrates but the

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substrate specificity differs markedly from the inducer specificity; e.g. lactamide is a very poor substrate but has the same inducing capacity as propionamide. Some N-substituted amides which are not hydrolysed, such as N-methylacetamide and N-acetylacetamide, can act as gratuitous inducers. A few amides, e.g. cyanoacetamide and thioacetamide, are able to compete with substrate or non-substrate inducers and prevent amidase induction ; this has been termed amide analogue repression. Amidase synthesis is also subject to catabolite repression particularly by succinate. The enzyme is very active as an acyl transferase as well as an amide hydrolase and transfers the acyl group of acetamide and propionamide to hydroxylamine. The transferase activity extends also to the related acids and esters, e.g. acetate, propionate and ethyl acetate are substrates for the transferase reaction. Ethyl acetate is also hydrolysed by the amidase but the esterase activity is about one hundredth of the hydrolase and transferase activities with acetamide as substrate. The particular advantage of the transferase reaction is that it provides it sensitive, simple and rapid method for enzyme assay. Throughout this review, and unless otherwise stated, amidase activities are expressed in amidase units as pmoles acethydroxamate formed per minute. Amidase specific activities refer to amidase units per mg. bacteria or per mg. protein where appropriate. Amide hydrolysis can be measured by estimating ammonia production by a suitable method (e.g. Conway’s microdiffusion method, reaction with ninhydrin reagent or reaction with Nessler’s reagent ; see references €or details). Selective media have been devised for the isolation of amidase mutants by employing the various amides as nitrogen or carbon sources and in some cases combining them with a high concentration of a catabolite repressor such as succinate. These media have been used to isolate amidase-negative mutants, mutants producing amidases with altered substrate specificities, constitutive mutants and mutants with other altered regulatory characteristics. Genetic transfer has been effected by transduction.

B. REGULATION OF SYNTHESIS 1. Inducer Xpecijicity

All the earlier experiments concerned with inducer specificitywere done with cultures grown overnight in succinate medium containing the test amide. I n Table 1 the amidase activities are expressed as pmoles propionamide hydrolysed/min./mg. dry wt. bacteria grown in the presence of 10 mM-amide. A few of the inducing amides had some effect at 1 mM but none was effective at 0.1 mM. Under these conditions, formamide

THE ALIPHATIC AMIDASES OF

Pseudomonas aeruginosa

185

did not induce amidase synthesis but it was found later by more sensitive methods that it was in fact a weak inducer. The values for acetamide and propionamide do not reflect the true inducing capacity of these amides since they are hydrolysed during growth. Figure 1 compares the effect on

+

TABLE. 1 Amidase induction in succinate ammonium chloride medium using Pseudomonas aeruginosa 8602. From Kelly and Clarke (1962) Amide added to medium None Formamide Acetamide Propionamide N-Methylformamidc N-Ethylformamide N-Methylacetamide N-Ethylacetamide N-Acetylacetamide N-Phenylacetamide N-Dimethylacetamide N-Methylpropionamide N-Ethylpropionamide Cyanoacetamide GIy cinamide Sarcosinamide Glycollamide 8-Hydroxypropionamidc Lactamide Fumaramide Methyl carbamate Urea Thioacetamide a

Propionamide hydrolysisa pmoles/mg. dry wt. bacteria/min. 0.1 0.1 2.0 1.3 7.6 0.2 12.7 2-0 14.0 0.1 0.1 3.0 1.5 0.1 0.1 0.1 0.2 0.1 13.0 0.1 1.7 0.1 0.1

Enzyme assayed by Conway's microdiffusion method. Cultures grown overnight at 37".

amidase specific activity after overnight growth in succinate medium of various concentrations of acetamide (substrate inducer), lactamide (poor substrate, good inducer) and N-methylacetamide (non-substrate inducer ; Kelly and Clarke, 1962). This preliminary screening made it possible to select gratuitous inducers for more detailed studies and, in spite of the limitations inherent in making single measurements on batch cultures, it remains a very convenient method for testing mutants or fresh isolates.

186

PATRICIA H. CLARKE

L

I

Amide inducer concentration (mM)

FIG.1. Effect of amide inducer concentration on the amidase activity of Pseudomonas aeruginosa 8602 (wild type) after overnight growth in succinate medium. The activity with acetamide as inducer is indicated by A ;with lactamide as inducer by o ; and with N-methylacetamide as inducer by A.

2. Amidase Induction During Growth

Figure 2 shows the differential rates of amidase synthesis by P. aeruginosu 8602 in succinate medium at four different concentrations of N-acetylacetamide. The amidase activity was measured as acetamide transferase (pmoles acethydroxamate formed/ml. culture) and is plotted against the bacterial growth (dry wt. bacteria/ml. culture). The system appeared to be saturated a t 10 mM since there was no increase in the rate of amidase synthesis a t higher concentrations. Amidase induction by acetamide could be detected with as little as 0.01 mM-acetamide although amidase synthesis ceased as soon as the acetamide had been hydrolysed. With both N-acetylacetamide and acetamide as inducers in succinate medium there was a lag of about one generation time before a constant differential rate of amidase synthesis was achieved. The induction lag was markedly affected by the carbon source for growth and, in pyruvate or glycerol medium, the lag was only a quarter of a generation whereas in citrate or malate medium, thelag was the same as in succinate medium. As will be discussed later the duration of the induction lag was related to the effectiveness of these compounds as catabolite repressors (Brammar and Clarke, 1964).

THE ALIPHATIC AMIDASES OF

Pseudornonas awuginosa

187

When acetamide was used as the sole carbon source the amidase was essential for growth. Non-induced bacteria, previously grown in succinate medium, were inoculated into acetamide medium and were found to synthesize amidase before any growth of the culture could be observed. As growth and amidase synthesis progressed, the acetamide disappeared from the medium and the specific amidase activity fell off after a few

Bacterial density (mg dry wt /mi )

FIG.2. Amidase induction by various concentrations of N-acetylacetamide (as shown on graph) in succinate medium. N-acetylacetamide was added at the time indicated by the arrow t o exponentially growing cultures. From Brammar and Clarke (1964).

hours since the inducer was no longer present. Rather surprisingly it was found that several hours later, when the culture was reaching the stationary growth phase, the specific amidase activity increased very rapidly. At this time, there was apparently no acetamide remainingin the medium ; the culture was presumed to be growing on ammonium acetate and amidase was not needed. It was found that when P.aeruginosa 8602 was grown in acetate medium it also synthesized amidase at the end of the exponential growth phase although there was of course no amidase synthesis at the earlier stage of growth. Growth and amidase synthesis in acetamide andin acetate medium are shown in Figure 3 (Brammar, 1965).

188

PATRICIA H. CLARKE

Amidase synthesis was not detected at the end of growth when any of the other aliphatic acids were used as growth substrates and the reason for this gratuitous synthesis is obscure. W. J. Brammar (personal communication) has suggested that sufficient acetamide can be formed from

2'or-----

P

i7

Hours

FIG.3. Comparison of growth and amidase synthesis in acetamide and acetate medium. Growth is expressed as 1 + loglo ( E 6 7 0 mp) in acetamide medium (A) or acetate medium ( 0 ) . Amidase specific activity is indicated in acetamide medium ( A ) and acetate medium ( 0 ) .

acetate and ammonia by spontaneous chemical reaction to induce amidase synthesis under the conditions of general derepression which are known to occur as cultures enter the stationary phase. This hypothesis has not been tested but it is interesting to note that the aliphatic amidase induced by acetamide in Hycobacterium smegmatis (Draper, 1967) is also induced by growth on acetate but not on propionate or butyrate. 3. Repression by Amide Analogues

Kelly and Clarke (1962) had found that several amides prevented amidase induction by the non-substrate inducer N-methylacetamide. This was confirmed with exponentially growing cultures and Figure 4

THE ALIPHATIC

AMIDASES OF

Pseudomonas aeruginosa

189

shows repression by 10 mM-thioacetamide, and cyanoacetamide, of amidase induction by 10 mM-acetylacetamide in succinate medium. Cyanoacetamide was very effective as an amide-analogue repressor and produced marked repression of N-acetylacetamide induction even at one hundredth this concentration (0.1 mM: 10mM). Repression of acetamide

Bacterial denslty (mg dry w t 'ml

FIG.4. Repression of amidase induction by amide analogues. 10 mM-N-acetylacetamide was added as inducer and, a t the time indicated by the arrow, the culture was divided into three parts to which were added IOmM-thioacetamide ( O ) , 10 mM-cyanoacetamide (A); control culture (0). From Brammar and Clarke (1964).

induction, however, required a higher ratio of cyanoacetamide to acetamide (50 mM:l m M ) . One interesting consequence of this amideanalogue repression was that cyanoacetamide, which has no effect on amidase activity or on growth generally, was able to prevent growth on acetamide of succinate-grown bacteria, but not of acetamide-grown bacteria which already contained sufficient enzyme (Brammar and Clarke, 1964). Kelly and Clarke (1962) had shown that formamide and N-phenylacetamide repressed amidase induction by N-methylacetamide, and Brammar (1965) showed that butyramide and hexanoamide

I80

PATRICIA H. CLARKE

repressed induction by N-acetylacetamide. These findings suggested that the various amides were all able to combine with an amidase inducer binding site but that only the inducing amides were able to bring about the reactions which resulted in amidase synthesis. Brammar et ul. (1966) showed that P . ueruginosa 8602 produced a constitutive amide permease and that the uptake of acetamide or N-acetylacetamide was not affected by cyanoacetamide. The possibility that some of the other amide analogues act at the permeability barrier has not been formally excluded. N

0

71-

Reciprocal of acetamide concentration ( m M ) x

lo-'

FIG. 5 . Effect of cyanoacetamide on the rate of amidase induction by acetamide in carbon-starved bacteria. Rate of induction in the absence of cyano-acetamide is indicated by (o), in the presence of 5 x 10-2 mM-cyanoacetamide by (A). From Brammar (1965).

More exact values for the affinities for the amidase inducer sites of the various amides were obtained by measuring the rates of amidase synthesis by carbon-starved bacteria. The bacteria were grown in minimal medium with limiting succinate until growth had ceased and the rates of amidase synthesis were measured during the period between 10 and 40 min. after the inducer had been added. The Kinducer value was obtained by plotting the reciprocal of the rate of amidase synthesis against the reciprocal of the inducer concentration. This method was also used to examine repression of induction by cyanoacetamide. With both acetamide and N-acetylacetamide as inducers the results obtained with cyanoacetamide were similar to those for a typical competitive enzyme inhibition (Clarke and Brammar, 1964). Figure 5 shows the results of an

THE ALIPHATIC

AMIDASES OF

Pseudomonas aeruginosa

191

experiment with acetamide as inducer and cyanoacetamide as analogue and Krepressor constants calculated. repressor, and Table 2 lists the Kinduoer It can be seen that the values are in good agreement with the apparent TABLE2. Inducer and Repressor h i d e s : Constants determined for Rates of Induction and Repression. Experiments were carried out with carbon-starved bacteria obtained by growing Pseudomonas aeruginosa 8602 in minimal medium with a growth-limiting concentration of succinate. From Brammar (1965). Acetamide Kinaucer N-acetylacetamide Kinducer Cyanoacetamide Krepressor

7 x IO-3mM 8 to 9 x 10-1 mM 4to 5 x mM

selative affinities for acetamide, cyanoacetamide and N-acetylacetamide which had been deduced from the experiments on induction and repression of exponentially growing cultures. 4. Catabolite Repression Many inducible enzymes have been shown to be subject to catabolite repression (Clarke and Lilly, 1969). It was found that in succinate medium the induction of P. aeruginosa 8602 amidase by N-acetylacetamide was repressed by acetate, propionate, glutamine and intermediates of the tricarboxylic-acid cycle. I n this system catabolite repression could be reversed by increasing the inducer concentration. When acetamide and propionate were added to cultures growing exponentially in succinate medium, it could be shown that the amount of repression by propionate was dependent on the ratio of the concentrations of propionate and acetamide (Brammar and Clarke, 1964). Similarly, Moses and Prevost (1966) showed that glucose repression of the induction of ,8-galactosidase could be relieved by IPTG (isopropyl-fi-D-thiogalactoside) and Mandelstam and Jacoby (1965)showed that catabolite repression of the aromatic pathway enzymes of a pseudomonad could be overcome by the addition of more inducer. Although P. aeruginosa 8602 grows well in succinate and can be induced to synthesize amidase, the differential rate of synthesis by fully induced cultures was found to be much greater in pyruvate medium. Succinate is a very effective repressing metabolite and 10 mM-succinate added to 1% pyruvate medium repressed amidase induction by 10 mM-Nacetylacetamide by 98-100%. These conditions were used as the standard test for catabolite repressibility by Brammar et al. (1967).

192

PATRICIA H. CLARKE

C. ENZYME CHARACTERISTICS 1. Substrate Speci$city It was established that amide hydrolase and transferase activities were due to a single enzyme protein by showing that (a) both activities were co-ordinately induced under avarietyof different growth conditions, (b) the ratio of amide hydrolase to amide transferase activities remained unchanged throughout purification, (c) during purification both amide hydrolase and transferase activities were associated with a single protein fraction, and (d) the pure enzyme catalysed both reactions. Kelly and Kornberg (1964) confirmed that the enzyme formed at TABLE3. Substrate Specificity of a Partially Purified Preparation of the Aliphatic Amidase of Pseudomonas aeruginosa 8602. From Kelly and Kornberg ( 1 964) Specific activities : pmoles substrate transformed/ min/mg protein Substrate

Formamide Acetamide Propionamide Butyramide Acetate Propionate Acrylamide Glycollamide Lactamide j3-Hydroxypropionamide Glycinamide

'-

7

Hydrolase

Transferase

36 260 698 0

14 1280 212 tl 100 91 565 221 t l 3 3

446 410 24 3 13

the end ofgrowth on acetate was identical with that formed during the early exponential phase of growth on acetamide and the data given in Table 3 on amide substrate specificity were compiled from their paper. It can be seen that acetamide and propionamide are the optimal substrates for both reactions but the specific hydrolase rate is highest for propionamide and the specific transferase rate is highest for acetamide. Later, Brown et al. (1969)found that butyramide was hydrolysed slowIy. Of the amides with side-chain substitutions only glycollamide and acrylamide were comparable with acetamide and propionamide as substrates. Measurements with acrylamide were complicated by inhibition of the enzyme by both acrylamide and acrylate. There was significant transferase activity with both acetate and propionate as substrates. No

THE ALIPHATIC AMIDASES OF

Pseudomonas aeruginosu

193

activity could be detected with mono or di-N-substituted amides or with thioacetamide where the carbonyl oxygen had been replaced by a sulphur atom (Kelly and Clarke, 1962). McFarlane et al. ( 1 965) found that during purification esterase activity was invariably associated with the amidase activity. A few esters were substates for both hydrolase and transferase reactions but the activity for the best ester substrate, ethyl acetate, was only about 1% of that for acetamide. The hydrolysis of ethyl acetate was activated by NH,+ ions and both ester hydrolysis and ester transfer reactions were inhibited by ethyl alcohol. Ethyl acetate inhibited the transferase reaction with acetamide as substrate (McFarlane, 1967). These effects are probably all due to interactions at the active site of the enzyme. 2. Enzyme Inhibitors

The compounds which were very effective as catabolite repressors (acetate, propionate, malate, citrate and succinate) had no inhibitory effect on amidase activity. The amide analogue repressors, cyanoacetamide and thioacetamide, were also non-inhibitory so that inhibition of amidase action was clearly not a complication of the experiments carried out on enzyme repression by these compounds. Kelly and Clarke (1962) found that propionamide hydrolysis by intact bacteria was inhibited by glycinamide but McFarlane (1967) found later that glycinamide had no effect on the hydrolase or transferase activities of the pure enzyme. It seems probable that glycinamide inhibits amide uptake by competing with the amide permease (Brammar et al., 1966). The following inhibitors have been studied in some detail. (a) Urea was found to inhibit propionamide hydrolysis by the partially purified enzyme in a non-competitive manner giving a K i of 1.1 mM (Kelly and Kornberg, 1962,1964).The enzyme appeared to be protected from inhibition by urea by hydroxylamine, and McFarlane (1967)showed that acetamide and ethyl acetate transferase reactions were inhibited by only 3 0 4 0 % by 100-250 mM urea. Kelly and Kornberg (1964) found that, in the presence of hydroxylamine, both the hydrolysis and the propionyl transfer reactions with propionamide were relatively insensitive to urea inhibition. Although urea may be considered as an amide substrate analogue it is likely that its effect on amidase activity is less direct. Inhibition by urea of the hydrolase reaction may be due to its known effect on the hydrogen bonding of proteins, and it is possible that when hydroxylamine is present and bound to the amidase the change in conformation of the enzyme protein may make it less vulnerable to attack by urea. (b) Fluoride was shown to have little effect on amide hydrolysis but Kelly and Kornberg (1964) reported that propionamide transferase was 7

194

PATRICIA H. CLARKE

inhibited competitively in the presence of limiting propionamide and excess hydroxylamine, while with excess propionamide and limiting hydroxylamine the fluoride inhibition was dependent on the hydroxylamine concentration. McFarlane ( 1967) concluded that the fluoride inhibition was due to non-specific attack on the enzyme protein and suggested that in this case the binding of hydroxylamine by the enzyme made it more susceptible to inhibition by fluoride. McFarlane (1967) also showed that ester transferase was inhibited in the same manner as amide transferase with exactly the same relationship between fluoride inhibition and hydroxylamine concentration (Figure 6).

1.0

10

100

Potassium fluoride concentration ( m M )

FIG.6. Fluoride inhibition of transferase activity with propionamide (A) or ethyl acetate ( 0 )as substrates. From McFarlane (1967).

(c) Thiol reagents inhibit amidase activity and it has been found necessary to add cysteine or mercaptoethanol to protect the enzyme from inactivation during purification and storage. McFarlane (1967) showed that with acetamide both hydrolase and transferase reaction were inhibited 96% by 5 x lop3 mH-PCMB (p-chloromercuribenzoate). Mercaptide formation could be detected by an increase in the absorption a t 250 nm of the enzyme solution incubated with PCMB. A series of experiments a t different enzyme concentrations indicated that there were 20-22 thiol groups reacting with PCMB per molecule of enzyme protein. Similar experiments were carried out with DTNB (5,5'-dithiobis-2nitrobenzoate) measuring the rate of formation of 5-thio-2-nitrobenzoate by the increase in absorption a t 512 nm. The reaction with DTNB was rapid, reaching completion in 5-10 min. and was linear with enzyme concentration. The number of reactive thiol groups per enzyme molecule

THE ALIPHATIC AMIDASES OF

Pseudomonas aeruginosa

195

calculated from experiments with DTNB was calculated to be 20-25 which agreed well with the PCMB determinations. Inhibition by iodoacetamide is probably due to its alkylating reaction rather than to its resemblance to the amide substrates. Amide hydrolase and amide and ester transferase activities were equally susceptible to iodoacetamide inhibition. Whereas inhibition by PCMB and DTNB could be reversed by cysteine, the inhibition by iodacetamide was irreversible. Acetamide or ethyl acetate had little effect on iodoacetamide inhibition but a mixture of acetamide and hydroxylamine a t normal substrate concentrations gave significant protection. 3. Amiduse Reactions The reactions which can be catalysed by this enzyme are listed in Table 4.The mechanism of action most likely involves the formation of a thiol ester as an acyl-enzyme intermediate which is subjected to nucleophilic attack by water or hydroxylamine. If this suggested mechanism TABLE4. Reactions catalysed by the amidase produced by Pseudomonas aeruginosa 8602

Reaction

Optimum substrate

Amide hydrolysis RCONHz HzO = RCOO- NH4f Amide transfernse RCONHz + NHzOH + H+ = RCONHOH + NH4+ Acid transferase RCOO’ + NHzOH H+ = RCONHOH HzO Ester hydrolysis RCOOR’ HzO = RCOO’ H+ + R’OH Ester transferase RCOOR’ + NHzOH = RCONHOH + R’OH

+

+

+

+

+

+

Propionamide Acetamide Acetate Ethyl acetate Ethyl acetate

is correct, the relative overall rates of these reactions will depend on the rates of formation of the acyl intermediates and the relative rates a t which these intermediates are broken down to release the products. I t is of considerable interest that the relative rates of amide hydrolysis, propionamide > acrylamide > glycollamide > acetamide, are reversed for the transferase reactions with these substrates, acetamide > acrylamide > glycollamide = propionamide. The size of the aliphatic sidechain is very important in determining whether or not an amide will be a good substrate. The C.) amide glycollamide, with a hydroxyl group, has a side-chain similar in size to that of propionamide and is intermediate in activity as a substrate between acetamide and propionamide. However, cyanoacetamide is not a substrate and, while Iactamide (a-hydroxypropionamide) is a poor substrate, its isomer P-hydroxypropionamide is

196

PATRICIA H. CLARKE

rather worse. The physicochemical properties of the pure wild-type enzyme are discussed in more detail in Section I11 when it is compared with several mutant amidases which have been isolated.

111. Amidase Mutants A. REGULATOR MUTANTS 1. Constitutive Mutants Mutants of Pseudomonas aeruginosa 8602 producing amidase in the absence of inducer (C mutants) were first isolated on S/F agar plates (succinate 1% + formamide 0.1%; Brammar et al., 1967). TABLE5. Mutants of Pseudomonas aeruginosa 8602 with Altered Regulation of Amidase Synthesis. From Brammar et al. (1967) and J. E. Brown (1969). Growth on selective media,a S / F S/F/CN

S/L

B

74 Cat. Rep.6 Ind. RatioC

l a . Magno-constitutive mutants isolated on SF plates from WIT ++ 80-90 C1, C8, C10, C18 c4 80 84-85 c11, c 2 2 63 C24

++ ++ ++ ++

++ ++ ++

-

+

l b . Semi-constitutive mutants isolated on S Q plates from C15, C20 c2 c5 C9, C17

++ ++ ++ ++

++

1 1 1 0.3

WT

5 55, 80

3, 2 2 3 9, nt

2. lf70rmamide-induciblemutants isolated on SIF plates from W T F1, F2, F4, F 6 60-90

>loo

++

+

55,93 90

++

3 . Butyramide-resistant mutants isolated on B plates a. From WT CU 6 - CR 12 nt b. From strain C11 ++ nt CB 1 - CB 5 4 . Catabolite-resistant mutanfs isolated on SIL plates from L5, L11 L9, L10

++

++

++ ++

++

++ ++ ++

nt nt

WT 56, 60 15,lO

1 1

>loo 1

a Relative growth after 3 days at 37" expressed as ++ or +, n t = not tested. Plate ),(,: + formamide (0.1 %)); S/F/CN, (succinate (1%) + formmedia: S/F, (succinate (1 amide (0.05%) cyanoacetamide (1%)); S/L, (succinate (1%) lactaniide (0.05%)); R, butyramide (0.1%). Cat. Rep., % catabolite repression when 10 mM-succinate was added to cultures growing in pyruvate medium, 10 m M N-acetylacetamide was added t o inducible strains only. Ind. Ratio, Induction ratio measured in succinate medium Rate + 10 mM-N-acetylacetamide Rate without inducer

+

'

+

THE ALIPHATIC AMIDASES OF

Pseudomonas aeruginosa

197

The wild type did not grow on this medium since formamide was a poor substrate and a very weak inducer. The strain is unable to use C1 compounds as growth substrates so that formamide provided only the nitrogen source and succinate the carbon source. Succinate exerts an additional selection pressure since it acts as a catabolite repressor of amidase synthesis. Most of the mutants isolated were magno-constitutive, synthesizing amidase at about the same rate as, or slightly greater than, the fully induced wild-type strain and could not be induced further by the addition of amides to the growth medium. Others were semiconstitutive with differential rates of amidase synthesis in succinate medium ranging from l0-50% of the rate of the fully induced wild type. The semi-constitutives could all be fully induced by 10 mM-N-acetylacetamide and were repressed by cyanoacetamide. S/F/CN plates (succinate 1 % + formamide 0.05% + cyanoacetamide 1 %) could be used to distinguish between the magno-constitutive and semi-constitutive mutants. The finding that none of the semi-constitutives grew on S/F/CN plates suggested that growth of these strains on S/F plates required amidase to be induced and that the formamide had some inducing activity. One magno-constitutive mutant, C 24, was unable to grow on S/F/CN plates and it was found that amidase synthesis by this strain was repressed, in cultures growing exponentially in succinate medium, by both cyanoacetamide and N-acetylacetamide. The induction ratio for this m u t m t growing in the presence or absence of Nacetylacetamide was 0.3 which meant that amidase synthesis was repressed by an amide which acted as an inducer for the wild type and semi-constitutive strains; in other words, it could be said that the mutation had changed the regulation of amidase synthesis from being inducible by amides to being repressible by amides. 2. Formamide-Inducible Mutants Some of the mutants isolated from S/F plates were not constitutive and could be induced by N-acetylacetamide and other amide inducers. When the differential rates of amidase synthesis in the presence of various amides were compared with those of the wild-type strain it was found that these mutants differed markedly from the wild type in inducer specificity. While amidase induction of the wild-type strain by formamide was very slight, the inducible mutants (F mutants) isolated from S/F plates could be induced to synthesize amidase a t a significant rate by 10 mM-formamide. Mutant F 6 had a higher rate of amidase synthesis with formamide as the inducer than with acetamide and propionamide (Figure 7). Other formamide-inducible mutants had rather lower induction rates with formamide than strain F 6 but, for all those tested, the induction rates for formamide, acetamide and propionamide differed markedly from those of the wild type (Branimar et al., 1967).

198

PATRICIA H. CLARKE

The earlier results on induction and amide-analogue repression of amidase synthesis in growing cultures had suggested that various amides were capable of binding to an amidase inducer site and that this could result in induction or competitive repression. The properties of the constitutive and formamide-inducible mutants would suggest that all these mutations have occurred in a regulator gene of the lac i-type producing in some cases cytoplasmic repressors with altered structures.

A

x

c ._

g

2.0

~

U ._

E

a

1.0

~

P 0.2

0.3

0.4

-

-A

0.5

6,

I

I

0.1 0.2 0.3 0.6 0.7 Bacterial density (extinction at 670 rnp)

I

0.4

5

FIG.7 . Induction of amidase in succinate medium in Pseudomonas aeruginosa 8602 wild type a n d formamide-inducible mutant F 6. Amides were added to give a final concentration of 10 mM; formamide (A), acetamide ( 0 )and propionamide (0). From Brammar et al. (1967).

For most of the magno-constitutive mutants it could be suggested that the cytoplasmic repressor was absent or if present had in some way lost its affinity for an operator site and was unaffected by amides. The magnoconstitutive mutant C 24 was particularly interesting and it would seem reasonable to conclude that the mutation was such that the altered cytoplasmic repressor could bind amides in such a way that a conformational change occurred which increased its affinity for the operator site, thereby repressing amidase synthesis. For the formamide-inducible mutants on the other hand the basal level of amidase synthesis remained unchanged and it would seem that the mutations had affected only the amide-binding properties of the cytoplasmic repressor.

THE ALIPHATIC AMIDASES OF

Pseudomonas aeruginosa

199

For the lac operon of E . coli, Jayaraman et al. (1966)isolated a number of constitutive P-galactosidase mutants which could be repressed by 2-nitrophenyl-P-~-fucoside. This P-galactoside analogue represses induction by the non-substrate inducers such as methyl-P-D-thiogalactoside. These repressible, constitutive /3-galactosidase mutants appear to be analogous to such amidase constitutive mutants as C 24 which is repressed by cyanoacetamide and N-acetylacetamide. Mutants isolated by Gilbert and Miiller-Hill ( 1966) produced lac cytoplasmic repressors which had a higher affinity for the inducer isopropy~-P-~-thiogalactoside than the lac repressor produced by the parent strain, and the amidase F mutants may be of this type. I n looking for mutations which alter enzyme regulation one should look for changes in inducer specificity, interactions between induction and repression by inducer analogues as well as the simple mutation from inducibility to constitutivity. 3. Butyramide-Resistant Mutants

Mutants were isolated from butyramide-agar plates which, unlike the wild-type Pseudomonas aeruginosa 8602, were able to grow well with butyramide as the carbon source for growth. The butyramide-positive phenotype was in some cases due to the production of an amidase with altered substrate specificity and these mutants will be discussed in Section 111.Others were able to grow on butyramide because they had certain specific regulatory mutations. Butyramide was a very poor substrate for the amidase produced by the wild type and C strains. The rate of butyramide hydrolysis was only about 2% of that of acetamide. Butyramide did not induce amidase synthesis and like cyanoacetamide it repressed amidase induction by N-acetylacetamide. A group of spontaneous butyramide-positive mutants isolated directly from the wild-type strain proved to have acquired also a constitutive phenotype and these are listed in Table 5 as CB 6 to CB 12. Mutant C 11 which had been isolated from S/B' plates did not grow on butyramide and was the parent strain of another group of butyramidepositive mutants listed in Table 5 as CB 1 to CB 6. A few of the constitutive C mutants previously isolated (four magno-constitutives and two semi-constitutives) also grew well on butyramide plates while others grew slightly or not at all (see Table 5). When a few of these mutants were examined in more detail it was discovered that the difference between the constitutive mutants growing on butyramide and those unable to do so could be traced to the amount of repression exerted by butyramide on the rate of amidase synthesis by each strain. Amidase synthesis by strain C 11 growing in succinate medium was almost completely repressed by 10 mM-butyramide. The other strains were not completely insensitive to butyramide repression, but the

PATRICIA H. CLARKE I

I

25

I

50

I

1

75

I

Butyramide concentration ( m M )

FIG.8. Butyramide repression of amidase synthesis by constitutive mutants of Pseudornonas aeruginosa 8602 in succinate medium (C 11, CB 6, CB 4, L 10). From J. E. Brown (1969).

concentration at which butyramide repression became significant varied greatly. Figure 8 shows the effect of different concentrations of butyramide on the rates of amidase synthesis by strain C 11 (unable to grow on butyramide), strains CB 4 and CB 6 (isolated from butyramide plates) and strain L 10, a constitutive strain which will be discussed in more

Reciprocal of butyramide concentration (mM)

FIG.9. The effect of butyramide concentration on repression of amidase synthesis expressed as the reciprocal of the percentage repression plotted against the reciprocal of the butyramide concentration. Data as in Fig. 10. From J. E. Brown (1969).

THE ALIPHATIC AMIDASES OF

Pseudomonas aeruginosa

201

detail in the next section. It can be seen that butyramide repression of amidase synthesis by strain C 11 reached 50% at a concentration of butyramide which has little effect on amidase synthesis by strains CB 4 and L 10. As well as the differences in sensitivity to repression at the lower butyramide concentrations, it can be seen that there were differences in the maximum amounts of repression. These data are replotted in Figure 9 as the reciprocal of the repression of amidase synthesis against the reciprocal of the butyramide concentration, to obtain approximate values for a Krepressor for butyramide. The values for the butyramide repression constants for the four mutants are given in Table 6. It was thought that repression of certain constitutive strains by butyramide resulted from the interactionof butyramide with the mutant TABLE 6. Butyramide Repression Constants for Mutants of Pseudornonas aeruginosa 8602 growing in Succinate Medium in the Presence of Various Concentrations of Butyramide. From J. E. Brown (1969) Maximum repression Mutant

%

Apparent K mM -

c 11 CB 6 CB 4 L 10

92 87 87 56

5

12 28 20

cytoplasmic repressors. Those particularly sensitive to butyramide repression, like strain C 11, behaved as butyramide-repressible mutants and would not grow on butyramide and those less sensitive, like strains CB 4 and CB 6, behaved as butyramide-resistant mutants and were able to grow on butyramide. It was thought that the low activity of the amidase produced by these latter strains on butyramide as a substrate was zompensated by the very large amounts of enzyme which they produced. The other type of bntyramide-utilizing mutant (producing an altered enzyme) was obtained by a further mutation of strain C 11 and it was of interest to see if its sensitivity to repression was the same as its parent strain. It was not possible to test this directly since any butyramide added to growing cultures of mutants of this class was rapidly hydrolysed. The strains were therefore compared for their sensitivities t o repression of amidase synthesis by cyanoacetamide which was known to repress some constitutive mutants. Figure 10 shows that strains C 11

202

PATRICIA H. CLARKE

and its mutant B 6 were equally sensitive to repression by cyanoacetamide. Strain CB 2 which, like strains CB 4 and CB 6, was less sensitive to butyramide repression was also much less sensitive than C 11 to repression by cyanoacetamide. The other properties of mutant B 6 will be discussed in Section 111, p. 208.

21 -

-

0 0

0.3

0

~

0

i0

1

I

I

0.3 C 0.3 Bacterial concentration (extinction a t 670 m p ) (

0

C

FIG.10. Cyarioacetamide repression of amidase synthesis by constitutive mutants (a) mutant C 11 (constitutive, produces A amidase, does not grow on butyramide); (b) mutant CB 2 (constitutive, produces A amidase, resistant to butyramide repression); (c) mutant B 6 (derived from C 11, produces B amidase). Cyanoacetamidc was added to cultures growing exponentially in succinate medium; 80 mM (o), 40 mM ( A ) and control culture ( 0).From J. E. Brown (1969).

4. Catabolite-Resistant Mutants Succinate had been found to be very effective in producing catabolite repression of amidase synthesis by constitutive strains as well as by the inducible wild-type Pseudomonas aeruginosa 8602. When succinate was used as the carbon source and an amide such as lactamide, which is a poor substrate but a good inducer, as the nitrogen source (S/Lplates) this provided a suitable selection medium for isolating mutants which were less sensitive to cataboliterepression by succinate (Brammaretal., 1967). The wild-type strain and most constitutive mutants grew very slowly on this medium but the catabolite-resistant (L mutants) produced large colonies within 48 hr. Some of the L mutants were found to be inducible while others were constitutive, It was clear however that the catabolite

TIIE ALIPHATIC AMIDASES OF

Pseudomonas aeruginosa

203

repressibility of the constitutive L strains was not an essential feature of their constitutivity. By genetic analysis it could be shown that the constitutivity character of strain L 10 could be transferred by transduction to a recipient without it becoming a t the same time more resistant to catabolite repression by succinate. It appears therefore that, although catabolite repression of the wild type can be relieved by increasing the concentration of the amide inducer, the particular type of cataboliteresistant mutants so far isolated from S/L plates are probably miitants in which alterations have occurred in enzymes concerned with succjnate metabolism. A similar technique has been used to select mutants resistant to catabolite repression by glucose and malate. The standard test for catabolite repressibility consisted of adding 10 mM-succinate to cultures growing exponentially in pyruvate medium with N-acetylacetamide added as inducer if required. This method gave 8 0 - 1 0 0 ~ orepression of amidase synthesis by the wild type and most of the constitutive strains. All of the L mutants were much Iess sensitive to repression by succinate in this test. A few of the constitutive strains had significantly lower sensitivities to catabolite repression but have not yet been fully investigated. The values are given in Table 5. Loomis and Magasanik (1967) isolated a mutant of Escherichia coli which was resistant to catabolite repression of p-galactosidase. The mutation was not in the lac operon but mapped near the tryptophan locus. It appeared later that this mutation was not specific for the lac genes. Mutations in the promotor region of the lac operon have been found to affect catabolite repressibility of p-galactosidase in a specific manner, and it has been suggested that the initiation of m-RNA transcription may be involved (Pastan and Perlman, 1968; Silverstone et al., 1969). The mechanism of catabolite repression of the amidase of Pseudomonas aeruginosa 8602 has not been investigated in detail. Most of the catabolite-resistant mutations which have been examined appear to be of the non-specific class but some of the variations in catabolite repression exhibited by the constitutive strains may be due to mutations in the amidase genes. 5 . Amidase Xynthesis in Continuous Culture by the Wild-Type Xtrain and Regulator Mutants The results obtained with plate growth and batch cultures had shown that amidase synthesis by the wild-type Pseudomoms aeruginosa 8602 was subject to dual regulation. It was induced by amides and repressed by metabolites and therefore, with a substrate amide such as acetamide as the sole carbon source for growth, induction of amidase would result in hydrolysis of the inducer followed by repression by metabolites. This dual control had beenapparent in the batch-culture experiments but was

204

PATRICIA H. CLARKE

shown up more dramatically when amidase synthesis was examined in continuous culture. Boddy et al. (1967) measured amidase synthesis during the transition period to the new steady state when incoming medium for a carbonlimited culture of Pseudomonas aeruginosa 8602 was changed from 10 mM-succinate to 10 mM-succinate + 20 mM-acetamide. Amidase was required to utilize the additional carbon source and it was found that the induction lag during the transition period depended on the growth rate of the culture. At a dilution rate of 0.22 hr-l, amidase synthesis started

9

,Acetomide

% ,’

Ol 0

I

0.2

I

0.4

succinate

I

0.6

1

0.8

Dilution rate (hr.-’)

FIG.11. Amidase synthesis by Pseudomonas aeruginosw 8602 wild-type growing in chemostat culture at various dilution rates. The minimal salts medium contained 20 mM-acetamide (A) or 20 mM-acetamide + 10 mM-succinate (0). From Clarke et al. (1968).

soon after the change-over to the new medium but, when the dilution rate was increased to 0.76 hr-l, there was almost complete repression of amidase synthesis during the first four hours. Oscillations were observed in both amidase specific activity and in bacterial cell density and it was concluded that these oscillations were due to the interactions of induction by acetamide and catabolite repression by succinate derivatives. The dual control of amidase synthesis was also apparent in the steadystate level of amidase of cultures grown either onlimiting acetamide or on limiting succinate + acetamide. Figure 11 shows the effect of the dilution rate on amidase synthesis by the wild-type strain. Clarke et aZ. (1968) considered that the increase in amidase specific activity as the dilution

Pseudomonas aeruginosu

THE ALIPHATIC AMIDASES OF

205

rate was increased was due to induction by acetamide and that, at a critical growth rate, the concentration of catabolite repressor in the metabolic, pool became sufficient to dominate the control of amidase synthesis. The falling off in the amidase specific activity at the higher dilution rates was considered to be due to the increasing effects of catabolite repression. Some support for this view was given by the finding that the addition of succinate increased the repression at higher dilution rates without affecting the rates of amidase synthesis at the lower dilution rates. When various regulator mutants were examined in

01 0

I

I

0.2

I

I 0.4

I

( 3

Dilution rate (hr.-')

FIG.12. Amidase synthesis by constitutive mutants of Pseudomonas aeruginosa 8602 growing in chemostat culture a t various dilution rates. The minimal salts medium contained 20 mM-acetamide + 10 mM-succinate. Mutant C 11 is magnoconstitutive ( 0); mutant L 9 is magno-constitutive with decreased sensitivity to catabolite repression (A). From Clarke et al. (1968).

this system, the results fitted in with this interpretation. Figure 12 shows the amidase specific activity at various dilution rates of strain C 11, a magno-constitutive mutant, and strain L 9 a magno-constitutive mutant with decreased sensitivity to catabolite repression. Acetamide was synthesized by these strains in the absence of inducer at low dilution rates. It can be seen that the repression curve for strain L 9 is displaced significantly to higher dilution rates as would be expected from its resistance to catabolite repression by succinate in batch culture. The particular shape taken by the wild-type curve is dependent on the relative affinities of the system for acetamide as inducer and the catabolite

206

PATRICIA H. CLARKE

repressor derived from succinate. It was not possible to do the same experiment with the non-substrate inducer N-acetylacetamide since it was not sufficiently stable under these conditions. However, i t was used in experiments of the transition type and, when the specific activities of cultures were compared four hours after the introduction of 10 mMN-acetylacetamide to a steady-state culture growing on limiting succinate, it was clear that the specific activity decreased steadily as the dilution rate was increased (Clarke et al., 1968). The relative Kinducer values determined for acetamide and N-acetylacetamide were approximately 1 0 P mlM and 1 m M respectively (Brammar, 1965) and it was concluded that the very weak inducer activity of N-acetylacetamide could only be expressed a t the very low dilution rates.

PRODUCING ALTEREDENZYME PROTEINS B. MUTANTS 1. Amidase-Negative Mutants

Pseudomonas aeruginosa 8602 is not sensitive to penicillin so that the penicillin-enrichment method could not be used to select mutants lacking amidase activity. This method is in any case less suitable for seIecting mutants defective in catabolic enzymes than for those with biosynthetic defects. The method which was adopted, and found to be successful though tedious, was to treat the parent strain with mutagenic agents, usually ethylmethane sulphonate (EMS) or N-methyl-"-nitroN-nitroso-guanidine (NMG), and to plate out on minimal medium containing acetamide as the sole carbon source to give about 100 colonies on each of about 60 plates. The wild-type strain produced normal colonies and by hydrolysing the acetamide were able to leak out sufficient acetate for the amidase-negative mutants to produce minute or shadowy colony growth. I n some experiments a trace of succinate was added to encourage the growth of the mutants. Some mutants selected by this method had non-specific defects and were discarded, and others were unable to grow on acetamide because they had defects in enzymes concerned with acetate metabolism such as isocitrate lyase, malate synthase or acetic thiokinase (Skinner and Clarke, 1968). Brammar et aZ. (1967) isolated a number of amidase-negative mutants from the wild-type strain (Am mutants) and J. E. Brown (1969) isolated others from the constitutive strain C 11 (CAm mutants). These were used mainly for genetic studies (see Section IV, p. 217). P. R. Brown (1969) isolated a further series of amidase-defective mutants which had a more complex provenance. The magno-constitutive strain L 10 was also resistant to butyramide repression and relatively insensitive to catabolite repression. It was able to grow well on butyramide media because being derepressed it produced a very high level of the wild-type amidase.

THE ALIPHATIC

AMIDASES OF

Pseudornonas aeruginosa

207

P. 1%.Brown (1969) selected, from strain L 10, mutants which produced minute colonies on butyramide plates (LAm mutants). Mutants which were able to produce significant amounts of amidase in succinate medium were discarded and those appearing to possess arnidase-enzyme defects were investigated further. Table 7 shows that some of the LAm mutants were able to grow on acetamide but others had become acetamide-negative in phenotype. A few of the LAm mutants produced cross-reacting material to the antiserum prepared against the wild-type amidase, and two of the Am TABLE7. Amidase-Defective Mutants of Pseudomonas aeruginosa 8602 selected from t h e Constitutive Mutant L 10. From P. R. Brown (1969)

-

Growth o n plates containing r--

Mutant

Acetamide

Butyramide

Production of crossreacting material t o wild-type amidase

LAm 1 LAm 2 LAm 3

LAm LAm 1,Am LAm LAm LAm LAm LAm LAm LAm

-

tr

P -

4 5 11 12

13 15 16 17 18 21

++ + ++ + tt ++ ++ tr

+ + tr -

tr

-

P

++; +;

Helative growth after three days a t 37" is expressed as tr, trace; -, no growth. Immunodiffusion tests were carried out as described by Brown et al. (1969). Complete coss-reaction is expressed by +; partial cross-reaction by P; -, no reaction detected.

mutants also did so. This interesting group of mutants may be presumed t o produce amidase proteins which are less efficient for growth on these amides than is the wild-type enzyme. The mutants which were acetamidepositive but cross-reacting material-negative were a little puzzling but there are two possible explanations. They may, like the valeramide mutants discussed in Section 111, p. 196, produce amidases which are unstable in the cell-free state and may therefore be difficult to detect in immunodiffusion tests carried out under the standard conditions, or they may have additional regulator mutations and produce the normal wild-type enzyme but in such low quantities that again it is difficult t o detect. The LAm mutants have not yet been investigated in any depth. The value of using strain L 10 as the parent strain for this series

208

PATRICIA H. CLARKE

is that it produces such very high levels of thewild-type amidase that it might be expected also to produce high levels of mutant amidase proteins. 2 . Mutants Producing B Amidase

The magno-constitutive mutant C 11, which was unable to grow on butyramide because it was severely repressed, was used as the parent strain for the isolation of mutants producing amidases with significantly higher activities on butyramide as a substrate. Brown et al. (1969) treated strain C 11 with NMGandplatediton butyramide agar. Coloniesappeared after a few days and the larger ones were picked off and tested for enzyme production (B mutants). The mutants which appeared later and grew more slowly on butyramide agar were probably similar to the CB mutants described in Section 111,p. 199 which grew on butyramide because they had acquired additional regulator mutations making them resistant to butyramide repression. The B mutants were probably as sensitive to butyramide repression as the parent strain C 11. This was difficult to test directly since they hydrolysed any butyraniide in the growth medium fairly rapidly. J. E. Brown (1969) showed that, when mutant B 6 was grown in a minimal medium containing 40 mM-butyramide as growth substrate, the concentration dropped to 20 mM by one hour after inoculation. On the other hand, mutant CB 4,which was able to grow on butyramide because it was resistant to butyramide repression, hydrolysed butyramide much more slowly, and in a similar experiment the concentration of butyramide in the medium was still 35 mM six hours after inoculation although at this time the bacterial density of the two cultures was similar. As was discussed in Section 111,p. 202 mutant B 6 resembled its parent strain C 11 in sensitivity to repression by cyanoacetamide. Mutants B 1 to B 6 produced amidases which differed in electrophoretic mobility from the wild-type amidase produced by strain C 11. When cell-free extracts were subjected to starch gel electrophoresis the amidase could be seen as a major protein band. Figure 17a (see p. 220) is from a starch gel stained with naphthalene black to show the protein bands. Extract a was from strain C 11, extract c from strain B 6 and b was a mixture of the C 11 and B 6 extracts. Other slices of the gel were stained for acetamide hydrolase and transferase activities, and the protein bands marked with the arrows gave strong enzyme reactions. The amidase band in the extract from strain B 6, which can also be seen as the slower moving band in the mixture of the two extracts, gave strong butyramide hydrolase and transferase reactions. It was possible to detect slight butyramide hydrolase activity with extracts of C 11 by loading the gels very heavily. The amidases from all six B mutants gave the same electrophoretic pattern.

THE ALIPHATIC AMIDASES OF

Pseudomonas aeruginosa

209

The substrate profiles of the B mutants were all similar and differed markedly from that of the wild-type and its constitutive mutants. Figure 13 compares the amide hydrolase activities of washed suspensions of strain C 11 and strain B 6. The activities are all expressed relative to acetamide assigned an arbitrary value of 100. The specific acetamide

J F

A

P

21 L

, 20 is0 B

B

FIG.13. Substrate profiles for amide hydrolase activity. Relative rates of amide hydrolysis are shown for washed suspensions of (a)mutant C 11, producing A amidase and (b) mutant B 6, producing B amidase. Substrates were formamide (F),acetamide (A), propionamide (P), lactamide (L), isobutyramide (isoB) and butyramide (B).Rates are expressed relative to acetamide assigned an arbitrary value of 100. From Brown et al. (1969).

Mutant B I

L

50

100

a,

c

9 .+

25

0 a,

LL

A

P

B

isoB

FIG.14. Substrate profiles for amide transferase activity in (a) mutant C 11, (b) mutant B 1. Refer to Fig. 13 for descriptions. From Brown et al. (1969).

hydrolase activities of the two strains did not differ markedly after overnight batch growth. It can be seen that, whereas the butyramide hydrolase activity of the C 11 suspensions was 2% of that for acetamide under the test conditions, with the B 6 suspension the rate was 30% of that with acetamide. Figure 14 shows that strains C 11 and B 6 differed also in the relative rates of amide transferase activities. While only trace activity could be detected with strain C 11 for butyramide the rate with strain B 6 was 12% of the acetamide rate. It was concluded that

210

PATRICIA H. CLARKE

the B mutants produced a mutationally altered enzyme protein which was termed the B amidase to distinguish it from the wild-type A amidase. The differences in the rates of hydrolysis of acetamide and butyramide by the pure A amidase, prepared from strain C 11, and the pure B amidase prepared from strain B 6, are shown in Figure 15. The ratio of the rates of hydrolysis of acetamide and butyramide for the A enzyme was 100:2 and for the B enzyme was 100:30 which agreed well with the values

Time (rnin.)

FIG. 15. Rates of amide hydrolysis by purified A and B amidases. Data show hydrolysis by A amidase of acetamide ( 0 )and butyramide 0;hydrolysis by B amidase of acetamide ( 0 ) and butyramide 0.From Brown et al. (1969).

obtained with the bacterial suspensions. These determinations were all done under the standard assay conditions (Brown et aZ., 1969)using a concentration of 200 mM-amide. Since the A amidase has a much higher apparent K , for butyramide than the B amidase (Table lo), it is clear that if the assay were to be carried out at a higher butyramide concentration the A arnidase would appear more active. However, at the low substrate concentrations (40 to 80 mM) normally present in the growth media, the greater activity of the B amidase is of advantage to strain B 6 not only in making butyrate available as a carbon source but also in relieving butyramide repression of amidase synthesis. The physicochemical properties of the B enzyme are discussed in more detail on page 214 when it is compared with the wild-type and other mutant amidases.

THE ALIPHATIC AMIDASES OF

Pseudomonas aeruginosa

211

3. Valeramide- Utilizing Mutants

Valeramide did not support growth of the wild-type strain of Pseudomonas aeruginosa 8602 or either of the two groups of butyramideutilizing mutants. The mutation of the constitutive strain C 11 to produce mutant B 6 had been successful in extending the effective substrate range of the amidase to include butyramide and it was thought that it might be possible by further mutational steps to obtain a mutant amidase which could hydrolyse C5 or CBaniides. Starting with mutant TABLE8. Properties of Valeramide-UtiIizirig Mutants of Pseudornonas aeruginosa 8602. From Brown et al. (1969). Growth on plates containing amides

Mutant

V1 v2 v 3 v4 v 5 V6 V7 V8 v9 v 10

v 11 l36

c 11

Acet amide

t +t+

+++ tr

+t

+ +++ + + -t +++ +++

Butyramide Valeramide

+++ +t+ +++ +++ +++ +++ +t+ +++ + + + +++

-

++ ++ ++ ++ ++ t-t ++ ++ ++ ++ ++ -

-

Amide hydrolysis rate (pmoles NH3/mg. bact./min.) Acetamide

0.49 nt 3.0 3.1 0.85 3.7 0.68 3.4 0.8 0.55 0.83

'3.4 15.5

Butyramide

Valeramide

2.7 nt 6.5 8.1 3.1 9.3 3.1 8.1 0.98 0.57 0.56 3.3 0.3

0.44 nt 0.61 0.56 0.64 0.78 0.7 0.62 0.48 0.38 0.42 0 0

Growth after three days a t 37". +++, ++, + and tr, indicate relative growth and -, no visible growth. nt indicates not tested. Amide hydrolysis was measured by the ninhydrin method. Cultures were grown overnight in pyruvate medium. Mutant V 8 was isolated from strain CB 6 and other V mutants from strain B 6.

B 6, J. E. Brown (1969) isolated seven mutants from valeramide plates following treatment with EMS and four others arose spontaneously (V mutants). Another valeramide-utilizing mutant was derived from mutant CB 6 but none was obtained from C 11 or the wild-type strain either by spontaneous mutation or after mutagen treatment. The V mutants formed a heterogeneous group. Table 8 shows that, while almost all the mutants grew well on butyramide plates, some had lost the ability to grow on acetamide plates. Although they had all retained the constitutive character none of them grew as well on S/F plates as their parent strains and the V mutants derived from B 6 had also lost the

212

PATRICIA H. CLARKE

ability to grow on S/L plates (CB 6 did not grow on S/L plates so its V mutant would not be expected to do so). None of the V mutants utilized hexanoamide. Washed suspension of the V mutants had both hydrolase and transferase activity towards valeramide. Table 8 shows that there were wide differences in the amide hydrolase profiles of pyruvate-grown cultures of the V mutants, and mutants B 6 and C 11. All the V mutants hydrolysed acetamide less readily than strain B 6 but most of the V mutants were more active than strain B 6 on butyramide. The amide transferase activities of the V mutants also showed considerable variation. The results suggested that the valeramide mutants were probably of independent origin and that a number of different mutational sites were involved. The V amidases appeared to be very unstable in cell-free extracts. When the bacteria were disrupted in a French pressure cell or by ultrasonication about 95-100% of the amidase activity was lost. Although most of the V mutants had a comparatively high amidase activity with the intact bacteria, this instability in cell-free extracts made it impossible to detect amidase bands on starch gels or to proceed with detailed enzyme studies. I n immunodiffusion tests with antisera prepared against the pure A and B amidases, the extracts of several of the V mutants gave partial crossreaction (see Fig. 17c). 4. Acetanilide- Utilizing Mutants The wild-type strain of Pseudomonas aeruginosa 8602 was unable to hydrolyse N-substituted amides a t a detectable rate and did not grow in media containing either phenylacetamide or acetanilide (N-phenylacetamide) as the carbon source. Attempts to obtain acetanilideutilizing mutants directly from the wild-type strain were unsuccessful. P. R. Brown (1969) decided to use mutant L 10 for this purpose since it produced a high amidase activity constitutively, was resistant to catabolite repression and also was relatively insensitive to repression by amide analogues including acetanilide. It had previously been shown that acetanilide repressed amidase induction by N-acetylacetamide with the wild-type strain (Kelly and Clarke, 1962) and constitutive amidase synthesis by mutant C 11 (J.E. Brown, 1969). The acetanilide-utilizing mutants (A1mutants) were all isolated from plates containing 0.1% acetanilide as carbon source from strain L 10 after treatment with NMG. The A1 mutants grew on acetamide and S/F plates but none of them grew on B plates although the parent strain L 10 was able to do so. The relative hydrolase and transferase activities of an amidase preparation obtained from one of the A1 mutants are given in Table 9. The substrate profiles differ very much from those of the A and B amidases. The rate of propionamide hydrolysis was more than ten

THE ALIPHATIC AMIDASES OF

Pseudomonas aeruginosa

213

TABLE9. Properties of A1 3 Amidase Isolated from Strain A1 3, an AcetanilideUtilizing Mutant of Pseudomonas aeruginosa 8602. From P. R. Brown (1969) Hydrolase activity (pmoles NH3 produced/ min./mg. protein)

Amide Acetamide Propionamide Butyramide Acetanilide (N-phenylacetamide)

Transferase activity (pmoles acylhydroxymate produced/min./mg. protein)

17.9 320.5 0.99 ND

657 590 1-1 4.7

Hydrolase activity was determined by the ninhydrin method.

times the rate of acetamide hydrolysis. The rate of acetanilide hydrolysis wasnot determined but wasobviouslyhighenough for thegrowth to occur. The rate of transferase activity for acetanilide was determined and although low it was significantly higher than that for butyramide. The

::Ii 0

0

2

I

,

4 6 Time at 57 5' (min )

8

10

FIG.16. Heat inactivation of wild-type and mutant amidases. Data are given for cell-free extracts of mutant L 10, producing A amidase, ( x ), and mutants A1 2 (0) and A1 8 (A), pure enzyme preparations of I3 amidase (v),and A1 3 amidase ( 0 ) .From P. It. Brown (1969).

214

PATRICIA H. CLARKE

mutations of the A1 mutants have clearly resulted in completely different alterations in the amidase protein from those which produced the B and V mutations. Extracts of the A1 mutants were subjected to electrophoresis on starch gels and all gave amidase bands which ran slightly faster than the A amidase. There were no apparent differences in the electrophoretic patterns of extracts of the different A1 mutants but the extracts differed somewhat in the sensitivity of the amidases to heat denaturation (Fig. 16). It is possible that there is more than one A1 amidase enabling Pseudomonas aeruginosa 8602 mutants to grow on acetanilide. The amidase from mutant A1 3 was purified and examined in more detail. 5 , Comparison of the Wild-Type and Mutant Amidase Proteins

a. The physicochemical properties of the wild-type A amidase were investigated by P. R. Brown (1969). The pure enzyme was prepared from strain C 11. The molecular weight of the enzyme, determined by the sedimentation equilibrium method, is in the region 200,000 to 210,000. Evidence from end-group determinations suggested that the enzyme is composed of identical subunits. Methionine was the only N-terminal amino acid detected by the fluorodinitrobenzene method ; the cyanate method (Stark and Smyth, 1963) gave slightly more than four N-terminal methionine residues per molecule of enzyme protein. The only C-terminal amino acid determined after hydrazinolysis and hydrolysis by carboxypeptidase A was alanine. The exact number of subunits remains to be determined but may be four or six. b. The mutant B and A I 3 amidases probably do not differ significantly from the A amidase in overall physicochemical properties, such as molecular weight and number of subunits, although they can be distinguished from the A amidase by the differences in their electrophoretic mobilities a t pH 8.5 (see Fig. 17). The A amidase was very heat stable and the B amidase was only slightly less so, but the A1 3 enzyme lost over 50% of its activity on heating for 10 min. a t 60" (Fig. 16). These differences in structure were not sufficient to alter their antigenic specificity and both the B and the A1 3 amidases gave complete cross-reactions with the antiserum prepared against the A amidase (Fig. 17b). I n the reciprocal tests the A amidase gave complete cross-reactions with antiserum prepared against the purified B amidase. When the V amidases, which were enzymically so unstable in the cell-free state, were used in the immunodiffusion tests any cross-reactions which could be detected were only partial (Fig. 17c). To obtain extracts capable of giving crossreactions it was necessary to carry out the experiments a t 4" and the reason why the V amidases were so unstable may be that the enzyme

THE ALIPH-4TIC AMIDASES O F

Pseudomonas aeTuginoSa

215

dissociated into inactive subunits very readily when it was released from the bacterial cell. Attempts were made to identify the amino-acid differences between the wild-type and mutant amidase proteins by ‘fingerprint’ analysis of the peptides appearing on the chromatography electrophoresis maps after digesting the pure enzymes with trypsin and chymotrypsin. I n spite of the marked differences in electrophoretic mobilities of the enzyme proteinsit was not possible to find any differences in the peptides obtained from the A and B amidases. For the A I 3 enzyme, however, the peptide map prepared from a combined tryptic and chymotryptic digestion showed changes in the location of one of the peptides when compared with the map obtained from the A amidase. The amino-acid sequences of these two peptides are shown below and indicate that in the A1 3 amidase an isoleucine residue has replaced a threonine residue of the wild-type A amidase protein.

+

Ser-Leu-Thr-Gly-Glu-Arg

A amidase (from strain C1 1)

Ser-Leu-lle-Gly-Glu-Arg

A1 3 amidase (from strain A1 3)

The substrate differences of the A, B and AI 3 enzymes with respect to their amide substrates have already been discussed. These differences extended also to their ester substrates although the esterase activity was about 1% of the acetamidase activity for the A and B amidases and about 4% for the A1 3 amidase. The esterase activities of the three amidases are compared in Table 10. Determinations were made of the TABLE10. Esterase Activity of Pseudornonas aeruginosa 8602 Wild-Type and Mutant Amidases. From Brown et al. (1969) and P. R. Brown (1969) A amidase

Methyl acetate Ethyl acetate n-Propyl acetate iso-Propyl acetate Uutylacetate Ethyl formate n-Propyl propionate iso-Propyl propionate

Hydrolase

Transferase

70 100 62 0 ND ND 0

72 100 67 10 147 65 1.2 0

0

k3 amidase A1 amidase Transferase Transferase ND 100 109

ND 178 ND 9.2 0

190 100 210 0 420 ND ND ND

The esterase activity is expressed relative to that for ethyl acetate arbitrariIy assigned a valuc of 100. ND = Not determined. A amidase was isolated from strain C 11; B amidase was isolated from strain B 6; A1 amidase was isolated from strain A1 3.

216

PATRICIA H. CLARKE

apparent K,values in the amide transferase reaction for the three enzymes (Table 11). The relative insolubility of acetanilide made it difficult to carry out K , determinations with this substrate but it can be seen that the mutant enzyme A1 3 which can attack acetanilide has a much lower affinity than the A amidase for the two aliphatic amides. Both the A1 3 amidase and the B amidase may be considered as examples of enzyme evolution in that the mutant is able to utilize for growth a substrate which is unavailable to the parent strain. The A1 3 enzyme is less active than the A amidase in respect to the original substrates of the wild-type strain and might therefore be classed as a defective enzyme, but when the B amidase is considered the picture is quite different. Pollock (1965) compared the penicillinases produced by two Bacillus strains and observed that whereas one strain had a high V,,, and a fairly high K , the TABLE11. Comparison of A, I3 and A1 3 Amidases of Pseudomonm aeruginosa 8602 : Apparent Michaelis Constants

B amidase AT 3 amidase A amidase Apparent K , ( m M ) Apparent K , (mM) Apparent K , ( m M ) Acetamide Propionamide Butyramide

19 55 500

15 9.5 73.5

52.6 208

ND

Determined for the transferase reaction in the presence of excess hydroxylamine.

other strain had both a lower V,,, and a lower K , for penicillin. This meant that, at a fairly low concentration of penicillin which might well occur in nature, both strains could hydrolyse the substrate at about the same rate. He introduced the concept of “physiological efficiency” measured as Vmax/Km and on these terms the two penicillinases could be classed as equally efficient. If this is applied to the A and B amidases, then, since for butyramide the K , of the B amidase is about 10 times lower and the V,,, is about 10 times greater than the A amidase, it could be said that the physiological efficiency of the B amidase for butyramide is about one hundred times greater than that of the A amidase. The K , determinations were actually carried out for the transferase reaction and the assumption also has to be made that the values can be extrapolated to the hydrolase reaction which enables growth to occur on butyramide. The wild-type A amidase hydrolyses propionamide more readily than other amide substrates and when the specific activities of the A and B amidases are compared with respect to propionamide it can be seen that the B enzyme is also more active on propionamide than the A enzyme. If the values obtained from K , and V,,, determinations in the transferase reaction (Tabie 9) can again be extrapolated to the hydrolase

THE ALIPHATIC AMIDASES OF

Pseudomonas aeruginosa

217

reaction it would appear that the B amidase has also a higher physiological efficiency for propionamide than the parent enzyme. The mutation from A to B amidase is therefore put forward as an example of a positive enzyme mutation.

IV. Genetic Analysis The genetic study of pseudomonads is much less developed than that of Escherichia coli or Salmonellu typhimuriurn (Holloway, 1969) but transfer of genetic material by conjugation has been known for some years (Holloway, 1955; Holloway and Pargie, 1960; Loutit and Marinus, 1968). Genetic linkage has been studied by transduction (Fargie and Holloway, 1965; Pearce and Loutit, 1965; Mee and Lee, 1967). Brammar et ul. (1967) found that the pseudomonad phage F 116 isolated by Holloway et al. (1960) could transduce the amidase-positive character from the wild-type strain to amidase-negative mutants. There was no cotransduction of the amidase character with the genes determining enzymes of acetate metabolism, isocitrate lyase, acetic thiokinase or citrate synthase (Skinner and Clarke, 1968). When magno-constitutive or semi-constitutive strains were used as donors, Brammar and coworkers (1967) found that the constitutivity marked was cotransduced with the amidase-positive character a t a very high frequency. Formamide-inducibility was also cotransduced a t a high frequency. The interpretation of these results was that a regulator gene and a structural gene for amidase were closely linked. When constitutive catabolite-resistant strains such as L 10 were used as donors, it was found that most of the amidase-positive transductants were constitutive and grew on X/F plates but none grew rapidly on X/L plates so that they were not catabolite-resistant. This indicated that the resistance to catabolite repression exhibited by these strains was not due to mutations in the amidase regulator genes. The properties of the various regulator mutants have suggested that the regulation of amidase synthesis is controlled by a regulator gene of the lac i type producing a cytoplasmic repressor which in the wild type prevents amidase synthesis unless an inducer is present. Many of the regulator mutations appear to result in the production of altered cytoplasmic repressors rather than in the absence of regulator-gene products. The resolution of the genetic system has not yet allowed any mapping of the regulator gene and since it has not been possible to make partial diploids it is not known whether any of the regulator mutants can be assigned to an operator site. There is no evidence to suggest that any other enzymes are subject to co-ordinate regulation in this system. The amidase structural gene has been assigned the genetic description

218

PATRICIA H. CLARKE

of amiE and the regulator gene amiR following the recommendations of Demerec et aZ. (1966). The mutants which have been discussed in detail in previous sections are listed in Table 12 with their genetic descriptions TABLE12. Phenotypic and Genotypic Characteristics of Mutants of Pseudomonas aeruqinosa 8602 Amidase genotype Selection Type of Strain media amidase _ _ ~

c---------T

amiR

-

Regulatory phenotype

-

~~~

8602 WT

amiE

A

+

i-

Inducible, catabolite- repressible Constitutive, butyramideresistant Constitutive, butyramidesensitive Inducible

c1

SIJf

A

m

-t

c 11

S/F

A

m

F 6

S/F

CB 6

I3

A A

m m

L 10

sir,

A

m

+ + + +

B 6 (C 1 1 )

B

B

m

V-type

m m

Constitutive, butyramideresistant Constitutive, butyramideand cataboliteresistant Constitutive

m

Constitutive

AI-type

m

m

Constitutive

V 1 (B 6)

V A1 3 (L 10) A1

Parent strains of mutants not isolated directly from the wild-type strain are given in brackets. Amidase genotypes : amiR, regulator gene ; amiE, amidase structural gene. Wild type given as and one or more mutations as m. For other details see text. Selection Media: S/F, succinate (1%)t formamide (0.1%); S/L, succinate (176) + lactamide (0.05%); B, butyramide (0.1%); V, valeramide (0.1%); AI, acetanilide (0.1%).

+

and main phenotypic characteristics. J. E. Brown (1969) was able to obtain intragenic crosses among the amidase-negative mutants by transduction, but intensive mapping of this gene awaits the isolation of further mutants.

V. Genetic Homology among Pseudomonas spp. The nutritional versatility of pseudomonads offers many possible biochemical variations of metabolic pathways and regulatory mechanisms. The genetic and biochemical relationships of the enzymes concerned with the breakdown of aromatic compounds have been studied in some detail by several groups of workers (see Stanier, 1968, and also Chakrabarty et al., 1968; Kemp and Hegeman, 1968; Rosenfeld and

THE ALIPHATIC AMIDASES OF Pseudornonas

aeruginosa

219

Fiegelson, 1969). The pseudomonads can be grouped according to whether the mechanism of cleavage of the aromatic ring is ortho or meta. The species in which the ortho-cleavage pathway occurs, producing ,%-ketoadipate,are also alike with respect to the regulation of biosynthesis of these enzymes, but this regulatory pattern differs from that of Moraxella spp. although they also produce P-ketoadipate. Stanier (1968) described the immunological reactions of extracts of Pseudomoms spp, metabolizing aromatic compounds via the P-ketoadipate pathway with antisera prepared against two of the aromatic pathway enzymes purified from a strain of P. putida biotype A. The two enzymes were the muconate lactonizing enzyme and muconolactone isomerase, and the tests were carried out with extracts prepared from cultures grown in the presence and absence of benzoate as inducer. No cross-reaction was detected with extracts of P. multivorans. Extracts of P . stutzeri reacted weakly with antiserum to the lactonizing enzyme. Three species ( P .aeruginosa, P.jluorescens and P. putida) had previously been recognized within the fluorescent group (Stanier et al., 1966) and extracts of P. jluorescens strains gave cross-reactions with heavy spurring against the anti-serum to the lactonizing enzyme and very weak reactions against the antiserum to the isomerase. The P. aeruginosa extracts gave strong reactions with both antisera but heavy spurs were always present. When extracts of other strains of P. putida biotype A were tested against the antiserum for the standard strain the crossreaction was complete in almost every case. The results indicated very close genetic homology of these enzymes within the P. putida biotype A group and a closer relationship with P. aeruqinosa than with the other species tested. Some strains of P. putida biotype A, and all strains of P. aeruginosa, were reported by Stanier et al. (1968)to grow on acetamide. (One strain of P. putida biotype B, listed as acetamide-positive, has now been found to be unable to do so). P. H. Clarke and H. Dewhurst (unpublished) found that extracts prepared from strains of P . aeruginosa (kindly provided by R. Y. Stanier) gave complete cross-reactions with antiserum prepared against the pure A amidase produced by P. aeruginosa 8602 wild type. Extracts of acetamide-positive strains of P. putida biotype A also gave strong cross-reactions but these were partial only and gave spurring. Typical reactions are shown in Fig. 17d and e. These results again suggest a very high degree of genetic homology within each of the two species and a fairly close relationship between them with respect to the aliphatic amidase. Stanier (1968) pointed out the value of qualitative and quantitative studies of antigenic divergence among bacterial species. When this is combined with detailed analysis of metabolic pathways and regulatory

220

PATRICIA H. CLARKE

mechanisms, it may provide valuable clues in attempts to unravel evolutionary relationships. Studies of the antigenic relationships of single enzymes may also contribute to an understanding of the extent of (a)

I

2

3

FIG. 17. (a) Starch-gel electrophoresis of cell-extracts from Pseudomonas aeruginosa 8602 mutant strains C 11 and B 6. (1) Extract from B 6, (2) mixture of extracts of C 11 and B 6, (3) extract of C 11. Amidase bands are indicated by arrows. (b) Precipitin reactions of cell-extracts from P. aeruginosa 8602 mutant strains C 11 and B 6 and purified A and B amidase proteins with antiserum t o purified A amidase (centre well). (c) Precipitin reactions of cell-extracts from P. aeruginosa 8602 mutant strains C 11, V 4 and V 7 with antiserum to purified B amidase (centrewell). (d) Precipitin reactions of cell-extmcts from P. aeruginosa 8602 mutant L10, P. aeruginosa 45 and P. putida A 87 and A 51 with antiserum to purified Aamidaso (ccntre woll). ( e ) Precipitin reaction of cell-extracts from P. aeruginosa 8602 mutant L 10, P. aeruginosa 277, P. putida A 87 and A 51 with antiserum t o purified A amidase (centre well).

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structural variations which may occur among protein molecules carrying out similar catalytic functions.

VI. Acknowledgements It is a pleasure to acknowledge the contributions of my collaborators especially Drs. Jane E. Brown, P. R. Brown, W. J . Brammar, M. Kelly, Norma D. McFarlane and A. J . Skinner. I am grateful to Dr. Pauline Meadow for advice and discussion and to Mrs. Heather Dewhurst and Mrs. Renee Tata for technical assistance. The Medical Research Council and the Science Research Council have provided generous support for Research Training Grants and for scientific apparatus. REFERENCES Batchelor, .’!I K., Chain, E. B., Richards, M. and Rolinson, G. N. (1961). Proc. R. Soc., B. 154, 522. Hergmann, M. (1942). Ad v. Enzymol. 2, 49. Hoddy, A., Clarke, P. H., Houldsworth, M. A. and Lilly, M. D. (1967). J . gem. Microbiol. 48, 137. Ronicke, R. (1960). Zentlb. Bakt. ParasitKde (Abt. I ) 179, 209. Urammar, W. J. (1965). Ph.D. Thesis. University of London. Brammar, W. J. and Clarke, P. H. (1964).J . gen. Microbiol. 37, 307. Brammar, W. J., Clarke, P. H. and Skinner, A. J. (1967).J . gen. Microbiol. 47, 87. Brammar, W. J., McFarlane, N. D. and Clarke, P. H. (1966). J . gen. Microbiol. 44, 303. Bray, H. G., James, S. P., Thorpe, W. V., Wasdell, M. R. and Wood, P. B. (1949). Biochem. J . 45, 467. Bray, H. G., James, S. P., Thorpe, W. V. and Wadell, M. R. (1950). Biochem. J . 47, 294. Brown, J . E. (1969).Ph.D. Thesis: University of London. Brown, P. R . (1969).Ph.D. Thesis: University of London. Brown, J. E., Brown, P. R. and Clarke, P. H. (1969).J. gen. Microbiol. 57, 273. Biihlman, X., Vischer, W. A. and Bruhin, H. (1961).J . Bact. 82, 787. Chakrabarty, A. M., Gunsalns, C. F. and Gunsalus, I. C. (1968).Proc. natn. Acad. Sci. U.S.A. 60, 168. Chiang, C. and Bermett, R. E. (1967).J. Bact. 93, 302. Christensen, W. B. (1946).J . Bact. 52,461. Clarke, P. H. and Brammar, W. J. (1964). Nature, Lond. 203, 1153. Clarke, P. H. and Lilly, M. D. (1969). I n “Microbial Growth”, S y m p . Soc. gen. Microbiol. 19, 113 (P.Meadow, and S. J. Pirt, eds.). Cambridge University Press. Clarke, P. H., Houldsworth, M. A. and Lilly, M. D. ( 1 9 6 8 ) . J .gen. Microbiol. 51,225. Demerec, M., Adelberg, E. A., Clark A. J. and Hartman, P. E. (1966). Genetics, I’rrinceton 54, 61. den Dooreri de Jong. L. E. (1926). Bijdrage tot de kennis van het mineralsatieproces. Nijgh and Van Ditmar, Rotterdam. Draper,P. (1967).J.gen.MicrobioL46,lll. Durrell, J. and Fruton, J. S. (1954).J . biol. Ghem. 207, 487. Fargie, €3. and Holloway, I3.W. (1965). Genet. Res. 6, 284. Folk, J . E. and Cole, P. W. (1966). Biochim. biophys. Acta, 122, 244. Gilbert, W. and Muller-Hill, B. (1966). Proc. natn. Acad. Sci. U.S.S. 56. 1891. Gorr, G. and Wagner, J. (1933). Bot. Ztg. 266, 96. Grossowicz, N. and Halpern, Y. S. (1957).J . biol. Chem. 228,643. Hagihara, B. (1960). I n “The Enzymes”, vol. 4, p. 193 (P. D. Boyer, H. Landy and:K. Myrbiick, eds.), New York, Academic Press Inc.

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Halpern. Y. S. and Grossowicz, N. (1957). Biochem. J. 65, 716. Holloway, B. W. (1955).J. gen. Microbiol. 13, 572. Holloway, B. W. (1969). Bact. Rev.in press. Holloway, B. W. and Fargie, B. (1960).J. Bact. 80, 362. Holloway, B. W., Egan, 5. B. and Monk, M. (1960). Aust. J. exp. Biol. med. Sci. 38, 321. Holt, R. J. and Stewart, G. T. (1964).J . gen. Microbiol. 36, 203. Hughes, D. E. and Williamson, D. H. (1953). Biochem. J. 55, 851. Jacoby, W. €3. and Fredericks, J. (1964).J . biol. Chem. 239, 1978. Jayaraman, K., Muller-Hill, B. and Rickenberg, H. V. (1966). J . molec. Biol. 18, 339. Johnson, R. B., Myeck, M. J. and Fruton, J. S. (1950).J. biol. Chem. 185, 629. Joshi, 5. G. and Handler, P. (1962).J. biol. Chem. 237, 929. Juhlin, I. (1960).Acta path. microbiol. scand. 50, 195. Kelly, M. and Clarke, P. H. (1960). Biochem. J. 74, 21P. Kelly, M. and Clarke, P. H. (1962).J. gen. Microbiol. 27, 305. Kelly, M. and Kornberg, H. L. (1962). Biochens. biophys. Acta, 64, 190. Kelly, M. and Kornberg, H. L. (1964). Biochem. J . 93, 557. Kcmp, M. B. and Hegeman, G. D. (1968).J. Bact. 96, 1488. Kimura, T. (1959a).J. Biochem., Tokyo 46, 973. Kimura, T. (1959b).J. Biochem., Tokyo 46, 1133. J. Biochem., Tokyo 46, 1271. Kimura, T. (1959~). Kimura, T. (1959d).J. Biochem., Tokyo 46, 1399. Lamaire, Y. and Brunel, A. (1951). C.r.hebd. Sdanc. Acad. Sci. Paris, 232, 872. Loomis, W. I?. and Magasanik, B. (1967).J. molec. Biol. 23, 487. Loutit, J. S. and Marinus, M. G. (1968).Genet. Res. 12, 37. Mandelstam, J. and Jacoby, G. A. (1965). Biochem. J. 94,569. McFarlane, N. D. (1967). Ph.D. Thesis : University of London. McFarlane, N. D., Brammar, W. J. and Clarke, P. H. (1965). Biochern. J . 95, 24c. Mee, E. J. and Lee, T. 0. (1967). Genetics, Princeton 55, 709. Meister, A., Levintow, L., Greenfield, R. E. and Abendschein, P. A. (1955).J . biol. Chem. 215, 441. Moses, V. and Prevost, C. (1966). Biochem. J. 100, 336. Mycek, M. J. and Waelsch, H. (1960).J . biol. Chem. 235, 3513. Pastan, I. and Perlman, R. L. (1968). Proc. natn. Acad. Sci. U .S.A. 61, 1336. Pearce, L. E. and Loutit, J. S. (1965).J. Bact. 89, 58. Petrack, B., Greengard, P., Craston, A. and Sheppy, F. (1965).J. biol. Chern. 240, 1725. Pollock, M. R. (1965). Biochem. J. 93,557. Rosenfeld, H. and Feigelson, P. (1969).J. Bact. 97, 697. Schneidau, J. D. (1963). Am. Rev. resp. Dis. 88, 563. Silverstone, A. E., Magasanik, R., Reznikoff, W. S., Miller, J. H. and Beckwith, J. R. (1969). Nature, Lond. 221, 1012. Skinner, A. J. and Clarke, P. H. (1968).J. gem. Microbiol. 50, 183. Smith, E. L. and Slonkim, N. B. (1948).J. biol. Chem. 176, 835. Stanicr, R. Y. (1968). I n “Chemotaxonomy and Serotaxonomy”, Vol. 2, p. 201 (J.G. Hawkes, ed.) Academic Press, London. Stanier, R. Y., Palleroni, N. J. and Doudoroff, M. (1966).J.gen. Microbiol. 43, 159. Stark, G. R. and Smyth, D. G. (1963).J. biol. Chem. 238, 214. Sumnor, J. B. (1951). In “The Enzymes”, Vol. 1, p. 873 (J. B. Sumner and K. Myrback, eds.), Academic Press, New York. Urabe, K., Takei, N. and Saito, H. (1965).Am. Rev. resp. Dis. 91, 120. Zittle, C. A. (1951). I n “The Enzymes”, Vol. 1, p. 922 (J. B. Sumner and K. Myrback, eds.) Academic Press, New York.

The Place of Continuous Culture in Microbiological Research D. W. TEMPEST Microbiological Research Establishment, Porton, N r . Salisbury, Wiltshire, England A thing ma,y look specious in theory, and yet be ruinous in practice; a thing may look evil in theory, and yet be in practice excellent. Edmond Burke (1788).

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I. Introduction 11. Microbial Growth in a Closed System: T h e “Batch Culture” . 111. Microbial Growth i n an Open System : The Continuous-Flow Culture IV. T h e Chemostat as a Research Tool . A. Use of a Chemostat i n Studies of Bacterial Cation Metabolism. R. Use of a Chemostat i n Studies of Bacterial Cell-Wall Synthesis C. Use of a Chemostat i n Studies of Microbial E n z y m e Synthesis. V. Some Inadequacies of Continuous Culture as a Research Tool . VI. Operational Problems . A. Foaming B. W a l l G r o w t h . V I I . Conclusions . V I I I . Acknowledgements References

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I. Introduction

It is now almost twenty years since the fundamental principles of “continuous culture” were expounded by Monod (1950) and by Novick and Szilard (1950a). But although research in microbiology, as in biochemistry, has expanded enormously during this post-war period, the obvious potentialities of continuous culture, both as a research tool and as a production tool, have remained largely unexploited. The reasons for this are not immediately obvious. Of course, scientific papers generally only indicate why certain procedures were adopted ; they seldom if ever state why the alternative and, perhaps, more promising methods were discounted. So one cannot deduce from the published literature why continuous culture methods have found so little favour amongst microbiologists ; one can only base opinions on hearsay and, in my experience, 223

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the root cause would seem to be more often emotional than rational. This being so, it may be of some value to take a fresh “down to earth” look a t the philosophy underlying the use of continuous culture as a research tool; to try and assess its real potentialities and to expose its undoubted short-comings. I n doing this I shall try to avoid a detailed (and, I am sure, now boring) re-iteration of continuous culture theory. This has been exhaustively covered in successive publications over the past twenty years (see, for example, the papers of Herbert et al., 1956; Herbert, 1958; Powell, 1965; Fencl, 1966; Tempest, 1969a) and would be of little added value here. What I wish to do here is concentrate on the basic concepts pertaining to microbial growth and physiology (the facts and the myths), and to show how further progress in microbiological research can be greatly facilitated by the use of continuous culture.

11. Microbial Growth in a Closed System: The “Batch Culture” If, as some may argue, the traditional methods of culturing bacteria were wholly adequate, then there would be no point in going to the extra trouble of setting up and maintaining continuously growing cultures of organisms. So, before attempting to assess the value of continuous culture in microbiological research, it is necessary to point out some of the failings of the “batch culture” method of growing micro-organisms. No doubt all will agree that a landmark in the history of microbiology was the development of the now classical procedures whereby specific microbes could be isolated and cultured free from the “contaminating” organisms with which they were naturally associated. Isolation of organisms in a “pure” state was, of course, necessary in order to investigate and rationalize the many biological phenomena, ranging from disease and putrefaction t o fermentation, that could be observed in Nature. But, in order to study the physiological properties of pure strains, larger quantities of organisms were required ;this, it was found, generally could be accomplished by simply inoculating cells of the isolated strain into batches of nutrient medium contained in a closed vessel, incubating a t a suitable temperature (with or without aeration) and allowing events to run their course. The success of this process-the “batch culture” processwassuch that it quicklybecame theroutine methodfor culturing microbial cells, which it remains today. It is ironic, however, that many microbiologists now seem to look upon this “batch” process as being essentially natural whereas this it clearly is not. The environment in a batch culture generally is far removed from that likely to be found in Nature (see Section 11, p. 2 2 7 ) and there is good reason for believing that the behaviour of the organisms, so cultured, also is considerably different from that expressed in natural environments. Indeed so much is this the

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case that, as the late Professor Kluyver is reported to have argued, one should look upon all pure cultures as “laboratory artefacts’’ (see Postgate, 1969). Clearly, a factor which must have encouraged the widespread adoption of batch-culture methods is the ease with which they generally can be set up and maintained free from contaminating organisms. Conversely, the difficulties encountered in setting up and maintaining continuously growing cultures free from contaminating organisms probably has, over the years, repeatedly militated against their ready exploitation. But fundamentally the success of both processes stems from the enormous physiological “plasticity” of prokaryotic cells ; that is their ability to adapt to sudden shifts in environment. Underlying this adaptability is the facility with which these cells effect changes in their genetic content and, more significantly, in the expression of their genetic content (that is through processes such as repression, derepression, and allosteric effects). I n fact, bacterial cells are able to change themselves phenotypically to such an extent that it is quite impossible to define them chemically (or structurally or functionally) without reference to the growth environment (see Herbert, 1961a). This is an enormously important concept to which I shall return shortly. First let us examine critically the processes of microbial growth in a batch culture. We are all fully aware that, when microbial cells are inoculated into a nutrient medium and incubated at a suitable temperature, a characteristic sequence of changes occurs. After a period of metabolic adjustment (the “lag phase”), the organisms increase in mass and then divide. As this growth and division process proceeds (which it generally does a t a more or less constant rate), the culture population density increases exponentially. During this “exponential growth phase” nutrients are taken up from the medium and end-products of metabolism are excreted into it at a rate which increases exponentially with the biomass. Thus the processes of growth cause the environment to change progressively; the organisms adapt continuously to these changes but eventually the environment becomes so changed that it is unable to support further growth and, at this time, the culture enters the so-called “stationary phase”. This sequence of changes ((‘lag’’to ‘(exponential7’ to “stationary” phases) has been analysed in great detail (Monod, 1942, 1949)and collectively is referred to as the growth cycle. But i t is essential to realize that, unlike the division cycle (whereby one organism ultimately gives rise to two progeny), this growth cycle is in no way a fundamental property of the organism but an inevitable consequenceof the interaction of the organisms with their environment in a closed system. This important fact is not always appreciated by microbiologists, particularly those who persist in using expressions such as “young cells”, “old cells”, “mid 8

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log-phase cells” and “stationary-phase cells” to define the physiological state of their experimental material. Clearly these terms are not only imprecise, they engender a complete misunderstanding of the real situation. For example, we all know that bacteria divide by binary fission which means that the mother cell is consumed as the daughter cells are created. So what is an “old” cell and a “young” cell? Usually (and quite misleading) these terms are used to define the organisms in terms of the age of the culture. Thus, what is meant by a “young” cell is one growing rapidly in an environment where all nutrients are present in excess of requirement. An “old” cell, on the other hand, is by this definition one suspended in a toxic or nutrient-depleted environment. But logically, the age of a cell can only be fined in relation to the division cycle, that is in terms of the time interval subsequent to cell division. It might be argued that for practical purposes it does not matter that identical terms are used to define different events, but this is valid only so long as the basic concepts are clear and correct; in this particular instance they are not! At the risk of being tedious, let me amplify this point by taking another example, the expression “mid log-phase cells”. This defines the organisms in terms of a particular moment of time during the culture growth-cycle. But as pointed out above, the growth cycle is not an inherent property of the organisms and since the environment changes continuously throughout the “exponential phase”, and will be different for different cultures, the terms is no more meaningful than the expression “x-hour cells”. Of course it is often claimed that the use of “mid log-phase cells” is justifiable on the grounds that these organisms are in “balanced growth” (i.e. that all the molecular species composing the population of cells are increasing at the same rate). But this is unlikely to be true since, throughout the “exponential phase”, the environment changes continuously and so, too, it is reasonable to suppose (and relatively easy to show), does the physiology of the organisms. Thus, it is unlikely that fully “balanced growth” is ever regularly attained in batch-type cultures although theoretically this is possible if one maintains the organisms a t a sufficiently low population density, during the period of the experiment, so that their growth and metabolism cause only an insignificant shift in the chemical environment. So it must be recognized that organisms in a batch-type culture are, from the moment of inoculation to the moment of “harvesting”, in a state of continual physiological change, and frequently this matters when attempting to interpret experimental data. This is particularly likely to matter when dealing with organisms from the so-called “stationary phase” which, physiologically speaking, is anything but stationary! But what of the organisms in the “exponential phase”?

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How representative are they of organisms growing naturally? Since, in a batch-type culture, all nutrients are initially in excess of requirement, growth rate during the early exponential phase is limited only by tbe rate at which these essential nutrients can be assimilated and converted into cell substance. Although the maximum growth rate, so expressed, is an intrinsic property of the organism, it can and does vary with the nutritional complexity of the environment. Thus, in a simple salts medium in which growth requires first the synthesis of monomers and then their polymerization into the cellular macromolecules, growth rate is generally much slower than when many of these monomeric constituents are added to the medium. But, irrespective of the nutritional status of the environment, there is a limit to the rate at which microbial populations can grow and multiply. With cultures of Escherichia coli (various strains) this appears to be about three doublings of mass per hour. This is, of course, a phenomenal rate of growth which, if it continued unabated for as little as two days, would give rise to a mass of organisms far in excess of the mass of the Earth. Clearly, since the Earth’s microbial population accounts for only a tiny fraction of its total mass (< 250 Kg. acre of good agricultural soil), growth for any significant period of time at a rate of three doublings per hour (or even at a rate of one doubling per hour) must be an exceedingly rare event in Nature. So it is clear that, as mentioned earlier, the environment in most batch cultures is far removed from that likely to be encountered in Nature and so too, one must conclude, is the physiology of the organisms. Three features in particular frequently must be different : in Nature (i) organisms rarely would be confined in a closed and protected environment; (ii) rarely would nutrients be present in concentrations sufficient to support growth at its maximum possible rate; and (iii) the natural environment generally would contain other species of organisms which would compete more or less effectively for the available nutrients. Since, in Nature, the environment generally will contain insufficient nutrient to permit growth of micro-organisms at their potentially maximum rates, it is important to establish whether natural populations grow for extended periods at low rates or spend long periods more or less dormant with brief intermittent periods of rapid growth when the environment so permits. In other words (and borrowing the terminology of the electronic engineer) is growth subject to “on-off control” or
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organisms/ml., each organism scavenging for the residual traces of growth-limiting nutrient. And removal of these last traces of essential nutrient would cause no detectable increase in the culture populationdensity (bearing in mind that the substrate concentration at which growth rate would be affected is likely to be less than 0.1 mM for a nutrient such as glucose). But what would happen if, as in Nature, organisms were suspended at a much lower concentration (say, less than 1 x 1O6/ml.)in media containing extremely low concentrations of some essential nutrient? Here, growth would not significantly change the chemical environment, at least for the period of the first few doublings of biomass. It was found (Monod, 1942) that under these conditions growth occurred at a sub-maximal rate, a rate depending on the actual concentration of growth-limiting nutrient in the medium. Monod suggested that the relationship between substrate concentration and growth rate could best be expressed by a Michaelis-Menten type equation, thus :

where p was the measured growth rate (that is, l/x.dx/dt), pmaxthe maximum value of p (that is when the growth-limiting substrate was no longer limiting growth), X was the growth-limiting substrate concentration in the extracellular fluid, and K , was a constant which was numerically equal to the value of X when p = &pmax. This formulation is almost certainlyinadequate (seePowell, 1967) but it is important to realise that the basic concept which it embodies, and its factual basis, are unquestionably true : bacteria can and do grow at sub-maximal rates when some essential component of the medium is present in a low, enzyme sub-saturating concentration. A n d this i s the situation most likely to prevail in Nature. The present day theory of continuous culture and its practicability stem from this dependence of growth rate on nutrient concentration. It is appropriate, therefore, to consider now the behaviour of organisms growing in a “continuous” culture. 111. Microbial Growth in an Open System: The Continuous-Flow Culture

Although occasional attempts have been made to equate organisms growing in a continuous culture with similar organisms in a batch culture at specific stages of the growth cycle, basically the two situations are entirely different. Nevertheless, all continuous cultures start their existence as batch cultures in that medium, contained in a suitable vessel,

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is inoculated with organisms which then proceed to grow and divide as described earlier. Again, the environment changes progressively with time but if, during the period of exponential growth, fresh medium is added t o the culture at arate sufficient to maintain the culture population density at some fixed sub-maximal value, then the environment similarly would cease to change with time; consequently growth would not ultimately cease, as in a batch culture, but continue indefinitely. Clearly, as the total biomass accumulated (exponentially), the medium input rate and culture volume would have to increase exponentially, unless provision was made for the removal of culture at a rate equal to that of the medium flow. I n practice this is easy to arrange (see Fig. 1) and is essentially the operating principle of one type of continuous culture apparatus-the “turbidostat” (Bryson, 1952). Medium-

Culture

FIG. 1. Diagram showing the essential features of a continuous-flow culture vessel. The vessel must (i)be able t o contain the culture free from contaminating organisms, (ii) allow addition of fresh medium (and air with aerobic cultures) a t controlled rates, (iii) provide for the rapid mixing of the inflowing nutrient with the culture, and (iv) provide for the continuous removal of culture a t a rate equal to the rate of addition of fresh medium.

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But let us consider what would happen if, with the continuous-flow culture described above, the medium flow rate was progressively lowered. Since the rate of dilution of the culture would now be less than the growth rate of the organisms, the culture population density would increase. Clearly the culture density could not increase indefinitely, for the same reason that it cannot do so in a batch culture, simply because of the growth-associated changes in the environment. As in a batch culture, the environment ultimately would become so altered that it would be unable to support further growth of organisms at their maximum rate. But growth would not cease completely, as in a batch culture, since the continuous flow of fresh medium to the culture would constantly correct the environment to an extent sufficient to allow some further growth, although only at a rate equal to the rate of dilution of the culture. With a complex medium, such as a tryptic digest of meat or acidhydrolysed casein, it would be difficult to ascertain the precise nature of the environmental change which ultimately would limit growth ;indeed, this probably would vary with parameters such as temperature and medium-flow rate. However, with a defined “simple salts” medium, the situation is much clearer and it is easy to arrange for some essential nutrient (e.g. NH,+, K+, Mg2+,Po43-, S042-, glucose) to be present in the medium at a concentration sufficient to allow only a limited amount of growth; thus, if this medium was used in a batch culture, it would be the depletion of the chosen essential nutrient which would cause growth ultimately to cease. Using such a medium in a continuous culture, it would be the availability of this particular nutrient that limited growth ; the culture population density would be prescribed by the initial concentration of this nutrient in the medium and growth rate would be limited by the rate at which this growth-limiting nutrient (in effect, fresh medium) was added to the culture. This is the basic operating principle of a second type of continuous culture apparatus-the “chemostat” (Novick and Szilard, 1950b). It is the philosophy underlying the use of the chemostat as a research tool that I now wish to consider in detail.

IV. The Chemostat as a Research Tool With the experimental arrangement depicted in Fig. 1, in which medium flows through a culture of fixed volume ( V ) ,the growth rate of organisms will depend not merely on the rate of flow medium (f)but on the dilution rate (D=fly, that is the number of culture volumes of medium passing through the vessel per unit time). I n a turbidostat, the dilution rate is adjusted automatically to maintain the culture growing

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at a near-maximum rate, but in a chemostat the dilution rate is fixed a t some lower value and the bacterial growth rate automatically adjusts to it. But, in either apparatus, equilibrium conditions are established in which the culture population density does not change with time, and nor does the environment. True “steady state” conditions prevail in which the organisms really are in “balanced growth”. This is most particularly so with the chemostat cultures since these are inherently self balancing. Thus, providing the dilution rate is kept constant (and at a value less than the critical dilution rate; D, N pmax),steady state conditions inevitably will be established. A further point which may be added here is that the growth rate of organisms in a chemostat culture can be varied at will between wide limits, thereby providing yet another parameter for study. Of course, growth rate can be varied in a batch culture by varying either the temperature or the nutritional complexity of the medium. But only in a chemostat can the growth rate be varied independently, that is, dissociated from all environmental parameters other than the concentration of growth-limiting nutrient. At this point it may not be obvious to some readers why the chemostat is such a valuable research tool, so let me remind you of some earlier statements. The most important one is that of Herbert (1961a) in which he pointed out that, since micro-organisms are able quickly to change themselves chemically (structurally and functionally) in response to sudden changes in environment, then it is difficult (in most cases impossibIe) to study the physiology of growing, functioning, organisms without culturing them in a rigidly controlled environment. And this one simply cannot achieve with batch cultures or, indeed, with any cultures maintained in a closed system. The only effective way of maintaining the chemical environment constant is to use an “open” system (Herbert, 1961b) that is, continuous-flow culture. Now, if continuous culture could do no more than provide a single controlled environment, that would be a substantial advance on the use of batch-culture methods for studies of microbial physiology. But the chemostat type of continuous culture can do much more. It can provide a whole range of controlled environments ; it can provide a whole range of unique environments, environments that are never obtained in a batch culture. Therefore, by using a chemostat, one might expect to produce (and,indeed,canproduce) a whole range of cultures containing phenotypically different organisms, organisms with some properties that are never expressed (or only transiently expressed) in batch cultures. By studying the changes in cell physiology associated with specific (individual) changes in environment, the functional significance of precise associated changes in cell structure can be assessed.

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So, at least theoretically, the advantages of using continuous-culture methods in microbiological research (particularly in studies of microbial physiology) would seem to be immense; but “the proof of the pudding is in the eating” and so it is now necessary to illustrate some of the ways in which continuous culture has been used in practice, that is, to solve particular research problems.

A. USE OF A CHEMOSTAT IN STUDIES OF BACTERIAL CATION METABOLISM In order to grow and multiply, all micro-organisms require a source of energy and an adequate supply of the essential elements-C, N, 0, H, S, P, Mg, K and a variety of “trace” elements. Since the first six elements here listed are structural components of cell proteins and nucleic acids, it is clear why they are essential for growth. But the functional basis (or bases) of the cellular requirement for various cations is less obvious. Although each of the different cations commonly found in bacteria can function as an activator of some enzyme-catalysed reaction (see Dixon and Webb, 1964), spectrochemical analyses of these elements in vegetative organisms (Rouf, 1964; Humphrey and Vincent, 1962 ; Curren et al., 1943) indicate that potassium and magnesium are present in much higher concentrations than one would expect for them to be functioning in the growing cell solely as enzyme activators. And, since magnesium and potassium are present in bacteria in concentrations that make them major cellular components, it is easy to arrange chemostat conditions in which each is the growth-limiting component of the medium. When growth of any organism is limited by the availability of such a nutrient, the organism may be expected to contain the minimum concentration of that element necessary for growth at the imposed rate in the prescribed environment. Thus, any change in this minimum requirement would be indicated by a change in the “growth yield” (that is the number of g. of organisms synthesized per g. growth-limiting nutrient consumed) and, since at growth rates less than the maximum value (pmax), nearly all the growth-limiting nutrient in the steady-state chemostat culture may be expected to be contained within the organism, a change in yield would be evident as a change in the steady-state concentration of organisms in the culture. Therefore, simply by studying the changes in culture,population density in response to changes in the growth condition (e.g. temperature, pH value, medium salinity, growth rate), one may obtain valuable information regarding the minimum requirement of organisms for a particular substance when growing in a specified environment at a specified rate. By coupling this information with observed change in cell structure and function, fundamental

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relationships may be discovered from which the functional role (or roles) of each particular growth-limiting nutrient in the cell may possibly be derived. This is the way my colleagues and I set about studying cation metabolism in bacteria. We set about trying to answer the question ‘what roles do magnesium and potassium serve in bacteria that demands their presence in high concentrations within the growing cell’? This work has been reviewed recently (Tempest, 1969b) so that it is only necessary here to select 8 few individual experiments, data which illustrate particularly well the value of using a chemostat. 1. Magnesium

With Mg2+-limited cultures of Aerobacter aerogenes (and of Pseudomonas putida), it was found that the concentration of bacteria in the culture varied with the growth rate (that is, the dilution rate) in a manner that indicated a substantial increase in the cell’s minimum requirement for magnesium with increasing growth rate (Fig. 2). This suggested a quantitative relationship between magnesium and some growth ratedependent cellular process, although changes in growth yield could have been due to the synthesis at low growth rates of substances (such as

Dilution rate ( h r - ’ 1

FIG.2. Influence of dilution rate on the steady-state concentration of Aerobacter aerogenes in chemostat cultures (maintained at 35” and pH 6.5) limited by the supply of Mg2+ (0) and K+ ( A ) .

234

D . W. TEMPEST

glycogen) that added to the bacterial weight but not to their magnesium requirement (see Herbert, 1961a). However, analysis of Mg2+-limited A. aerogenes (and, for comparison, glycerol-limited organisms) showed that, a t all growth rates, over 80% of the bacterial dry weight could be accounted for as protein, RNA and DNA indicating the absence of storage polymers. The only other obvious growth rate-linked change in the composition of Mg2+-limited bacteria was in their RNA content which varied in a manner that was strikingly similar to the variation in cellular magnesium content. Thus, although with Mg2+-limitedorganisms the cell-bound magnesium and RNA contents varied considerably (over two-fold with changes in dilution rate from 0.1 to 0-8 hr.-I), their molar ratios (RNA-nucleotide :magnesium) remained almost constant. This relationship strongly implied an interdependence between RNA and magnesium in the growing organisms. Since the bulk of the RNA in a bacterial cell is associated with ribosomes (Wade and Morgan, 1957; Ecker and Schaechter, 1963; Kjeldgaard and Kurland, 1963) the above findings suggested that this is where the bulk of the cellular magnesium also was located in the growing cell. But, in order to establish unequivocally a direct relationship between bacterial magnesium and ribosome contents, it was necessary to show that, under all conditions where the ribosome content varied, the bacterial magnesium content varied proportionately. For example, irrespective of the nature of the growth limitation, the magnesium content of bacteria should vary with growth rate in a parallel manner to changes in their RNA content. With differently limited cultures of A . aerogenes, this was invariably found to be true (Table 1).But, to be absolutely certain of a stoichiometry between the bacterial ribosome and magnesium contents, it was necessary to dissociate changes in ribosome content from changes in growth rate. I n batch-type cultures, it is impossible to change the bacterial ribosome content without simultaneously affecting the growth rate although one can alter the growth rate without altering the ribosome content (Schaechter et ab., 1958). I n a chemostat culture, however, growth rate can be maintained at any desired sub-maximal value and, under these conditions, the bacterial ribosome content can be caused to vary simply by changing the incubation temperature (Tempest and Hunter, 1965). When this experiment was carried out it was found (Table 2) that the magnesium content of the cell varied stoichiometrically with the RNA and ribosome contents. Thus it could be concluded that, in A . aerogenes at least, the bulk of the cell-bound magnesium was located in the ribosomes and that i t is this ribosomal requirement that demands the presence of high concentrations of magnesium in the growing cell.

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CONTINUOUS CULTURE I N MICROBIOLOGICAL RESEARCH

TABLE1. Influence of Growth Rate on the Magnesium and RNA Contents of Chemostat Cultures of Aerobacter aerogenes Limited by Mg2+, Glycerol or K+. g. Mgz+/100 g. dry weight bacteria

,------

Dilution rate (hr.-1) 0.1 0.2 0.4 0.6 0.8

g. RNA/100 g. dry weight bacteria -7

Mg2+limited

Glycerollimited

0.11 0.16 0.21 0.25 0.27

0.13 0.18 0.21 0.24 0.30

K+-

7--

7

limited

Mg2+limited

Glycerollimited

0.13 0.16 0.19 0.22 0.24

8.5 10.6 14.9 16.8 17.0

8.1 9.4 12.8

K+limited 9.1 11.4 15.0 18.7 19.7

14.4 16.5

The temperature was controlled at 35" and the culture pH value adjusted automatically to 6.5.

TABLE2. Effect of Iricubatiori Temperature on the Magnesium, RNA and Ribosome Contents of Aerobacter aerogenes, Growing in a MgZ+-Limited Chemost,at Culture a t a Fixed Growth Rate (D= 0.2 hr-1) and pH Value (6.5).From Tempest (1969b).

Tcmpcrttture

("1 40 35 30 25

-

g./100 g. dry weight bacteria

Ribosomes

RNA

Magnesium

Molar ratio* RNA/Mg2+

-

9.4 10.7 12.4 14.9

0.14 0.15 0.17 0.21

4.8 5.0 5.1 5.0

I -

26.5 -

33.1

7

* The molecular weight of RNA was taken to be 340 (i.e. the average molecular weight of an RNA nuoleotide). This may now seem all very obvious, but what was less explicable at the time were reports in the literature of wide differences in magnesium requirements, particularly between Gram-positive and Gram-negative organisms (Webb, 1949,1966; Rouf, 1964). If the bulk of the magnesium in a cell is required to satisfy the ribosomal requirements, and if the ribosomal requirements are similar when growing a t similar rates in similar environments (see Herbert, 1958), then why these differences? I think it is true to say that the chemostat really comes into its own when it is required to make a comparative study of two or more different organisms. Because the growth conditions are precisely defined and rigidly controlled, all the possibly troublesome phenotypic variations

236

D.

W. TEMPEST

can be eliminated and a meaningful assessment made of any observed differences in physiology. A comparison of the minimum magnesium requirements of a Gram-positive (Bacillus subtilis) and a Gram-negative ( A . aerogenes) organism, grown a t a variety of rates, provides a good example. By growing B. subtilis var. niger in a simple-salts Mg2+-limitedmedium in chemostat culture, and varying the growth rate, data could be obtained which could be compared directly with those obtained from similarly grown A . aerogenes. The results (Fig. 3) showed clearly and unequivocally that essentially there were no large differences between =I 0 - 3 0

U

cn 0

0 L

a,

c 0

:: 0 . 2

L

0 t

a,

c

c V

0

I

I

I

I

0.1

0.2

0.4

Dilution rate ( h r

-' )

I 0.8

FIG.3. Influence of dilution rate on the steady-state iritracellular content of magnesium in Aerobacter aerogenes organisms (0) and Bacillus subtilis var. niger organisms ( A ) growing in MgZ+-limitedchemostat cultures (35", pH 6.8).

the magnesium requirements of the two species. Undoubtedly, the previously reported differences were due t o the different conditions employed in culturing the various species, and in particular to the effects of these conditions on the growth rate which, being batch cultures, were uncontrolled. 2. Potassium

The above results clearly vindicated the use of continuous culture for studies of magnesium metabolism in bacteria and much of the subsequent work done to elucidate the bases of bacterial requirements for potassium has similarly been greatly facilitated by the use of chemostats (Tempest and Dicks, 1967; Tempest and Meers, 1968, Dicks and Tempest, 1966;

CONTINUOUS CULTURE I N MICROBIOLOGICAL RESEARCH

237

Tempest et ab., 1966, 1968). With cultures of A . aerogenes growing in simple salts media, the potassium content of the organisms, like the magnesium content, was found to vary with the ribosome content (see Fig. 2 ) . Irrespective of the growth rate, the ratio of cell-bound magnesium :potassium :RNA remained approximately constant a t 1:4 :5 and therefore it seemed, as with magnesium, that the presence of high concentrations of potassium in growing organisms also was due t o a requirement for this cation by the functioning ribosomes. But again, as with magnesium, many reports of gross differences in cellular potassium content could be found in the literature. I n particular, there seemed once more to be gross differences between the potassium contents of Grampositive and Gram-negative organisms. At first it seemed that these TABLE3. Influence of Dilution Rate on the Potassium, Magnesium, Phosphorus and RNA Contents of Aerobacter aerogenes and Bacillus subtilis var. niger Growing in K+-Limited Chemostat Cultures a t a Fixed Temperature (35")and p H value (6.7). Dilution rate (hr.-I)

______

g./100 g. dry weight bacteria n

c -

Potassium

0.2 0.4

3.7 4.9

0.2 0.4

1.08 1.42

Magnesium

Phosphorus

Bacillus subtilis var. niger 0.18 3.2 0.22 3.5 Aerobacter aerogenes 0.16 0.21

1.7 2.2

RNA

12.8 14.0 11.7 15.0

reported differences were due to the uncontrolled growth conditions used by the various investigators. But a brief examination of a K+-limited culture of B. subtilis var. niger and, for comparison, a K+-limited culture of A . aerogenes, clearly showed that the differences were real (Table 3). Although when grown a t similar rates in similar environments the bacterial magnesium contents and RNA contents were similar, their potassium contents and phosphorous contents were markedly different. Thus, the Gram-positive bacillus contained about three times as much potassium, and 50% more phosphorus, than the Gram-negative organism ; and this was in an environment where the organisms would be expected to contain the minimum concentration of potassium necessary for them to function (that is, grow) at the imposed rate. Therefore either the Gram-positive organism had a different ribosomal requirement for potassium, which seemed unlikely since in vitro they were not

238

D. W. TEMPEST

obviously different from the ribosomes of other species (Taylor and Storck, 1964), or else these Gram-positive organisms contained much “extra” potassium in order to fulfil some non-ribosomal function. Since B. subtilis also contained much “extra” (non-nucleic acid) phosphorus, it was reasonable to assume that the “extra” potassium was directly associated with those cellular components containing the non-nucleic acid phosphorus. However, the bulk of the non-nucleic acid phosphorus turned out to be present in the cell-wall teichoic acid and, since this polymer is located outside the plasma membrane (which is the supposed site of cation specificity), it seemed unlikely that any potassium could be physically associated with it. Nevertheless, the presence of teichoic acid in the walls of Gram-positive bacteria may in some other way prescribe the level of “extra” potassium within the cells. The techniques described above for studying bacterial cation requirements can (and should) be applied, almost without modification, to elucidating the quantitative requirements of organisms for specific amino acids, purines, pyrimidines and vitamins. But is seems likely that, in the future, continuous-culture methods will be used extensively in unravelling the many complex relationships between environment and the mechanisms that control the precise composition, structure and functioning of specific cellular components. I n this connection some small success has already attended the application of these methods to studies of bacterial cell-wall synthesis; this work is outlined briefly in the following section. B. USE OF

A

CHEMOSTAT IN STUDIES OF BACTERIAL CELL-WALL SYNTHESIS

The walls of both Gram-positive and Gram-negative bacteria are complex structures, each composed of several types of heteropolymer. But the precise function which the various wall components serve are largely unknown. Of course, many of the listed functions (e.g. “phage receptor sites”, “antigen sites”,) would seem, on reflection, to be of very doubtful value to the organisms. Nevertheless, all seemingly agree that one important function which the wall must serve is the provision of a mechanically tough envelope within which the cell contents may be contained and structurally organized. Since removal of the mucopeptide layer produces osmotically fragile organisms, it is concluded that this cell-wall component provides its mechanical strength. But even in this case it is difficult to understand why such a complex polymer should be required simply to confer mechanical strength. It is more reasonable to suppose that first and foremost this, and the other wall components, provide a selective barrier between the intracellular and extracellular

239

CONTINUOUS CULTURE I N MICROBIOLOGICAL RESEARCH

environments. This being so, one might expect that gross changes in the extracellular environment would elicit significant changes in the functioning of the wall and, possibly, significant changes in its structure. Using a chemostat it is easy to provide a wide range of environmental conditions and therefore to test this hypothesis ;also, hopefully, one may thereby gain some insight into the functioning of some of the wall polymers. Most of our work has been done on Gram-positive bacteria (Bacillus subtilis, Micrococcus lgsodeikticus and Staphylococcus aureus). Apart from the presence of small amounts of protein, the walls of many Gram-positive bacteria contain only mucopeptide, teichoic acid and/or teichuronic acid. But, although teichoic and teichuronic acids may account for over 70% of the dry weight of the wall (Dr. D. C. Ellwood, personal communication), the functional role(s) which they serve in growing bacteria, is, as yet, unknown. Since, in a chemostat culture, organisms may be expected to contain the minimum concentration of any compound when their growth is limited by the availability of that compound, then by limiting the growth of Gram-positive bacteria with some nutrient that provides a component of teichoic acid (e.g. carbon, nitrogen or phosphate) one may expect to limit production of this polymer to a level where it is just able to perform its necessary function with the required efficiency. With a culture of B. subtilis var. niger, growing in a chemostat a t a dilution rate of 0.3 hr.-I ( 3 5 O , pH 7.0), the wall content and gross wall composition varied only insignificantly when the growth-limiting nutrient was changed from the carbon source (glucose) to the nitrogen source (NH4+),sulphur source (S042-), or finally K+ (Table 4). Significantly, perhaps, when the growth was limited TABLE 4. Influence of Growth Condition on the Cell-Wall Content and Composition of Bacillus subtilis var. nigev, Grown in a Chemostat a t a Dilution Rate of 0.3 hr.-1 (35O, pH 7.0). Unpublished data of D. C. Ellwood and D. W. Tempest.

Growthlimiting nutrient

Cell-wall content (% dry weight bacteria)

Glucose NH4+

13 21 21 15 18 21

so42-

K+ Mgz+ Po43-

Content (in g. per 100 g. dry weight, isolated cell walls) of r

1

Phosphorus

Teichoic Acid*

Uronic Acid

3.8 4.9 5.0 4.5 G.9 0.2

41 52 54

<3 t 3 t 3

48 74

t 3 t3

t 3

24

* Assuming the teichoic acid t o be fully glucosylated but without alanine.

240

D . W. TEMPEST

by the availability of Mg2+,the amount of phosphorus which the polymer contained was much increased. But, more significantly, when growth was limited by the availability of phosphate, the amount of phosphorus in the wall decreased to an extremely low level, suggesting the virtual absence of teichoic acid ;this was confirmed by chromatographic analysis of acid and alkaline hydrolysates of isolated walls (Tempest et al., 1968). Thus, organisms were apparently able to function and grow even though their walls contained no teichoic acid. It was possible, however, that the teichoic acids had been functionally replaced by some other polymer, and this seemed likely since the walls still were strongly anionic and their mucopeptide content still did not account for more than 50% of their weight. Extraction of the isolated walls with 10% (w/v) trichloroacetic acid ( 3 7 O , 24 hr.) removed a polymer which, on analysis, was found to be an acidic polysaccharide (teichuronic acid). Therefore it seemed reasonable to suppose that the teichuronic acid had functionally replaced teichoic acid in the walls of phosphate-limited B. subtilis. Although the nature of the functions which these anionic polymers serve in the growing organisms remains obscure, two pieces of data indicate a possible connection between teichoic acid and magnesium metabolism. Firstly, whenever growth of Gram-positive organisms was limited by the availability of Mg2+,their walls contained increased amounts of teichoic acid (Table 4); thus, even cultures of M . lysodeikticus, which previously were not known to synthesize a teichoic acid, were found to contain considerable amounts of this polymer when growth was limited by the availability of Mg2+ (Ellwood and Tempest, 1969a). Secondly, when a constraint was applied to the uptake of Mg2+by adding sodium chloride (up to 4%, w/v) to the growth medium, organisms responded by synthesizing increased amounts of teichoic acid ; this was particularly true with phosphate-limited B. subtilis var. niger whose walls otherwise contained no teichoic acid (Meers and Tempest, 1969). But definitive evidence of teichoic acid participation in the uptake of Mg2+ by Grampositive bacteria is still lacking. Although it was reasonable to assume that gross changes in cell-wall composition of Gram-positive organisms that occurred when cultures were changed from being Mg2+-limited to being P0,3--limited were entirely phenotypic, they could conceivably have resulted from the selective growth of different genotypes. This is particularly likely to occur in a chemostat culture since the chemostat provides a strongly competitive selective environment (see Powell, 1958;Meers and Tempest, 1968). The clearest way of distinguishing between the two possibilities is to study the kinetics of the transition from one limitation to the other growth-limiting condition. The data obtained when cultures of B. subtilis var. niger were changed from being Mg2+-limitedto being P043--

CONTINUOUS CULTURE I N MICROBIOLOGICAL RESEARCH

24 1

limited are shown in Fig. 4.The speed of the transition process indicates clearly and unequivocally that not only were the changes in cell-wall composition phenotypic in nature but that, during the period of transition from fully Mg2+-limited growth t o fully P0,3--limited growth, there was substantial turnover of cell-wall material in the growing bacteria (Ellwood and Tempest, 196913). This experiment illustrates particularly well the value of continuous culture in studying transient phenomena in growing populations. Obviously the bacterial cell wall must be in a dynamic state during growth, but the large shifts in its

L

0

In m C

c

c

0

V

0

I

2

3

4

5

Time following change -over ( hr)

FIG.4. Changes in the contents of cell-wall (0) phosphorus (indicative of teichoic acid) and ( 0 )uronic acid (indicative of teichuronic acid)following-changeover from Mg2f-limiting conditions t o PO&--limiting conditions in a chemostat culture of Bacillus subtilis var. niger. The broken line indicates the theoretical rates.

chemical composition, in response to relatively small changes in the environment, suggest that the wall possibly is one of the most dynamic and phenotypically variable components of the whole cell. The relevance of such variability to problems of vaccine production, as well as to problems of interpreting the structural complexity of cell walls of organisms grown in undefined and uncontrolled environments (that is, in batch-type cultures), is obvious.

C . USEOF A CHEMOSTAT IN STUDIES OF MICROBIAL ENZYME SYNTHESIS The precise composition of any micro-organism is influenced markedly by the growth environment (see Herbert, 1961a; Neidhardt, 1963;

242

D . W. TEMPEST

Tempest, 1969b) and this is particularly evident when one examines the content of enzymes. The effects of changing the chemical environment on the enzymic make-up of micro-organisms may be either “qualitative” (i.e. production in some environments of enzymes that are totally absent from organisms grown in other environments) or “quantitative” (i.e. production of more or less of an enzyme that invariably is present in the organism), although this distinction may not be real so far as the underlying control mechanisms are concerned. Qualitative changes in the enzyme content of organisms can be readily effected with cultures growing in uncontrolled (batch-type) environments, but meaningful quantitative studies generally can only be made with cultures growing under rigidly controlled (steady-state) conditions. A single example should suffice to illustrate this point. I n order to grow in media in which the carbon and energy source is provided by a Cz compound such as acetate or ethanol, micro-organisms must synthesize enzymes that can effect a net synthesis of tricarboxylic acid (TCA)-cycleintermediates from the C2substrate. For Pseudomonas ovalis growing on acetate, the necessary enzymes are isocitrate lyase and malate synthase, and the anaplerotic route (Kornberg, 1966a) is via the glyoxylate cycle (Kornberg, 1966b).It has been found that synthesis of isocitrate lyase ceased when small amounts of succinate were added to cultures growing on acetate and, in these batch cultures, no synthesis of isocitrate lyase occurred until the succinate had been largely, if not entirely, metabolized. Since the concentrations of organism and substrate varied continuously with time in these cultures, it was impossible to assess quantitatively the relationship between acetate and succinate in the control of synthesis of isocitrate lyase. Many of the problems could be overcome, however, by studying the synthesis of this enzyme in continuously growing steady-state populations of Ps.ovalis. Thus it was found (Table 5 ) that, when succinate was added to acetate-limited cultures growing in a chemostat at a fixed rate, synthesis of isocitrate lyase either was unaffected or only partially repressed. Indeed, even when succinate was the sole carbon and energy source, these carbon-limited organisms still synthesized substantial amounts of isocitrate lyase. Thus, growth on succinate (and, consequently the absence of a need for the net synthesis of TCA-cycle intermediates) alone was insufficient to effect a cut-off in the operation of the glyoxylate cycle. Presumably, in these carbon-limited cultures, “pool” substances could not be accumulated intracellularly in concentrations sufficient to affect synthesis of isocitrate lyase. This conclusion was supported by the further finding (Table 6) that, when growth of Ps. ovalis was limited by the availability of the nitrogen source (NHa+)and acetate was present in excess ofrequirement, synthesis of isocitrate lyase was severely repressed by the addition of

CONTINUOUS CULTURE I N MICROBIOLOGICAL RESEARCH

243

TABLE5. Influence of Succinate on the Synthesis of Isocitrate Lyase by Pseudomonas ovalis Growing in a Carbon-Limited Chemostat Culture (30°, p H 7.0) a t a Fixed Dilution Rate. Unpublished data of H. L. Kornberg, D. Herbert and D. W. Tempest.

Acetate

Succinate

Specific activity of isocitrate lyase (pmoles glyoxylate formed per mg. protein per hr)

0.40 0.40 0.39 0.40

84 82 78 79

0 0 1.0 4.0

11.2 12.6 9.2 10.3

Experiment 2 0.52 0.52 0.53 0.54 0.54

81 82 81 80 81

0 8.0 8.1 38.9 38.7

4.5 4.8 5.3 2.0 2.6

Experiment 3 0.30 0.30 0.29 0.31

164 159 166 160

0 32.1 55.6 86.5

1.9 2.4 1.8 1.1

Substrate input concn Dilution rate (hr.-I)

(mM)

r---------7

Experiment 1

Organisms were disrupted by sonication at 25kc. per sec., for 2 min. using a 600 w Mullard magnetostrictor oscillator operating at 3.5 amps. Isocitrate lyase activity was assayedon thenon-particulatefractionby themethod described by Kornbergetal.(1958).

TABLE6. Influence of Succinate on the Synthesis of Isocitrate Lyase by Pseudomonas ovalis Growing in an NHst-Lirnited Chemostat Culture (30", p H 7.0, D = 0.3 hr.-l) Containing Acetate. Unpublished data of H. L. Kornberg, D. Herbert and D. W. Tempest. Substrate input concn

Supernatant substrate concn

(mM) c

7

r-

h

Acetate

Succinate

Acetate

75 82 85 85

0 15 30 45

29 49 60 70

Succinate

0 t l


Specific activity of isocitrate lyase

1.6 0.05 0.04 0.03

Enzyme assays were carried out on extracts of organisms prepared as described in Table 5.

244

U. W. TEMPEST

proportionately small amounts of succinate to the medium, even though succinate was metabolized in preference to acetate and its steady-state extracellular concentration therefore was extremely low (< 1 mM). For example addition of succinate (15 mM) to an NH,+limited medium containing 82 mix-acetate caused the isocitrate lyase content ofPs. ovalis to decrease to less than one-twentieth of the initial steady-state value, and to less than one-two hundredth of the steadystate maximum value found in acetate-limited organisms. Although, in these NH,+-limited cultures, the steady-state extracellular succinate level was undetectably small, presumably the “pool7’level of succinate (or repressor substance derived from succinate) was considerably greater than in the carbon-limited organisms. It is probable that syntheses of all of the enzymes present in growing organisms are regulated by the intracellular levels of “p00l” metabolites whose concentrations are in turn prescribed by their rates of synthesis and utilization (Vogel, 1957). With organisms growing in a batch-type culture, the size of the intracellular ‘‘pool” is likely t o be relatively large for most intermediates, since the environment contains an excess of all of the nutrients essential for growth. But, with chemostat cultures, where growth rate is limited by the supply of some essential nutrient, the intracellular concentration of ‘ L p ~ ~ growth-limiting l” metabolite is likely to be very low. Thus, the enzyme content of organisms growing in such chemostat cultures will be very different from that of the same

a r

- 600 0 W

%400 -

.I

-

I

0 C

._

lo

t) 0

.-

5 zoo

------

o/,

0

&

-

L

0

...w c?

0-

0 Time following change-over from acetate metabolism to succinate metabolism ( h r )

8

16

24

Time following change-over from succinate metabolism to acetate metabolism ( h r )

FIG. 5 . Changes in the acetate thiokinase activity (0)and acetate-oxidizing capacity ( 0 ) of Pseudomonm ovalis Chester following change-over from growth on acetate to growth on succinate (a)and vice versa ( b )in a carbon-limited chemostat culture. The broken lines represent the theoretical rates of change.

CONTINUOUS CULTURE IN MICROBIOLOQICAL RESEARCH

245

organisms growing in a batch culture, and so will be their response to changes in the environment. Although in a chemostat culture the population is continuously changing (through synthesis of new organisms and, correspondingly, loss of culture via the overflow tube), it is not difficult to analyse and interpret changes in the levels of enzymes following a change in the steady-state growth condition. For example, when carbon-limited cultures of Ps.ovalis were changed from an acetate-containing medium to one containing succinate and vice versa., the changes in acetate thiokinase activity and acetate-oxidizing capacity followed closely a plot which indicated that enzyme synthesis ceased (and was initiated) at the moment of medium change-over, and was closely associated with the synthesis of new organisms (Fig. 5 ) . I hope that the above few examples adequately illustrate the varied ways in which continuous culture can be used to facilitate research in microbial physiology. But is it now necessary to strike a sour note and detail some of the inadequacies of this technique.

V. Some Inadequacies of Continuous Culture as a Research Tool In order to exploit continuous-culture methods to the full, it is essential to understand not only the ways in which this technique can succeed but also the ways in which it may possibly fail. The greatest single advantage to be gained from growing cultures in a chemostat is that the environment is rigidly controlled and invariant with time ;thus phenotypic variations in the population are minimized. But the chemostat environment also is fiercely selective and the selective pressures (due primarily to the low concentration of growth-limiting nutrient in the culture) are enhanced by the continuous flow of biomass from the growth chamber. Thus, organisms best adapted to the prescribed environment quickly establish themselves in the culture, whereas those less well adapted are equally rapidly lost from the growth vessel. A natural feature of prokaryotic cells is the facility with which their genetic content can be varied. Therefore substantial genetic changes may be expected to occur in any bacterial population that is maintained for extended period of time (say, greater than 1000 generation times) in a chemostat culture. As a consequence of this fact, all results obtained with chemostat-grown cultures should be carefully checked to ensure that no important genetic changes have occurred during the course of the “run” that might have contributed to the experimental findings. Furthermore, as a “rule of thumb” the duration of individual “runs” should be limited to less than four weeks, whenever possible. Finally, organisms from a chemostat culture never should be used to start a fresh chemostat

246

D . W. TEMPEST

culture ; in order to ensure consistent results a new culture always should be inoculated with organisms from a “primary stock culture” (preferably a spore suspension or freeze-dried culture). In studying the relationship between environment and the structure and functioning of microbial cells, it is frequently assumed that organisms can exist in only one stable physiologicalstate in each prescribed environment. Thus, the history of the culture can be ignored; no matter what the starting point may have been, once in equilibrium at a particular dilution rate, temperature and pH value, the physiological state of the culture should be fixed. However, frequently this assumption has not been vindicated in practice. For example, when the dilution rate a t which a culture of Aerobacter aerogenes was being grown was progressively increased to a value near to the critical dilution rate (D,) and then progressively lowered to the starting value, the changes in steady-state macromolecular composition and metabolic activity of the organisms showed hysteresis (D. W. Tempest, unpublished observation). Consequently the precise quantitative data obtained from this experiment were of rather less value than the broader conclusions, and this is always likely to be the case. Thus, although it would be erroneous to claim that A . aerogenes should contain, say, 11.25% RNA when grown (glycerollimited) at a dilution rate of 0.2 hr.-I ( 3 5 ” ,pH 6.5), nevertheless it would generally be safe to state that, all other conditions being equal, A . aerogenes organisms growing at a dilution rate of 0.2 hr.-I should contain more RNA than when growing at a rate of 0.1 hr.-I but less RNA than when growing at a rate of 0.4 hr.-l. The RNA content of microbial cells is a function of their growth rate, but at any particular growth rate the cellular RNA content may vary significantly (i.e. &lo% of the mean value). It is fair to claim that continuous culture always should be the preferred method when studying actively growing organisms ; it is useless, of course, for studying non-growing cultures of organisms except as the second stage of a two-stage system. And although, as shown earlier, continuous-culture methods can easily be used to study transient phenomena, they cannot be used to study transitions from a non-growing state to a growing state (i.e. growth initiation). Clearly, continuous-culture methods do have their limitations but nevertheless they can be applied effectively to most research projects involving growing organisms.

VI. Operational Problems A detailed description of the difficulties likely to be encountered in operating a chemostat is out of place in this article. But, since potential users of this technique frequently are inhibited from setting up a

CONTINUOUS CULTURE I N MICROBIOLOGICAL RESEARCH

247

chemostat because o f the supposed “expertise)’ necessary to keep one functioning for a prolonged period of time, some brief comments here may be of value. Substantial advances have been made in the design of continuousculture equipment over the past ten years (see Evans et al., 1969) and consequently need no longer be looked upon as the “diabolical machine” complained of by one of its inventors. But not all the operational difficulties have as yet been totally eradicated; some are seemingly intractable. So far as I am aware, nothing can be done to prevent the formation of mutant organisms, or to suppress their selection if they happen to be better adapted to the chemostat environment. When selection of a mutant strain occurs, all that can be done is to stop the experiment and restart with a fresh population from the primary stock culture. Fortunately mutant selection rarely occurs in experiments of relatively short duration (i.e. less than one month). Two other major (although less serious) problems are likely to be encountered; these are foaming and wall growth. A. FOAMING Foaming usually can be controlled by the periodic addition of a suitable defoaming agent to the culture. I n some cases, however, huge doses of antifoam are required and these may, and usually do, severely interfere with subsequent experimental procedures (e.g.processing of the biomass and analysis of various environmental components). When this situation arises, it is better to seek some other solution. Lowering the concentration of organisms in the culture (by lowering the input concentration of growth-limiting substrate) usually is effective, and so too is adding sodium chloride (1-2%, w/v) to the medium, although this may lead to a substantial change in the physiology of the organisms (Epstein and Schultz, 1965; Tempest and Meers, 1968).

B. WALLGROWTH Conditions which facilitate foaming of the culture generally also precipitate wall growth (that is the accumulation of a mass of microorganisms on the walls of the growth vessel and associated projections). Addition of antifoam to the culture generally accelerates the build up of biomass on the walls of the growth chamber; ultimately large pieces of this “crust)’begin to fall into the culture (thereby affecting the steadystate conditions) and may block the effluent line. Again the only way of dealing with this problem is to arrange conditions such that wall growth is minimized (e.g. by avoiding an excessive concentration of organisms in the culture and/or an excessive rate of addition of antifoam).

248

D. W. TEMPEST

But for the above technical difficulties (particularly wall growth and mutant formation), there is no reason why cultures could not be maintained in a continuously growing state for indefinite periods of time; mechanical breakdown is a rare event with modern equipment. However, in my experience, few experiments demand that the culture be kept growing continuously for more than two months and therefore the main operational difficulties, described above, seldom manifest themselves.

VII. Conclusions It is now beyond doubt that further progress in many areas of microbiology can be greatly facilitated by, and occasionally may only follow, the application of continuous-culture techniques. Therefore inevitably the chemostat ultimately will become a standard piece of equipment in a11 laboratories where micro-organisms are studied. But why, one may ask, has this technique been largely ignored up to the present time? Why are so many microbiologists still inexperienced in its usage? Why is it generally taught t o students only a t the theoretical level? Clearly it would be facile to suppose that the basic cause is conservatism and prejudice amongst microbiologists. I n my view a more likely factor is the virtual lack of ready-made equipment of a suitable size. Of course, continuous-flow culture apparatus of various types have been marketed for many years now but, almost without exception, these units have been of too large a capacity to be of practical value as research tools. Possibly this unsatisfactory situation has arisen from the belief, prevalent many years ago, that the real value of continuous culture lay in its capacity to provide vast quantities of biomass. Whereas chemostats are good “production tools”, when used as “research tools” the requirements are different; generally only the culture actually in the growth vessel is of importance, the effluent accumulated in the receiver vessel being an embarrassment. Thus, the ideal volume of a chemostat culture (when required for research purposes) is that from which adequate amounts of biomass can be drawn directly (without upsetting unduly the steady state conditions) yet sufficiently small not to require more than a modest daily supply of medium, even when growing at a fast rate. The facilities available in most small laboratories would be severely overloaded if called upon t o process more than 25 litres of medium each day, seven days every week. And, since a chemostat may frequently be operated at a dilution rate of 1.0, clearly a vessel of one litre capacity is the maximum size of any practical use. I n fact the ideal research-type chemostat probably would be one with a capacity of 0.5 litres, but with an aeration system capable of sustaining a population equivalent to 5-10 mg. dry weight organismlml. (see Herbert et al., 1965).

CONTINUOUS CULTURE I N MICROBIOLOGICAL RESEARCH

249

Since suitably sized chemostats have not in the past been available commercially, microbiologists have been forced to design and construct their own models. Consequently the setting up of continuous culture invariably has required an initial thorough understanding of the basic principles and theory. This, I am sure, has often provided an insuperable barrier to the practice of continuous culture. This is a pity because, in many research activities where complex manufactured equipment is routinely used (and, indeed, in other every-day activities), “theory” is not an essential prerequisite to “practice”. If it were so, how many people today would be capable of driving a motor car! No, theory is important, but only in so far as it explains the practice, not dictates it. This is no less true for the chemostat than it is for the computer, the spectrophotometer or the motor car. Clearly there is an urgent need for the manufacture and marketing of a simple (and cheap) chemostat that is suitable for teaching purposes. Only then can students acquire early experience in the practice of continuous culture and thereby overcome the many doubts and inhibitions generated by exposure to the theoretical complexities of the system. Familiarity with the practice of continuous culture will then be carried into their research activities and the chemostat thus take its rightful place as a basic tool in the armoury of the microbiologist.

VIII. Acknowledgements This paper contains ideas and impressions acquired over the past ten years. Although my colleagues at Porton may not agree with all that I have written, I am conscious of the enormous debt I owe to them for their many wise counsels. REFERENCES Bryson, V. (1952).Science, N.Y. 116, 45. Burke, E. (1788). Impeachment of Warren Hastings; House of Commons. Curren, H. R., Brunstetter, 13. C. and Myers, A. T. (1943).J. Bact. 45, 485. Dicks, J. W. and Tempest, D. W. (1966).J. gen. Microbiol. 45, 547. Dixon, M. and Webb, E. C. (1964). “The Enzymes”, 2nd ed. London, Longmans Green. Ecker, R. E. and Schaechter, M. (1963). Biochim. biophys. A c t a 76, 275. Ellwood, D. C. and Tempest, D. W. (1969a). Proc. 6th. F.E.B.S. Meeting, No. 153. Ellwood, D. C. and Tempest, D. W. (1969b). Biochem. J. 111, 1. Epstein, W. and Schultz, S. G. (1965).J. gen. Physiol. 49, 221. Evans, C. G. T., Herbert, D. and Tempest, D. W. (1969). I n “Methods in Microbiology”, (J.R. Norris and D. W. Ribbons, eds.), Vol. 2. Academic Press, London. Fencl, Z. (1966).I n “Theoretical and Methodological Basis of Continuous Culture”, (I.Malek and Z. Fencl, eds.), p. 69. Academic Press, New York.

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Herbert, D. (1958). I n “Recent Progress in Microbiology”, V l l t h Int. Gongr. Microbiol. p. 381. University Press, Toronto. Herbert, D. (1961a).Symp. SOC. gen. Microbiol. 11, 391. Herbert, D. (1961b). In “Continuous Culture of Microorganisms”, p. 21. S.C.I. Monograph No. 12. Butterworths, London. Herbert, D., Elsworth, R. and Telling, R. C. (1956).J . gen. Microbiol. 14, 601. Herbert, D., Phipps, P. J. and Tempest, D. W. (1965). Lab. Pract. 14, 1150. Humphrey, B. andvincent, J. M. (1962).J . gen. MicrobioZ. 29, 557. Kornberg, H. L. (1966a). Essays in Biochemistry 2, 1. Kornberg, H. L. (196613). Biochem. J . 99, 1. Kornberg, H. L., Gotto, A. M. and Lund, P. (1958). Nature, Lond. 182, 1430. Kjeldgaard, N. 0. and Kurland, C. G. (1963).J . molec. Biol. 6, 341. Meers, J. L. and Tempest, D. W. (1968).J . gen. Microbiol. 52, 309. Meers, J. L. and Tempest, D. W. (1969).J . gen. Microbiol. 55, x. Monod, J. (1942). “Recherches sur la Croissance Bacterienne”. Masson, Paris. Monod, J. (1949). A. Rev. Microbiol. 3, 371. Monod, J. (1950). A n n l s Inst. Pasteur, Paris 79, 390. Neidhardt, F. C. (1963). A . Rev. Microbiol. 17, 61. Novick, A. and Szilard, L. (1950a). Proc. natn. Acad. Sci. U.S.A. 36, 708. Novick, A. and Szilard, L. (1950b). Science, N.Y. 112, 715. Postgate, J. R. (1969). “Microbes and Man”, p. 85. Penguin Books, London. Powell, E . 0. (1958).J . gen. Microbiol. 18, 259. Powell, E. 0. (1965). Lab. Pract. 14, 1145. Powell, E. 0. (1967). I n “Microbial Physiology and Continuous Culture”, (E. 0. Powell, C. G. T. Evans, R. E. Strange and D. W. Tempest, eds.), p. 34. H.M.S.O., London. Rouf, M. A. (1964).J . Bact. 88, 1545. Schaechter, M., Maalne, 0. and Kjeldgaard, N. 0. (1958).J . gen. Microbiol. 19,592. Taylor, M. M. and Storck, R. (1964). Proc. natn. Acad. Sci. U.S.A. 52, 958. Tempest, D. W. (1969a). In “Methods in Microbiology”, (J.R. Norris and D. W. Ribbons, eds.), Vol. 2. Academic Press, London. Tompest, D. W. (1969b).S y m p . SOC. gen. Microbiol. 19, 87. Tempest, D. W. and Dicks, J. W. (1967).In “Microbial Physiology and Continuous Culture”, (E. 0. Powell, C. G. T. Evans, R. E. Strange and D. W. Tempest, eds.), H.M.S.O., London. Tempest, D. W. and Hunter, J. R. (1965).J . gen. Microbiol. 41, 267. Tempest, D. W. and Meers, J. L. (1968).J . gen. Microbiol. 54, 319. Tempest, D. W., Dicks, J. W. and Hunter, J. R. (1966).J . gen. Microbiol. 45, 135. Tempest, D. W., Dicks, J. W. and Ellwood, D. C. (1968). Biochem. J . 106, 237. Vogel, H. J. (1957). In “The Chemical Basis of Heredity”, p. 276. Johns Hopkins Press, Baltimore. Wade, H. E. and Morgan, D. M. (1957). Biochem. J . 65, 321. Webb, M. (1949).J . gen. Microbiol. 3, 410. Webb, M. (1966).J . gen. Microbiol. 43, 401.

Catabolite Repression and Other Control Mechanisms in Car bohyd rate Ut iI izat ion KENNETH PAIGEN and BEVERLY WILLIAMS Department of Experimental Biology, Roswell Park Memorial Institute, Buflulo, New York 14203, U.S.A. To baptize a phenomenon is not to understand it any further. H. POINCAR&.

To the ignorant, the great results alone are admirable, to the knowing, rather the infinite device and sleight of hand that made them possible. LOUISSTEVENSON. RORERT

I. Introduction . . A. The Control of Carbohydrate Utilization . . B. The Choice of Alternative Substrates . . C. Historical Review . . 11. Catabolite and Transient Repression . . A. Quantitative Factors . . B. Occurrence of Catabolite Repression . . C. Environmental Conditions Which Produce Catabolite Repression D. Occurrence of Transient Repression . . . 111. The Mechanisms of Transient and Catabolite Repression . A . Transcriptional or Translational Control . . B. Repression in Regulatory Mutants . . C. Models of Repression . . D. Identity of the Effector . . IV. Catabolite Inhibition . . A. Definition and Properties . . B. Examples of Catabolite Inhibition . . C. Mechanism of Catabolite Inhibition . . V. Control of Inducer Concentration . . A. Gratuity . . B. Long-Term Adaptation . . C. Inducer Entry . . D. Effector Synthesis . . E. Summary . VI. Diauxie . . VII. Acknowledgements . . References . . 25 1

252 252 252 253 254 254 256 271 276 281 281 285 291 293 298 298 300 302 303 303 304 305 306 308 308 311 311

252

KENNETH PAIGEN AND BEVERLY WILLIAMS

I. Introduction

A. THE CONTROLOF CARBOHYDRATE UTILIZATION Bacteriologicaletiquette calls for growing cultures in simple chemically defined media when physiological experiments are undertaken. We provide compounds containing phosphorus, sulphur, nitrogen (preferably as inorganic salts), any essential organic growth factors, and a few cations, chiefly K+, Mg2+,Ca2+and Fe3+, and we trust to impurities in our reagents to supply the necessary trace elements. Finally, we add a simple compound, rarely containing more than a dozen carbon atoms, in order to provide carbon and energy. Both tradition and experience tell us that the simpler our culture conditions, the more likely we are to arrive at valid physiological conclusions. In nature, though, bacteria reproduce under scientifically scandalous circumstances, growing in mixed cultures on a miscellany of sources of carbon, nitrogen, phosphorus, and sulphur. In that world, survival may depend on utilization of the optimum substrate, for the cell that saves the best carbon source for last may well find that its neighbour has not been so forbearing. I n the context of survival, it is the total ensemble of devices by which microbial cells choose their carbon source that is significant. This review will consider three of these devices ; catabolite repression, transient repression and catabolite inhibition, which regulate the utilization of many carbohydrates. Induction and specific repression of individual enzymes will not be considered. Catabolite repression is a reduction in the rate of synthesis of certain enzymes, particularly those of degradative metabolism, in the presence of glucose or other readily metabolized carbon sources. I n addition to this repression observed during steady-state growth in glucose, a period of more intense repression occurs immediately after cells are exposed to glucose. This effect, which may last up to one generation, is termed transient repression. Catabolite inhibition is a control exerted by gIucose on enzyme activity rather than on enzyme formation, analogous to feedback inhibition in biosynthetic pathways. Enzymes involved in the utilization of other carbohydrates are inhibited by glucose. B. THE CHOICE OF ALTERNATIVE SUBSTRATES The ability to choose between alternative carbon sources is a special case of the general ability of micro-organisms to choose a preferred substrate. For example, in Hydrogenomonas, certain electron donors are used preferentially. I n this organism which can grow autotrophically on a mixture of hydrogen and carbon dioxide, exposure to molecular hydrogen represses adaptation t o a variety of organic substrates (Blackkolb

CATABOLITE REPRESSION AND OTHER CONTROL MECHANISMS

253

and Schlegel, 1968). Synthesis of a t least three enzymes of the EntnerDoudoroff pathway, which function in the utilization of gluconate and fructose, (gluconokinase, 6-phosphogluconate dehydrogenase and 2keto-3-deoxyphosphogluconate aldolase) is repressed by molecular hydrogen (Schlegel and Triiper, 1966; Blackkolb and Schlegel, 1968). The repression is exerted by molecular hydrogen alone and does not require carbon dioxide; thus, when hydrogen but not carbon dioxide is present, growth is inhibited on many organic substrates that could otherwise be utilized. Similar devices exist for selecting other preferred substrates. Glucose prevents carbon dioxide fixation in Chlorelka and Euglena by inhibiting chlorophyll production (Shugarman and Appleman, 1966; Matsuka and Hase, 1966; Buetow, 1967). I n Hydrogenomonas, synthesis of two enzymes whose only known function is carbon dioxide fixation (ribose phosphate pyrokinase and ribulosediphosphate carboxylase) is decreased when cells are grown on some organic substrates (McBadden and Tu, 1967). Synthesis of both the alkaline phosphatase of Escherichia coli (Torriani, 1960) and a permease for organic phosphonates in Bacillus cereus (Rosenberg and LaNauze, 1967) is repressed by inorganic phosphate, a preferred source of phosphorus. Similarly, ammonia is preferred as a source of nitrogen in Aerobacter, and a mixture of ammonia and glucose represses synthesis of histidase, which can supply both carbon and nitrogen (Neidhardt and Magasanik, 1957a).

C. HISTORICAL REVIEW During the late nineteenth and early twentieth centuries, a number of papers appeared reporting the ability of glucose to inhibit the development of various fermentative capacities in bacterial cultures (for early reviews, see Berman and Rettger, 1918; Monod, 1942; Gale, 1943). Perhaps the earliest of these were the reports by Katz (1898) that amylase induction in Penicillium, and by Dienert ( 1900) that galactozymase induction in yeast, were prevented by glucose. This early phase of work on the subject was limited to observations on the fermentative capacities of intact cells. However, in 1936, Happold and Hoyle demonstrated that in cell-free extracts of E. coli the content of the enzyme now recognized as tryptophanase was much reduced if the cells had been grown in the presence of glucose. Shortly afterwards, Stephenson and Gale (193713) found a similar effect of growth in glucose on the in vitro activity of several amino-acid deaminases. The glucose effect, as the phenomenon had become known, was established in its modern sense by the important paper of Epps and Gale

254

KENNETH PAIGEN AND BEVERLY WILLIAMS

(1942). I n it they demonstrated that repression by glucose was limited t o certain degradative enzymes and was not an incidental result of acid production during fermentation. I n the same year, Monod published his monograph on enzyme adaptation in micro-organisms (Monod, 1942), which included a description of the diauxie phenomenon, now recognized as another manifestation of catabolite repression. I n diauxie, bacteria in the presence of two carbon sources defer the formation of enzymes for using one until the other has been exhausted from the culture medium. I n subsequent years, several basic aspects of the glucose effect were described. Magasanik summarized these in a review (1961) which had an important heuristic influence in clearly focusing the problems involved and in which he renamed the phenomenon, catabolite repression. The discoveries of transient repression and catabolite inhibition as significant mechanisms in the control of carbohydrate utilization are more recent. The recognition of transient repression as a distinct phenomenon came in 1966 in papers by Paigen (1966a) and Moses and Prevost (1966), although a milder form of the same effect had already been noted by Boezi and Cowie (1961). The discovery that microorganisms use catabolite inhibition, a mechanism for regulating enzyme activity in choosing between carbon sources, was first recognized for sorbitol utilization by Gaudy et al. (1963) and for galactose utilization by Stumm-Zollinger (1966). McGinnis and Paigen (1969) have extended these observations to show that catabolite inhibition is a geperal phenomenon in the control of carbohydrate utilization.

11. Catabolite and Transient Repression

A. QUANTITATIVE FACTORS 1. Rates of Enzyme Synthesis

Expressing the rate of synthesis of a specific protein relative to the rate of synthesis of all protein, that is as a differential rate, was first introduced by Monod et al. (1952). The differential rate is preferable to comparing amounts of enzyme synthesized as a function of time since it avoids the complications arising from variations in rates of protein synthesis under different conditions. For example, the apparently autocatalytic nature of enzyme induction was shown to be an artifact arising from the use of chronological time as the measure of rate when induction occurred under non-gratuitous conditions. A further refinement of the differential rate was introduced by Paigen (1966a) to avoid the confusion resulting from the fact that the synthesis of one mg. of new cell protein occurs in a small fraction of a generation if a culture contains many cells, but may occupy a n entire generation if a culture contains few ceIIs. For this purpose, the behaviour of a growing culture is expressed as

CATABOLITE REPRESSION AND OTHER CONTROL MECHANISMS

255

the increment in enzyme synthesized at any time (AE) divided by the initial mass of cells present (Mo) and plotted against the mass of cells present at that time (Mt) also divided by the initial mass of cells (i.e. dE/Mo v. Mt/Mo). The use of relative growth introduces a measure of physiological time, and makes the shape of the resulting curve independent of the initial cell density. The slope of this curve is the same as that obtained by plotting denzyme versus Amass as originally performed by Monod et al. (1952). The application of this modification becomes particularly significant in experiments which examine transient effects during the first generation or so of bacterial growth. This is illustrated in Fig. 1 which describes the behaviour of three bacterial cultures at high, medium and low cell densities during a transient repression which lasts for half a 1

I

I

(a) Mo=O.lO

(d)AlI cultures

0

AM

AM

0.1

AM

0 0.1 Mt/Mo (relative growth)

FIG.1. The significance of using relative growth ( A M ) in the study of enzyme induction ( A E ) .At the beginning of a hypothetical experiment, inducer was added to an exponentially growing culture. The further addition of glucose one-fifth of a generation later produced a transient repression lasting half a generation. Cultures a, b, and c differ only in their cell density at the start of the experiment. The apparent effect of glucose addition is quite different for the three cultures when A E is plotted against A M . Reducing each measurement t o a value relative to the initial cell density, as in d, produces identical curves.

generation followingthe addition of glucose. Unless the data are corrected for the initial cell density (as in Fig. Id), the apparent duration of the lag will depend on the experimental conditions chosen. Several other methods for determining rates of enzyme synthesis are still used although they may give misleading results. A plot of enzyme specific activity (units per mg. cell protein) as a function of growth is valid only if very short times are used. The resulting curve increasingly deviates from linearity at longer times ; this deviation is already significant in experiments as short as one-fifth of a generation time. Another error which is still encountered is the use of lapsed time as a measure of the amount of protein synthesized by a culture. This is particularly erroneous in medium-shift experiments or when studies are carried out on cells with only a Iimited ability to synthesize protein, as in the case of

256

KENNETH PAIGEN AND BEVERLY WILLIAMS

an auxotrophic mutant deprived of a required amino acid. Changes in culture conditions, such as providing a carbohydrate substrate, may markedly affect the low levels of protein synthesis which remain; and these must be measured directly by isotopic incorporation. Theoretically, even the use of the differential rate of enzyme synthesis is quantitatively valid only if the differential rate is independent of the total rate of protein synthesis. If anything changes the total rate of protein synthesis (AM) without proportionately changing the rate of enzyme synthesis (AE), there will be an apparent induction or repression, as measured by the differential rate, even though no change in the regulatory state has occurred. Fortunately, this limitation usually is not encountered. 2. Metabolic Concentrations

Many experiments on induction of enzyme synthesis are concerned with transient changes in inducibility extending as long as one generation. Such transient effects usually are interpreted as changes in the internal concentration of various effectors. However, a bacterial cell only has a volume of approximately 1 p3 or litres and a dry mass of approximately 2 . 10-13g. If organic substrates are converted to cell material with an efficiency approaching 40%, then the rate of substrate consumption when the doubling time is 60 min. will be 2.10-13/0.4.3600 or 1.4. 10-ls g./sec. With glucose as a substrate this corresponds to 4.6. l o 6 molecules/sec. If the internal concentration of glucose during steadystate growth is 1 mM, then a cell contains 6. lo5 molecules at any one time; enough glucose to support growth for just over one second. The flux of metabolites through the intermediary pools is therefore very rapid compared with the absolute size of the pools. Changing any of the parameters used, such as growth rates, pool sizes and cell volumes, within conceivable limits, will not affect this conclusion. Hence, changes in metabolite concentration which occurs over periods of experimentally measurable time must reflect the operation of homeostatic mechanisms which adjust metabolite concentrations quite slowly to the new balance of rates at which various reactions proceed.

B. OCCURRENCEOF CATABOLITEREPRESSION Catabolite repression influences many aspects of microbial growth and metabolism. I n addition to the well known repressions of carbohydrate utilization and amino-acid degradation in bacteria and yeast, catabolite repression affects the formation of enzymes that function in the tricarboxylic acid cycle, glyoxylate cycle, fatty acid degradation, carbon dioxide fixation, and the respiratory chain. It affects cell struc-

CATABOLITE REPRESSION AND OTHER CONTROL MECHANISMS

257

ture by preventing flagella production in E. coli, budding in Saccharomyce8, hyphae formation in Mucor, and mitochondria1 genesis in Saccharomyces. The specialized cell functions of sporulation in Bacillus, chlorophyll production in Euglena and Chlorelb, and antibiotic production in several species are all sensitive to catabolite repression. I n higher organisms, catabolite repression has been observed in sugar cane, rats, and man. Although, in almost all systems, glucose is effective in producing catabolite repression, in Arthrobacter crystallopoietes it is succinate which represses glucose utilization and glucose permease (Krulwich and Ensign, 1969). I n this organism, growth on a mixture of glucose and succinate is diauxic with succinate utilized first. Table 1 summarizes the occurrence of catabolite repression as far as we have been able to determine. For the better known examples, we have tended to give references which describe definitive and recent work rather than citations of the original discoveries. 1. Substrate Utilization

The largest group of enzymes observed to be sensitive to catabolite repression are those required to bring a substrate into one of the main pathways of energy metabolism. Included are the enzymes which bring carbohydrates into the Embden-Meyerhof pathway, hexose monophosphate shunt, or Entner-Doudoroff pathway ; the enzymes required for the generation and utilization of Cz fragments from fatty acids ; and the enzymes that degrade amino acids whose carbon skeletons can be used to support growth. 2. Tricarboxylic Acid Cycle

The tricarboxylic acid cycle is an amphibolic pathway (Davis, 1961) which functions both in the catabolic generation of ATP and in the anabolic synthesis of important biosynthetic precursors (Pig. 2). The major biosynthetic products are derived from a-ketoglutarate, which serves as the source of glutamate via the NADP-linked glutamate dehydrogenase, and oxaloacetate, which provides aspartate via transamination with glutamate. Each of these amino acids in turn gives rise to a family of products :glutamine, arginine, and proline from glutamate; asparagine, diaminopimelate, lysine, threonine, methionine, and pyrimidines from aspartate. The other essential product of the tricarboxylic acid cycle is succinyl-CoA, a precursor of both methionine and porphyrins. Such a diversity of function has produced a complex set of repressive controls for the synthesis of the enzymes involved. These controls can be 9

N

ur

TABLE1. Examples of Catabolite Repression Substrate, Enzyme or Structure Enzyme

Organism

00

Reference

A. Substrate Utilization

Lactose /3-galactosidase(EC 3.2.1.23) j3-galactoside permease galactoside acetyl transferase (EC 2.3.1.18) Galactose galactokinase (EC 2.7.1.6) galactose 1-phosphateuridylyltransferase (EC 2.7.7.10) UDP glucose epimerase (EC 5.1.3.2) galactose oxidase (EC 1.1.3.9) CTlyceroZ glycerol kinase (EC 2.7.1.30) glycerophosphate transport glycerol dehydrogenase (EC 1.1.1.6) Arabinose arabinose isomerase (EC 5.3.1.3) Fructose fructokinase (EC 2.7.1.4) Sucrose invertase (EC 3.2.1.26) Maltose Xylose Rhamnose

Numerous papers

A !

M

‘d

Escherichia coli

Stephenson and Gale (1937a); Adhya and Echols (1966); Lengeler (1966)

z t-

3W Dactylum dendroides Escherichia coli Mycobacteriurn Aerobacter

Markus et al. (1965) Cozzarelli et al. (1968); Koch et a2. (1964) Bowles and Segal(l965) Neidhardt and Magasanik (1956b)

Lactobacillus plantarurn

Chakravorty (1964)

Aerobacter aerogenw

Sapico et al. (1968)

Sacchromyces fragilis Saccharornyces cerevisiae Bacillus subtilis, E . coli Escherichia coli Escherichia coli

Davies (1956) McMurrough and Rose (1967) Monod (1947) Monod (1947) Monod (1947)

M

9td 2

3r,

E 3

T r e h l ose ribokinase (EC 2.7.1.15) gluconokinase (EC 2.7.1.12) hexokinase (EC 2.7.1.1) a-glucosidase (EC 3.2.1.20) p-glucosidase (EC 3.2.1.21)

.

Escherichia coli Staphylococcus aureus Staphylococcus aureus Bacillus subtilis Candidu stellatoidea Saccharomyces sp. Saccharomyces sp.

amylomaltase (EC 2.4.1.3)

Escherichia coli

inositol dehydrogenase (EC 1.1.1.18)

Aerobacter aerogenes

Sorbitol Dulcitol alcohol dehydrogenase (EC 1.1.1.1) Mannitol ribitol dehydrogenase (EC 1.1.1.56) arabitol dehydrogenase Camphor paraoxygenase lactate oxidase (EC 1.1.3.2) tartrate permease pyruvate oxidase Acetate Benzoate acetate kinase (EC 2.7.2.1) acetamide amidase (EC 3.5.1.4)

Bacillus subtilis Escherichia coli Escherichia coli Escherichia coli Staphylococcus sp. Aerobacter aerogenes Aerobacter aerogenes Pseudomonaa putida Pseudomonas aeruginosa Streptococcus faecium Penicillium chrlesii Smharomyces cerevisiae Sphaerotilus sp. Sphaerotilus sp. Escherichia coli Pseudomonas aeruginosa

Ernbden-Meyerhof Pathway

Escherichia coli

Monod (1947) Strasters and Winkler (1963) Strasters and Winkler (1963) Moses and Sharp (1968) Bradley and Creevy (1966) Grts (1967); Wijk (1968a, b, c, d, e) Hauge et al. (1961); MacQuillan et al. (1960); MacQuillan and Halvorson (1962a) Hsie and Rickenberg (1967); Rickenberg et al. (1968) Monod (1947) ; Neidhardt and Magasanik (1956b) Monod (1947) Monod (1947) Monod (1947) Epps and Gale (1942) Murphey and Rosenblum (1964) Hulley et al. (1963) Lin (1961) Gunsalus et al. (1967) Eyk and Bartels (1968) London (1968) m a t t and Gander (1968) Polakis and Bartley (1965) Stokes and Powers (1967) Stokes and Powers (1967) Halpern et al. (1964) Brammar and Clarke (1964); Brammar et al. (1967); Clarke and Brammar (1964); Clarke et al. (1968) Wright and Lockhart (1965)

0

2 k0

E

2 9

8b! 8r

P 2 U

1 0

0 0

14

[ E d

w

P

3

v,

t.3

UI

CB

TABLEl--contin.ued Substrate, Enzyme or Structure

Organism

Fatty acids enoyl-CoAhydratase (EC 4.2.1.17) Escherichia COG acyl-CoA synthetase (EC 6.2.1.3) 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) 43-42-enoyl-CoA-isomerase (EC 5.5.3.2.) 3-hydroxy butyryl-CoA epimerase (EC 5.1.2.3) acyl-CoA dehydrogenase (EC 1.3.2.2) thiolase (EC 2.3.1.9) Schizophyllum commune A m i n o sugars Bacillus subtilis glucosamine 6-phosphate deaminase Escherichia coli Aspergillus parasiticus N-acetylglucosamine 6-phosphatedeacetylase N-acetylglucosamine kinase acetohydroxy acid synthase Escherichia coli

E

Reference

OverathandRaufuss (1967); Weeksetal. (1969)

3H x

w

k

0

M

Wilson and Niederpruem (1967) Bates and Pasternak (1965a, b) White (1968) McGarrahan and Maley (1965)

urocanase formiminoglutamic hydrolase

Bacillus subtilis Aerobacter aerogenes Pseudomonas aeruginosa Pseudomonas jluorescens Staphylococcus a u r e w Salmonella typhimurium

3td M 4

E*

Coukell and Polglase (1969)

B. Amino Acid Degradation Histidine histidase (EC 4.3.1.3)

z

b-

3

sF 5

Chasin and Magasanik (1968) Neidhardt and Magasanik (1956a, b, 1957a); Magasanik et al. (1965). Lessie and Neidhardt (1967) Jacoby (1964) Strasters and Winkler (1963) Magasanik (1963)

Arginine arginase (EC 3.5.3.1) ornithine-8-transaminase pyrroline-8-carboxylate dehydrogenase Tryptophan tryptophanase

Bacillus licheniformis Sphaerotilus sp. Pseudomonas jluorescens

Laishley and Bernlohr (1966, 1968) Stokes and Powers (1967) Jacoby (1964)

Pseudomonas jluorescens Escherichia coli

threonine deaminase (EC 4.2.1.16)

Escherichia coli Bacterium cadaveris Pseudwmonas fiuorescens

Jacoby (1964) P Epps and Gale (1942); Happold and Hoyle W (1936) ; Boyd and Lichstein (1955) 0 CI Boyd and Lichstein (1951) Boyd and Lichstein (1951) !a Jacoby (1964) La

Tyrosine p-hydroxyphenylpyruvate hydroxylase (EC 1.14.2.2) homogentisate oxygenase (EC 1.13.1.5) Aspartic Acid Escherichia coli

Glutarnic Acid glutamate dehydrogenase (NAD) (EC 1.4.1.3)

L-serine deaminase (EC 4.2.1.13)

Bacterium cadaveris Staphylococcus aureus Pseudomonas fluorescens Aerobacter aerogenes Proteus vulgaris Escherichia coli Staphylococcus aureus Sphaerotilus sp. P s e u d o m o w fluorescene Saccharomyces cerevisiae Escherichia coli Bacterium cudaveris Staphylococcus aureus

d

x

8

(I,

Boyd and Lichstein (1951, 1953) ; Epps and Gale (1942); Gale (1938) Boyd and Lichstein (1951) Strasters and Winkler (1963) Jacoby (1964) Boyd and Lichstein (1953) Boyd and Lichstein (1953) Stephenson and Gale (1937b); Epps and Gale (1942)

P

2

U 0

1 (1

3La 8

Strasters and Winkler (1963) Stokes and Powers (1967) 0 Jacoby (1964) Polakis and Bartley (1965); Beck and Meyen3(I, burg (1968) K Gale and Stephenson (1938); Epps and Gale ca (1942) ;Boyd and Lichstein (1951) Boyd and Lichstein (1951) N Strasters and Winkler (1963) Qs

5

P

I-

c3 Q,

t s

TABLEl-continued Substrate, Enzyme or Structure

Organism

Sphaerotilus sp. Pseudomonas jluorescens n-serine deaminase Escherkhia coli Bacillus lichenifarmis Proline pyrroline-6-carboxylate dehydrogenase Staphylococcus aureus Pseudomonas jluorescens Escherichia coli Ornithine ornithine decarboxylase (EC 4.1.1.17) Staphylococcus aureus Escherkhia coli Alanine

Glycine Phenylalanine Asparagine Glutamine Hydroxyproline Isoleucine Leucink Lysine Valine

Staphylococcus aureus P s e u d o m o m Juorescens Sphaerotilua sp. Escherichia coli Staphylococcus aureus Pseudomonas jluorescens Sphaerotilua sp. Pseudomonas jluorescens Heterogeneous population Pseudomonas jluorescens Pseudomonas jluorescens P s e u d o m o m jluorescens Pseudomonas jluorescens P s e u d o m o m jluorescem Pseudomonas jluorescens Pseudomonas jluorescens

Reference Stokes and Powers (1967) Jacoby (1964) McFall (1964a, b; 1967a, b, c) Laishley and Bernlohr (1966, 1968) Strasters and Winkler (1963) Jacoby (1964) Epps and Gale (1942)

3 x

w

&

Strasters and Winkler (1963) Stephenson and Gale (1937b); Epps and Gale (1942) k% Strasters and Winkler (1963) U Jacoby (1964) m M Stokes and Powers (1967) Stephenson and Gale (1937b) 1 Strasters and Winkler (1963) tr Jacoby (1964) Stokes and Powers (1967) Jacoby ( 1964) Stumm-Zollinger (1966) Jacoby (1964) Jacoby (1964) Jacoby (1964) Jacoby (1964) Jacoby (1964) Jacoby (1964) Jacoby (1964)

4

;F E

C. Glyoxylate Cycle isocitrate lyase (EC 4.1.3.1)

Escherichia coli Saccharomyces cerevisiae

malate synthase (EC 4.1.3.2)

Rhizopus n.igriCalzs Hydrogenomom sp. Smharomyces cerevisiae

malate dehydrogenase (EC 1.1.1.37)

Hydrogenomonas sp. Saccharomyces cerevkhe

Kornberg (1966); Hsie and Rickenberg (1967) o Polakis and Bartley (1965); Gorts (1967); Witt et al. (1966a); Beck and Meyenburg (1968) 0 Wegener and Romano (1964) M Schlegel and Triiper (1966) w Polakis and Bartley (1965); Witt et al. (1966a); Schlegel and Triiper (1966); Beck and Meyenburg (1968) ro Schlegel and Triiper (1966) 0 Polakis and Bartley (1965);Witt et ul. (1966a, b); Zink (1967); Zink and Shaw (1968); Beck tj and Meyenburg (1968) U

z 8 w

-

D. Tricarboxylic Acid Cycle

YP i

B

o 0

citrate synthase (EC 4.1.3.7) aconitate hydratase (EC 4.2.1.3)

Saccharomyces cerevisiae Escherichia coli Saccharomyces sp. Escherkhia coli Bacillus subtilis

isocitrate dehydrogenase

Bacillus licheniformis Saccharomyces sp.

NAD-dependent (EC 1.l.1.41)

Escherichia coli

2

Polakis and Bartley (1965) Gray et al. (1966) Polakis and Bartley (1965); Gray et al. (1966); Wijk (1968~) 0 Gorts (1967); Hanson and Cox (1967) Hanson et al. (198313, 1964); Hanson and Cox (1967);Cox and Hanson (1968) Hanson and Cox (1967) MacQuillan and Halvorson (196213); Polakis et al. (1964); Polakis and Bartley (1965); Gorts (1967); Wijk (1968~) tQ 43 Halpern et al. (1964); Gray et al. (1966) 0

E

8 5

m

TABLEl - c o n t i n m d Substrate, Enzyme or Structure

NADP-dependent (EC 1.1.1.42) succinate dehydrogenase (EC 1.3.99.1)

Organism

Bacillus subtilis Staphylococcus aureus

Saccharomyces sp. Escherichia coli Staphylococcus aureus

fumarate hydratase (EC 4.2.1.2)

Bacillus subtilis Saccharomyces sp.

malate dehydrogenase (EC 1.1.1.37)

Escherichia coli Staphylococcus aureus Bacillus subtilis Saccharomyces cerevisiae Escherichia coli Staphylococcus aureus

a-ketoglutarate dehydrogenase

Bacillus subtilis Staphylococcus aureus

citrate permease

Escherichia coli Salmonella typhirnurium

Q, I&

Reference

Hanson et al. (1964) Gershanovich and Burd (1964); Gershanovich et al. (1964); Collins and Laacelles (1962) MacQuillan and Halvorson (1962b); Polakis et al. (1964, 1965); Gorts (1967); Beck and Meyenburg (1968);Wijk (196%) Epps and Gale (1942); Halpern et al. (1964);

4

3 8

'd

k8

Gray et al. (1966) Gershanovich and Burd (1964); Gershanovich et al. (1964); Collins and Lascelles (1962); % Strasters and Winklar (1963) U Hanson et al. (1963b) W M Polakis and Bartley (1965); Gorts (1967); Wijk ( 1 9 6 8 ~ ) Halpern et al. (1964); Gray et al. (1966) Strasters and Winkler (1963) Hanson et al. (1963b) t3 Polakis and Bartley (1965); Beck and Meyenburg (1968) Halpern et al. (1964); Gray et al. (1966) Gershanovich and Burd (1964) ; Gershanovich et al. (1964) Hanson et al. (1963b) Gershanovich and Burd (1964); Gershanovich et al. (1964) ; Polakis and Bartley (1965) Amarasingham and Davis (1965) Englesberg et al. (1961)

4

3

E 5

E. Respiratory Enzymes Cytochromes

Saccharornyces cerevisiae

cytochrome oxidase (EC 1.9.3.1)

Staphylococcus aurew Salmonella typhimurium Saccharomyces cerevisiae

NADH-cytochrome c oxidoreductase (EC 1.6.2.1) NADH oxidase

Saccharomyces cervisiae Saccharomyces cerevisiae

NADH dehydrogenase (EC 1.6.99.3) catalase (EC 1.11.1.6)

Saccharomyces cerewisiae

Strittmatter (1957) ; Gorts (1967); Jayaraman d et al. (1966) Strasters and Winkler (1963) ti Richmond and Maal0e (1962) 0 Polakis et al. (1964, 1965); Jayaraman et al. (1966); Strittmatter (1957); Tustanoff and Bartley (1964a, b) w Polakis et al. (1964, 1965) ; Jayaraman et al. (1966) Polakis et al. (1964, 1965); Jayaraman et al. g 0 (1966) Z Gorts (1967) bSulebele and Rege (1967, 1968a, b)

5

!$

3

1

0

F. Miscellaneous Enzymes

n

z formate dehydrogenase (EC 1.2.1.2) ribulose diphosphate carboxylase (EC 4.1.1.39) phosphoenolpyruvate carboxykinase succinyl-CoAsynthase (EC 6.2.1.5) acetyl-CoA kinase (EC 6.2.1.1)

Pseudomonas oxalaticus Escherkhia coli Pseudomonas oxalaticus

Blackmore and Quayle (1968) Epps and Gale (1942) Blackmore and Quayle (1968)

Escherichia coli Rhodotorula glutinis Hansenula anomala Saccharomyces cerevisiae Saccharomyces cerevisiae Hydrogenornow

Hsie and Rickenberg (1966, 1967) De Torrontegui et al. (1966) De Torrontegui et al. (1966) Polakis and Bartley (1965) Polakis and Bartley (1965) Schlegel and Triiper (1966)

T1F

Pni

FZ Y

Gn

B El Q,

or

E3

TABLEl-continued Substrate, Enzyme or Structure urease (EC 3.5.1.5)

Reference

Organism

extracellular protease arginine biosynthetic enzymes

Proteus rettgeri Proteus vulgaris Bacillus lichen~ormis Escherichia coli

acid phosphatase (EC 3.1.3.2)

Escherichia coli

Q, Q,

Magaiia-Plaza and RuinHerrera (1967) Passmore and Yudkin (1937) Laishley and Bernlohr (1968) Gorini and Gundersen (1961); Gorini et al. (1961) Hsie and Rickenberg (1967)

E

z

G. Miscellaneous Processes ~~~

~

Spwulation Flagella Production Budding Hyphae Formation Mitochondria Formation

Bmillus subtilis Escherichia coli Saccharornycescerevisiae Mueor sp. Saccharomyces cerevisiae

Chlorophyll Production

Chlorella sp. Euglena gracilis

Antibiotic Formtion acetonomycin bacitracin

Streptomyces antibioticus Bacillus licheniformis

Schaeffer et al. (1965) B Adler and Templeton (1967) BeckandMeyenburg (1968); Meyenburg (1968) Bartnicki-Garcia (1968) Polakis et al. (1964, 1965); Jayaraman et al. (1966) Shugarman and Appleman (1966) Matsuka and Hase (1966) ; Buetow (1967)

3

Marshall et 01. (1968) Laishley and Bernlohr (1968)

H. Higher Organisms invertase (EC 3.2.1.26) histidase (EC 4.3.1.3)

Sugar cane Rat liver

Glasziou and Waldron (1964) Myasoedova (1966)

urocanase threonine dehydrase (EC 4.2.1.16)

Rat liver Rat liver

Myasoedova (1966) Pitot and Peraino (1963) ;Peraino and Pitot

ornithine-6-transaminnase(EC 2.6.1.13)

Rat liver

Pitot and Peraino (1963); Peraino and Pitot

(1964) (1964)

phosphoenolpyruvate carboxykinase dimethylaminoazobenzene reduotase mginase (EC 3.5.3.1) I-aminolaevulinatesynthase I-aminolaevulinatedehydratase (EC 4.2.1.24) 6-aminolaevulinateproduction in mute intermittent porphyria variegate porphyria

Rat liver Rat liver Liver cells (Changs) Rat liver Rat liver

Shrago et al. (1967) Jervell et d.(1965) Eliasson (1965) Marver et al. (1966) ;Tschudy et al. (1964) Marver et al. (1966)

d

5 .k 0

5 M

1 M

+d

w

HUm2LIl Human

Perlroth et al. (1966) Perlroth et al. (1968)

!$

w0 z

d 0

268

KENNETH PAIGEN AND BEVERLY WILLIAMS

SUCCINATE SUCClNYL-CoA

e@

a- KETOGLUTARATE

a

FIG.2. The tricarboxylic acid cycle and the glyoxylate bypass. The open arrows indicate material leaving the cycle to supply biosynthetic precursors.

related to the various functions of the cycle and may be summarized as follows : (a) I n any medium, synthesis of the tricarboxylic acid cycle enzymes is repressed during anaerobic growth when the cycle cannot function catabolically (Englesberg et al., 1954; Umbarger, 1954; Gray et al., 1966). (b) During aerobic growth, the lowest enzyme levels are observed when both glucose and a supply of biosynthetic precursors (e.g. casamino acids) are available (Hanson et aZ., 1963a, 1964; Gray et al., 1966). Repression therefore is most intense when the requirements for both catabolic and anabolic functions of the cycle are minimal. (c) If the cycle must function anabolically because biosynthetic precursors are not provided, the levels of tricarboxylic acid enzymes may be increased. This increase occurs in E . coli even when a substrate such as glucose, which is relatively active in producing catabolite repression, is present (Gray et al., 1966). Among the enzymes of the cycle, cc-ketoglutarate dehydrogenase is an exception in that synthesis of this enzyme remains repressed when the cycle must function anabolically, apparently to conserve a-ketoglutarate by limiting its irreversible oxidation to succinyl-CoA (Amarasingham and Davis, 1965; Gray et al., 1966). I n yeast, the need for anabolic functions has no influence, and the amounts of tricarboxylic acid cycle enzymes are the same whether or not biosynthetic precursors are provided (Polakis and Bartley, 1965). (d) When biosynthetic precursors are available, and the only function of the cycle is catabolic, the amounts of tricarboxylic acid cycle enzymes

CATABOLITE REPRESSION AND OTHER CONTROL MECHANISMS

269

present reflect the energy source that is provided. I n both yeast and E. coli, the lowest levels are observed with glucose as substrate; higher levels are produced during growth on less readily utilized substrates such as glycerol, galactose or pyruvate; and the highest levels are observed when the substrate is itself a member of the tricarboxylic acid cycle, such as malate. Very high enzyme levels are also found when no carbohydrate is provided and energy must be obtained by oxidation of amino acids through the cycle (Strasters and Winkler, 1963; Gray et al., 1966; Gorts, 1967; Beck and Meyenburg, 1968). Synthesis of each enzyme apparently is controlled independently since these variations in enzyme content are not strictly co-ordinate. I n E. coli, a weak co-ordinacy exists within the group of enzymes concerned with tricarboxylic acid metabolism and also within the group involved in the metabolism of four-carbon dicarboxylic acids (Gray et al., 1966). As we have mentioned, synthesis of a-ketoglutarate dehydrogenase is especially sensitive to repression in order to conserve a-ketoglutarate. I n Bacillus the reaction catalysed by aconitate hydratase appears to be an important control point since synthesis of this enzyme is especially sensitive to repression by a mixture of glucose and glutamate (Hanson and Cox, 1967; Cox and Hanson, 1968). 3. Glyoxylate Cycle Whenever acetate is the sole carbon source, cells must generate C4 units to replenish the tricarboxylic acid cycle intermediates which are removed for synthesis of cellular material. This function is performed by the glyoxylate cycle, which contains most of the enzymes of the tricarboxylic acid cycle but omits the two oxidative reactions which convert isocitrate to a-ketoglutarate. Instead isocitrate lyase cleaves isocitrate (C,) into succinate (C,) and glyoxylate (C2). Malate synthase then condenses glyoxylate (C,) with acetyl-CoA (C,) to give malate (C,) with a net gain of one C, unit (Fig. 2 ) . I n Escherichia coli, isocitrate lyase is the key enzyme in the control of this pathway. Its activity is inhibited by phosphoenolpyruvate and pyruvate and its synthesis repressed by glucose and pyruvate (Kornberg, 1966). Examination of mutants suggests that the pyruvate repression is distinct from that exerted by glucose, and that the glucose repression is probably mediated through the formation of gluconate or 6-phosphogluconate (Kornberg, 1967). I n Saccharomyces, synthesis of three enzymes of the glyoxylate pathway, namely isocitrate lyase, malate synthase, and malate dehydrogenase, is repressed by glucose (Witt et al., 1966a). The regulation of malate dehydrogenase is particularly interesting. Both Newrospora

270

KENNETH PAIGEN AND BEVERLY WILLIAMS

(Zink and Shaw, 1968) and Saccharomyces (Witt et al., 1966b) possess at least two isozymes of this enzyme. One of these isozymes is soluble, functions in the glyoxylate bypass, and is repressed by glucose. The other isozyme is located in the mitochondria, functions in the tricarboxylic acid cycle, and is not subject to catabolite repression. Hence, cells growing on acetate have both the mitochondrial and soluble isozymes while cells growing on glucose have primarily the mitochondrial isozyme.

4. Respiratory Enzymes I n general, micro-organisms growing on glucose obtain their energy by glycolysis and do not utilize the respiratory chain. For example, in Salmonella (Richmond and Maabe, 1962) and Staphylococcus (Strasters and Winkler, 1963) glucose-grown cells have a decreased content of cytochromes. I n yeast, not only the cytochromes but several other enzymes that have respiratory functions are repressed by either glucose or anaerobiosis (Table 1 ; Strittmatter, 1957 ; Tustanoff and Bartley, 1964b; Polakis et al., 1964, 1965; Polakis and Bartley, 1965). I n yeast cells growing on glucose, the development of new mitochondria is arrested so that the number drops from 6-12 per cell to 0-2 per cell (Polakis et al., 1964, 1965; Jayaraman et al., 1966) and the few mitochondria present have poorly developed cristae. Polakis et al. (1964) have presented a series of electron micrographs showing the various stages of mitochondrial formation as cells recover from glucose repression. A vacuolar structure first appears which evolves in stages to fully developed mitochondria with cristae ; only when fulIy mature mitochondria are formed do the cells acquire the ability to oxidize acetate. When exposed to oxygen, yeast that has been grown anaerobically on galactose can respire immediately while glucose-grown yeast cannot (Tustanoff and Bartley, 1946b; Polakis et al., 1965). On the basis of this observation, Polakis et aE. (1965) suggest that oxygen is not a true inducer of respiratory adaptation and mitochondrial formation, but rather that aerobiosis provides a partial release from the catabolite repression produced by glucose. 5. Sporulation

Bacterial cells undergo marked changes in enzymatic content when they sporulate after exhausting the carbon source from a glucoseminimal medium. Although these changes have been regarded as part of the process of sporulation, they may stem from the release of catabolite repression. For example, the three enzymes involved in arginine degradation appear in Bacillus licheniformis when vegetative growth stops

CATABOLITE REPRESSION AND OTHER CONTROL MECHANISMS

271

due to the exhaustion of glucose from the medium and sporulation begins. However, these enzymes can be produced by vegetative cells in the absence of glucose (Laishley and Bernlohr, 1966) suggesting that, although they may have a role in sporulation, they are also under catabolite repression control. Similarly, Hanson and his coworkers (1963a, b) have reported that functional tricarboxylic acid and glyoxylate cycles are not present during vegetative growth of Bacillus subtilis and appear at the beginning of sporulation. Although they have suggested that the activities of these cycles are necessary for sporulation, it is difficult to know whether this conclusion is correct since these cycles are generally under catabolite repression control. The possibility that some catabolite-repressible enzymes are required for sporulation is suggested by the studies of Schaeffer et aZ. (1965) who examined the number of spores present in cultures during exponential growth under various conditions, The fraction of cells which underwent sporulation was greatly decreased in the presence of glucose, malate, casein hydrolysate, or aspartate, suggesting that one or more sporulation enzymes are catabolite repressed. 6. Porphyrin Synthesis The mitochondria1 enzyme 6-aminolaevulinate synthase is thought to be the rate-controlling enzyme in porphyrin biosynthesis. Levels of this enzyme are elevated in rats and guinea pigs after administration of compounds which produce porphyria in these animals. The effect is counteracted by high concentrations of carbohydrate (sucrose or glucose) in the diet (Tschudy et al., 1964; Marver et al., 1966). I n humans, the disease’s acute intermittent porphyria and variegate porphyria are characterized by seven-fold elevated levels of 6-aminolaevulinate synthase and the excretion of porphyrin and porphyrin precursors. The levels of dietary carbohydrate influence the course of this disease by its effect on the control of this enzyme, since restricting the dietary intake of carbohydrate exacerbates the symptoms and biochemical findings (Perlroth et aZ., 1966, 1968).

C. ENVIRONMENTAL CONDITIONSWHICHPRODUCE CATABOLITE REPRESSION The enzymes described in the preceding section are all relatively repressed when cells are grown on glucose and certain other carbon sources. Synthesis of the same enzymes is also repressed when cells are subjected to a variety of other influences such as exposure to radiation, antibiotics, or deprivation of a required growth factor. The variety of circumstances that produce repression are most easily related by assuming that each modifies the coiicentratctionof one or more intermediary

272

KENNETH PAIGEN AND BEVERLY WILLIAMS

metabolites which are regulatory effectors controlling the synthesis of these sensitive enzymes. This view was first clearly put forth by Magasanik (1961) and has since served as the unifying explanation for the diversity of circumstances that can produce catabolite repression. Obviously, the concentration of effectors will reflect both their rates of formation and subsequent utilization. Any factor which changes either rate is likely to affect the intensity of catabolite repression. 1. Growth Substrates

a. Carbohydrates. I n general, carbon sources which support a rapid rate of growth are most effective in producing catabolite repression (Neid-

\

gluconate

0

0.4 0.8 Rate of growth

1.2

FIG.3. Relationship between growth rate and catabolite repression. The differentiaI rate of @-galactosidasesynthesis is plotted as a function of growth rate for a series of cultures of Escherichia coli growing on different carbon sources. Redrawn from Okinaka and Dobrogosz (1967b).

hardt and Magasanik, 1956a, 1957a, b ; Neidhardt, 1960; Magasanik, 1961; Mandelstam, 1962; Okinaka and Dobrogosz, 1967b). This relationship is illustrated for p-galactosidase synthesis in Escherichia coli cultures growing on various carbohydrate substrates (Fig. 3). Growth rate alone, however, is not an adequate predictor of catabolite repression, as some media which permit rapid growth give very little repression. Since substrates which rapidly feed into the early reactions of glycolysis are usually the best repressors, the critical factor is probably changes in the concentrations of certain key metabolites. Glucose, glucose 6phosphate and gluconate are strong repressors, and mixtures of these produce even more intense repression (Okinaka and Dobrogosz, 1966; Hsie and Rickenberg, 1967).

CATABOLITE REPRESSION AND OTHER CONTROL MECHANISMS

273

b. Casamino acids. Although casein hydrolysate is commonly employed as a growth stimulant, its use in studies of catabolite-repressible enzymes presents problems since this material itself produces catabolite repression (Paigen, 1963; Beggs and Lichstein, 1965). This repression probably results from a combination of factors including partial growth inhibition by valine, serine, histidine and leucine ;provision of additional carbon substrates in the form of degradable amino acids; and replacement of a major drain (amino acid synthesis) on the intermediary pool. Casamino acids have been used, for example, t o repress amino-acid biosynthesis in order to determine whether a diminution in the variety of species of enzymes that a cell makes will alter the differential rate of ,8-galactosidase synthesis (Moses and Yudkin, 1968). Such experiments are vitiated by the simultaneous catabolite repression that is produced. The interpretation of experiments which include casamino acids in the medium is further complicated by the fact that, as the supply of various amino acids becomes exhausted, the intensity of repression decreases (Paigen, 1963). This effect has been shown to depend on the concentration of casamino acids present (Paigen, 1963))and probably accounts for the increased rate of synthesis of enzymes of the gal operon in the late log phase of growth in casamino acids medium (Jordan et al., 1967). 2. Factors Limiting Synthesis and Growth The size of the catabolite pool is influenced both by the entry of intermediates and by the rate at which intermediates are utilized for synthesis of cellular material. The balance between these rates is affected by the availability of required growth factors, the presence of metabolic inhibitors, cell damage resulting from irradiation or radioactive decay and perhaps by temperature changes. a. Growth factors. Strong catabolite repression is produced whenever an auxotroph is starved of its growth requirement in the presence of a carbon source. For example, in a thymine-requiring mutant starved of thymine in the presence of glycerol, the catabolite pool builds up and strong catabolite repression ensues. If, however, glycerol is also omitted from the medium, there is neither entry nor exit from the catabolite pool, and a catabolite-sensitive enzyme such as /?-galactosidasebecomes fully inducible (McFall and Magasanik, 1960, 1962; Nakada, 1962a, b). This effect has also been seen with other auxotrophic mutants starved of their growth requirements, including mutants unable to synthesize uracil (Pardee, 19551, methionine (Yanagisawa, 1962a, b), leucine (Mandelstam, 1961),and threonine (Nakada and Magasanik, 1962,1964). I n all cases, the differential rate of /%galactosidasesynthesis was higher in the absence of a carbon source than in its presence. Similar resuIts have

274

KENNETH PAIQEN AND BEVERLY WILLIAMS

been obtained by limiting the supply of phosphate (McFall and Magasanik, 1962) or nitrogen (Wainwright and Nevill, 1956; Mandelstam, 1957, 1961, 1962; Palmer and Mallette, 1961). Here again the presence of a carbon source produces severe catabolite repression if the supply of other nutrients is limited. The presence of a carbon source during limitation of growth produces a much more intense catabolite repression than that seen during unrestricted growth. Indeed, even carbon sources such as lactate and succinate, which ordinarily cause little if any repression, become strong repressors. b. Antibiotics and metabolic inhibitors. Any agent which reduces utilization of the catabolite pool while still allowing entry causes catabolite repression. The preferential repression of a catabolite-sensitive enzyme has been observed following exposure to partially inhibitory doses of several antibiotics, including puromycin (Sypherd et al., 1962; Paigen, 1963; Sells, 1965), streptomycin (Anand and Davis, 1960; Sypherdet al., 1962),actinomycinD (Paigen, 1963),mitomycinC (Paigen, 1963), tetracycline (Sypherd et al., 1962) and chloramphenicol (Hahn and Wisseman, 1951; Nakaya and Treffers, 1959; Sypherd et at., 1962; Sypherd and Strauss, 1963a, b; Sypherd and DeMoss, 1963; Paigen, 1963). Since the preferential repression by puromycin only occurs in the presence of a carbon source, its effect is most likely due to catabolite repression (Sells, 1965). The same is true of mitomycin C (Basu et al., 1965). The case of chloramphenicol is more complicated. Paigen (1963) found that concentrations of chloramphenicol which had no effect on growth rate repressed synthesis of both 8-galactosidase and galactokinase by more than 50%, and that concentrations which inhibited the growth rate by only 5% produced almost complete repression. Sypherd and his coworkers (Sypherd and Strauss, 1963b; Sypherd and DeMoss, 1963) have suggested that this remarkably intense repression derives from an over-production of RNA, and is distinct from catabolite repression. They reported that the chloramphenicol repression of /3-galactosidasewas not relieved by anaerobiosis although that by glucose was, and that chloramphenicol continued to repress during carbon starvation. Curiously, one lac I - constitutive strain (3.300) was insensitive to chloramphenicol repression while another (ML308) was not. Since cultures recover from chloramphenicol inhibition (Sypherd and Strauss, 1963a), the effects studied may have been due to the subsequently discovered phenomenon of transient repression. As expected, antibiotics are not the only growth inhibitors that cause catabolite repression. Some compounds, such as 2,4-dinitrophenol, azide, malonate and iodoacetate, are predictably complex in their effects

CATABOLITE REPRESSION AND OTHER CONTROL MECHANISMS

275

(Mandelstam, 1962; Paigen, 1963) since they affect both the flow of carbon metabolism and the supply of energy available for biosynthetic processes. Several compounds which have been used in attempts to study various aspects of protein synthesis illustrate the dangers inherent in drawing physiological conclusions from experiments using catabolitesensitive enzymes as representative proteins. Kepes and Beguin (1966), for example, have suggested that hydroxylamine decreases /3-galactosidase synthesis by acting as an inhibitor of chain initiation. However, Basu et al. (1967) suggested that this effect is partly due to catabolite repression resulting from growth inhibition by hydroxylamine, since repression is much stronger in the presence of a carbon source. Although hydroxylamine does decrease translation in the absence of a carbon source, it cannot be used as a specific inhibitor of chain initiation with catabolite-sensitive enzymes. 5-Fluoro-uracil, which is incorporated into m-RNA, inhibits the synthesis of /3-galactosidase. It has been postulated that this effect is due to the production of fraudulent messenger which codes for an enzymically inactive protein, an interpretation supported by the detection of a protein immunologically related to j3-galactosidase in cells growing in the presenceof 5-fluoro-uracil(Bussardetal., 1960; Gros et al., 1961; Nakada and Magasanik, 1964). However, 5-fluoro-uracil decreases the rate of total protein synthesis by SO%, and it is doubtful that the effect of 5-fluoro-uracilon /3-galactosidaseis due solely to the production of fraudulent messenger. Horowitz and Kohlmeier (1967), in re-examining the question, concluded that catabolite repression accounts for much of the inhibition. They report that the effect of 5-fluoro-uracil was more pronounced in the presence of carbon sources which produced catabolite repression than with carbon sources such as succinate which did not. Conditions which relieved catabolite repression (removal of carbon source or anaerobic shock) also relieved repression by 5-fluoro-uracil. Of of the six enzymes that have been examined, synthesis of only P-galactosidase and D-serine dehydrase, the two catabolite-sensitive enzymes (Horowitz et al., 1960), were inhibited by 5-fluoro-uracil; succinate dehydrogenase (Horowitz et al., 1960), catalase (Horowitz et al., 1960), alkaline phosphatase (Naono and Gros, 1960), and glucose 6-phosphate dehydrogenase (Bussard et al., 1960) were not affected. Thus, 5-fluorourecil causes both catabolite repression and fraudulent messenger production. Some indication of the relative effects can be calculated from the data of Nakada and Magasanik (1964). For every 100 molecules of correct j3-galactosidasemessenger made by a control culture, a total of 77 messengers, 45 valid and 22 fraudulent, were produced in the presence of 5-fluoro-uracil. c. Physical agents. Physical damage t o cells may produce catabolite

276

KENNETH PAIGEN AND BEVERLY WILLIAMS

repression. This was first observed in cells undergoing "suicide" from decay of incorporated 32P.I n this case, the ability to produce 13-galactosidase decreased a t the same rate as viability in media containing glycerol, but decreased much more slowly than viability if glycerol was absent (McFall et al., 1958; McFall, 1961). Bowne and Rogers (1962) have shown that the repression of 8-galactosidase synthesis which occurs after ultraviolet irradiation is reversed if the carbon source is omitted, an effect which has been confirmed by others (Pardee and Prestidge, 1963; Brownell and Witt, 1965) and most likely explains the earlier results of Torriani (1956), Kameyama and Novelli (1962) and Masters and Pardee (1962). Catabolite repression may also explain the repression of P-galactosidase synthesis in cells damaged by X-rays (Moore, 1965) or growing in the presence of deuterium (Henderson, 1962). Several laboratories have compared the intensity of catabolite repression in cultures growing at different temperatures (Ng et al., 1962 ; Marr et al., 1964; Heath and Brown, 1967). The differential rate of /I-galactosidase synthesis, both in constitutive mutants and in wild-type cells maximally induced by thiomethylgalactoside, decreased at lower temperatures in cells grown on succinate. At lo", the differential rate of /I-galactosidase synthesis was only 50% of that in the range 20-43'. Repression by glucose was also more intense a t lower temperatures (Marr et al., 1964). A different result was obtained in cultures that were only partially induced by thiomethylgalactoside (Marr et al., 1964). I n this case, the rate of S-galactosidase synthesis in lac Y- cells growing on succinate decreased with temperature until 20". Below 20", the rate of /3-galactosidase synthesis increased, a result that was also claimed by Heath and Brown (1967). This effect is probably explained by the observation of Kepes (1960) that higher internal concentrations of inducer are maintained at lower temperatures.

D. OCCURRENCE OF TRANSIENT REPRESSION 1. The Phenomenon

If microbial cultures are exposed to glucose after growth on another carbon source, synthesis of ,8-galactosidase may be severely repressed for as long as one generation, after which enzyme synthesis starts at the reduced rate characteristic of glucose-adapted cells (Fig. 4). This phenomenon of transient repression was first reported for certain mutant strains of E. coli whenever glucose was substituted for glycerol (Paigen, 1966a) and for several other strains of E. coli whenever glucose was added to glycerol-containing media (Moses and Prevost, 1966). Further studies

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have shown that at least three types of response are possible (Tyler et ab., 1967). Some strains of E.cobi, such as W3110, (2600 and ML, show no transient repression whether glucose is substituted or added as a carbon source; these are TR+ strains (Paigen, 1966a). Some strains, such as Q-22 derived from W3110 (Paigen, 1966,) or LA-12G (Tyler et al., 1967), show transient repression when glucose is substituted for glycerol ;these are TR* strains. Finally, many strains of E. coZi, such as HfrH 3.000, show transient repression when glucose is added to glycerol-containing

/

+

150 0

Q

n.

"1.0

1.5

2.0

2.5

3.0

3.5

Relative growth FIG.4. Transient repression in a TRS strain of Escherichia COXCells were pregrown on glycerol ( A - A , A-A) or glucose (0-0, 0-0) and then induced for /3-galactosidasein either glycerol-(A-A, 0- 0)or glucose-(A-A, 0-0) containing medium. The difference between open and closed circles indicates the extent of catabolite repression ;the difference between closed circles and triangles demonstrates the additional effect of transient repression seen when cells are transferred from glycerol to glucose. Reprinted from Paigen (1966a).

medium, but not when glucose is substituted for glycerol ; these are TR" strains. Following the period of transient repression, cells of either the TR" or TR" phenotypes increase their rate of /3-galactosidase synthesis to that observed in cells in steady-state growth in the presence of glucose and inducer. Several lines of evidence suggest that, in transient repression, glucose does not act by preventing the entry of inducer. The intensity and duration of transient repression following substitution of glucose are the same whether or not the cells contain a high concentration of Zac proteins including the h c permease (Paigen, 1966a). Transient repression following addition of glucose is the same whether the inducer is added before,

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simultaneous with, or after glucose (Mosesand Prevost, 1966).Transient repression of the addition type also can occur in mutants deficient in galactoside permease (Moses and Prevost, 1966) and in some lac I mutants in which synthesis of /3-galactosidase does not depend upon inducer entry (Palmer and Moses, 1967; Tyler et al., 1967).Moreover, the addition of glucose to a fully induced culture growing in the presence of 14C-thiomethylgalactosidedoes not chase inducer out of the cells (Tyler et al., 1967). The limited period of intense repression probably reflects changes in the balance of metabolic reactions that occur during the change-over to a new carbon source. The approach to a new balance may involve both changes in concentrations of metabolic intermediates consequent to feeding another compound (glucose) into the metabolic machinery, and in the relative amounts of various enzymes during adaptation t o a new carbon source. Transient repression as a phenomenon is not limited to E. coli, but has been observed in Salmonella typhimurium carrying an F l a c (Tyler et al., 1967).Enzymes other than those specified by the lac operon are sensitive to transient repression, notably tryptophanase (Paigen, 1966a),galactokinase (Paigen, 1966a), and D-serine deaminase (Moses and Prevost, 1966) in E. coli, and amidase in Pseudomonasaeruginosa (Clarke et al., 1968).However, considerable differences exist among sensitive enzymes. I n the TRS mutant strain of E. coli Q22, the transient repression of tryptophanase synthesis lasts several times as long as that of 8-galactosidase (Paigen, 1966,). I n E. coli ML, which shows almost no transient repression for P-galactosidase synthesis, the transient repression of synthesis of D-serine deaminase lasts approximately one-fifth of a generation (Moses and Prevost, 1966). 2 . Relation of Transient Repression to Catabolite Repression Originally, both Paigen (1966a) and Moses and Prevost (1966) considered transient repression to be a special case of catabolite repression, notable only for the intensity and brief duration of the effect. Indeed, further work has shown that the similarities between the two phenomena are considerable. I n both cases, the control is exerted on m-RNA synthesis, the same family of enzymes are sensitive, and glucose is an effective agent in producing both repressions. Nevertheless, on the basis of two experiments, Tyler et al. (1967)have suggested that the two phenomena may not be identical. I n the first experiment, strain HfrH of E. coli 3000 (TR' in phenotype) was grown on either glucose or glycerol. The carbon source was then removed and both cultures starved for 5 min. When glucose and glycerol were added to glucose-grown cells, there was a short period of release from catabolite

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repression. When glucose and glycerol were added to glycerol-grown cells, transient repression was immediately evident. Tyler and coworkers suggested that catabolite, but not transient, repression is lost immediately after carbon-source starvation. However, the idea that glycerolgrown cells contain a different complement of carbohydrate-metabolizing enzymes compared with glucose-growncells, and will respond differently to glucose, was used originally to account for transient repression (Paigen, 1966a). I n the second experiment, Tyler et al. (1967) demonstrated that, at high concentrations, a-methylglucoside and 2-deoxyglucose, two glucose analogues which do not support growth, can produce transient repression in two lac I- mutants of E. coli. Previously, Moses and Prevost (1966) had reported that lower concentrations of 2-deoxyglucose were without effect. Catabolite repression is thought to result from changes in the intermediary pool of metabolites. Since these compounds do not support growth of E. coli, and thus cannot contribute to the common pool of metabolic intermediates, Tyler and coworkers concluded that transient repression is distinct from catabolite repression. However, these compounds do influence the metabolic pool since they are growth inhibitors (Schick et al., 1958)and they can produce catabolite repression (Kessler and Rickenberg, 1964; Witt et al., 1966,). This is especially true of 2-deoxyglucose which is phosphorylated in E. coli (Gershanovich, 1962). The strongest evidence suggesting the non-identity of catabolite and transient repression is genetic. Pastan and Perlman (1968)have reported that one of the mutations affecting the lac promotor region, mutation L-8 of Scaife and Beckwith (1966))has lost transient, but not catabolite, repression. Revertants of this mutant, selected for increased promotor function, had regained transient repression. Of the other two promotor mutants examined, L-29 had not lost either repression (Pastan and Perlman, 1968)while strain L-1 had lost both (Perlman et al., 1969).The results obtained with the L-8 mutant would not be expected if transient repression only represented an intense transitory form of catabolite repression. Despite this difference, Perlman and Pastan have suggested that catabolite and transient repression may be the same phenomenon. They report (Perlman and Pastan, 1968b; Pastan and Perlman, 1968; Perlman et al., 1969) that, in wild-type strains, 1 mM cyclic AMP is sufficient to reverse transient repression but 5 mM cyclic AMP is needed to reverse catabolite repression. The effect of glucose was explained by the observation of Makman and Sutherland (1965) that addition of glucose to E . coli cultures immediately lowers the internal concentration of cyclic AMP. I n the promotor mutant L-8 of E. coli, which has lost transient repression, even higher concentrations of cyclic

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AMP were required to reverse catabolite repression. Perlman and his coworkers (1969) suggest that the promotor mutation in strain L-8 has made this strain less sensitive to stimulation of p-galactosidase synthesis by cyclic AMP. However, if the L-S mutant requires more cyclic AMP to produce the same effect as in wild type, then one would expect this mutant to show an enhanced transient repression following glucose addition rather than no transient repression. 3. Transient Repression and Respiratory Shock

If an aerobic culture of E . coli growing on glucose is subjected to anaerobic shock by transferring it t o an environment of nitrogen and carbon dioxide, growth continues after a slight lag, but repression of /?-galactosidasesynthesis is temporarily lost (Cohn and Horibata, 1959~). The role of respiratory processes in the control of p-galactosidase synthesis was studied extensively by Dobrogosz and Okinaka (Dobrogosz, 1965, 1966; Okinaka and Dobrogoz, 1966, 1967a, b) who established that the converse also occurs ; a culture growing anaerobically and subjected to aerobic shock by exposing it to oxygen undergoes an intense transient repression. The period of derepression or repression which follows one of these respiratory shocks lasts for approximately a generation after which the differential rate found in glucose cultures is re-established. It is not the presence or absence of oxygen per se which is important but the availability of an electron acceptor since nitrate, which can serve as an alternate electron acceptor in E . coli, is able to replace oxygen (Dobrogosz, 1965). This suggests that the transient changes in the differential rate of /3-galactosidase synthesis which follow respiratory shock result from switching between respiration and fermentation. Supporting this idea is the fact that cells grown on carbon sources other than glucose, gluconate, and possibly mannitol, do not change their rate of /?-galactosidasesynthesis after respiratory shock (Cohn and Horibata, 1 9 5 9 ~Dobrogosz, ; 1965). Several facts indicate that the rate of dissimilation of pyruvate is an important factor. After anaerobic shock, transient derepression does not occur if either pyruvate or gluconate, which readily generates pyruvate, is present. Moreover, pyruvate accumulates in the medium during the transient repression which follows aerobic shock, and the release of repression is co-incident with the disappearance of pyruvate (Okinaka and Dobrogosz, 1967a).Using pyruvate labelled in various carbon atoms, a correlation between the existence of repression and a rapid rate of oxidative decarboxylation of pyruvate was demonstrated (Okinaka and \ (Dobrogosz, 1967a, b).

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Cyclic AMP has been implicated as the effector of the transient repression which follows glucose addition (see section III.D, p. 297). A relationship between that form of transient repression and the one seen after aerobic shock wouId be established if it can be demonstrated that a connection exists between the rate of pyruvate decarboxylation and the internal concentration of cyclic AMP.

111. The Mechanisms of Transient and Catabolite Repression A. TRANSCRIPTIONAL OR TRANSLATIONAL CONTROL The question of whether catabolite repression acts to inhibit the transcription of DNA into m-RNA or to inhibit translation of messenger into protein has received conflicting answers. For the P-galactosidase system of E . coli, which has been analysed exhaustively by Nakada and Magasanik (1962, 1964) and Kepes (1963), the control appears to be at the transcriptional level. I n the yeast Xacch. carlsbergensis, catabolite repression of a-glucosidase synthesis acts on messenger translation (Wijk, 1968e).A decision as to whether these conflicting results reflect a fundamental difference between the regulatory devices of prokaryotes and eukaryotes, or whether the same organism can exert catabolite repression at either level depending upon the enzyme which is controlled, awaits analogous experiments in other systems. 1. Catabolite Repression of fi-Galactosidase Synthesis in Escherichia coli The key feature of the Nakada and Magasanik experiments (1962, 1964) was the development of a technique for experimentally separating in time the transcription and translation steps in 13-galactosidase synthesis. This technique consisted of exposing cultures to the inducer isopropylthiogalactoside for a few minutes and, before any appreciable amount of enzyme had been formed, removing the inducer by rapid filtration of the cells. A series of control experiments in which 5-flUOrOuracil was used to influence RNA synthesis, and chloramphenicol and amino-acid starvation were used to block protein synthesis, showed that, during the brief exposure to inducer, lac m-RNA was formed and could be translated subsequently into protein if the cells were resuspended in inducer-free medium. In the absence of inducer, translation continued at an exponentially declining rate for about 10 min. until the cells had exhausted the supply of preformed messenger. The half-life of the messenger was estimated by measuring the residual P-galactosidase-forming capacity remaining at any time after removal of inducer. The half-life of the messenger was 2.5 min. at 30°, whether or not the messenger was formed under a state

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of catabolite repression. It was also the same whether translation proceeded in a medium in which both glycerol and threonine were present or both absent (minimal catabolite repression), in a medium containing glucose and threonine (moderate catabolite repression) or only glycerol 1

-

I

I

4 Time (min)

6

t

\

-\

\ \

2

8

FIG. 5. Decay of /%galactosidase m-RNA in Escherichin coli as seen in a semi-log

plot of the differential rate of p-galactosidase synthesis existing a t various times after the cessation of m-RNA synthesis. The experimental procedure for this experiment and the justification for equating the rate of enzyme synthesis with messenger concentration are described in the text. The results show that the rate of decay is independent of the intensity of catabolite repression during the decay period since the rate is the same for cells suspended in the absence of glycerol and threonine (m), in the presence of glycerol and absence of threonine (A),and in the presence of both compounds (a).The fact that such a plot produces a straight line indicates that messenger decay is a random process. The slope of the line is a measure of the rate of decay. The arrow shows the differential rate of synthesis before inducer was removed at zero time. Since the line extrapolates back to this point, messenger decay probably proceeds continuously from the moment messenger is synthesized. Reprinted from Nakada and Magasanik (1964).

(intense catabolite repression ; Fig. 5). The differential rate of P-galactosidase formation was also measured during the period of translation to test whether catabolite repression influences the efficiency of translation of messenger molecules. For this purpose, the amount of accumulated enzyme was measured and plotted against the total amount of protein

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synthesized as estimated by incorporation of 14C-leucine. Under all conditions, the differential rate proved to be the same. Thus, the existence of a state of catabolite repression during translation of preformed messenger does not reduce the efficiency of translation, nor does it accelerate breakdown of m-RNA molecules. Furthermore, the rate of messenger decay is independent of its rate of translation and the absolute rate of protein synthesis. Although catabolite repression had no influence on the translation process, it markedly affected messenger synthesis. The quantity of messenger formed during the induction period was estimated from the total amount of /3-galactosidase that could be formed if cells were resuspended in a glycerol-containinggrowth medium during the subsequent translation period. If a state of catabolite repression existed during induction, messenger formation was decreased whether the repression was produced by the addition of glucose, by the removal of threonine in the presence of a carbon source, or by the addition of an inhibitor of protein synthesis such as 5-methyltryptophan or chloramphenicol. For the case of glucose addition, the decrease in messenger concentration accounted quantitatively for the reduced differential rate of /3-galactosidase synthesis seen during steady-state growth on glucose. I n experiments analogous to those of Nakada and Magasanik, Kepes (1963) separated the phases of transcription and translation by brief exposure of a culture to inducer followed by rapid dilution until the inducer concentration was below the level required to maintain a state of induction in the lac Y- strain used. Control experiments demonstrated that transcription into m-RNA took place during exposure to inducer, and translation into /I-galactosidaseoccurred after dilution of the culture. The presence of glucose during the translation phase had no effect on the amount of /3-galactosidase formed, but the presence of glucose during the transcription phase decreased j?-gaIactosidase m-RNA synthesis by 50%.

Thus, in the synthesis of /?-galactosidase in E. coli, catabolite repression acts to inhibit lac rn-RNA synthesis and has no discernible effect on its subsequent translation. However, in the synthesis of a-glucosidase in the yeast Sacch. carlsbergensis (Wijk, 1968e), catabolite repression appears to affect translation. 2 . Cutaboliite Repression of a-Glucosidase Xynthesis in Saccharomyces carlsbergensis

I n Succh. carlsbergensis, a-glucosidase synthesis proceeds a t a basal rate in a medium containing 0.2% glucose, is induced by adding maltose, and is catabolite-repressed in the presence of 1.0% glucose. I n yeast

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protoplasts, actinomycin D, shown by an independent assay to inhibit RNA synthesis, had no effect on basal enzyme synthesis, but puromycin and cycloheximide did prevent basal synthesis (Wijk, 1968e). These facts indicate that a stable m-RNA is responsible for the basal synthesis. Heating the protoplasts at 40" for 20 min. apparently destroys this messenger since actinomycin D prevented subsequent recovery at 30".

When synthesis of the enzyme was induced in protoplasts by maltose, there was a lag of 60 min. duration before activity appeared. Addition of actinomycin D at zero time blocked induction ;its addition at later times became progressively less effective and, at the end of the lag period, it had no influence. At all times, enzyme synthesis was sensitive to puromycin and cycloheximide. From these results it appears that maltose induction involves the synthesis of new stable m-RNA molecules. The effect of glucose on messenger synthesis was tested by inducing protoplasts with maltose in media containing either 0-2 or 1.0% glucose, centrifuging to remove the inducer, and resuspending the cultures in media containing the lower glucose concentration. The same differential rate of enzyme synthesis was obtained whether 0.2 or 1.0% glucose was present during induction, indicating that glucose has no effect on the transcription of u-glucosidase messenger. Most significantly, 1.0% glucose appears to inhibit the translation of preformed messenger. Protoplasts were induced in the presence of maltose and 0.2% glucose, and then centrifuged and resuspended in inducer-free medium. If this medium contained 0.2% glucose, a-glucosidase was synthesized and the synthesis was insensitive to actinomycin D ;if the medium contained 1.0% glucose, no enzyme synthesis occurred. Thus, catabolite repression acts on the transcription, but not the translation, of /I-galactosidase messenger in E . coli, and on the translation, rather than transcription, of u-glucosidase messenger in Sacch. carbbergensis. 3. Transient Repression of /I-Galactosidase in Escherichia coli

Like catabolite repression, transient repression appears to act on the synthesis of m-RNA. Tyler and Magasanik (1969) examined the expression of preformed lac m-RNA in E . coli strain LA-12G, a strain sensitive to transient repression but not catabolite repression. When cells were induced for three minutes with isopropylthiogalactoside in a medium containing glycerol, filtered, and resuspended in inducer-free medium, the presence of glucose had no effect on subsequent translation of the preformed m-RNA. Since glucose did not influence the translation of

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m-RNA, it was concluded that glucose must act on the transcription of DNA. Perlman and Pastan (1968b)determined the levels of m-RNAproduced under various conditions by assaying for subsequent ,6-galactosidase formation after removal of the inducer. When cyclic AMP was present during induction in the presence of isopropylthiogalactoside, glucose and chloramphenicol, the amount of m-RNA formed was increased. Cyclic AMP also increased messenger formation if protein synthesis was blocked during the induction period by removal of a required amino acid. The strain used (E. coli C-600) is sensitive to both catabolite and transient repression. If, as these authors have proposed, the levels of cyclic AMP employed were sufficient to reverse only transient repression, but not catabolite repression, the experiments suggest that transient repression acts at the level of m-RNA synthesis. However, this interpretation depends upon the concentration dependence of the cyclic AMP effect. The result may also have been influenced by the fact that the experiments were performed under conditions of intense catabolite repression. To demonstrate conclusively that transient repression acts at the level of transcription requires an experiment in which the amount of lac m-RNA is measured before glucose addition, immediately after glucose addition during the period of intense transient repression, and some time later after the cells have recovered from transient repression and only catabolite repression remains.

B. REPRESSION IN REGULATORY MUTANTS I n the preceding section we have presented evidence for the lac operon of E. coli that both catabolite repression and transient repression act a t the level of transcription to block messenger RNA synthesis in a manner analogous to the repressions that operates on biosynthetic pathways. This analogy suggests that apo-repressors and corresponding operator genes may exist through which the repression is mediated. If these aporepressors and operators exist, we can ask whether they are identical with those involved in specific induction and repression and, if they are not, where their genetic determinants are located. I n several systems, mutants which are constitutive for enzymes that are normally inducible and catabolite repressible have been tested for sensitivity to catabolite repression. I n general they remain sensitive, although sometimes less sensitive than the inducible synthesis in wildtype cells. For example, the constitutive synthesis of ribitol dehydrogenase in Aerobacter aerogenes is only partially repressed by glucose, while the induced synthesis is completely repressed to the basal level (Hulley et al., 1963). The same is true of the gal operon in E . coli where

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both R- and 0"mutants are relatively resistant to catabolite repression (Adyha and Echols, 1966; Williams and Paigen, 1969). I n this last case, increasing the concentration of inducer partially overcomes catabolite repression (Williamsand Paigen, 1969)thereby supporting the suggestion of Adhya and Echols (1966) that glucose repression of wild-type cells is partly due to inhibition of inducer entry. Examples of enzymes whose inducible and constitutive synthesis are equally sensitive to catabolite repression include the enzymes of histidine degradation and lactose metabolism (which are discussed in more detail below), the /3-glucosidase of yeast (MacQuillan et al., 1960;MacQuillan and Halvorson, 1962a), the amidase of E. coli in which succinate rather than glucose is the most effective repressor (Brammar and Clarke, 1964; Brammar et al., 1967), and the three enzymes concerned with glycerol utilization in E. COG,namely glycerol kinase, a-glycerophosphate permease and a-glycerophosphate dehydrogenase. The last three enzymes, although not genetically linked, appear to be controlled by the same regulator gene (Koch et al., 1964; Cozzarelli et al., 1968).With synthesis of amidase (Brammar and Clarke, 1964; Clarke et al., 1968) and the galactose enzymes (Williams and Paigen, 1969), increasing the concentration of inducer partially overcomes catabolite repression, although this is not true for /3-glucosidase (MacQuillan et al., 1960; MacQuillan and Halvorson, 1962a) or /?-galactosidase. Those enzymes which have been most extensively studied with respect to genetic modification of catabolite repression are D-serine deaminase, histidine-degrading enzymes, leucine biosynthesis enzymes, and those of the lac operon. The evidence from these bearing on the identity of the genetic determinants of catabolite repression, together with the evidence for a locus determining generalized catabolite repression, are summarized below. 1. D-Xerine Deaminase

Synthesis of D-serine deaminase is very weakly sensitive to catabolite repression in E. COG.Although not repressed by glucose under normal growth conditions, the enzyme is repressed if growth is limited in the presence of a carbon source. Among six mutants known to be constitutive for this enzyme, two are far more sensitive to catabolite repression than the parent. Both mutants are partial constitutives and have been placed in a cistron, dsd C, which maps adjacent to the D-serine deaminase structural gene, dsd A (McFall, 1964a, b). Both of these dsd C mutants are also temperature sensitive, with a low rate of constitutive synthesis at 20" and 41" and a maximal rate between 30" and 35" (McFall, 1964b), indicating that this gene probably specifies a protein. The existence of a

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protein product is also suggested by the partial dominance of the dsd C+ he levels of uninduced allele in dsd C-ldsd C+ diploids. I w i l d - t y p d and enzyme are intermediate betwe tivity of the diploids the constitutive levels of haplo en that of the two to catabolite repression is als haploid parents (McFall, 1967a, b). 2 . Enzymes Involved in Histidine Degradation

Synthesis of the four enzymes which catalyse the degradation of histidine in Bacillus subtilis is induced by histidine and repressed by glucose. Structural mutants have been isolated for two of the enzymes, namely histidase and formimino-glutamate hydrolase ; the structural genes are closely linked and presumably form an operon. Chasin and Magasanik (1968) have selected and mapped four classes of regulatory mutations affecting these enzymes : (1) those constitutive and sensitive to catabolite repression; (2) those inducible and resistant to catabolite repression ; (3) those constitutive and resistant to catabolite repression ; and (4) those which are pleiotropic-negative for the three enzymes assayed. Crosses between mutants in the first two classes established that the genetic factors were closely linked and the gene order was catabolite repression-inducibility-histidase-formimino-glutamate hydrolase. The one mutant found for class 3 and the one for class 4 seemed to map at the site for catabolite repression. Revertants of the pleiotropic-negative mutant (class 4) were usually inducible and sensitive to catabolite repression, but 2% were catabolite repression-resistant and 0.2% were both catabolite repression-resistant and constitutive. 3. Enzymes Involved in Leucine Biosynthesis

Catabolike repression is a control system which usually affects catabolic enzymes. However, a mutation in the presumed operator region of the leucine biosynthetic operon appears to have brought the leu enzymes under catabolite repression control (Friedman and Margolin, 1968).The leu400 mutation, an operator-negative which affects the entire leucine operon in E. COG,can revert to a leu+ phenotype. Most of the revertants are wild-type in character, but one of them, GD-1 which mapped at or very close to the original leu-500 mutation site, was leu+ in citrate medium but leu- in the presence of glucose. Enzyme assays confirmed that the leu enzymes in GD-1 were repressed by glucose. The authors postulate that the original leu-500 mutation made the leu operon intensely sensitive to catabolite control (so that leu-500 was leu- under all conditions), while strain GD-1 was less sensitive so that GD-1 was leu-

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only on glucose. By analogy with the his operon, it is also possible that leu-500 is an operator-negative and that only the GD-1 revertant has become catabolite-repression sensitive. It is interesting to note that leu-500 is reverted by 2-aminopurine, suggesting that this mutant differs from wild type only by a single base pair. 4. Enzymes of the Lac Operon Tests in a variety of lac I gene mutants of E. coli show that this gene is not required for catabolite repression. Lac I- constitutive mutants are as sensitive to catabolite repression as are their induced parental wildtype strains (Cohn and Horibata, 1959c; Brown, 1961; Mandelstam, 1962; Clark and Man, 1964; Palmer and Moses, 1967).Especially significant is the fact that I-sus mutants, which carry an amber codon and produce only a fragment of the repressor, are also subject to catabolite repression (Hsie and Rickenberg, 1967; Palmer and Moses, 1968). Catabolite repression also occurs in the Zac It' mutant, which makes a thermolabile repressor (Palmer and Moses, 1968), and when a lac I+Z+ chromosome enters an F- lac deletion strain (Loomis and Magasanik, 1964). Further evidence that catabolite repression does not act through the lac I gene product comes from experiments in which attempts were made to reverse catabolite repression in the presence of high concentrations of inducer. If a glucose catabolite reacted with the same apo-repressor as lac inducers, their effects should be competitive. However, Mandelstam (1962) found that 0.5 mM-isopropylthiogalactoside, a concentration 10-foldhigher that that required to induce maximally a wild-type strain, did not reverse even mild catabolite repression in lac I-. Very high concentrations of the same inducer (1mM and 1OmM)do not reverse glucose repression in wild-type strains (Brown, 1961; Colby et aZ., 1968; B. Williams and K. Paigen, unpublished data). Just as catabolite repression does not require a functional 1 gene product, neither does it require a functional operator region. Glucose represses constitutive enzyme synthesis in many Oc mutants (Brown, 1961; Loomis and Magasanik, 1964; Hsie and Rickenberg, 1967; Tyler et al., 1967; Palmer and Moses, 1967). Transient repression persists in at least some Zac I- and Zac Oc mutants of E. coli. These include the two lac I-mutants; HfrH 3300 (Tyler et al., 1967; Palmer and Moses, 1967) and I-522(Pastan and Perlman, 1968; Tyler and Magasanik, 1969).Likewise the 0 ' mutants 2000 (Tyler et al., 1967; Pal er and Moses, 1967)and OCl5and OC3,,,(Pastan and Perlman, 1968) are ensitive to transient repression. Tyler and Magasanik (1969) have reported that transient repression in a strain carrying the Oo6,

T

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mutation is quite variable in both the intensity and duration of repression. This variability may be the reason that Palmer and Moses (1967)reported OCe7 to be resistant to transient repression while Perlman and Pastan (1968b) have found the same strain to be sensitive to transient repression. In contrast to these findings, Palmer and Moses (1968) reported that both an I-sus strain, which produces only a fragment of the repressor, and the It' strain which produces a thermolabile repressor inactive at 42", were resistant to transient repression, and concluded from their results that transient repression requires intact lac repressor. The first of these results was questioned by Tyler and Magasanik (1969) who carefully reexamined the same 1-sus and found it to be sensitive to transient repression. The conclusions drawn from the behaviour of the lac It' are also open to question. Palmer and Moses (1968) tested this mutant and its parental wild-type strain at 32" and 42" and concluded that transient repression is lost when the lac I gene product is inactivated at 42" in the lac It' mutant. However, examination of the data shows that both strains were less sensitive to transient repression at 42"than at 32", and that, at 42", the behaviour of the two strains was not significantly different, indicating that the effect of raising the temperature was not primarily on the I gene product. Taken together, the majority of the evidence suggests that, like catabolite repression, transient repression requires neither a functional I gene product nor operator region. Several recent papers (Pastan and Perlman, 1968; Silverstone et al., 1969)have implicated the promotor region as the site at which catabolite and transient repression are exerted. Pastan and Perlman (1968)report that a lac promotor mutant (L-8)of E. coli has lost sensitivity to transient repression, but has retained sensitivity to catabolite repression. Three separate revertants of L-8, selected for increased production of /3-galactosidase in the presence of inducer, simultaneously had regained sensitivity to transient repression. Thus, the site at which transient repression control is exerted appears to be within the lac promotor region. Another promotor mutation (strain L-1), which is probably a short deletion extending into the I gene, has lost sensitivity both to catabolite repression (Silverstone et al., 1969),and to transient repression (I.Pastan, personal communication), suggesting that the site for catabolite repression control is also within the lac promotor. However, the promotor mutant (L-29), which is very closely linked to L-8, is still sensitive to both repressions. The various results obtained with these three promotor mutants indicate that the precise genetic limits of the three functions (promotion, sensitivity to catabolite repression, and sensitivity to transient repression) determined by this region remain to be determined. Moses and Yudkin (1968), from a study of an E. coli strain which 10

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KENNETH PAICEN AND BEVERLY WILLIAMS

carries a deletion fusing the lac and pur operons, concluded that the promotor, the regulator, and the operator regions are not involved in catabolite repression, and hence that, in contrast to the results cited previously (Nakada and Magasanik, 1962,1964; Kepes, 1963))catabolite repression must act at the level of translation. The mutant used carried a deletion extending from within the Z gene to the pur E gene, removing the 0, P, and I regions. I n this strain, the lac enzymes are under control of the purine regulatory system, and their synthesis is repressed by adenine. When derepressed by removing adenine, glucose-grown cells produced thiogalactoside transacetylase at only 50% of the rate of glycerol-grown cells, suggesting that catabolite repression of the lac enzymes does not require participation of the lac 0, P or I regions. However, the possibility that glucose affects the purine regulatory system was not tested. I n an analogous experiment, Silverstone et al. (1969)examined a similar strain in which lac was fused to the trp operon. Here, glucose also had some effect in repressing synthesis of lac proteins, but this was identical to its effect on the synthesis of anthranilate synthase, the first protein of the trp operon. I n view of the results of Silverstone et al. (1969) and the effects obtained with lac promotor mutants, the promotor region probably is involved in both transient and catabolite repression. Colby and his coworkers (Colby et al., 1968; Colby and Hu, 1968a, b) have moved F'lac episomes from E. coli into Proteus mirabilis, and found several anomalies in the regulation of the lac operon. Although P'lac+ wild type was sensitive to glucose repression in P. mirabilis, episomes carrying lac I- and lac 0"were not. The fact that the lac permease was competitively inhibited by glucose in P. mirabilis might explain the existence of an apparent catabolite repression with wild-type episomes, where glucose could inhibit inducer entry, but not with the Ior 0" episomes where enzyme synthesis is not dependent upon an exogenous supply of inducer. Although catabolite repression has not been tested for in P. mirabilis, it does occur in Proteus rettgeri and Proteus wulgaris (Passmore and Yudkin, 1937; Boyd and Lichstein, 1953). 5. Catabolite Repression Locus

If catabolite repression and transient repression are not mediated by the specific apo-repressor of each operon, there must be another protein which recognizes the low molecular-weight effector. One possibility would be an apo-repressor which functions only for catabolite repression. Loomis and Magasanik (1965)have looked for mutants of a gene determining such a protein by selecting for the ability to produce high levels of /3-galactosidase in the presence of glucose. The selection procedure

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utilized the fact that E . coli can only use N-acetyllactosamine as a source of nitrogen if this compound is first hydrolysed by ,8-galactosidase. Cells were selected for the ability to grow with N-acetyllactosamine as sole nitrogen source in the presence of isopropylthiogalactoside as an inducer and glucose as a catabolite repressor of P-galactosidase. A strain was obtained (LA12)which formed P-galactosidase at maximal rates in the presence of glucose, although it had only 80% of the normal growth rate on glucose, gluconate or glycerol. From this original isolate, a fast-growing mutant (LAlSG) was selected. The mutation producing loss of catabolite repression in LAl2G mapped near trp and was recessive to wild type (Loomis and Magasanik, 1967b). Loomis and Magasanik originally suggested that the gene involved, which they called CR, determines a regulatory protein involved in the catabolite repression of sensitive operons. More recently, however, Rickenberg et al. (1 968) have shown that both LA12G and L-9, another catabolite-repression mutant isoIated by Loomis, probably carry defects in glucose metabolism, rather than lacking a specific apo-repressor. They report that, in these strains, synthesis of tryptophanase and amylomaltase, as well as P-galactosidase, is resistant to catabolite repression by glucose, and that synthesis of all three enzymes remains sensitive to catabolite repression by glucose 6-phosphate or a mixture of glucose and gluconate. These findings have been confirmed by Moses and Yudkin (1968) who found that, in LAlBG, syntheses of P-galactosidase, tryptophanase, and Dserine deaminase are all resistant to repression by glucose.

C. MODELSOF REPRESSION Genetic evidence suggests two models of catabolite repression. The first was originally proposed by Magasanik (1961) and is a variation of the model postulated by Jacob and Monod (1961)to account for specific induction and repression. I n this model, catabolite repression results from the action of an apo-repressor on an operator region a t the beginning of each catabolite-sensitive operon. The low molecular-weight effector which mediates catabolite repression could act as a co-repressor, as proposed by Magasanik, or as a co-inducer which is always required and which is depleted during catabolite repression. If such an apo-repressor exists, genetic evidence indicates that it is not identical with the apo-repressor active in specific induction and repression, and that it does not use the same operatorregion. Thus, if this model is correct, two regions of operator function are required at the beginning of each catabolite-repressible operon. Since the genetic data also suggest that the promotor region may be involved in catabolite repression, it is possible that this region provides the second operator function.

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KENNETH FAIGEN AND BEVERLY WILLIAMS

An alternative model, in which catabolite repression acts a t the level of m-RNA synthesis but does not involve an apo-repressor, can be suggested. RNA polymerase has been shown to contain a subunit which determines its substrate specificity (Burgess et al., 1969). If more than one form of subunit occurs, so that a family of RNA polymerases exists, each may recognize a different set of promotor regions. Cataboliterepressible operons may contain the same promotor sequence, sharing a common species of RNA polymerase which contains the control site for catabolite repression. Such a polymerase (c-RNA polymerase) could be inhibited by a low molecular-weight metabolite, or more likely, may require cyclic AMP as a cofactor for activity. I n the absence of cyclic AMP, the activity of this species of RNA polymerase would be decreased and the transcription of catabolite-repressible operons would be inhibited. Promotor mutants which have lost the ability to bind RNA polymerase are unable to synthesize any enzymes of that operon. Revertants selected for operon function may recover the original promotor site, again using the c-RNA polymerase and being sensitive to catabolite repression; or they may mutate to the ability to recognize another form of RNA polymerase, and become insensitive to catabolite repression. Both classes of revertants were obtained in the histidine operon (Chasin and Magasanik, 1968). If changes in the promotor occur by small deletions extending into the adjacent operator, revertants of pleiotropic negatives could become constitutive. I n the gal operon, constitutive mutants were obtained as revertants of apleiotropic-negative by Hill and Echols (1966) and Morse (1967). The GD-1 mutation of Friedman and Margolin (1968) in the leu operon may represent the reverse sequence, going from a promotor site which recognizes an insensitive polymerase to one which recognizes the c-RNA polymerase. Mutants which lose the c-RNApolymeraseshould be unable to ferment a variety of carbohydrates. Such pleiotropic fermentation mutants are common. Some of these have been shown to lack either Enzyme I or the heat-stable protein which functions in many carbohydrate transport systems (see Sections V.C, p. 305). I n others, these two components are present and the defect has not been identified. Some of this latter group may be pleiotropic negatives because they have low c-RNA polymerase activity and are unable to make all catabolite-repressible proteins, including a number of metabolic enzymes and specific carbohydrate permeases. From such mutants, two types of revertants able to ferment all sugars would be expected: those which have regained the original form of c-RNA polymerase, and those in which an altered c-RNA polymerase insensitive to catabolite repression is produced. Mutations should also occur which restore the function of individual operons by

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altering their promotor regions to permit recognition of other forms of RNA polymerase. Such mutants would be resistant to catabolite repression and, if they are small deletions covering an adjacent operator, may be constitutive as well. Indeed, Wang and Morse (1968) have reported that mutants of E. coli selected for lac function from a pIeiotropic fermentation-negative strain are often constitutive. Silverstone et al. (1969))assuming the existence of a single form of RNA polymerase, have suggested that catabolite repression acts on the complex of the polymerase with the promotor region.

D. IDENTITY OF THE EFFECTOR 1. Generul Considerations

I n both catabolite repression and transient repression, the rate of enzyme synthesis is thought to reflect the concentrations of one or more effectors that can be identified with components of the pool of intermediary metabolites. The non-committal term “effector” is preferable to the frequently used expression “co-repressor” since (a) catabolite repression does not always involve control of m-RNA synthesis, (b) as yet there is no evidence that an apo-repressor is involved in the catabolite repression of P-galactosidase, and (c) the recent work on cyclic AMP suggests that onset of catabolite and transient repression may reflect the absence rather than the presence of a key compound. It is not certain whether any micro-organism uses a single effector to control synthesis of all catabolite-repressible enzymes. At least one experiment suggests that different effectors may be involved. I n Aerobacter aerogenes, repression of histidase synthesis is more effective when both glucose and ammonia are present, although the presence of ammonia has no effect on the repression of synthesis of inositol dehydrogenase (Neidhardt and Magasanik, 1957a). Thus, the effector for histidase synthesis is probably a nitrogen-containing derivative of glucose, but the one for synthesis of inositol dehydrogenase is not. McFall and Mandelstam (1963a, b) have proposed that catabolite repression does not result from the action of a few generalized effectors. Instead, catabolite repression is the sum of a series of specific end-product repressions, each sensitive enzyme being repressed by its own metabolic products. Glucose is effective as an exogenous repressor simply because a variety of metabolites are easily derived from it. This hypothesis stemmed from their observation that galactose, an end-product of P-galactosidase action, was a better catabolite repressor for this enzyme than pyruvate, and that pyruvate, an end-product of action of both tryptophanase and D-serine deaminase, was a better repressor for these enzymes than was galactose. However, data from several laboratories

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KENNETH X’AIOEN AND BEVERLY WILLIAMS

do not support this view, and it is unlikely that the effectors for catabolite repression can be identified with a set of specific metabolic end-products. Englesberg et al. (1961), using mutants of Salmonella typhimurium, concluded that glucose does not repress induction of citrate permease by citrate or induction of histidase by histidine, by giving rise to metabolic products of these reactions. Moreover, among the metabolites which might reasonably be considered as specific end-products of lactose metabolism, neither galactose nor galactose 1-phosphate represses ,&galactosidase synthesis (Paigen, 1966b; Beggs and Rogers, 1966). L -ARABiNOSE LACTOSE

P

L-RiBULOSE

J

L-RIBULOSE-5-P

6-P-GLUCONATE

2

GLUCOSAMINE-6-p

FRUCrOSE-I 6-dj-P

N-ACETYLGLUCOSAMINE-6-P

N-AC-GLUCOSAMINE-I-P

SEOOHEPTULOSE-7-P

1.3-Dl-P-GL YCERATE

2-KErO-3-DEOXY-6-P- GLUCONArE

3-P-GL YCERATE ACETYL-CoA

c PYRUVATE

a

*

P-ENOLPYRUVATE

@2-P-GLYCERAE

FIG.6. Carbohydrate intermediary metabolism. Double arrows indicate reactions in which separate enzyme systems are responsible for reaction in each direction. Open arrows indicate major exit routes. P indicates a phosphate residue.

UDP-Galactose, which is also an end product of lactose metabolism, does function as a specific repressor (Paigen, 1966b). However, this specific repression is distinct from catabolite repression since glucose continues to repress the lac operon in strains unable to synthesize UDP-galactose. I n the case of the gal operon, repression by glucose exists in mutant strains unable to synthesize any members of the gal pathway, including gaIactose I-phosphate, UDP-galactose, and UDP-glucose, or any metabolite derived from these compounds (Paigen et al., 1967). I n no system has the effector been positively identified, nor is it certain that each system has only a single effector. The interpretation of experiments in this area is made difficult by the “looped” rather than simple linear pathways of carbohydrate metabolism (Fig. 6), the complex and

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295

poorly understood mechanisms by which these reactions are regulated, the sparing effects that one carbohydrate may have on the metabolism of another, and the roles which metabolic intermediates play as regulatory effectors for other reactions. For example, formation of glyceraldehyde 3-phosphate from glucose 6-phosphate in E . coli can proceed via direct glycolysis (Embden-Meyerhof pathway), oxidation and cleavage (Entner-Doudoroff pathway), or oxidation and re-arrangement (hexosemonophosphate shunt). The reverse reaction, gluconeogenesis, occurs by by-passing one irreversible reaction in glycolysis (that catalysed by phosphofructokinase) with another irreversible reaction (catalysed by hexosediphosphate phosphatase). Moreover, these reactions serve not only to provide for the catabolism of sugars and the generation of ATP and pyruvate, but also to supply important biosynthetic intermediates in the form of ribose 5-phosphate for nucleic acid biosynthesis, hexoses and hexosamines for cell-wall synthesis, and glucose 1-phosphate for glycogen synthesis. Within this network, it is probable that changing the rate at which any reaction proceeds, whether it be by mutation, addition of an inhibitor, or changing carbon sources, will have considerable effects on the concentrations of a number of metabolic intermediates. Despite these difficulties, the present data suggest that the effector for /3-galactosidase is one of a limited group of metabolites. 1. Cutabolite Repression of /3-Guluctosiduse Synthesis

The use of mutants, blocked in various reactions, has provided an important tool in the analysis of this problem. As we have mentioned, glucose continues to produce catabolite repression in mutants unable to synthesize UDP-galactose or UDP-glucose (Paigen et ul., 1967),thereby permitting us to conclude that the effector cannot be either of these compounds or any other sugar nucleotide derived from them. A further limitation is set by studies in mutants lacking glucosephosphate isomerase, and hence the ability to interconvert glucose 6-phosphate and fructose 6-phosphate (Loomis and Magasanik, 1966). Such a mutant is unable to form glucose 6-phosphate or 6-phosphogluconate from compounds beyond fructose in metabolism. Since this strain continues to show catabolite repression with fructose, xylose, lactate, and succinate, we can further eliminate glucose 1-phosphate, glucose 6-phosphate, 6-phosphogluconolactoneand 6-phosphogluconate. Parenthetically, it should be noted that there are two unusual features of the behaviour of the glucosephosphate isomerase-negative mutant. One is that this strain has lost catabolite repression by glycerol; the other, that glucose and gluconate continue to repress. There is no ready explanation for the first observation. The second is probably accounted

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KENNETH PAIGEN AND BEVERLY WILLIAMS

for by the operation of the Entner-Doudoroff pathway and hexosemonophosphate shunt in this organism. These reactions, together with those catalysed by aldolase and hexosediphosphate phosphatase, will permit the cell to form any intermediate from glucose that can be formed from fructose. This interpretation is supported by the fact that in Salmonella which is induced for enzymes of the Entner-Doudoroff pathway only after growth on gluconate (Loomis and Magasanik, 1966), a similar mutant deficient in glucosephosphate isomerase lacks catabolite repression by glucose (Fraenkel et al., 1963, 1964). Within the same area of metabolism, a mutant of Aerobacter which oxidized glucose to 6-phosphogluconate poorly was relatively resistant to catabolite repression of histidase synthesis (Neidhardt, 1960). This difference may reflect the use of another organism, the measurement of a different catabolite repression-sensitive enzyme, or the ambiguity regarding the precise metabolic block in this mutant (Magasanik and Bojarska, 1960). Tricarboxylic acid cycle intermediates are probably not responsible for catabolite repression since a mutant lacking lipoate acetyl-transferase, and hence unable to synthesize these intermediates, retains glucose repression of P-galactosidase synthesis (Loomis and Magasanik, 1966). A mutant of Salmonella which was also unable to form tricarboxylic acid cycle intermediates from hexoses, glycerol, or pyruvate retained glucose repression of synthesis of both citrate permease and histidase (Englesberg et al., 1961). Strains resistant to glucose repression were selected from this mutant, the majority of which proved to be defective in glucose transport. Many of these glucose-resistant mutants also had elevated levels of acid phosphatase, a fact later accounted for by the observation that this enzyme is sensitive to catabolite repression (Hsie and Rickenberg, 1967). Intermediates on several ancillary pathways also have been eliminated by the use of mutants ;these include galactose and galactose 1-phosphate (Paigen, 1966b; Beggs and Rogers, 1966) and L-arabinose, L-ribulose and ribulose 5-phosphate (Eichliorn and Nolte, 1965). The studies with mutants are consistent in suggesting that the P-galactosidase effector lies a t or below the level of fructose 6-phosphate and D-ribulose 5-phosphate, and at or above pyruvate. Based on the observation that N-acetylglucosamine is effective in producing catabolite repression in wild-type cells, Dobrogosz (1968a, b) has suggested that the effector is a derivative of fructose 6-phosphate, namely a phosphorylated N-acetylhexosamine. Additional evidence for this view was obtained from studies of two mutants blocked in the dissimilation of N-acetylglucosamine, one unable to deacetylate N-acetylglucosamine and the other unable to deaminate glucosamine (Dobrogosz, 1969).

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These mutants can incorporate N-acetylglucosamine into cell-wall material, but are unable to utilize it as a growth substrate as do wild-type cells. During growth of the mutants on gluconate, addition of N-acetylglucosamine to the medium at low concentrations produced catabolite repression, suggesting that N-acetylglucosamine 6-phosphate or a derivative of this compound is the effector. Some ambiguity is introduced into the interpretation of these experiments by the fact that N-acetylglucosamine 6-phosphate is not physiologically inert. Although it does not produce deleterious effects during growth on gluconate, it is a severe growth inhibitor when other substrates are used, and must be presumed to interfere with reactions in carbohydrate metabolism. 3. Transient Repression of fl-GalactosidaseSynthesis

I n Escherichia COGstrain Q22, only glucose, among a large number of carbohydrates tested, was able to produce transient repression (Paigen, 1966a).Moreover, cells were sensitive t o transient repression by glucose after growth on all carbon sources tested except glucose and, to some extent, gluconate. These observations suggest that the effector of transient repression is closely related to glucose in metabolism. Prevost and Moses (1967) have examined the metabolic behaviour of strain Q22, which is TRs, and its wild-type counterpart, which is TR+, immediately after a shift from glycerol-containing to glucose-containing medium. During growth on glycerol, both TR+ and TRs cells had similar levels of glucose 6-phosphate, fructose diphosphate, ATP and NADPHz ; however, TRa cells had twice as much NADP and appreciable amounts of 6-phosphogluconate, a compound not found in TR+ cells. After the shift to glucose-containing medium, TRs but not TR+ cells showed transient rises in the concentrations of glucose 6-phosphate, fructose diphosphate, and 6-phosphogluconate. These results suggest that the effector molecule might be either glucose 6-phosphate or fructose diphosphate. 6-Phosphogluconate is an unlikely candidate since both TRs and TR+ cells synthesized P-galactosidase a t equal rates in glycerol-containing medium even though these strains contained very different concentrations of 6-phosphogluconate. Perlman and Pastan (1968a) reported that cyclic AMP reverses the glucose repression of P-galactosidase and tryptophanase synthesis in E . coli. Transient repression was reversed by 1mM-cyclic AMP (Perlman and Pastan, 1968b) while catabolite repression was reversed only at higher concentrations (Perlman et al., 1969). The ability to reverse transient repression was specific to cyclic AMP; ATP, ADP, 5’-AMP, 3’-AMP and fructose 1,6-diphosphate were inactive (Perlman and Pastan, 1968a).

KENNETH PAIGEN AND BEVERLY WILLIAMS 298 The ability of cyclic AMP to reverse glucose repression has been confirmed by two other laboratories (Goldenbaum and Dobrogosz, 1968; Ullmann and Monod, 1968)although a clear distinction between transient and catabolite repression was not made. Goldenbaum and Dobrogosz (1968)also showed that a concentration of cyclic AMP which completely reversed repression by glucose only partially reversed the stronger repression produced by glucose 6-phosphate or a mixture of glucose and gluconate. Perlman and his coworkers (1969) have confirmed that higher concentrations of cyclic AMP are required to reverse more intense repression. Ullmann and Monod (1968)report that the presence of cyclic AMP eliminated diauxie. These results suggest that either cyclic AMP is the effector for transient repression or that cyclic AMP affects an enzyme which is involved in metabolism of the effector. The results obtained with the TR" mutant of E . coli implicate the sequence of reactions between glucose 6-phosphate and fructose diphosphate in the control of transient repression. I n this sequence, glucose 6-phosphate and fructose 6-phosphate are in equilibrium with each other. Thereactions from fructose 6-phosphate to fructose diphosphate and from fructose diphosphate to fructose 6-phosphate are both irreversible and are catalysed by two independent enzyme systems. I n many organisms, both enzymes are regulated in part by cyclic AMP. Either cyclic AMP is the effector of transient repression, and the changes seen in glucose 6-phosphate and fructose diphosphate concentrations in the TR" mutant are caused by altered concentrations of cyclic AMP, or one of the three hexose phosphates is the effector, and cyclic AMP acts by inducing a change in its concentration. I n this regard, Makman and Sutherland (1965) demonstrated that the concentration of cyclic AMP in E . coli varies inversely with the concentration of glucose in the growth medium. Addition of glucose not only suppresses the formation of cyclic AMP, but causes the cells to excrete 99% of their internal cyclic AMP into the medium. Glucose was not specific since several other sugars were able to produce the same effect.

IV. Catabolite Inhibition A. DEFINITION AND PROPERTIES The significance of a control mechanism which influences the activity as opposed to the concentration of a carbohydrate-metabolizing enzyme is readily appreciated since bacteria have a limited ability to change enzyme concentrations. Protein turnover is relatively slow, reaching its maximum value of approximately 5% per hour only under non-growing conditions (Mandelstam, 1963).I n order to decrease the concentration of

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an enzyme, cells must dilute it by growth; consequently, the concentration of a repressed enzyme will be decreased to only one-half at the end of one generation and to one-quarter after two generations. Attainment of an optimal enzyme concentration is equally slow, Although the maximum rate of induced enzyme synthesis can be achieved within a few minutes, the total concentration of enzyme reaches one-half of the maximum after only one generation of growth in inducer and three-

c

._ c

08

._ Fi ._ c 3

m 0 c

04

-u 0

Lc 0

a,

. I -

02

0

30 60

90

I20

0

30

90 120 Time(min.)

60

0

30

60

90

120 I!

FIG.7. Catabolite inhibition of lactose utilization in Escherichia coli. The rate of lactose utilization is plotted on a logarithmic scale as a function of time for three exponentially growing cultures. Cultures a and b were grown in lactose labelled with 1% in the glucose moiety, and the formation of either radioactive carbon dioxide (a)or acid-insoluble products (b)was used t o measure substrate utilization. I n experiment c, cells lacking galactokinase and unable to ferment galactose were grown on lactose labelled in the galactose moiety. I n each case, the rate of substrate utilization by a control culture ( 0- 0)increased exponentially as the culture grew. Addition of glucose to a parallel culture (e-.), a t the time indicated by the arrow, resulted in an immediate and similar inhibition in all three cultures. I n experiment b, after 90 min., the control and experimental cultures were centrifuged and the cells resuspended in fresh medium containing labelled lactose but no glucose. The control rate of lactose utilization was immediately recovered indicating that the inhibition is completely reversible. After McGinnis and Paigen (1969).

fourths of the maximum after two generations. Accordingly, if a bacterium is to make a rapid and effective choice between alternate substrates, it must employ a mechanism that acts directly on the activities of preformed enzyme molecules. Catabolite inhibition is such a mechanism in which glucose inhibits the activity rather than the formation of enzymes involved in the utilization of other carbohydrates. A clear demonstration that catabolite inhibition is physiologically significant is seen in the response of an exponentially growing culture of E. coli when glucose is added to a lactose-containing medium (Pig. 7).

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KENNETH PAIGEN AND BEVERLY WILLIAMS

The incorporation of labelled carbon from lactose into either carbon dioxide (Fig. 7a) or acid-insoluble cell material (Fig. 7b) is immediately inhibited. Several facts indicate that this effect is due to inhibition of pre-existing enzyme molecules rather than to repression. First, the rate of lactose metabolism falls to a level lower than that seen prior to glucose addition. Secondly, the original rate is immediately recovered if glucose is removed (Fig. 7b). Thirdly, during the period of inhibition, the number of /3-galactosidase molecules estimated by enzyme assay remains constant. The site at which glucose inhibits lactose metabolism was tested in cells unable to metabolize galactose. I n this strain, hydrolysis of lactose labelled in the galactose moiety is accompanied by the accumulation of free labelled galactose. This accumulation also was inhibited by glucose (Fig. 7c), indicating that the inhibition must be exerted either on the entry of lactose into the cell or its hydrolysis by /3-galactosidase. Glucose is able to inhibit the metaboIism of all other carbohydrates tested including lactose, galactose, maltose, mannose, xylose, arabinose and glycerol. Only glucose and, to a lesser extent, glucose 6-phosphate act as inhibitors. Using mutants blocked at later stages of galactose metabolism, the site of inhibition also was shown to be either at the level of entry into the cell or a t the first enzyme, namely galactokinase (McGinnis and Paigen, 1969).

B. EXAMPLES OF CATABOLITE INHIBITION The existence of catabolite inhibition as a control mechanism was clearly recognized by Gaudy et al. (1963)and Stumm-Zollinger (1966)in their studies of carbohydrate utilization by mixed populations of bacteria derived from the activated sludge of sewage treatment plants. When such heterogeneous microbial populations were grown in a mixture of glucose and sorbitol, glucose inhibited sorbitol utilization even after prior adaptation to sorbitol (Gaudy et al., 1963). A similar result was obtained using a pure culture of E . coli. Stumm-Zollinger (1966)reported that glucose inhibited galactose utilization in a mixed culture from sewage which was first adapted to galactose and then fed a mixture of the two sugars. The same phenomenon had been observed even earlier (Table 2, p. 301). The earliest example of catabolite inhibition probably was the report by Woods (1935)that production of indole from tryptophan by washed cell suspensions of E. coli was inhibited by glucose, an observation confirmed by Mastafa (1937),Silberstein (1941))and Evans et al. (1942).I n a Hydrogenomonas, molecular hydrogen inhibits fructose oxidation (Blackkolb and Schlegel, 1968)and fructose can, in turn, inhibit oxidation of aspartate or glutamate (Schlegel and Triiper, 1966). I n the anaerobe Clost-

TABLE2. Examples of Catabolite Inhibition

Metabolic Reaction Sorbitol utilization Galactose utilization Carbohydrate utilization (lactose, maltose galactose, mannose, xylose, L-arabinose, glycerol) Glucose utilization

Organism

Comments

Reference

Heterogeneous population Heterogeneous population Escherichia coli

Catabolite inhibition by glucose in Gaudy et al. (1963) activated sludge Catabolite inhibition by glucose in Stumm-Zollinger (1966) activated sludge Inhibition by glucose and glucose McGinnis and Paigen (1969) 6-phosphate

Escherichia coli

Inhibition by glucose 6-phosphate and gluconate Utilization of glucose, but not glutamate, inhibited by yeast extract Inhibition following exposure to molecular hydrogen, probably at the glucose 6-phosphate dehydrogenase reaction I n &tro inhibition by fructose diphosphate I n vitro inhibition by gIucose Inhibition of indole production from tryptophan by washed cell suspensions Inhibition by fructose

McGinnis and Paigen (1969)

Inhibition of antibiotic synthesis Inhibition of incorporation into cell-wall material Glucose inhibits hydroxyproline reduction, but not alanine oxidation, in washed cell suspension

Kimura (1967a, b) Bates and Pasternak (1965b)

Glucose utilization

Clostridium tetanomorphum

Fructose oxidation

Hydrogenomonas sp.

Glycerol kinase

Escherichia coli

j?-Glucosidase Tryptophan degradation

Schizophyllum commune Escherichia coli

Aspartate and glutamate oxidation Siomycin formation N-Acetylglucosamine uptake Hydroxyproline reduction

Hydrogenomonas sp. Streptmyces sioyaensis Bacillus subtilis

Clostridiurn sporogenes

Anthony and Guest (1968) Blackkolb and Schlegel (1968)

Zwaig and Lin (1966) Wilson and Niederpruem (1967) Mastafa (1937); Woods (1935); Silberstein (1941); Evans et al. (1942) Schlegel and Truper (1966)

Raynaud and Macheboeuf (1946)

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ridium tetanomorphum, yeast extract inhibits glucose utilization (Anthony and Guest, 1968).These observations suggest that a generalized system of catabolite inhibition exists by which microbial cells choose a preferred carbon source. This inhibition acts on pre-existing enzymes and is distinct from catabolite repression. Catabolite inhibition probably explains the ability of glucose to relieve the growth stasis that can be induced in certain mutants defective in carbohydrate metabolism. Fermentation mutants are often severely inhibited by the sugar whose catabolism is blocked, particularly if phosphorylated intermediates accumulate. This effect has been observed, for example in transferase- and epimerase-deficient mutants of galactose metabolism in E. coli (Kurahashi and Wahba, 1968; Yarmolinsky et al., 1959; Fukasawa and Nikaido, 1961; Nikaido, 1961; Sundararajan et al., 1962; Elbien and Heath, 1965),and in glycerophosphate dehydrogenasedeficient mutants of glycerol metabolism (Cozzarelli et al., 1965). The addition of glucose relieves the growth stasis of sensitive mutants in media containing glycerol (Cozzarelliet al., 1965)or galactose (Fukasawa and Nikaido, 1961) probably by preventing the formation of inhibitory products. Glucose also inhibits some synthetic processes. Although, strictly speaking, these are not examples of catabolite inhibition as defined above, since alternative growth substrates are not involved, it is nevertheless likely that they involve the same basic mechanism. For example, production of the antibiotic siomycin by resting-cell suspensions of Streptomyces sioyaensis is inhibited by glucose (Kimura, 1967b) and, in Bacillus subtilis, incorporation of labelled glucosamine and N-acetylglucosamine into cell-wall material also is inhibited by glucose (Bates and Pasternak, 1965b). c . MECHANISM O F CATABOLITE INHIBITION

It is not known whether inhibition is exerted a t the level of entry of substrates into the cell or on the first enzyme that catalyses metabolism of the substrate; adequate precedents exist for both. Observations of enzyme inhibition in. vitro have been reported for the P-glucosidase of Schizophyllum commune by glucose (Wilson and Niederpruem, 1967)and for the glycerokinase of E. coli by fructose 1,6-diphosphate (Zwaig and Lin, 1966).Inhibition by glucose of uptake of various sugars is common and may proceed by competition between various sugar permease complexes for a limited supply of the phosphorylated heat-stable protein which serves 8s the energy donor for many transport systems (Kundig et al., 1964). For a more detaiIed discussion of permease inhibition, the reader is referred to the sectiononinducer transport (Section V.C, p. 305).

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Glucosenot only inhibits enzyme formation by catabolite and transient repression, but also may do so indirectly through the operation of catabolite inhibition. For those operons in which the product of the first metabolic enzyme is the effective inducer (lactose, glycerol and histidine catabolism), catabolite inhibition will always act to inhibit enzyme synthesis by preventing the accumulation of effective inducer molecules. Por those operons in which the first substrate is the effective inducer, as in galactose catabolism, enzyme synthesis will also be inhibited if glucose prevents entry of inducer. The evolution of operons that are induced by the first reaction product, rather than the first substrate, may reflect the selective advantage of having a second level of control over enzyme induction. Catabolite inhibition is a control exerted on enzyme activity rather than on enzyme formation, analogous to feedback inhibition in biosynthetic pathways. However, these two inhibitions differ in an important regard in their secondary effects on enzyme synthesis. I n biosynthetic pathways, feedback inhibition Iimits production of the metabolic endproduct, and this effect is partially compensated for by derepression of the sensitive pathway. The combined action of feedback inhibition and end-product repression tends to maintain an intermediate level of enzyme activity. I n contrast, catabolite inhibition tends to repress further the sensitive pathway. The combined action of catabolite inhibition and substrate induction produces an all-or-none state of enzyme biosynthesis.

V. Control of Inducer Concentration A. GRATUITY When a cell is exposed to exogenous inducer, among the conditions which must be fulfilled before the appearance of induced enzyme are: (a) the inducer must penetrate the cell either by passive diffusion, facilitated diffusion or active transport ; (b) inducer must be retained within the cell; (c) internal inducer may be required to undergo a metabolic conversionto generate the true effector (lactose, glycerol, histidine) ; and (d) an energy-generating system must be present. These conditions will not be met if one or more of these activities requires the participation of the enzyme to be induced or if glucose inhibits one of these activities. Circumstances under which the induced enzyme does not participate in its own induction are referred to as gratuitous conditions (Benzer, 1953). Such would be the case for P-galactosidase if isopropylthiogalactoside were added at high concentration to cells adapted to and growing on glycerol. Non-gratuitous conditions for P-galactosidase synthesis would exist if cells were first grown on glycerol and then transferred to lactose.

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I n this case, the lac permease is required to transport lactose into the cell, P-galactosidase is required t o transgalactosylate lactose to form the effective inducer, and both activities are required to generate glucose from lactose to provide a source of energy for transport and protein synthesis. Since both catalytic activities are very low in uninduced cells (approximately 1-2 molecules per cell), cells experience a considerable delay in initiating a rapid rate of enzyme synthesis. Indeed, a population of cells transferred t o minimal lactose medium responds in a very heterogeneous fashion. Any cell which, by chance, has a slightly higher activity has a much better chance of making more enzyme, and any additional enzyme greatly increases the rate of induction. The kinetics of this process have been elegantly analysed by Benzer (1953) who showed that, for individual cells, transition from the basal to the induced rate of enzyme synthesis (an increase of approximately a thousand-fold) is very rapid. During the early stages of induction, a culture consists of a mixture of uninduced and fully induced cells. This heterogeneity is duo to the considerable variation in time at which cells, by chance, get started. B. LONG-TERM ADAPTATION The difficulty of becoming induced under non-gratuitous conditions has proved to be the explanation for a form of cultural differentiation in micro-organisms.First discovered by Winge and Roberts in yeast (1948), it was extensively studied in yeast by Spiegelman and his coworkers (Spiegelman, 1946, 1951, 1954; Spiegelman and DeLorenzo, 1952; Spiegelman et al., 1950,1951) and in E . coli by Novick and Weiner (1957, 1959) and by Cohn and Horibata (1959a, b, c). The phenomenon is best described by considering an experiment in which a culture of E. coli, grown in glycerol minimal medium, is divided into three parts. To one part, a low concentration of the inducer thiomethylgalactoside, just sufficient to induce the maximal rate of enzyme synthesis, is added. To the second part, glucose is added 15 min. after the inducer. Due to catabolite repression, this culture will maintain a rate of induction only half that of the first culture. To the third part, glucose and inducer are added simultaneously. I n this culture, no induction results. The difference between the second and third cultures, both growing in media of the same composition, is due to formation of some permease molecules in the second culture before glucose is added and catabolite repression begins. Additional permease activity allows a higher internal concentration of inducer to form which in turn generates the capacity further to induce the permease. When glucose is added simultaneously, the cells never have a chance t o get started. The different cellular phenotypes can be maintained for many generations of growth, even in mixed culture. I n

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yeast, the different phenotypes are transmitted through the cytoplasm during sexual recombination.

C. INDUCER ENTRY Glucose can inhibit the uptake of other carbohydrates, including galactose (Horecker et al., 1960;Adhya and Echols, 1966;Kamogawa and Kurahashi, 1967),P-galactosides (Kepes, 1960; Kessler and Rickenberg, 1963;Koch, 1964;Winkler and Wilson, 1967),maltose (Egan and Morse, 1966)and sucrose (Egan and Morse, 1966).Glucose also inhibits partially the uptake of tryptophan (Boezi and DeMoss, 1961) and histidine (Ames, 1964).Little information is available concerning the effect of glucose on the entry of other compounds such as amino acids or fatty acids whose catabolism is subject to catabolite repression. Inhibition of carbohydrate uptake by glucose in some cases requires prior growth of the cells on glucose (Koch, 1964)implying that external glucose does not directly inhibit other specific permeases. Rather, the data suggest a competition between active permeases for a limited quantity of a common factor required for carbohydrate transport (Kepes, 1960;Kessler and Rickenberg, 1963;Koch, 1964).This common factor has been tentatively identified (Tanaka and Lin, 1967;Tanaka et al., 1967)with the phosphotransferase system of Kundig and his coworkers (1964,1966) that is lost in some pleiotropic carbohydrate non-fermenting mutants (Doudoroff et al., 1949;Asensio et al., 1963;Egan and Morse, 1965;Tanaka andLin, 1967;Tanaka e t a l . , 1967;WangandMorse, 1968). In this system, enzyme I catalyses the transfer of a high-energy phosphate from phosphoenolpyruvate to a heat-stable protein (HPr). The product, P-HPr, acts as the high-energy phosphate source for a set of carbohydrate-specific enzymes, enzymes 11, which catalyse the phosphorylation and, presumably, the transport of each sugar. Different sugarenzyme I1 complexes probably compete for a limited supply of P-HPr. If so, the amount of glucose-enzyme II,,,, that can be formed, and its affinity for P-HPr, compared to the amounts and affinity of another sugar-enzyme I1 complex, will determine the intensity with which the two sugars inhibit each other’s entry. Since enzymes I1 are generally inducible, this would explain why cells sometimes must be grown in a carbon source for that compound to inhibit the uptake of another sugar (Kessler and Rickenberg, 1963). The contribution which inhibition of inducer entry makes to the intensity of glucose repression varies markedly from one system to another. For the lac operon of E. coli, it is insignificant when analogue inducers, like thiomethylgalactoside and isopropylthiogalactoside, are used a t saturating concentrations (< 0.1 mM). Glucose does not repress j3-galac-

KENNETH PAICEN AND BEVERLY WILLIAMS 306 tosidase synthesis to any greater extent in a wild-type strain exposed to a saturating concentration of inducer than it does in a constitutive lac I mutant which does not depend upon an external supply of inducer. It is not known whether the glucose inhibition of /3-galactoside entry is strong enough significantly to increase the concentration of inducer required for 50% induction in lac+ cells. Conflicting data have been reported for a lac Y- strain, ML-3. Although Clark and Marr (1964)have reported that glucose increased the requirement for inducer in a catabolite repression-sensitive strain, Herzenberg (1959) reported that the intensity of catabolite repression was the same at all inducer concentrations, and that the concentration of isopropylthiogalactoside required to half-saturate induction was the same in media containing glucose or maltose. Boezi and Cowie (1961) found no increased requirement in a substrain insensitive to catabolite repression. Although it is of minimal significance for the lac operon, glucose inhibition of inducer entry is a very significant factor in the regulation of the gal operon. The major part of the sensitivity of the gal operon to glucose repression results from an inhibition of inducer entry (Adhya and Echols, 1966).This finding explains why induced enzyme synthesis in the gal operon is much more sensitive to glucose repression than that in the lac operon (Williams and Paigen, 1969). It also explains why gal constitutive mutants, either gal R- or gal 0") are only repressed about 50% by glucose (Adhya and Echols, 1966; Williams and Paigen, 1969), the same level to which lac is repressed, rather than the 90% or greater repression seen with induced wild-type cells. I n brief, both lac and gal are catabolite sensitive to apprdximately the same extent when inducer entry is not a significant factor, but the ability of glucose to inhibit galactose (or fucose) entry produces an additional inhibition of gal enzyme synthesis in induced wild-type cultures. Inhibition of inducer entry probably does not play a significant part in catabolite repression of tryptophanase in E. coli (Freundlich and Lichstein, 1960),or of inositol dehydrogenase and histidase in Aerobacter aerogenes (Magasanik, 1955; Neidhardt and Magasanik, 1956b).

D. EFFECTOR SYNTHESIS For some operons, the external and internal inducers are chemically identical. However, in several cases the product of the first metabolic step is the effector; examples include urocanic acid for histidine dissimilation (Schlesinger et al., 1965)) a-glycerophosphate for glycerol metabolism (Koch et al., 1964)) and a transgalactosylation product (possibly glycerol ,8-~-gdactoside) for lactose breakdown (Burstein et al., 1965).

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The experiments of Cozzarelli et al. (1965) on the formation of aglycerophosphate in E.coli provide the strongest indication of a possible control over effector synthesis. The reaction scheme involved is outlined in Fig. 8. Mutants lacking a-glycerophosphate dehydrogenase accumulate internal a-glycerophosphate when exposed to either glycerol or a-glycerophosphate, and the accumulated phosphorylated compound causes growth stasis. Dehydrogenase-negative mutants can become resistant to glycerol if a second mutation causes loss of glycerol kinase, and resistant to a-glycerophosphate if a second mutation causes loss of a-glycerophosphate permease. The growth stasis of dehydrogenasenegative single mutants can be relieved by adding glucose. Although GLYCEROL

a - GLYCEROPHOSPHATE

FIG.8. The metabolism of glycerol and a-glycerophosphatein Escherichia coli.

Cozzarelli et al. (1965) originally suggested that glucose acted by increasing the levels of other phosphorylated intermediates and relieving the metabolic inhibition by a-glycerophosphate, it seems more probable that catabolite inhibition prevents the accumulation of internal aglycerophosphate. Catabolite inhibition may act by inhibiting the transport of glycerol or a-glycerophosphate or by inhibiting glycerol kinase, an enzyme known to be inhibited by fructose diphosphate (Zwaig and Lin, 1966). There is some suggestion that /?-galactosidase, which is required for the synthesis of active inducer from lactose, may possess binding sites other than for its substrate. Pructose diphosphate, and to a lesser extent glucose 6-phosphate, increase the thermolability of /?-galactosidase, suggesting that these compounds can induce a conformational change in the enzyme (Gest and Mandelstam, 1966).A variety of related

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compounds were without effect, including lactose, glucose, glucose 1-phosphate, galactose, fructose, fructose 6-phosphate, 3-phosphoglycerate, melibiose, thiomethylgalactoside and isopropylthiogalactoside. The last two compounds can certainly complex with the active site of the enzyme since they act as competitive inhibitors of enzyme activity. Brewer and Moses (1967) subsequently tested a more extensive series of compounds with different results. They report that, in addition to fructose diphosphate, several other sugars and sugar phosphates were active, the most effective being fructose. These discrepancies would be accounted for if increased thermolability resulted from the oxidation of enzyme-SH groups by heavy-metal contaminants present in some reagents. This interpretation is supported by the observations that sugars and sugarphosphates readily form heavy-metal complexes and are easily contaminated during isolation, that the presence of mercapto-ethanol protected against the fructose diphosphate-induced lability (Gest and Mandelstam, 1966), and that only some preparations of glucose 6-phosphate were effective (Brewer and Moses, 1967).

E. SUMMARY Our understanding of the quantitative aspects of control over internal effector concentration is primarily derived from studies on the effects of gratuity and non-gratuity on induction. Historically, the early emphasis was on demonstrating that catabolite repression was a distinct and real phenomenon, not accounted for by inhibition of inducer entry by glucose. It is now known that glucose inhibition of enzyme synthesis can be achieved both by reducing the concentration of active internal inducer molecules and by inhibiting biosynthesis of enzyme protein. For most enzyme systems, the relative importance of these two mechanisms under natural circumstances remains an open question. For the gal operon, control of effector concentration predominates. For the lac operon, no answer is available since most experimental work has been carried out with saturating concentrations of analogue inducers. The discovery of catabolite inhibition suggests that control of effector concentration is widespread and physiologically important in regulating the synthesis of induced enzymes in other operons. Catabolite inhibition provides an efficient set of controls for choosing between competing metabolic pathways when these already exist in the cell. An important consequence of the operation of these controls is a decrease in effector concentration and a secondary inhibition of induced enzyme synthesis.

VI. Diauxie As first observed by Monod (1941,19421, bacterial cultures exposed to two carbon sources may use one until it is exhausted from the medium,

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and then undergo a growth lag before starting to utilize the other (Fig. 9). When more than two carbon sources are available, multiple growth cycles may be observed. During the growth lag, rates of oxygen uptake and macromolecular synthesis are low. Growth in this manner in the presence of two carbon sources is termed diauxic. Diauxic growth occurs whenever two essential conditions are met. First, adaptation to the less preferred or secondary carbon source is completely prevented in the presence of the preferred compound ;and second, the process of adaptation to the second substrate occurs under nongratuitous conditions. Combinations of substrates which do not meet these requirements are used either simultaneously or sequentially with

Time (hours)

FIG.9. Glucose-sorbitol diauxie in Escherichia coli. The optical density of a growing culture is plotted against time for E . coli growing in glucose-sorbitol mixtures containing (mg./l.): (A) 50 glucose and 150 sorbitol; (B) 100 glucoseand 100 sorbitol; (C) 150 glucose and 50 sorbitol. Glucose is consumed first and there is a growth lag before growth resumes on sorbitol. Reprinted form Monod (1947).

no intervening lag. Among those combinations which do meet the requirements for diauxic growth, the primary substrate is usually glucose or another readily utilizable compound and the secondary substrate is one whose utilization requires the synthesis of an inducible pathway. Microbial species, and even their substrains, vary considerably in their handling of different substrates. A combination of carbon sources which gives diauxic growth in one strain may not in another, and a compound which is a preferred substrate for one organism may act as a secondary source for another. Complete repression of the enzymes in a pathway for utilization of the secondary substrate may be achieved by catabolite repression itself or any other mechanism which decreases the internal concentration of active inducer. The requirement for non-gratuitous induction of the secondary pathway is met either if the substrate per-

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mease is required to generate internal inducer or if the induced enzymes are required to metabolize the carbon and energy supply. The glucose-lactose diauxie of E. coli provides a convenient model system for an appreciation of this phenomenon. Cells placed in a mixture of glucose and lactose begin growth immediately with glucose as the preferred substrate. A combination of factors prevents lactose induction of the lac operon proteins. Initially, effective levels of enzymes needed to generate internal inducer, lac permease and /I-galactosidase, are absent. Moreover, any ability to metabolize lactose is subject t o catabolite inhibition by glucose. Finally, if any internal inducer were made, the synthesis of lac operon proteins would be subject to catabolite repression. When the supply of glucose is exhausted, the culture behaves as if it were freshly transferred to lactose, with a heterogeneous response and a considerable delay before all of the cells resume growth. A change in conditions which either provides for lac protein synthesis in the presence of glucose or relieves non-gratuity after glucose exhaustion will eliminate the lag phase and will permit immediate sequential utilization of lactose. Thus, diauxie is missing in strains lacking catabolite repression (Englesberg, 1959; Loomis and Magasanik, 1967a) and in lac+strains when either an efficient inducer, such as isopropylthiogalactoside (Moses and Prevost, 1966), or a very high concentration of lactose (Loomis and Magasanik, 1967a) is present during growth on glucose. The requirement that a permease functions in promoting its own induction is often the major factor causing non-gratuitous induction. However, if another mechanism for inducer entry can be provided, nongratuity and hence one of the conditions necessary for diauxic growth disappears. Because the lac permease is also able to transport galactose, the pronounced glucose-galactose diauxie, present in wild-type E. coli strains, is absent in lac I-Y+mutants (Adhya and Echols, 1966;Lengeler, 1966) which have high levels of lac permease. It remains in lac 1- Ymutants which lack the permease. The differential rate of /I-galactosidase synthesis during the lag period between growth cycles is high suggesting the “preferential)) synthesis of the lac operon proteins during diauxic lag (Rickenberg and Lester,1955). However, as Mandelstam (1957) has pointed out, the rate of enzyme formation during the lag can be fully accounted for by the observed rates of protein turnover. Epstein and his coworkers (1966) measured the absolute rate of protein synthesis during the lag by isotope incorporation, and concluded that the differential rate of enzyme synthesis during the lag phase of glucose-lactose diauxie is indeed higher than during steady-state growth on lactose. However, it was not higher than the rate during growth in the presence of a strong inducer and a “neutral”

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carbon source such as glycerol. The decline in differential rate at the end of the lag period results from the onset of catabolite repression once cells are fully adapted to lactose utilization and an internal supply of glucose is generated. The earlier results of Attardi et al. (1963) and Naono et al. (1965), reporting a preferential synthesis using isotope incorporation as a measure of protein synthesis, are probably accounted for by the use of cells whose protein had become labelled some time before the onset of the lag period. If protein is prelabelled, turnover is not detected, and the absolute rate of protein synthesis is underestimated. If two growth substrates are provided and the essential conditions are met, diauxic growth may result whether the substrates are used as a source of carbon or for any other purpose. Bourgeois et al. (1960) have demonstrated diauxie in prototrophic cultures provided with both an amino acid and an inorganic nitrogen source from which the cells can makethe amino acid. When the amino acid is exhausted from the medium, a growth lag occurs because the cells lack a sufficient supply of the amino acid to manufacture the enzymesrequired for its biosynthesis. Eventually the process of protein turnover permits some enzyme to be made, and growth resumes. Stumm-Zollinger (1966, 1968), who compared natural populations of micro-organisms, found that diauxie exists if the heterogeneous population is derived from a sewage plant but not if it is derived from a stream, suggesting that rapid growth rate and its attendant pressures may have been an important evolutionary factor in the acquisition of the control factors that produce diauxie.

VII. Acknowledgements The authors wish to thank Drs. Irwin Olsen, Shiro Tomino, and Herbert Weinfield for their heIpful comments and criticisms of the manuscript. The literature search for this review was completed in January, 1969. REFERENCES Adhya, 6. and Echols, H. (1966). J . Bact. 92, 601. Adler, J. and Templeton, B. (1967). J . gen. Microbiol. 46, 175. Amarasingham, C. R. and Davis, B. D. (1965). J . biol. Chem. 240, 3664. Ames, G. F. (1964). Archs Biochem. Biophys. 104, 1. Anand, N. and Davis, B. D. (1960). Proc. S y m p . Proteins. Mysore, India, pp. 100106. Anthony, C. and Guest, J. R. (1968). J . pn. Microbiol. 54, 277. Asensio, C., Avigad, G. and Horecker, €3. L. (1963). Arch8 Biochem. Biophys. 103, 299.

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N O T E ADDED I N P R O O F The papers which have appeared since our original survey was completed have been concerned with the role of cyclic AMP, the question of transcriptional or translational control, and additional reports of catabolite repression. Cyclic A M P . Chambers and Zubay (1969) have succeeded in demonstrating that cyclic AMP markedly stimulates /I-galactosidase synthesis in a cell-free system. Moreover, the cyclic AMP-stimulated synthesis was very sensitive to inhibition by purified lac repressor while synthesis in the absence of cyclic AMP was inhibited only by 50%. They suggest that the “irrepressible” synthesis results from initiation of m-RNA synthesis a t improper sites and that cyclic AMP enhances initiation at the promotor site. De Crombrugghe et al. (1969) have extended the range of cyclic AMP effects to several enzymes and organisms. Cyclic AMP stimulates the catabolite repressed synthesis of D-serine deaminase, thymidine phosphorylase, galactoside and arabinose permeases, and enzyme I1 of fructose transport. I n addition to E. coli, cyclic AMP is effective in Salmonella typhimurium, Serratia marcescens, Proteus inconstans, and Aerobacter aerogenes. Considerable progress has been made toward elucidation of the metabolism of cyclic AMP in bacteria. Tao and Lipmann (1969) and Ide (1969) have identified the hitherto elusive adenyl cyclase activity which synthesizes cyclic AMP in E . coli. ATP is the substrate, the pH optimum is 9.5, and the enzyme requires Mg2+and is bound to the cell membrane from which it can be released. Tao and Lipmann (1969) have purified the enzyme 100-fold and find that it is inhibited by fluoride and inorganic pyrophosphate. Ide (1969) reports that it is inhibited by pyridoxal phosphate and oxaloacetate and is weakly stimulated by phosphoenolpyruvate and guanosine monophosphate. A mutant deficient in adenyl cyclase activity has been isolated by Perlman and Pastan (1969).This strain does not ferment lactose, maltose, arabinose, mannitol, or glycerol and grows poorly on glucose, fructose, and galactose. The addition of cyclic AMP restores the ability t o utilize all of these carbon sources. Monard et al. (1 969) have reported a cyclic AMP degrading system in E. coli with three essential components. Component I, a protein of molecular weight above 200,000, is present in greatly decreased amounts in strain AB 267Pcc-1which is resistant to catabolite repression. Component 11, a protein of molecular weight less than 100,000, is missing from another strain resistant to catabolite repression, LA 12G. Component I11 is dialysable and its activity can be partially, but not

320

KENNETH PAIGEN AND BEVERLY WILLIAMS

completely, replaced by glucose 6-phosphate, fructose 6-phosphate, ribose 5-phosphate or 3-phosphoglyceraldehyde. The question of whether cyclic AMP acts directly on the regulatory machinery or indirectly by influencing the metabolism of the effector is not completely settled. The demonstration of an effect in vitro by Chambers and Zubay (1969) strongly suggests the effect is direct, an interpretation consistent with the properties of the cyclase-negative mutant of Perlman and Pastan (1969). Among other possible effectors, JaneZek and Rickenberg (1969) have shown that glucose 6-phosphate is the most efficient in producing catabolite repression and does so less than one minute after its addition to a culture. I n the same laboratory, Hsie et al. (1969) measured the steady-state concentration of glucose 6-phosphate, 6-phosphogluconate, and reduced NADP during growth in glycerol, glucose, and glucose plus gluconic acid in three strains sensitive to catabolite repression and in two strains resistant to catabolite repression. Since there was no correlation between the levels of any of these compounds and the degree of catabolite repression, the authors concluded that none of these compounds is a direct effector of catabolite repression. I n dealing with this same question, Pastan and Perlman (1969b) suggest that sugars need not be metabolized in order to produce repression and that they repress by interacting with the phosphotransport system to drive out cyclic AMP by some unknown mechanism. The suggestion is based on their data that glucose and a-methylglucoside continue to produce transient repression in mutants lacking Enzyme I or heat-stable protein, and thus unable to phosphorylate the sugars effectively, and that the same compounds can no longer repress a mutant lacking the glucose-specific Enzyme 11. Transcriptional or translational control. Efforts to identify the site(s) of action of catabolite repression have intensified. Wijk et al. (1969)have modified the earlier conclusion (Wijk, 1968e) that glucose represses a-glucosidasesynthesis in Saccharomyces carlsbergensis only by controlling the translation of a stable m-RNA. They now report that glucose also causes some decrease in the formation of the m-RNA. Glucose represses the synthesis of a sugar phosphatase in Neisseria meningitides (Lee et al., 1967). Since the synthesis, which occurs in the absence of glucose, is not inhibited by actinomycin D, Lee and Sowokinos (1969) conclude that regulation occurs during translation and assembly of enzyme subunits. This conclusion must remain open, however, since actinomycin D was not shown to enter the cells and had no effect on growth of the organism. Pastan and Perlman (1 969a) have reported that cyclic AMP stimulates tryptophanase synthesis in E. coli, at least in part, by increasing the rate

CATABOLITE REPRESSION AND OTHER CONTROL MECHANISMS

321

of messenger translation. This is in contrast to their earlier conclusion that cyclic AMP stimulation of ,8-galactosidase synthesis is solely at the level of transcription. Jacquet and Kepes (1969) agree with earlier conclusions that, in the case of 13-galactosidase,catabolite repression acts exclusively on transcription. Based on experiments using rifampicin as an inhibitor of RNA chain initiation and actinomycin D as an inhibitor of RNA chain elongation, they report that glucose represses and cyclic AMP stimulates the rate of m-RNA initiation, and that neither compound influences the rate at which m-RNA synthesis progresses, the efficiency of m-RNA translation, or the half-life of the messenger. Yudkin and Moses (1 969) have presented additional evidence to support their view that glucose does affect the translation of ,8-galactosidase messenger in E . coli. They examined the yield of ,8-galactosidase in cells where further transcription was blocked shortly after the addition of inducer either by the addition of phage T6 or by removal of inducer by filtration. Using either method, the addition of glucose during the subsequent translation period decreased the yield of enzyme. However, both experiments are open to methodological question. I n the filtration experiments, ,8-galactosidase synthesis was expressed as a function of time rather than total protein synthesis, and in comparing cultures no allowance was made for changes in protein synthetic capacity immediately after a shift from one carbon source to another. I n the case of T6 infection, glucose was effective in decreasing P-galactosidase synthesis only if added within 3 min. after T6 infection. If glucose was added 5 min. after T6, it had no effect upon subsequent translation even though half of the total enzyme translation had not yet occurred. Thus, the earlier addition of glucose was effective probably because messenger synthesis did not completely stop until several minutes after T6 addition. Yudkin (1969a) has concluded that the relative contributions of transcriptional and translational control vary between strains. He has also presented evidence that the prior growth history of strains affects their sensitivity to transient repression (Yudkin, 1969~). To eliminate any incidental strain differences that might obscure comparison of lac mutants, Yudkin (1969b)has introduced the principle of comparing the sensitivity to catabolite repression of ,8-galactosidase synthesized from a lac operon carried on an episome with transacetylase synthesized from a lac operon carried chromosomally.Appropriate structural gene defects assured that active enzymes of each type were produced by only one chromosome. I n the course of these experiments, he presented evidence for the co-ordinate catabolite repression of P-galactosidase and transacetylase and showed that a deletion in the /I-galactosidase gene did I J

322

KENNETH PAIGEN AND BEVERLY WILLIAMS

not affect the catabolite repression of transacetylase on the same chromosome. Occurrence of catabolite repression. Jensen and Neidhardt (1969) examined the level of histidase and the net consumption of histidine in chemostat cultures both a t different growth rates and during the transition from one growth rate to another. Catabolite repression of histidase is controlled by the balance between synthetic and degradative reactions and, at low growth rates, by an inhibition of in vivo enzyme activity analogous to catabolite inhibition. They have evaluated their experiments as a general model for the physiological problem of regulating metabolic pathways which produce intermediary metabolites, phosphate bond energy, and reducing capacity as three different products. Silver and Mateles (1969) used chemostat cultures to study the utilization of substrates in a mixture of glucose and lactose. Catabolite repression alone is not enough to cause diauxie. Diauxie only occurs if the culture is initially uninduced for lactose utilization and induction occurs under non-gratuitous conditions ; it does not occur in the presence of a gratuitous inducer or in lac constitutive mutants. Two additional extracellular enzymes are reported to be sensitive to catabolite repression, chitinase (Monreal and Reese, 1969) and polygalacturonic acid transeliminase (Hsu and Vaughn, 1969). I n Aerobacter aerogenes, synthesis of the citrate transport system is repressed by glucose, succinate, and mannitol (Villarreal-Mogueland Ruiz-Herrera, 1969), and in the yeast Rhodotorula, isocitrate lyase synthesis is repressed by glucose and 2-deoxyglucose (Guerritore et al., 1969). The latter authors also report that isocitrate lyase is inhibited by 6-phosphogluconate, suggesting the existence of an allosteric control mechanism. The glucose repression of phosphoenolpyruvate carboxykinase in both E. coli and Tetrahymena pyriformis has been studied by Shrago and Shug (1969)using wild-type strains and strains mutated in the glycolytic or pentose phosphate pathways. As mentioned previously, many proteins of the electron-transport system are subject to catabolite repression. Ubiquinone, a non-protein component of the mitochondria, is also decreased in amount during catabolite repression (Gordon and Stewart, 1969). This control is probably exerted at the biosynthetic pathway for ubiquinone since petite mutants, which lack functional mitochondria and are particularly sensitive to catabolite repression (Reilly and Sherman, 1965; Bowers et al., 1967; Mounolou et al., 1966), contain normal amounts of ubiquinone when grown under conditions of derepression. Catabolite repression of a biosynthetic process in a higher plant has been described in the thiamin pathway of the angiosperm Arabidopsis. This organism can grow on mineral agar without an organic carbon

CATABOLITE REPRESSION AND OTHER CONTROL MECHANISMS

323

source ;however growth is more rapid in the presence of glucose. If plants with leaky mutations a t three loci concerned with synthesis or coupling of the thiazole moiety of thiamin are exposed to glucose, growth is severely retarded. Normal growth resumes if thiamin is added. Mutants of a fourth locus that is concerned with synthesis of the pyrimidine moiety of thiamin are not sensitive to glucose. Since the enzymes involved cannot be assayed, it is not known whether this glucose effect is due to repression or inhibition (Li and Redei, 1969). Another possible example of glucose repression in man has been reported (Bacchus, 1969). Glucose infusion decreased the elevated excretion of certain hydroxylated metabolites of cortisol that occurred in a patient recovering from surgical removal of a pituitary tumour. The hydroxylated products involved are formed by the action of the hepatic microsomal 6j3- and 2cr-hydroxylases. I n Pseudomonas putida, succinate inhibits urocanase, the second enzyme in the histidine degradative pathway (Hug et al., 1968).Urocanate in turn inhibits histidase, the first enzyme of the pathway, leading to a sequential feedback inhibition that functions analogous to catabolite inhibition of carbohydrate breakdown. The literature search for this note was completed October 31, 1969. ADDITIONAL REFERENCES Bacchus, H. (1969). Metabolism 18, 277. Bowers, W. D. Jr., McClary, D. 0. and Ogur, M. (1967).J. Bact. 94, 482. Chambers, D. A. and Zubay, G. (1969). Proc. natn. Acad. Sci. U.S.A. 63, 118. de Crombrugghe, B., Perlman, R. L., Varmus, H. E. and Pastan, I. (1969).J. biol. Chem. 244, 5828. Gordon, P. A. and Stewart, P. R. (1969). Biochim. biophys. Acta 177, 358. Guerritore, A., Hanozet, G. M. and Cocucci, M. C. (1969). Experientia 25, 131. Hsie, A. W., Rickenberg, H. V., Schulz, D. W. and Kirsch, W. M. (1969). J. Bact. 98, 1407. Hsu, E. J. and Vaughn, R. H. (1969).J. Bact. 98, 172. Hug, D. H., Roth, D. and Hunter, J. (1968). J. Bact. 96, 396. Ide, M. (1969). Biochem. biophys. Res. Commun. 36, 42. Jacquet, M. and Kepes, A. (1969). Biochem. biophys. Res. Commun. 36, 84. Janedek, J. and Rickenberg, H. V. (1969). Polio microbiol., Praha 14, 287. Jenscn, D. E. and Neidhardt, F. C. (1969). J. Bact. 98, 131. Lee, Y-P. and Sowokinos, J. R. (1969).J. biol. Chem. 244, 1711. Lee, Y-P., Sowokinos, J. R. and Erwin, M. J. (1967).J. biol. Chem. 242, 2264. Li, S. L., and Redei, G. P. (1969). PI. Physiol., Lancaster 44, 225. Monard, D., Janedek, J. and Rickenberg, H. V. (1969). Biochem. biophys. Res. Commun. 35, 584. Monreal, J. and Reese, E. T. (1969). Can. J. Microbiol. 15, 689. Mounolou, J. C., Jakob, H. and Slonimski, P. P. (1966). Biochem. biophys. Res. Commun. 24, 218. 11*

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Pastan, I. and Perlman, R. L. (1969a).J. biol. Chem. 244, 2226. Pastan, I. and Perlman, R. L. (1969b). J. biol. Chem. 244, 5836. Perlman, R. L. and Pastan, I. (1969). Biochem. biophys. Res. Commun. 37, 151. Reilly, C. and Sherman, F. (1965). Biochim. biophys. Acta 95, 640. Shrago, E. and Shug, A. L. (1969). Archs Biochem. Biophys. 130, 393. Silver, R. S. and Mateles, R. I. (1969).J. Bact. 97, 535. Tao, M. and Lipmann, F. (1969). Proc. natn. Acad. Sci. U.S.A. 63, 86. Wijk, Van R., Ouwehand, J., Van Den Ros, T. and Koninsberger, V. V. (1969). Biochim. biophys. Acta 186, 178. Villarreal-Moguel,E. I. and Ruiz-Herrera, J. (1969). J. Bact. 98, 552. Yudkin, M. D. and Moses, V. (1969). Biochem. J. 113, 423. Yudkin, M. D. (1969a). Biochem. J. 114, 307. Yudkin, M. D. (196913). Biochem. J. 114, 313. Yudkin, M. D. (1969c). Biochim. biophys. Acta 190, 220.

AUTHOR INDEX Numbers in italics refer to the pages on which references are listed at the end of each article.

A Abbott, J., 37, 41 Abdulaev, N. D., 71, 9.9 Abendschein, P. A,, 182, 222 Abraham, E. P., 12, 36, 39 Abram, D., 50, 96 Abrams, A., 82, 85, 86, 96, 98 Acker, R. I?., 27, 39 Adam, K. M. G., 106, 128 Adelberg, E. A., 218, 221 Adhya, S., 258, 286, 305, 306, 310, 311 Adler, J., 266, 311 Adye, J. C., 28, 29, 42 Aggag, M., 95, 96 Agtarap, R.,78, 79, 96 Albers, R. W., 82, 84, 96, 97 Aldanova, N. A., 69, 71, 103 Alford, J. A., 8, 10, 18, 19, 20, 42 Allen, S. I,., 149, 155, 176 Allmann, D. W., 83, 100 Alonso, P., 148, 177 Amarasingham, C. R., 264, 268, 311 Amcs, G. F., 305, 311 Anagnostopoulus, C., 23, 26, 39 Anand, N., 274, 311 Anchel, M., 3, 39 Anderson, B., 305, 315 Anderson, R. L., 258, 317 Andreoli, T. E., 63,70,71,75, 77,78,96,104 Angus, T. A., 21, 41 Anthony, C., 301, 302, 321 Antoine, A. D., 17, 27, 39 Antonov, V. K., 74, 103 Appleman, D., 253, 266, 317 Argaman, M., 55, 102 Arima, S., 38, 42 Amaud, M., 263, 264, 268, 314 Artmen, If., 259, 263, 264, 314 Asai, J., 51, 83, 98, 99, 100 Asano, A., 64, 67, 96 Asbell, M. A,, 52, 91, 96, 97 Asensio, C., 305, 311 Asscher, A. W., 52, 104 Aszalos, A,, 18, 41 Attardi, G., 275, 311, 312, 314

Aubert, J. P., 266, 271, 317 Austin, M. L., 158, 167, 169, 170, 172, 173, 176

Avi-Dor, Y., 64, 67, 97 Avigad, G., 258, 305, 311, 315 Azzone, G. F., 75, 102

B Baarda, J. R., 6G, 67, 68, 71, 75, 77, 79, 82, 85, 86, 98

Bach, M. L., 6 , 7 , 15, 21, 39 Balassa, G., 33, 39 Balbinder, E., 143, 150, 169, 171, 176, 178 Band, R. N., 106, 107, 108, 116, 119, 120, 121, 122, 123, 124, 126, 127, 128

Bangham, A. D., 5L, 96 Barabas, Gy., 16, 39 Barker, D. C., 116, 128 Baron, C., 82, 85, 86, 98 Rartels, T. J., 259, 313 Bartholomay, A. F., 35, 40 Bartlcy, W., 259, 261, 263, 264, 265, 266, 268, 270, 317, 318

Bartnicki-Garcia, S., 266, 312 Basu, S. K., 274, 275, 372 Batchelor, F. R., 180, 221 Bates, C. J., 260, 301, 302, 312 Baudet, P., 18, 39 Bauer, H., 114, 128 Baum, H., 89, 102 Bautz, E. K. F., 292, 312 Bayan, A. P., 28, 39 Beale, G. H., 134, 139, 144, 145, 148, 149, 150, 151, 152, 153, 156, 157, 166, 167, 168, 169, 170, 175, 176, 177 Beck, C., 261, 263, 264, 266, 269, 312 Beckwith, J. R., 132, 177, 203, 222, 279, 289, 290, 293, 317 Beechey, R. B., 64, 83, 84, 85,96,100,102 Beers, C. D., 127, 128 Begg, R. W., 69, 1 0 1 Beggs, W. H., 273, 294, 296, 312 Beguin, S., 275, 314 BeIoR, R. H., 24, 41 325

326

AUTHOR INDEX

Ucnnctt,, It. E., 180, 221 Bent, D. F., 19, 27, 42 Benton, W. F., 106, 107, 108, 110, 113, 114, 116, 117, 118, 119, 120, 121, 122, 124, 128, 129 Rcnzer, S., 303, 304, 312 Hergdoll, M., 19, 39 Berger, A., 59, 60, 99 Berger, J., 18, 39 Berger, J. D., 159, 176 Borman, N., 253, 312 Bergmann, M., 179, 221 Bcrgter, F., 23, 41 Bergy, M. E., 62, 96 Bernard, C. W., 267, 271, 317 Ehnheirner, A., 21, 22, 39 Bornlohr, It. W., 7, 8, 14, 15, 33, 39, 42, 261, 262, 266, 271, 325 HertJand, A. U. 11, 259, 314 Bhagavan, N. V., 36, 39 Bielawski, J., 66, 75, 96 Birch, A. J . , 2, 39 Birdsell, D. C., 52, 56, 96 Bishop, J. O., 134, 135, 139, 17G Bjorlrlund, M., 5 , 20, 44 Blackkolb, F., 252, 253, 300, 301, 312 Blackmore, M. A., 265, 312 Blake, A., 84, 96, 104 Blaskovics, J . C., 111, 128 Blicharska, J., 263, 264, 268, 314 Bodanszky, A., 94, 96 Bodanszky, M., 2, 42, 94, 95, 96 Boddy, A., 204, 221 Boozi, J. A., 254, 305, 306, 312 Rojarslra, A., 296, 335 Honicke, R., 181, 221 Boroff, D. A., 19, 39 Borowski, E., 62, 96 Boschwitz, C., 55, 102 Bose, S . K., 26, 29, 42 Bott, K. F., 23, 24, 26, 39, 44 Bourgeois, S., 311, 312 Hourgoignio, J., 67, 100 Bowen, S. M., 124, 125, 128 Bowers, B., 110, 111, 112, 113, 114, 115, 116, 118, 124, 128 Bowles, J. A., 258, 312 Bowne, S. W . Jr., 276, 312 Boyd, W. L., 261, 290, 312 Brack, A., 28, 39, 43 Bradley, S . G., 260, 312 Bradner, W. T., 82, 102 Bragg, P. D., 64, 96 Brammar, W. J., 183, 186, 187, 189, 190, 191, 193, 196, 197, 198, 202, 206, 217, 221,222, 259, 286, 312 Bray, H. G., 181, 221 Bray, M., 155, 177

Brecher, P. I., 21, 43 Brenner, M., 16, 39 Brewer, M . E., 308, 312 Bridgeman, A. J . , 126, 128 Brierley, G. P., 91, 96 Brock, T. D., 53, 95, 96 Brodie, A. F., 64, 67, 96 Brot, N., 27, 31, 39 Brown, A. D., 276, 314 Brown, C. M., 5, 41 Brown, D. D.. 288, 312 Brown, J. E., 192, 196, 200, 201, 202, 206, 207, 208, 209, 210, 211, 212, 215, 218, 221 Brown, M. R. W., 52, 96 Brown, P. R., 192, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 221 Brown, W. E., 28, 39 Brownell, A. S., 276, 312 Browning, C. H ., 56, 57, 96 Bruhin, H., 183, 221 Brunel, A., 180, 222 Brunstetter, E. C., 232, 249 Bryson, V., 229, 249 Bucker, N. S., 67, 100 Buetow, D. E., 253, 266, 312 Buhlman, X., 183, 221 Bu’Lock, J. D., 1, 2, 4, 5, 6, 7, 12, 16, 20, 32, 36, 40, 95, 97 Bulos, B., 83, 85, 97 Burd, G. I., 264, 313 Burger, M., 266, 313 Burgess, R. R., 292, 312 Burke, E., 223, 249 Burns, R. O., 259, 318 Burstein, C., 306, 312 Bussard, A., 275, 312 Butler, T. F., 48, 55, 98 Butow, R. A., 89, 97 Buzhinsky, E. P., 7 0 , 1 0 0

C Cafter, H. E., 27, 40 Callow, D. S., 5, 42 Campbell, A. M., 304, 317 Campbell, T. C., 4, 44 Canellakis, E. S., 24, 41 Cannon, L. T., 119, 129 Carafoli, E., 49, 84, 100 Casida, J. E., 88, 99 Casman, E. P., 19, 27, 40 Caspersson, T. O., 177 Cass, A., 63, 97 Caswell, A. H., 73, 97 Catlin, G., 72, 98 Cavari, B. Z., 64, 67, 97

327

AUTHOR INDEX

Cecchetto, A., 75, 102 Cesari, I. M., 53, 102 Chain, E. B., 180, 221 Chakrabarty, A. M., 218, 221, 274, 275, 312 Chakravorty, M., 258, 312 Chamberlin, J. W., 78, 79, 96, 103 Chance, B., 50, 66, 97 Changeux, J. P., 93, 97 Chao, K.-C., 26, 40 Chapman, D., 48, 97 Chappell, J. B., 68, 69, 70, 72, 73, 74, 75, 77, 78, 79, 80, 97, 99 Chargaff, E., 275, 314 Chasin, L. A,, 260, 287, 292, 312 Chen, J. W., 8, 42 Cherbuliez, E., 18, 39 Chestera, C. G. C., 27, 40 Chiang, C., 180, 221 Christensen, W. B., 180, 221 Christoffersen, T., 267, 314 Ciani, S. M., 76, 81, 97 Ciegler, A., 13, 40 Clark, A. J., 218, 221 Clark, D. J., 288, 306, 312 Clark, D. S., 28, 33, 40 Clark, G. D., 27, 40 Clarke, P. H., 183, 185, 186, 187, 188, 189, 190, 191, 192, 193, 196, 197, 198, 202, 204, 205, 206, 207, 208, 209, 210, 211, 212, 215, 217, 221, 222, 259, 278, 286, 312 Clarkson, T. W., 90, 101 Claybourn, B. E., 95, 98 Cline, A., 260, 314 Cline, S. G., 172, 176 Cockrell, R . S., 7 2 , 97, 98 Cohen, G. N., 49, 67, 99 Cohen-Bazire, G., 254, 255, 316 Cohn, M., 280, 288, 304, 306, 312 Colby, C. Jr., 288, 290, 312 Cole, P. W., 182, 221 Cole, R. M., 22, 41 Coleman, G., 21, 40 Coles, 6. J., 88, 97 Collins, A., 267, 271, 315, 317, 318 Collins, F. M., 264, 312 Conner, R. L., 172, 176, 177 Cook, P., 71, 75, 77, 78, 104 Cornforth, J. W., 89, 97 Corum, C. J . , 78, 98 Cota-Robles, E. H., 52, 56, 96 Cotman, C., 265, 266, 270, 314 Cotter, D. A., 126, 128 Coukell, M. B., 260, 313 Cowie, D. R., 254, 306, 312 Cox, C. B., 88, 103 Cox, D. P., 263, 269, 313, 314

s. T., 52, 9r Cozzarelli, N. R., 258, 286, 302, 307, 313 Craig, L. C., 58, 59, 102 Crane, F. L., 87, 88, 98, 99 Craston, A., 181, 222 Creevy, D. C., 260, 312 Crofts, A. R., 68, 69, 70, 72, 73, 74, 75, 80, 97, 99 Crombag, F. J. L., 62, 63, 97 Crombrugghe, B. de, 279,280,297,298,317 Curren, H. R., 232, 249 Curtis, R. W., 2, 3, 4, 40, 43 Cuthbert, A. W., 57, 97 Cybulska, B., 62, 96 COX,

D Dagley, S., 120, 128 Dainko, J. L., 61, 104 Dalidowicz, J. E., 21, 43 Daniel, R. M., 88, 100 Daniels, M. J., 8, 40 DasGupta, B. R., 19, 39 Davies, A., 56, 97, 258, 264, 313 Davies, G. E., 56, 97 Davies, R., 21, 40 Davis, B. D., 53, 103, 257, 268, 274, 311, 313 de Duve, C., 114,128 DeLorenzo, W. F., 304, 317 Demain, A. L., 11, 12, 35, 40 Demel, R. A., 62, 63, 97, 100 Demerec, M., 218, 221 DeMoss, J. A., 53, 54, 99, 274, 318 DeMoss, R. D., 305, 312 den Dooren de Jong, L. E., 183, 221 Desai, I. D., 120, 129 de Souza, N. J., 48, 103 de Torrontegui, G., 265, 313 Deutsch, K., 111, 128 de Zwaig, R. N., 57, 93, 104 Diana, A. L., 4 8 , 1 0 3 Dicks, J. W., 236, 237, 240, 249, 250 Dienert, F., 253, 313 Dilley, R. A., 61, 97 Dingle, J. T., 54, 96 Dippell, R. V., 147, 148, 176 Dixon, M., 120, 128, 232, 249 Dobrogosz, W. J., 18, 19, 42, 272, 280, 296, 298, 313,316 Doetsch, R. N., 67, 68, 75, 97 Domagk, G., 46, 97 Doorenbos, N. J., 56, 57, 103, 104 Doudoroff, M., 183, 219, 222, 305, 313 Draper, P., 181, 182, 188, 221

328

AUTHOR INDEX

Dring, G. J., 17, 42 Druyan, R., 35, 40 Dryl, S., 155, 176 Dunitz, J. D., 76, 100 Dunn, J. J., 292, 312 Durrell, J., 182, 221

E Eagon, R. G., 52, 91, 96, 97 Eaton, M . D., 18, 19, 20, 42 Eble, T. E., 62, 96 Echols, H., 258, 286, 292, 305, 306, 310, 311, 314 Eckcr, R. E., 234, 249 Egan, J. B., 217, 222, 305, 313 Ehrensvlird, G., 28, 36, 40 Eichhorn, M. M., 296, 313 Eisenberg, W. V . , 12, 43 Eisenman, G., 76, 81, 97 Elander, R. P., 5, 40 Elbien, A. D., 302, 313 Eliasson, E. E., 267, 313 Ellar, D. J . , 50, 82, 101, 102 Elliker, P. R., 52, 101 Ellwood, D. C., 237, 240, 241, 249, 250 Elsworth, R., 224, 250 Englesberg, E., 264, 268, 294, 296, 310, 313 Engley, F. B., Jr., 21, 40 Ensign, J. C., 21, 40, 257, 315 Epps, H. M . R., 253, 259, 261, 262, 264, 265, 313 Epstein, W., 132, 177, 247, 249, 310, 313 Erickson, S. K., 88, 100 Ernster, L., 83, 84, 85, 100 Estrada-0, S., 74, 75, 77, 78, 79, 80, 97, 98, 100 Evans, C. G. T., 247, 249 Evans, D. J., 21, 40 Evans, W. C., 300, 301, 313 Even-Shoshan, A., 259, 263, 264, 314 Evstratov, A. V., 74, 103 Eyk, J. van, 259, 313

F Fahn, S., 84, 97 Falcone, A, B., 75, 97 Falcoz-Kelly, F., 296, 313 Fales, H., 27, 31, 39 Fargie, B., 217, 221, 222 Faust, M. A., 67, 68, 75, 97 Feigelson, P., 219, 222 Feigina, M. Yu. A , , 69, 71, 103 Fend, Z., 224, 249

Few, A. V., 54, 55, 56, 60, 97, 98,102 Field, B. S., 56, 97 Fields, K. L., 92, 97 Finger, I., 134, 137, 140, 141, 142, 143, 146, 149, 152, 154, 155, 156, 157, 158, 162, 163, 165, 174, 175, 177, 178 Finkelstein, A., 48, 63, 97, 102 Fisher, R. R., 86, 97 Fitz-James, P. C., 54, 97 Fleck, U. S., 19, 39 Folk, J. E., 182, 221 Folkers, K., 87, 88, 98, 99 Foster, J . W . , 26, 28, 29, 40, 127, 128 Fox, C. F., 35, 40, 91, 98 Fraenkel, D. G., 296, 305, 313, 318 Francis, J . , 56, 97 Frank, P. F., 67, 101 Frazier, W. C., 28, 41 Fredericks, J., 183, 222 Freedberg, W. B., 258, 286, 313 Freer, J. H., 50, 82, 101, 102 Freese, E., 16, 43 Freundlich, M., 306, 313 Friedman, M. E., 20, 40 Friedman, S. B., 287, 292, 313 Fraholm, L. O., 37, 43 Fruton, J. S., 182, 221, 222 Fulrasawa, T., 302, 313 Furuya, A., 26, 40 Fukimbara, T., 8, 41 Fuwa, K., 35, 40

G Gale, E. F., 253, 258, 259, 261, 262, 264, 265, 313, 317 Galeotti, T., 67, 85, 98, 100 Gallichio, V., 27, 40 Gander, J. E., 259, 314 Garbus, J., 66, 104 Garibaldi, J. A., 38, 40 Gary, C. T., 18, 19, 20, 42 Gary, E., 16, 39 Gattenbeck, S., 36, 43 Gaucher, G. M., 37, 40 Gaudy, A. F. Jr., 254, 300, 301, 313 Gaudy, E. T., 254, 300, 301, 313 Gebler, B., 148, 178 Gel’man, N. S., 48, 50, 82, 87, 89, 98 Gerhardt, P., 47, 102 Gershanovich, V. N., 264, 279, 313 Gerwing, J., 18, 19, 20, 40 Gest, H., 307, 308, 313 Ghosh, S., 302, 305, 315 Gibor, A,, 268, 313 Gibson, I., 171, 117 Gilbert, W., 199, 221, 275, 314

AUTHOR INDEX

Gilby, A. R., 54, 55, 56, 98 Gilvarg, C., 6, 7, 15, 21, 39 Gladstone, G. P., 21, 40 Glasziou, K. T., 266, 313 Goldberg, M. W., 18, 39 Goldenbaum, P. E., 298, 313 Goldfine, H., 48, 98 Goldman, M., 119, 129 Goodey, T., 126, 128 Goodnight, S. A., 14, 30, 44 Goodwin, J., 27, 31, 39 Gordon, A., 48, 54, 104 Gorini, L., 266, 313 Gorman, M., 5, 40 Gorr, G., 180, 181, 221 Gorts, C. P. M., 260, 263, 264, 265, 269,314 Gottlieb, D., 12, 27, 36, 40, 47, 90, 95, 98 Gotto, A. M., 243, 250 Gourevitch, A., 13, 40 Graven, S. N . , 58, 74, 75, 76, 77, 78, 79, 80, 97, 98,100 Gray, C . T., 263, 264, 268, 269, 314 Green, A., 143, 177 Green, D. E., 51, 83, 98, 99, 100 Green, H., 37, 40 Greene, R., 58, 98 Greenfield, R. E., 182, 222 Greengard, P., 181, 222 Griffiths, A. J., 108, 109, 110, 111, 113, 117, 118, 119, 120, 121, 122, 123, 124, 125, 123, 128 Griffiths, D. E., 88, 97 Grollman, A. P., 35, 40 Gros, F., 275, 310, 311, 312, 313, 314, 316 Gross, E., 17, 41, 86, 98 Grossowiez, N., 18, 19, 20, 41, 64, 67, 97, 181, 182, 221, 222 Grove, J. F., 28, 36, 41 Grula, E. A., 48, 55, 98 Grunberg, E., 18, 39 Guest, J. R., 301, 302, 311 Guillory, R. J., 86, 97, 98 Gundersen, W . , 266, 313 Gunsalus, C. F., 218, 221 Gunsalus, I. C., 218, 221, 259, 314

H Haarhoff, K. N., 70, 97 Hadler, H. I., 75, 95, 97, 98 Hagihara, B., 180, 221 Hahn, F. E., 274, 314 Hahn, J. J., 22, 41 Hall, C., 87, 88, 98, 99 Hall, H. H., 13, 40, 43 Hall, T. B., 37, 39

329

Halpern, Y. S., 181, 182, 221, 222, 259, 263, 264, 314 Halvorson, H. O., 16, 41, 260, 263, 264, 268, 271, 286, 314, 315 Hamill, R. H., 5, 40 Hamilton, D., 5, 40 Hamilton, W. A., 64, 67, 98 Handler, P., 180, 222 Handley, W. C . R., 300, 301, 313 Haney, M. E., 78, 98 Hanlin, R. T., 4, 44 Hanson, R. S., 263,264, 268,269,271,313, 314 Hanson, T. E., 258, 317 Happold, F. C., 253, 261, 300, 301, 313, 314 Harned, R. L., 78, 98 Harold, F. M., 61, 66, 67, 68, 71, 75, 77, 79, 82, 85, 86, 92, 98, 101 Harris, E. J., 66, 69, 70, 71, 72, 73, 75, 78, 79, 80, 97, 98, 102 Harris, H., 132, 174, 177 Harris, R. A., 51, 98, 99 Hartman, P. E., 218, 221 Hase, E., 253, 266, 315 Haselkorn, R., 35, 40 Hassid, W. Z., 305, 313 Hatefi, Y., 88, 99 Hauge, J. G., 260, 314 Hawes, R. S . J., 109, 128 Hawkins, D., Jr., 26, 40 Haxby, J., 62, 63, 100 Hayashi, S., 258, 286, 302, 306, 307, 313, 314 Hayes, A. W., 4, 44 Hayes, R. E., 116, 129 Hayflick, L., 37, 41 Haynes, D. H., 71, 99 Heal, 0. W., 126, 129 Heath, E. C., 302, 313 Heath, M. J., 276, 314 Hedberg, M. A., 26, 44 Hedges, A. J., 92, 103 Hegeman, G. D., 218, 222 Heimpel, A. M., 21, 41 Heller, C., 140, 142, 143, 146, 149, 154, 156, 157, 158, 162, 163, 165, 175, 177, 178 Henderson, P. J. F., 74, 75, 77, 78, 79, 80, 99 Henderson, T . R., 276, 314 Hendlin, D., 35, 40 Henrici, A. T., 41 Herbert, D., 224, 225, 231, 234, 235, 241, 247, 248, 249, 250 Herriott, R. M., 24, 43 Herzenberg, L. A., 306, 314 Hess, B., 67, 85, 98, 100

330

AUTHOR INDEX

Hesseltine, C. W., 13, 43 Heumann, W., 67, 104 Heytler, P. G., 64, 99 Hiatt, H. H., 275, 314 Hickey, R. J., 27, 41 Hidy, P. H., 78, 98 Hill, C. W., 292, 314 Hirschmann, D. J., 26, 41 Hobson, P. N., 107, 128 Hockenhull, D. J. D., 12, 13, 16, 41 Hodes, L. J . , 64, 99 Hoehn, M. M . , 78, 98 Hofer, M. P., 66, 72, 75, 98, 99 Hoffee, P. A., 264, 294, 296, 313 Hofmann, A,, 28, 43 Holland, I. B., 91, 92, 93, 98 Holloway, B. W., 217, 221, 222 Holloway, C . T . , 83, 84, 85, 96, 100, 102 Holmstrom, B., 5, 41 Holt, R. J., 180, 222 Holtzer, H., 37, 41 Holtzer, S., 37, 41 Holzer, H., 263, 269, 270, 279, 318 Hopfer, U., 66, 99 Horecker, B. L., 296, 305, 311, 313, 314 Horgan, D. J., 88, 99 Horhold, C., 48, 102 Horibata, K., 280, 288, 304, 312 Horitsu, H., 28, 33, 40 Horner, W. H., 8, 41 Horowitz, J., 275, 314 Hotchkiss, R. D., 46, 58, 68, 75, 99 Hou, C., 64, 96 Hough, J. S., 5, 41 Houldsworth, M. A., 204, 205, 206, 221, 259, 278, 286, 312 Housewright, R. D., 14, 21, 24, 26, 30, 35, 42 Hoyle, L., 253, 261, 314 Hsie, A. W . , 259, 263, 265, 266, 272, 288, 291, 296, 314, 317 Hu, A. S . L., 288, 290, 312 Hubbard, J. S., 33, 34, 41 Hughes, D. E., 108, 109, 110, 111, 113, 117, 118, 119, 120, 121, 122, 123, 124, 125, 128, 180, 222 Hugo, W . B., 56, 86, 99 Hulley, S. B., 259, 285, 314 Hulme, M. A., 5, 40 Humphrey, R., 232, 250 Hunter, F. E., Jr., 15, 41, 58, 68, 70, 71, 74,99 Hunter, G. H., Jr., 267, 271, 318 Hunter, J. R., 234, 237, 250 Hurst, A., 17, 41, 95, 104 Hutchinson, D. W., 88, 97 Huybers, K., 26, 41 Hyman, L. H., 109, 126, 128

I Ichishawa, E., 21, 27, 32, 42 Iida, K., 60, 100 Ikekawa, N., 48, 102 Imaeda, T., 53, 102 Ingraham, J. L., 276, 315, 316 Inoue, Y., 90, 98 Inturrisi, C. E., 84, 99 Ishida, T., 35, 41 Ishikawa, S., 88, 100 Ito, A., 27, 42 Ito, K., 28, 33, 40 Ivanov, V. T., 71, 74, 99, 103 Ivanovics, G., 23, 41

J Jackson, F. L., 89, 100 Jackson, J. B., 80, 99 Jackson, R. W., 53, 54, 99 Jacob, F., 151, 177, 291, 311, 312, 314 Jacobson, L. A., 259, 314 Jacoby, G. A., 191, 222, 260, 261, 262, 314 Jacoby, H. M . , 21, 43 Jacoby, W. B., 183, 222 Jagendorf, A. T., 51, 66, 67, 99 Jagger, W. S., 69, 70,71, 72, 78,79, 80,102 James, A. T., 89, 97 James, S. P., 181, 221 Jamieson, J. D., 165, 177 J a n e k k , J., 259, 291, 317 Jansen, E. F., 26, 41 Jayaraman, J., 265, 266, 270, 314 Jayaraman, K., 199, 222 Jeng, M., 87, 88, 99 Jervell, K . F., 267, 314 Jindra, A., 37, 43 Johnson, C. L., 61, 99 Johnson, D., 58, 76, 77,82,83, 98,100,104 Johnson, J. H., 69, 70, 71, 72, 78, 79, 80, 702 Johnson, M., 28, 43 Johnson, R. B., 182, 222 Jones, E. A., 106, 109,129 Jones, I. G., 135, 136, 137, 138, 139, 142, 144, 149, 150, i r r Jones, K. L., 78, 98 Jonsson, A. G., 21, 22, 41 Jordan, D. C., 53, 99 Jordan, E., 273, 314 Jordon, E., 302, 318 Jorgensen, 8. B., 259, 285, 314 Joshi, J. G., 180, 222 Juhlin, I., 181, 222 Juni, E., 22, 43 Jurand, A., 148, 177

33 1

AUTHOR INDEX

K

Koizumi, S., 155, 177 Kolodziej, B. J . , 26, 41 Komai, H-., 28, 34, 41 Komolrit, K., 254, 300, 301, 313 Konisky, J., 92, 100 Kopaczyk, K., 83, 700 Korn, E. D., 48, 100, 110, 111, 112, 113, 114, 115, 116, 118, 124, 128 Kornberg, A., 33, 43 Kornberg, H. L., 192, 193, 222, 242, 243, 250, 263, 269, 315 Korzybski, T., 46, 100 Kosaka, T., 88, 100 Koiciuszko, H., 147, 177 KovaE, L., 67, 85, 98, 100 Koval, G. J., 84, 97 Kowalsky, A., 71, 99 Kowszyk-Gindifer, 46, 100 Kretschmer, S., 23, 41 Krichevsky, M. I., 120, 128 Kronau, R., 263, 269, 270, 279, 318 Krulwich, T. A., 257, 315 Kudo, R. R., 109, 128 Kurachi, M., 27, 41 Kundig, F. D., 305, 315 Kundig, W., 302, 305, 315 Kurahashi, K., 302, 305, 314, 315 Kurland, C. G., 234, 250 Kurylo-Borowska, Z., 7, 8, 9, 16,37,41,42 Kurylowicz, W., 46, 100 Kuiela, 8., 100

Kacser, H., 144, 176' Kagawa, Y., 83, 99 Kalckar, H. M., 302, 318 Kalyanasundaram, R., 28, 41 Kameyama, T., 276, 314 Kammen, H. O., 24, 41 Kamogawa, A., 305, 314 Kaplan, J. H . , 66, 99 Katchalsky, E., 59, 60, 99 Kates, M., 48, 99 Kato, I., 34, 43 Katz, E., 3, 6, 8, 11, 14, 15, 21, 27, 41, 42, 44, 266, 315 Katz, J., 253, 314 Kawamata, J., 60, 101 Keister, D. L., 80, 103 Kelleher, W. J., 28, 42 Kelly, M., 183, 185, 186, 188, 189, 192, 193, 212, 222 Kelner, A., 27, 41 Kemp, M. B., 218, 222 Kennedy, E. P., 91, 98 Kepes, A., 49, 67, 99, 275, 276, 281, 283, 290, 305, 306, 312, 314 Kessler, D. P., 279, 305, 314 Kilbourn, B. T., 76, 100 Kimball, R. F., 152, 159, 171, 176, 177 Kimura, A., 301, 302, 314 Kimura, T., 180, 181, 182, 222 Kindler, S. H . , 18, 19, 20, 41 King, R. D., 48, 55, 98 King, T. P., 58, 59, 102 Kingdon, H. S., 34, 41 Kinoshita, S., 26, 40 Lagoda, A. A., 13, 40 Kinsky, C. B., 62, 100 Laine, I. A., 71, 99 Kinsky, S. C., 54, 61, 62, 63, 97, 100, 104 Laishley, E. J., 33, 42, 261, 262, 266, 271, Kirsch, E . J., 8, 27, 41 315 Laland, S. G., 37, 43 Kirschbaum, J., 18, 41 Kiryushkin, A. A., 74, 103 Lamaire, Y., 180, 222 Kitada, M., 8, 41 Lampen, J. O., 61, 62, 100 Kjeldgaard, N. O., 234, 250 LaNauze, J. M., 253, 317 Klahr, S., 67, 100 Landau, B., 279, 317 Klatt, K. P., 259, 314 Lardy, H. A., 58, 74, 75, 76, 77, 78, 79, 80, Klein, R. L., 109, 123, 128 81, 82, 83, 97, 98, 100, 104, 267, 317 Larkin, D., 175,177 Klemner, H. W., 28, 42 Knight, I. G., 83, 84, 85, 96, 100, 102 LaSala, E . R., 18, 39 Lascelles, J., 27, 32, 42, 47, 100, 264, 312 Knight, S . G., 28, 41 Knowles, C. J., 88, 100 Lash, J., 37, 41 Latham, W. C., 19, 27, 42 Knox, N. C., 27, 43 Leader, D. P., 84, 96' Kobel, H., 28, 43 Koeh, A. L., 305, 314 Lechevalier, H., 27, 39 Koch, J. P., 258, 286, 302, 306, 307, 313, Lederberg, J., 305, 313 Lee, C . P., 50, 66, 83, 84, 85, 97, 100 314 Lee, T . O., 217, 222 Komer, H., 28, 41 Lehninger, A. L., 49, 66, 84, 96, 99, 100 Kohlmeier, V., 275, 314 Lein, J., 13, 40, 82, 102 Koike, M., 60, 100

L

332

AUTHOR INDEX

Leive, L., 52, 100 Lelouchier-Dagnelie, H., 311, 312 Lenard, J., 48, 100 Lengeler, J., 258, 273, 310, 314, 315 Lennarz, W. J., 48, 100 Lenny, J. F., 28, 42 Leonard, C. G., 14, 21, 24, 26, 30, 35, 42 Lessie, T. G., 260, 315 Lester, G., 57, 100, 310, 317 Le Suer, A., 169, 178 Lev, A. A., 70, 100 Levine, E., 57, 103 Levine, L., 19, 27, 42 Levintow, L., 182, 222 Levisohn, R., 92, 100 Levy, J. B., 268, 313 Liberman, E. A., 66, 70, 100 Lichstein, H. C., 261, 273, 290, 306, 312, 313

Lightbown, J. W., 89, 100 Lilly, H. D., 8, 10, 18, 19, 20, 42 Lilly, M. D., 191, 204, 205, 206, 221, 259, 278, 286, 312 Lin, E. C. C., 258, 259, 285, 286, 301, 302, 305, 306, 307, 313, 314, 315, 318 Lippe, C., 74, 100 Lipton, S. H., 89, 102 Lloyd, D., 123, 128 Locke, A., 24, 42 Lockhart, W. R., 259, 318 Loginova, N. F., 69, 71, 103 London, J., 259, 315 Longworth, A. R., 56, 86, 99 Loomis, W. F., 203, 222 Loomis, W. F. Jr., 277, 278, 279, 288, 290, 291, 295, 296, 310, 315, 318 Losada, M., 265, 313 Loutit, J. S., 217, 222 Love, L. L., 120, 128 Low, H., 88, 104 Lubin, M., 71, 100 Lucy, J . A., 48, 54, 96, 100 Lukoyanova, M. A., 48, 50, 82, 87, 89, 98 Lund, P., 243, 250, 260, 315 Luria, S. E., 57, 92, 93, 97, 104 Luse, S. A., 61, 62, 63, 100 Lutsch, G., 60, 104 Luzzati, V., 54, 100 Lyr, H., 95, 101

M Maalse, O., 234, 250, 265, 270, 317 Mabe, J. A., 5, 40 McConnachie, E. W., 106, 109, 116, 117, 119, 127, 128

McDaniel, L. E., 28, 29, 40 MacDonald, J. C., 2, 42 McElroy, W. D., 33, 42 McFadden, B. A., 253, 315 McFall, E., 262, 273, 274, 276, 286, 287, 293, 315, 316 McFarlane, N. D., 183, 190, 193, 194, 221, 222

McGarrahan, J. F., 260, 316 McGinnis, J., 254, 294, 295, 299, 300, 301, 316 McGivan, J. D., 74, 75, 77, 78, 79, 80, 99 MacGregor, D. R., 52, 101 Mach, B., 3, 13, 15, 42, 58, 101, 102 Macheboeuf, M., 301, 317 Macindoe, H., 146, 162, 163, 165, 17Y, 178 McKinnon, D. L., 109, 128 McLafferty, F. W., 2, 3, 43 McLean, R. A., 8, 10, 18, 19, 20, 42 MacLennan, D. H., 83,101 McMurray, W. C., 69, 82, 83, 100, 101 McMurrough, I., 258, 316 MacQuillan, A. M., 260, 263, 264, 286, 314 315

Magafia-Plaza, I., 266, 315 Magasanik, A. K., 296, 315 Magasanik, B., 58, 98, 203, 222, 253, 258, 259, 260, 272, 273, 274, 275, 278, 279, 281, 282, 284, 287, 288, 290, 291, 292, 293, 295, 296, 306,

254, 277, 289, 310,

312, 315, 316, 317, 318

Mager, J., 18, 19, 20, 41 Mahler, H. R., 265, 266, 270, 314 Main, E. R., 24, 42 Majer, J., 2, 43 Majumdar, M. K., 27, 42 Majumdar, S. K., 26, 27, 29, 42 Mak, R. A., 18, 19, 42 Makman, R. S., 279, 298, 315 Malenkov, G. G., 74, 103 Maley, F., 260, 316 Mallette, M. F., 274, 316 Mandelstam, J., 106, 128, 191, 222, 272, 273, 274, 275, 288, 293, 298, 307, 308, 310, 313, 315, 316 Margolin, P., 143, 177, 287, 292, 313 Marinus, M. G., 217, 222 Markus, Z., 8, 10, 18, 19, 20, 42, 258, 315 Marquis, R. E., 90, 101 Marr, A. G., 276, 288, 306, 312, 315, 316 Marshall, R., 6, 15, 21, 42, 266, 315 Martin, A. R., 56, 97 Martin, F. D., 288, 290, 312 Marver, H. S., 267, 271, 315, 317 Mastafa, A., 300, 301, 315 Masters, M., 276, 315 Mateles, R. I., 28, 29, 42 Matsuka, M., 253, 266, 315

333

AUTHOR INDEX

Matsuo, T., 60, 100 Mattoon, J. R., 83, 101 Mauritzen, C . M., 61, 99 Maxon, W. D., 8, 42 Mee, B. J., 217, 222 Meek, G. A., 263, 264, 265, 266, 270, 317 Meers, J. L., 236, 240, 247, 250 Meister, A., 182, 222 Mela, L., 50, 66, 91, 97, 101 Metcalf, R. L., 64, 104 Meyenburg, H. K . von, 261,263,264,266, 269, 312, 316 Meyer, C. E., 81, 101 Meyer, T. S., 67, 101 Meyers, E., 76, 101 Mikhaleva, I. I., 74, 103 Miller, G., 258, 315 Miller, I. M., 35, 40 Miller, J. H., 203, 222, 289, 290, 293, 317 Miller, N., 54, 96 Miller, P. A., 18, 19, 20, 42 Millet, J., 266, 271, 317 Mitchell, P., 49, 50, 63, 65, 66, 68, 72, 73, 82, 83, 101 Mitscher, L. A., 4, 42 Miyamoto, S., 87, 103 Mizusawa, K., 21, 27, 32, 42 Mdler, K. M., 125, 128 Monahan, M., 63, 96 Monk, M., 217, 222 Monod, J., 151, 177, 223, 225, 228, 250. 253, 254, 255, 258, 259, 275, 291, 298, 305, 306, 308, 309, 312, 314, 316, 318 Moore, C. E., 63, 67, 69, 101 Moore, J. L., 276, 316 Moore, M. O., 122, 129 Morell, J. L., 17, 41 Morgan, D. M., 234, 250 Morgan, R. S., 116, 128 Mori, R., 87, 103 Morland, J., 267, 314 Morowitz, H. J., 55, 102 Morell, R. W., 18, 19, 20, 40 Morrison, N. E., 17, 27, 39 Morse, M. L., 292, 293, 305, 313, 316, 318 Morse, S . A., 18, 19, 42 Morton, H. E., 27, 41 Mosely, F. T., 27, 29, 43 Moses, V., 191,222, 254,260,273, 276, 278, 279, 288, 289, 291, 297, 308, 310, 312, 316, 317 Mossman, M. R., 263, 264, 268, 269, 314 Mott, M. R., 135, 144, 145, 168, 176, 177 Moudrianakis, E. N., 82, 101 Moyle, J., 65, 66, 72, 73, 101 Mueller, J. H . , 25, 27, 29, 42 Mueller, P., 70, 71, 74, 75, 76, 78, 81, 101 Muller, M., 18, 39

Miiller, M., 125, 128 Muller-Hill, B., 199, 221, 222 Mufioz, E., 50, 82, 101 Murata, R., 27, 42 Murphey, W. H., 259, 316 Murrell, W. G., 12, 42, 106, 128 Musilek, V., 31, 42 Myasoedova, K. N., 266, 267, 316 Mycek, M. J., 182, 222 Myers, A. T., 232, 249

N Nagatsu, J., 87, 103 Nager, U. F., 28, 39 Nakada, D., 273, 275, 281, 282, 290, 316 Nakajima, K., 60, 101 Nakamura, H., 57, 101 Nakaya, R., 274, 316 Nanney, D. L., 148, 151, 155, 177 Naono, S., 275, 310, 311, 312, 313, 316 Nason, A., 33, 42 Neal, R. A., 106,128 Neff, R. H., 107, 108, 110, 114, 118, 124, 129 Neff, R. J., 106, 107, 108, 110, 113, 114, 116, 117, 118, 119, 120, 121, 122, 123, 124, 128, 129 Neidhardt, F. C., 241, 250, 253, 258, 259, 260, 272, 293, 296, 306, 315, 316 Neilands, J. B., 17, 28, 34, 41, 42 Nes, W . R., 48, 103 Nevill, A,, 274, 318 Newton, B. A., 55, 60, 101 Newton, G. G. F., 12, 36, 39 Ng, H., 276, 316 Ng, M. H., 50, 82, 101 Niederpruem, D. J., 260, 301, 302, 318 Nikaido, H., 302, 313, 316 Nishida, S., 20, 43 Nitz, R. M., 18, 19, 20, 40 Nolte, A., 296, 313 Nomura, M., 91, 92, 93, 100, 101 Novelli, G. D., 7, 8, 14, 15, 39, 276, 314 Novick, A., 223, 230, 250, 304, 316 Novick, R. P., 91, 101

0 Oda, T., 83, 100 Ogasawara, F., 150, 178 Ohno, H., 88, 99 Okami, Y., 38, 42 Okinaka, R. T., 272, 280, 316 Onorato, F., 140, 142, 149, 156, 163, 177

334

AUTHOR INDEX

Osborn, M. J., 296, 313 Ostrovskii, D. N., 48, 50, 82, 87, 89, 98 Ovchinnikov, Yu, A., 71, 74, 99, 103 Overath, P., 259, 316

P Page, F. C., 127, 129 Paigen, K., 254, 273, 274, 275, 276, 277, 278, 279, 286, 294, 295, 296, 297, 299, 300, 301, 306, 316, 318 PaIacikn, E., 265, 313 Palade, G. E., 165, 177 Palkina, N. A., 264, 313 Palleroni, N. J., 183, 219, 222 Palmer, I. S., 274, 316 Palmcr, J., 278, 288, 289, 316, 317 Palmer, J . G., 12, 43 Pansy, 3'. E., 76, 101 Pappenheimer, A. M . Jr., 8, 9, 18, 19, 20, 42, 44, 254, 255, 316 Pardee, A. B., 49, 67, 101, 273, 276, 315, 316, 317 Parker, J. H., 83, 101 Passmoro, R., 266, 290, 317 Passow, H., 90, 101 Pastan, I., 203, 222, 279, 280, 285, 288, 289, 297, 298, 317 Pastornak, C. A., 260, 301, 302, 312 Pasternak, J., 169, 170, 171, 172, 173, 174, 176,177 Paulus, H., 15, 16, 36, 39, 42 PavlasovB, E., 66, 67, 68, 92, 101 Pavlenko, I. A., 69, 71, 103 Pearce, L. E., 217, 222 Pedersen, C. J., 81, 101 Penefsky, H. S., 86, 102 Penniston, J. T., 51, 98, 9.9 Peraino, C., 267, 317 Perdue, S. W., 171, 177 P8rez-Silva, J., 148, 177 Perlman, D., 2, 37, 42, 76, 95, 96, 101 Perlman, R. L., 203, 222, 279, 280, 285, 288, 289, 297, 298, 317 Perlroth, M. G., 267, 271, 317 Peters, W. J., 17, 26, 42 Peterson, R. E., 13, 40 Pethica, B. A., 54, 55, 56, 60, 101, 102 Petrack, B., 181, 222 Phipps, P. J., 248, 250 Pienta, P., 27, 41 Pinkerton, M., 7 8 , 79, 96, 103 Pinska, E., 304, 317 Pioda, L. A. R., 74, 76, 100, I 0 4 Pirt, S . J., 5, 42 Pitot, H. C., 267, 317 Poe, M., 73, 101

Polakis, E. S., 259, 261, 263, 264, 265, 266. 268, 270, 317 Polglase, W. J . , 260, 313 Pollard, L. W., 37, 39 Pollock, M. R., 216, 222 Popov, E. M., 71, 99 Postgate, J. R., 225, 250 Potter, A. L., 305, 313 Powell, A. J., 5, 20, 40 Powell, E . O., 224, 228, 240, 250 Powers, M. T., 259, 261, 262, 317 Pramer, D., 5, 42 Preer, J . R., 134, 135, 139, 143, 144, 145, 146, 148, 151, 162, 169, 171, 176, 177 Preer, L. B., 134, 144, 146, 162, 177 Prescott, D. M . , 171, 177 Pressman, B. C., 15, 42, 58, 63, 66, 69, 70, 71, 72, 73, 74, 75, 78, 79, 80, 81, 97, 98, 99, 101, 102 Prestidge, L . S., 276, 317 Prevost, C., 191, 222, 254, 276, 278, 279, 297, 310, 316, 317 Price, K. E., 82, 102 Prichard, W. W., 64, 99 Pringle, C. R., 150, 177 Prnett, J. R., 172, 177 Pruett, P. O., 172, 177 Pullman, M. E., 50, 82, 83, 84, 102 Pursiano, T. A., 13, 40 Putman, E. W., 305, 313

Q Quayle, J. R., 265, 312

R Racker, E., 82, 83, 85, 86, 97, 99, 102 Raikov, I. B., 147, 177 Raistrick, H., 2, 42 Ramaley, R. F., 33, 42 Rao, P. M . , 37, 39 Rao, R. K., 37, 39 Rao, 8. S., 34, 43 Raper, K. B., 111, 126,128 Rapin, A. M. O., 302, 318 Ratledge, C., 27, 42 Ratner, A., 267, 271, 317 Raufuss, E. M., 259, 316 Ray, D. L., 116, 129 Ray, S. A., 106, 116, 117, 11S, 119, 120, 121, 122,129 Raynaud, M., 301, 317 Razin, S., 55, 102 Rechcigl, M . Jr., 267, 271, 315, 317 Redeker, A., 267, 271, 317

335

AUTHOR INDEX

Redfearn, E. R., 88, 100 Redfield, B., 6, 15, 21, 42, 266, 315 Reeves, P., 91, 92, 102 Rege, D. V., 265, 318 Reich, E., 13, 42 Reisner, A. H., 146, 156, 162, 163, 165, 177, 178 Rettger, L. F., 253, 312 Reusser, F., 5, 42, 81, 90, 101, 102 Reynolds, D. M., 27, 42 Reynolds, P. E., 53, 99 Reznikoff, W. S., 203, 222, 289, 290, 293, 31 7 Richards, C. S., 119, 129 Richards, M., 180, 221 Richards, R. M . E., 52, 96 Richardson, S. H., 21, 40 Richmond, M. H., 265, 270, 317 Rickenberg, H. V., 199, 222, 259, 263, 265, 266, 272, 279, 288, 291, 296, 305, 310, 314, 317 Rieber, M., 53, 102 Riemersma, J. C., 67, 102 Rieske, J. S., 87, 89, 102 Rightmire, B., 79, 80, 97 Roach, G. I., 123, 128 Robertson, A. M., 83, 84, 85, 96, 100, 102 Roberts, C., 304, 318 Robinson, W. S., 35, 40 Rockstroh, T., 60, 104 Rodwell, A. W., 55, 102 Rogers, F., 276, 294, 296, 312 Rolinson, G. N . , 27, 40, 180, 221 Romano, A. H., 35, 44, 263, 318 Rosazza, J. P., 28, 42 Rose, A. H., 258, 316 Rose, F. L., 56, 97 Rose, G., 48, 102 Roseman, S., 302, 305, 315 Rosenberg, H., 253, 317 Rosenblum, E. D., 259, 316 Rosenfeld, H., 218, 222 Ross, R., 165, 178 Rossi, C. S., 49, 84, 100 Roth, C., 91, 101 Rothfield, L., 48, 102 Rothstein, A., 90, 91, 101, 102 Rottem, S., 55, 102 Rouf, M . A,, 232, 235, 250 RouviBre, J., 311, 312, 316 Roy, S . C., 274, 275, 312 Rudin, D. O., 70, 71, 74, 75, 76, 78, 81, 101 Rudman, B. M., 169, 170, 172, 173, 176 Ruiz-Herrera, J., 266, 315 Ruttenberg, M. A., 58, 59, 102 Rutter, A., 58, 76, 77, 98 Ryabova, I. D., 69,71, 74,103

S Saedler, H., 273, 314 Saito, H., 181, 222 Salton, M. R. J., 47, 48, 50, 55, 56, 60, 82, 95, 101, 102 Sanada, I., 20, 43 San Pietro, A., 80, 86, 98, 103 Sapico, V., 258, 317 Saraswathi-Devi, L., 28, 41 Sardesai, 8. K., 34, 43 Sato, H., 34, 43 Saukkonen, J. J., 275, 314 Sawant, P. L., 120,129 Scaife, J., 279, 317 Scarpa, A., 75, 102 Schaechter, M . , 234, 249, 250 Schaeffer, P., 266, 271, 317 Schantz, E. J., 21, 43 Schatz, A., 17, 43 Schatz, G., 50, 82, 83, 84, 86, 102 Schemer, R., 47, 102 Schick, M., 279, 317 Schindler, A. F., 12, 43 Schlegel, H. G., 95, 96, 253, 263, 265, 300, 301, 312, 317 Schlein, A., 82, 102 Schlenk, F., 61, 104 Schlesinger, S., 306, 317 Schmitt, M. D., 56, 95, 102 Schmitt, R., 16, 43 Schneidau, J. D., 181, 222 Schor, M. T., 50, 82, 101 Schubert, K., 48, 102 Schulman, J. H . , 55, 56, 60, 102 Schultz, S. G., 247, 249 Schuster, F., 111, 115, 116, 129 Schwarting, A. E., 28, 42 Schwartz, A., 61, 99 Schwartz, D. T., 260, 315 Schwartz, L. S., 15, 41, 58, 68, 70, 71, 74, 99 Schwartz, M., 66, 102 Scotto, P., 306, 317 Sebek, 0. K., 43, 60, 103 Seed, J. R,., 143, 146, 162, 165, 178 Segal, W., 258, 312 Sela, M., 59, 60, 99 Sells, B. H., 274, 317 Senn, M., 2, 3, 4 3 Senyavina, L. B., 71, 99 Seshachar, B. R., 148, 178 Sessa, G., 62, 103, 104 Settlemire, C. T., 91, 96 Seufert, W. D., 56, 103 Shaeffer, P., 20, 43 Shafer, S., 143, 146, 162, 165, 178 Shannon, R., 92,103

336

AUTHOR INDEX

Shapiro, M., 259, 318 Sharp, C. W . , 265, 266, 270, 314 Sharp, E. L., 28, 43 Sharp, P. B., 260, 316 Shavit, N., 80, 86, 98, 103 Shaw, D. A., 263, 270, 318 Shaw, P. D., 36, 40, 47, 76, 83, 84, 85, 90, 95, 98,103 Shay, D. E., 56, 57, 103 Shemyakin, M. M., 69, 71, 74, 99, 103 Shepherd, D., 5, 7, 32, 40 Shepherd, M. C., 37, 40 Sheppy, F., 181, 222 Shizeo, A., 26, 40 Shkrob, A. M., 74, 103 Shotwell, 0. L., 13, 43 Shrago, E., 267, 317 Shu, P., 28, 43 Shugarman, P. M., 253, 266, 317 Siewert, G., 52, 103 Silbersteiu, W., 300, 301, 317 Silman, H. I., 59, 60, 99 Silver, S., 57, 103 Silverman, G. J., 8, 10, 18, 19, 20, 42 Silverstone, A. E., 203, 222, 289, 290, 293, 31 7 Simon, E. J., 58, 103 Simon, W., 74, 104 Sinden, R. E., 159, 163, 164, 178 Singer, S.J., 48, 100 Singer, T. P., 88, 99 Sinton, S. E., 148, 176 Sivak, A,, 27, 41 Sjoland, S., 36, 43 Skaar, P. D., 169, 178 Skinner, A. J., 191, 196, 197, 198, 202, 206, 217, 221, 222, 259, 286, 312 Skou, J. C., 49, 82, 103 Slater, E. C., 50, 65, 67, 73, 86, 97, 98, 103 Slayman, C . W., 58, 101 Slayter, H. S., 116, 128 Slein, J. B., 26, 44 Slepeclry, R. A., 26, 41 Slonkim, N. B., 179, 222 Smalley, IT. M . , 5, 40 Smarda, J., 93, 103 Smith, D. A., 76, 101 Smith, D. H . , 53, 103 Smith, E. L., 179, 222 Smith, F. G., 28, 43 Smith, G. L., 48, 55, 98 Smith, G. N., 5, 40 Smith, J. P., 140, 177 Smith, L., 50, 67, 87, 89, 103 Smith, M. L., 13, 43 Smith, R. F., 56, 57, 103 Smith, R. L., 8, 43 Smith, S. M., 296, 313

Smyth, D. G., 214, 222 Snake, J. E., 16, 43 Snoswell, A. X., 88, 103 Soda, S., 27, 42 Sommerville, J., 146, 159, 163, 164, 165, 174,178 Sonneborn, T. M., 132, 148, 150, 151, 169, 178 Spaeren, U., 37, 43 Spahr, P. F., 275, 314 Spector, W. S., 33, 43 Spencer, H. T., 24, 43 Spero, L., 21, 43 Spiegelman, S., 304, 317 Spizizen, J., 21, 23, 26, 39, 43, 44 Squires, C. L., 276, 315 Srinivasan, V. R., 263, 264, 268, 271, 314 Staba, E. J., 37, 43 Stadtman, E. R., 33, 34, 41 Standish, M. M . , 54, 96 Stanier, R. Y . , 183, 218, 219, 222 Starbuck, W. C., 61, 99 Stark, G. R., 214, 222 Stark, W. M . , 8, 27, 43 Starlinger, P., 273, 314 Stecker, H. C., 64, 99 Steenbergen, J. F., 14, 30, 43 Steenbergen, S. M . , 14, 28, 30, 43 Steers, E., 135, 136, 137, 138, 139, 144, 178 Stefanac, Z., 74, 104 Stefanye, D., 21, 43 Stein, W . D., 49, 54, 67, 70, 82, 103 Stcinrauf, L. K., 78, 79, 96, 103 Stent, 0. S., 276, 316 Stephenson, M., 253, 258, 281, 262, 313, 317 Sternbach, L. H., 18, 39 Sterne, M., 19, 43 Stevens, A., 56, 57, 104 Stewart, G. T . , 180, 222 Stewart, M . L., 35, 40 Stockton, J. R., 33, 43 Stokes, J. L., 259, 261, 2G2, 317 Stoll, A., 28, 43 Stoner, C. D., 89, 102 Storck, R., 238, 250 Stout, J. D., 126, 129 Strasters, K. C., 260, 261, 262, 264, 265, 269, 270, 317 Strauss, N., 274, 318 Strickland, A. G. R., 117, 127, 129 Strittmatter, C. F., 265, 270, 318 Strominger, J. L., 52, 103 Stubblefield, R. D., 13, 43 Stull, H. B., 23, 26, 43 Stumm-Zollinger, E., 254, 262, 300, 301, 311, 318 Stuy, J. H ., 24, 43

337

AUTHOR INDEX

Sueoka, N., 35, 41 Sugawara, S., 95, 103 Sulebele, G. A., 265, 318 Sumner, J. B., 180, 222 Sundararajan, T. A., 302, 318 Sussman, M., 106, 125,129 Sussman, R. R., 304, 317 Sussman, R., 106, 125, 729 Sntherland, E. W., 279, 298, 315 Suzuki, A., 87, 103 Suzuki, E., 8, 41 Suzuki, M., 38, 42 Suzuki, S., 87, 103 Svihla, G., 116, 128 Swain, G., 56, 97 Sweetman, A. J., 88, 97 Swift, H., 148, 178 Sykes, J., 120, 128 Sypherd, P. S., 274, 318 Szabo, G., 16, 39, 76, 81, 97 Szilard, L., 223, 230, 250 Szulmajster, J., 263, 264, 268, 314

T Taber, W. A., 8, 43 Takahashi, H., 87, 98 Takahashi, N . , 87, 88, 99, 103 Takei, N., 181, 222 Takeuchi, S., 2, 3, 43 Tal, M., 35, 43 Tamura, S., 87, 88, 98, 99, 103 Tanaka, H., 4, 40 Tanaks, S., 305, 318 Tanner, F. W . Jr., 28, 43 Tappel, A. L., 120, 129 Tatum, E. L., 3, 13, 15, 37, 42 Taubeneck, U., 93, 103 Taylor, C. V., 126, 129 Taylor, M. M . , 238, 250 Taylor, W. H. , 22, 43 Telling, R. C., 224, 250 Tempest, D. W., 224, 233, 234, 235, 236, 237, 240, 241, 242, 247, 248, 249, 250 Templeton, B., 266, 311 Terry, T. M., 55, 102 ThiBry, J., 93, 97 Thomos, J., 305, 314 Thompson, T. E., 66, 96, 99 Thore, A., 80, 103 Thorne, C. B., 14, 21, 23, 24, 26, 30, 42, 43 Thorpe, W . V., 181, 222 Threlfall, E . J., 92, 99 Tieffenberg,M., 70, 71, 75, 77, 78, 96, 104 Tien, H. T., 48, 103 Titus, E., 84, 99 Todaro, G. J., 37, 40

Tomlinson, G., 106, 108, 109, 124, 129 Tonnis, S. M., 7, 8, 26, 29, 33, 34, 44 Tono, H., 33, 43 Topaly, V. P., 66, 70, 100 Torriani, A-M., 253, 276, 318 Tosteson, D. C., 70, 71, 75, 76, 77, 78, 81, 96,103,104 Totter, J. R., 27, 29, 43 Trager, W., 106, 129 Travers, A. A., 292, 312 Treffers, H. P., 274, 316, 318 Trefts, P. E., 56, 102 Trimble, I. R., 83, 101 Triiper, H. G., 253, 263, 265, 300, 301, 317 Tschang, T. P., 95, 98 Tschudy, D. P., 267, 271, 279, 315, 317, 318 Tu, C-C. L., 253, 315 Tustanoff, E. R., 265, 270, 318 Tyler, B., 277, 278, 279, 284, 288, 289, 318 Tzagoloff, A., 83, 101

U Ueyama, H., 8, 41 Ullmann, A., 298, 318 Umbarger, H. E., 268, 318 Urabe, K., 181, 222

V Vallee, B. L., 34, 35, 40, 44 Vallin, I., 88, 104 van Dalen, A., 27, 36, 44 van Dam, K., 67, 97 Van Deenen, L. L. M., 61, 62, 63, 97, 100 VanBk, Z., 2, 43 van Heyningen, W . E., 19, 21, 27, 29, 40, 43 van Lanen, J. M., 28, 43 Van Praag, D., 58,103 Van Steveninck, J., 91, 102 Van Wagtendonk, W . J., 106, 107, 117, 129, 158, 178 Variechio, F., 56, 57, 104 Vernon, L. P., 51, 87, 89, 104 Vickerman, K., 111, 113, 115, I29 Vincent, 5. M., 232, 250 Vining, L. C., 8, 43 Vinogradova, E . I., 69, 71, 103 Vischer, W. A., 183, 221 Vloedman, D. A., 158, 178 Vogel, H. J., 244, 250 Vogt-Kohne, L., 177 Vojnovich, C., 28, 43 Volkonsky, M., 116, 129

338

AUTHOR INDEX

von Stedingk, L. V., 80, 99 Voss, J. G., 52, 104

W Wachtel, H., 48, 702 Wacker, W. E. C., 34, 35, 40, 44 Wade, H . E., 234, 250 Wadell, M . R., 181, 221 Waelsch, H . , 182, 222 Wagner, J., 180, 181, 221 Wahba, A. J., 302, 315 Wahn, K., 60, 104 Wainwright, S . D., 274, 318 Wakil, S. J., 259, 318 Waksman, S. A,, 27, 42 Waldron, J. C., 266, 313 Wallach, D. F . H., 48, 54, 97, 104 Walker, J. B., 6 , 37, 44 Walker, L. M., 158, 167, 170, 176 Walker, M . S., 37, 44 Walter, P., 82, 104 Walter, R. W., 258, 317 Wang, R. J., 293, 305, 318 Waring, W. S., 27, 44 Warren, R. A. J., 17, 26, 42 Watson, J. A., 264, 294, 296, 313 Watson, J. D., 275, 314 Wattiaux, V., 114, 128 Webb, E . C., 120, 128, 232, 249 Webb, M., 235, 250 Weber, M . M., 61, 104 Wedema, M., 27, 36, 44 Weeks, G., 259, 318 Wegener, W. S., 35, 44, 263, 318 Weinbach, E . C., 64, 66, 104 Weinberg, E . D., 3, 4, 7 , 8, 14, 15, 16, 20, 24, 28, 29, 30, 33, 34, 38, 43, 44, 52, 56, 57, 86, 104 Weiner, M., 304, 316 Weisenborn, F. L., 76,101 Weiser, R., 52, 104 Weisman, R. A., 122, 129 Weiss, S. B., 35, 40 Weissbach, H., 6, 14, 15, 21, 42, 44, 266, 315 Weissmann, G., 62, 103, 104 Wellend, F . H., 267, 271, 318 Weller, D. L., 116, 128 Wendt, L., 57, 103 Wensinck, F., 27, 36, 44 Werkman, C . H., 27, 44 Westhead, J. E., 27, 43 Wheeler, K. P., 84, 104 White, J. D., 20, 40 White, R. J., 95, 104, 260, 318 Whitehouse, M . W., 64, 104

Whittam, R., 84, 96, 104 Wiame, J. M., 311, 312 Widmayer, D., 158, 167, 170, 176 Wiesmeyer, H., 302, 318 Wijk, R. van, 260, 263, 264,281,283,284, 318 Wilborn, M., 106, 116, 117, 118, 119, 120, 121, 122,129 Wilcox, H. B., 140, 142, 149, 156, 163, 177 Wilkinson, B. E., 64,104 Wilkinson, J. F., 13, 44, 175, 176 Wilkinson, S . G., 52, 104 Williams, B., 286, 294, 295, 306, 316, 318 Williams, R. J . P., 33, 44 Williams, R. P., 26, 40 Williamson, D. H., 180, 222 Williamson, R. L., 64, 104 Wilson, B. J., 4, 44 Wilson, G. A., 23, 24, 26, 39, 44 Wilson, R. W., 260, 301, 302, 318 Wilson, T . H., 67, 104, 305, 318 Wimpenny, J., 52,104 Wimpenny, J.W.T., 263,264,268,269,314 Winder, F. G., 27, 42 Winderman, S., 260, 286, 315 Windisch, F., 67, 104 Winge, O., 304, 318 Winkler, H . H., 67, 104, 305, 318 Winkler, K . C., 260, 261, 264, 265, 269, 270, 31 7 Winnick, T., 36, 37, 39, 44 Wipf, H . K., 74, 104 Wisseman, C. L. Jr., 274, 314 Witt, D. M., 276, 312 Witt, I., 263, 269, 270, 279, 318 Witten, C., 93, 101 Wojnar, R. J., 24, 41 Wolfe, R. S., 21, 40 Wood, P. B., 181, 221 Wood, R. K. S., 2, 44 Woodard, J., 148, 178 Woodroffe, R. C. S., 64,154 Woodruff, H . B., 4, 13, 14, 17, 26, 29, 40, 44, 95,104 Woods, D. D., 300, 301, 318 Wright, B . E., 120, 125, 128, 129 Wright, D. N., 259, 318 Wright, G. G., 26, 44 Wu, M., 87, 98 Wurm, M., 91,104 Wyatt, H . V., 34, 44 wyss, o., 33, 43

Y Yamamoto, A,, 27, 42 Yanagisawa, K., 273, 318

339

AUTHOR INDEX

Yarmolinsky, M. B., 302, 318 Yoneda, M., 19, 20, 44 Yoshida, A., 22, 44 Yoshida, F., 21, 27, 32, 42 Yoshida, T., 14, 44 Young, F. E., 23, 44 Young, J. W., 267, 317 Young, M. R., 148, 177 Yphantis, D. A., 61, 104 Yudkin, J., 266, 290, 317 Yudkin, M. D., 273, 289, 291, 316 Yukioka, M., 36, 44

Z Zacharias, B., 5, 20, 44 Zaman, V., 111, 128 Zapf, K., 60, 104 Zeigel, R. F., 267, 271, 317 Zeydel, M., 89, 97 Zhdanov, 0.L., 74,103 Zink, M. W., 263, 270, 318 Zittle, C. A., 179, 222 Zopf, D., 63, 100 Zwaig, N., 301, 302, 307, 318

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SUBJECT INDEX A Acanthamoeba, encystment in, 108 micrograph of cyst of, 110 Acetamide and amidase, 181, 182, 183, 183, 185, 186, 187, 188-192, 195, 197, 198, 199, 203, 204, 205, 206, 207, 209, 210, 211, 212, 213, 215, 216, 219 Acetamide-induced serotype transformation, 170 Acetanilide-utilizing mutants, 212-214, 215,216,218 Acetate, catabolite repression in utilization of, 259 effect of on synthesis of isocitrate lyase by Pseudomonas ovalis, 243 Acetate kinase, catabolite repression in synthesis of, 259 Acetate-oxidizing activity of Pseudomonas ovalis,effect of carbon source on, 244 Acetate thiokinase activity of Pseudomonas ovalis, effect of carbon source on, 244 N-Acetylacetamide and amidases, 184, 185, 187, 189, 190, 191, 197, 199, 203, 206, 212 Acetyl-CoA kinase, catabolite repression in synthesis of, 265 Acid phosphatase, catabolite repression in synthesis of, 266 Aconitate hydratase, catabolite repression in synthesis of, 263 Actinomycins, 3, 6, 8, 11, 14, 27 effect on serotype transformation, 172, 173, 174 Actinomycin D and catabolite repression, 274 Actinorubin, 27 Active transport, 49 Acyl-CoA dehydrogenase, catabolite repression in synthesis of, 260 Acyl-CoA synthetase, catabolite repression in synthesis of, 259 Acyltransferase, 184, 186, 192 Acrylamide and aniidase, 183, 192, 195 Adenosine triphosphatase, activity of, 82 effect of dicyclohexylcarbodiimide on, 85 Adenosine triphosphatase and i-antigens, 158

341

Adenosine triphosphatase activity in bacteria, 81 Adenosine triphosphatases in bacteria, 49 Aeration, effect of on encystment in amoebae, 122 Aerobecter, ammonia as a preferred substrate in, 253 A . aerogenes, effect of dilution rate on composition of, 237 magnesium-limited cultures of, 233 ribonucleic acid composition of grown in a chemostat, 246 Aflatoxins, 3, 4, 12, 13, 28, 29 Age, effect of on encystment in amoebae, 120 Age of cells in batch culture, 225 Alamethicin, action of, 81 Alanine, catnbolite repression in degradation of, 262 Alcohol dehydrogenase, catabolite repression in synthesis of, 259 Alcohols, effect of on bacterial membranes, 54 Algae, encystment in, 106 Aliphatic amidases, 1S1, 182 Alkali metal ionophores, 68 Alkaline phosphatase, regulation of synthesis of in Escherichia coli, 253 Alkylguanidines, effect of on microbial membranes, 86 Alkyl quinoline oxides, effect of on bacterial membranes, 88 Alternariol, 36 Alternative substrates, choice of by microorganisms, 252 Amidase, A1 type, 212-214, 215, 216, 217 amino acids of, 214, 215 B type, 208-210,214,215, 216, 217 catabolite repression of, 191 esterase activity of, 193 genetic analysis of, 217, 218 heat-inactivation of, 213, 214 inhibitors of, 193-195 molecular weight of, 214 KInducer Of, 190, 191 Michaelis constants of, 216 mutants of, 196-217 reactions of, 195, 196

342

SUBJECT INDEX

Amidase-mnt. repression of, 188-192 specificity of induction, 186186 structure of, 214, 215 substrate specificity of, 192, 193 V type, 211, 212, 214 Amidase of Pseudomonas aeruginosa, 183217 B-Amidase mutants, 208-210, 217, 218 Amidase-negative mutants, 206-208, 217, 218 Amidases, 179-221 Amidase unit, 184 Amide transferases, 182-183 Amino-acid degradation, catabolite repression in, 256 Amino acids, catabolite repression in degradation of, 260 a-Aminoadipicacid, 1 1 Amino sugars, catabolite repression in utilization of, 260 Amoebae, encystment in, 105 Amphotericin B, action of on yeast membranes, 63 oc-Amylase, 21 Amylomaltase, catabolite repression in synthesis of, 259 Anaerobic shock and transient repression 280 Anaesthetics, effect of on bacterial membranes, 57 Anionic detergents, effect of on bacterial membranes, 55 Antibiotic formation, catabolite repression in, 266 Antibiotics and metal binding, 16, 17 and sporulation, 15, 16 as reagents in microbial physiology, 47 relation to catabolite repression, 274 Antifoams, use of with continuous cultures, 247 Antigen-determinant sitesof i-antigens,l37 Antimicrobial agents and membrane function, 45 Antimycin, effect of on bacterial respiration, 87 Antiserum-induced serotype transformation, 169-170 Arabinose epimerase, catabolite repression of, 258 Arabinose utilization, catabolite repression of, 258 Arabitol dehydrogenase, catabolite repression in synthesis of, 259 Arginase, catabolite repression in synthesis of, 261 Arginine, catabolite repression in degradation of, 261

Asparaginase, 181, 182 Aspartic acid, catabolite repression in degradation of, 261 Arsenate, effect of on encystment in amoebae, 123 Arthrobacter crystallopoites, repression of glucose utilization by succinate in, 257 Asparagine, catabolite repression in degradation of, 262 Aspartate oxidation, catabolite inhibition of, 301 Asynchronousencystment in amoebae, 118 ATPase, see Adenosine triphosphatase Autolysosomes in cysts in amoebae, 114 Aurovertin, effect of on cells, 85 Autogamy in Paramecium, 133 Autoradiography of i-antigen, 146 Autolysosome in encysting Hartlnnnnella castellanii, micrograph of, 114 N-Acetylglucosamine kinase, catabolite repression in synthesis of, 260 Axenic cultures, encystment in, 107, 118 Azasteroids, effect of on bacterial membranes, 56 Azotobacter sp., action of valinomycin on, 71

B Bacillin, 26 Bacillus antibiotics, 14-16 glutamyl polypeptide, 21 proteases, 21 B. anthracis protective antigen, 21,26 B. lichenifomis arginase, 33 B. cereus, synthesis of alkaline phosphatase in, 253 B. megaterium, action of antimycin on, 90 effect of vitamin A on, 54 penicillin amidase, 180 B. subtilis, u amylase, 21 effectof dilution rate on composition of, 237 growth of, under conditions of magnesium limitation, 236 regulation of histidine degradation in, 287 ribonuclease, 21 synthesis of cell walls by, 239 transformation, 23, 24 B. thuringiensis toxin, 21 Bacitracins, 3, 7, 8, 14, 19, 26, 29, 33, 34 action of, 52 Bacterial cation metabolism, use of chemostat in studies of, 232 Bacterial cell walls, use of chemostats in study of synthesis of, 238 Bacterial transport systems, 49

SUBJECT INDEX

Bacteriocins, 23 action of, 91 Balanced growth in chemostats, 231 Batch culture, a closed system, 224 unnatural nature of, 224 Benzamidase, 182 Benzoate, catabolite repression in utilization of, 259 Biguanidines, effect of on microbial membranes, 86 Biochemistry of encystment in amoebae, 123 Botulinus toxin, 18, 19, 20 Brij 59, effect of on Escherichia coli, 52 Budding, catabolite repression of in yeast, 266 Butanol, effect of on bacterial membranes, 54 Uutyramide and amidase, 181, 182, 185, 189, 192, 195, 196, 199-202, 206, 207, 208, 209,210,211, 212, 213, 216, 218 Butyramide-resistant mutants of amidase, 196, 199, 200,201, 202, 218

C Cadmium, effect of on ion accumulation, 91 Calcium and secondary metabolism, 25, 31 Camphor, catabolite repression in utilization of, 259 Candida amidase, 181, 183 Candicidin, 27 Carbohydrates, as wall components in cysts, 109 catabolite repression in utilization of, 272 control of utilization of, 252 Carbohydrate intermediary metabolism, 294 Carbohydrate utilization and catabolite inhibition, 301 control of in micro-organisms, 251 Carbonylcyanide, uncoupling action of, 64 Carboxylic polyethers, action of, 78 Casamino acids, catabolite repression and utilization of, 273 Catabolite inhibition, examples of, 300 discovery of, 254 identity of the effector, 293 locus, 290 nature of, 298 relation t o transient repression, 278 role of, 252 Catabolite repression, and amidase, 203206 of amidase. 191 of ,B-galactosidasesynthesis, 295 in micro-organisms, 251

343

Catabolite repression-cont. mechanism of, 281, 362 nature of, 254 origin of name, 254 relationship t o growth, 272 role of, 252 Catabolite-resistant mutants of amidase, 196,202-203,217,218 Catabolite RNA polymerase, 292 Cation metabolism, bacterial, use of chemostat in studies in, 232 Cation transport, 15 and energy generation in bacteria, 73 Cationic detergents, effect of on bacterial membranes, 56 Cell wall composition of Bacillus subtilis as affected by substrate limitations, 239 Cell walls, bacterial, function of, 239 bacterial, use of chemostats in study of synthesis of, 238 Cellular differentiation, encystment as, 106 Cellulose as a wall component in cysts, 109 Cellulose production as a measure of encystment in amoebae, 108 Cetyltrimethylammonium bromide, effect of on bacterial membranes, 56 Chelating agents, effect of on encystment in amoebae, 119 Chemical coupling in oxidative phosphorylation, 50 Chemical-induced serotype transformation, 170 Chemi-osmotic, coupling in oxidative phosphorylation, 50 hypothesis, 65 Chemostat, and amidase production, 203206 8s research tools, 229, 231 nature of, 229 Chitin as a wall component in cysts, 109 Chloramphenicol, 12, 20, 27 and catabolite repression, 274 effect of on encystment in amoebae, 124 effect on serotype transformation, 172, 173 Chlorella, inhibition of carbon dioxide fixation in, 253 Chlorhexidine, effect of on bacterial membranes, 56 effect of on microbial membranes, 86 Chloroform, effect of on bacterial membranes, 54 para-Chloromercuribenzoate, action of on bacteria, 90 Chlorophyll production, catabolite repression &, 266 Chloroplasts, proton conduction in, 66 Chromatophores, proton conduction in, 66

344

SUBJECT INDEX

Chromosomes of Paramecium, 147 Chymotrypsin-induced serotype transformation, 170 Cilia, i-antigen association with, 144, 145 Citrate permease, catabolite repression of synthesis of, 264 Citrate synthase, catabolite repression in synthesis of, 263 Cobalt and secondary metabolism, 28 Cobalt ions, effect of on bacterial membranes, 91 Colicins, action of, 91 Compounds which disorganize lipoprotein membranes, 53 Conformational coupling in oxidative phosphorylation, 51 Continuous culture, and amidase, 203-206 and secondary-metabolite production, 5 inadequacies of as a research tool, 245 place of in microbiological research, 223 Continuous flow cultures, 228 Control of carbohydrate utilization in micro-organisms, 251 of encystment in amoebae, 125 of inducer concentration, 303 Control genes for i-antigens, 150, 151, 152, 153, 154 Clostridium neurotoxin, 27 Copper and secondary metabolism, 26, 28, 31 Coproporphyrin, 27, 28 Cusohygrine, 37 Cyanamidase of Steiromagmatocystis, 180 Cyanoacetamide and amidases, 184, 185, 189, 190, 191, 193, 195, 197, 201, 202, 208 Cyclic AMP, and diauxie, 298 and glucose repression, 297 and transient repression, 285 Cysts, nature of in micro-organisms, 106 Cyst wall, in ameobae, 109 of Hartmannella castellanii, micrograph of, 111 Cytochrome oxidase, catabolite repression of synthesis of, 265 Cytoplasmic organelles in cysts in amoebae, 115 Cytoplasmic membrane, action of antibiotics on, 46 permeability, effect of gramicidins, 7 5

D Dactylum dendroides, catabolite repression of glucose oxidase in, 258 Decamethylenediguanidine, effect of on microbial membranes, 86

Decay of 8-galactosidase messenger RNA, 282 Defoaming agents, use of with continuous cultures, 247 Design of continuous culture equipment, 247 of continuous-flow culture vessels, 229 Detergents, effect of on bacterial membranes, 55 effect of on Escherichia coli, 52 Dianemycin, action of, 78 Diauxie, and cyclic AMP, 298 nature of, 308 Dicyclohexylcarbodiimide, action of on membranes, 8 5 Differential rate of enzyme synthesis, use of, 254 Dilution rate, in continuous cultures, 230 effect of on macromolecular composition of bacteria, 246 influence of on steady-state concentration of Aerobacter aerogenes in a chemostat, 233 relation of t o growth rate in continuous cultures, 231 Dinactin, action of, 76 Dinitrophenol, uncoupling action of, 65 DNA polymerases and edeine, 16 Dio 9, effect of on microbial membranes, 86 Diphtheria toxin, 8 , 9 , 18, 1 9 , 2 0 , 2 1 , 2 9 , 3 4 Dipicolinate synthase, 15, 21 Dipicolinic acid, 6, 12, 16 Disorganization, reversible membrane, 57 Disulphide bridges in i-antigens, 136, 137 Dulcitol, catabolite repression in utilization of, 259 Duration of runs in chemostats, 145 of secondary-metabolite synthesis, 8-12

E Edeine, 7, 8, 9, 16 biosynthesis, 37 Effector synthesis in catabolite repression, 306 Electrogenic cation pumps, 72 Electron acceptors and transient repression, 280 Electron transport and generation of ATP, 49 Embden-Meyerhof pathway enzymes, catabolite repression of synthesis of, 257,259 Encystment, experimental approaches used in studying, 107 in axenic cultures of amoebae, 118

345

SUBJECT INDEX

Encystment in amoebae, 105 biochemistry of, 123 control of by metabolites, 125 fate of major cell organelles during, 124 measurement of, 108 physiology of, I17 structural changes during, 109 Encystment in mixed cultures, 117 Endocyst in Acanthamoeba, 110 Endoplasmic reticulum and i-antigen synthesis, 164, 165 Energy coupling in solute transport, 49 Energy generation and cation transport in bacteria, 73 Energy transfer in bacteria, effect of Dio 9 on, 86 inhibitors of, 81 Enniatins, action of, 74 formula of, 69 Enoyl-CoA hydratase, catabolite repression in synthesis of, 259 Enterotoxin, staphylococcal, 18, 19, 20, 21 Entamoeba histolytica, encystment in, 106 Entner-Doudoroff pathway, catabolite repression of synthesis of enzymes in, 257 Entry of inducers into micro-organisms, 305 Environmental conditions which produce catabolite repression, 271 Enzymes of the lac operon, 288 Enzymes, microbial, use of chemostats in study of synthesis of, 241 synthesis in encystment in amoebae, 124 Ergolamine, 28 Ergoline alkaloids, 8 Erythromycin, 8 Eschmichia coli, action of colicins on, 91 eatabolite repression and synthesis of /3-galactosidasein, 281 catabolite inhibition of lactose utilization by, 299 effect of EDTA on, 52 induction of tryptophanase synthesis in, 253 oxidative phosphorylation in, 66 regulation of synthesis of alkaline phosphatase in, 253 transient repression in, 271 Estcrase activity of amidase, 193, 195 Ethylenediaminetetraacetic acid, effect of on Escherichia coli, 52 Eucaryotes, encystment in, 106 Euglena, inhibition of carbon dioxide fixation in, 253 Examples of catabolite inhibition, 300 Excystment in amoebae, 126 Exocyst in Acanthamoeba, 110

Experimental approaches used in studying encystment, 107 Exponential growth phase in batch culture, 225

F Factors limiting synthesis and growth, 273 Fate of major cell components in encystment in amoebae, 124 Fatty acid metabolism, catabolite repression in, 259 Ferrichrome, 28 Ferritin-conjugated antibodies, 145, 146 Ferritin-labelled i-antigen, 168, 1G9 Filipin, chemical structure of, 61 Fingerprinting of i-antigens, 137 Foaming as a hazard in continuous cultures, 247 Food reserves in amoebae, 115 Formamide-inducible mutants of amidase, 196,197,198,217,218 Formamide and amidase, 181, 182, 184, 185, 192, 195, 19G, 197, 198,209,218 Formate dehydrogenase, catabolite repression in synthesis of, 265 Formiminoglutamic hydrolyase, catabolite repression in synthesis of, 260 Fluorescein-conjugated antibodies, 144, 145 Fluorescent -labelled i-antigen, 168 Fluoresein, 27, 29 Fluoride and amidase inhibition, 193 Flagella production, catabolite repression in, 256 Flavensomycin, action of on electron transport, 90 Fructokinase, catabolite repression of, 258 Fructose oxidation and catabolite inhibition, 301 Fructose utilization, catabolite repression of, 258 Fulvic acid, 36 Fumarate hyclratase, catabolite repression of synthesis of, 264 Function of cysts in amoebae, 126 of microbial membranes, 47

G Galactokinase, eatabolite repression of, 258 Galactose, eatabolite inhibition and utilization of, 301 Galactose-phosphate uridylyl transferase, eatabolite repression of, 257 Galactose utilization, catabolite repression on, 258

346

SUBJECT INDEX

fi-Galactosidase,191, 199, 203 catabolite repression of, 258 fi-Galactoside permease, catabolite re. pression of, 258 P-Galactosidase synthesis, and catabolite repression, 295 and transient repression, 297 Galactoside acetyl transferase, catabolite repression of, 258 Galactozymase induction in yeast, 253 Gel-diffusionof i-antigens, 140 Generation of ATP and electron transport, 49 Genetics of serotype expression, 147-157 Genetic changes in organisms in a chemostat, 245 Gentisyl alcohol, 7, 28, 32, 36 Germination of cysts in amoebae, 126 Gibberellins, 2, 3 Glucokinasein utilization of gluconate, 253 Gluconokinase, catabolite repression in synthesis of, 260 catabolite inhibition and utilization of, 301 Glucose as an inhibitor of entry of other sugars, 305 Glucose effect, nature of, 253 a-Glucosidase, catabolite repression in synthesis of, 260 synthesis in yeast and catabolite repression, 283 8-Glucosidase, catabolite inhibition of, 301 Glutamate, effect of on encystment in amoebae, 125 Glutamate dehydrogenase, catabolite repression in synthesis of, 261 Glutamate oxidation, catabolite inhibition of, 301 Glutamic acid, catabolite repression in degradation of, 261 Glutaminase, 182 Glutamine, catabolite repression in degradation of, 262 synthesis, 33, 34 D-Glutamyl polypeptide, 21,26 Glycerol, metabolism of in Eschcrichia coli, 306 Glycerol dehydrogenase, catabolite repression of, 258 Glycerol kinase, catabolite inhibition of, 301 catabolite repression of, 258 Glycerol utilization, catabolite repression of, 258 a-Glycerophosphate, synthesis of in Escherichia coli, 307 Glycerophosphate transport, catabolite repression of, 258

Glycinamide and amidase, 181, 185, 192 Glycine, catabolite repression in degradation of, 262 Glycogen reserves in amoebae, 115 Glycollamide and amidase, 183, 185, 192, 195 Glyoxylate cycle, catabolite repression of enzymes on, 263 Golgi body in cysts in amoebae 113 Golgi vesicles in Acanthamoeba, 110 Gradient-coupled transport in bacteria, 49 Gramicidin, biosynthesis of, 36, 37 Gramicidin-S, effect of on bacterial membranes, 59 Gramicidin, action of, 68, 74 Gramicidme, 15 Gratuity in metabolic regulation, 303 Grisein, 27 Griseofulvin, 28, 36 Growth cycle in batch culture, 225 Growth factors, catabolite repression and starvation of, 273 Growth-limiting nutrients in continuous cultures, 230 Growth rate, in microbial cultures, 228 relation of to dilution rate in continuous cultures, 231 Growth stasis, relief of by glucose, 302 Growth substrates, catabolite repression in utilization of, 272 Growth temperature and catabolite repression, 276 Growth yield in continuous cultures, 232 Guanidines, effect of on bacterial membranes, 57

H 8-Haemolysin, 21 Haemophilus infuenzae competence, 24 Hartmannella astronyx;s, encystment in, 108 H . castellanii, encystment in, 108 micrograph of cyst wall of, 111 H . rhysodes, encystment in, 108 Hartmannellids, encystment in, 106 Heavy metals, interaction of with microbial membranes, 90 Heptyl quinoline oxide, effect of on bacteria, 89 structural formula of, 88 Hexanoamide and amidase, 189 Hexokinase, catabolite repression in synthesis of, 260 Hexose monophosphate shunt, catabolite repression of synthesis of enzymes of, 257 Hispidin, 16

347

SUBJECT INDEX

Histidase, catabolite repression in synthesis of, 260 Histidine, catabolite repression in degradation of, 260 effect of on encystment in amoebae, 125 Histidine degradation, regulatory mutants and, 287 Histones, effect of on bacterial membranes, 60 Historical review of regulation of carbohydrate utilization, 253 Homogeneity of cells in continuous cultures, 231 Hybrid i-antigens, 141-3 Hydrogen ion concentration, effect of on encystment in amoebae, 121 Hydrogenomonas, regulation of carbon dioxide fixation in, 253 use of electron donors by, 252 Hydroxamate synthesis, 182 Hydroxyproline, catabolite repression in degradation of, 262 Hydroxyproline reduction, catabolite inhibition of, 301

I I-antigens, cellular localization of, 144-147 effect on i-antigen synthesis, 174 formation of, 158-165 function of, 157, 158 hybrids of, 141-3 in ribosomes, 146 preparation of, 134-135 relationship of different ones, 139-141 secondary, 143-144, 157 structure of, 134-144 subunits of, 135 transport of, 164, 165 tryptic digest of, 137, 138 I-antigen-determining genes, 148-150 Identity of the effector in catabolite repression, 293 Immobilization-antigens of Paramecium, 132-175 Inadequacies of continuous culture as a research tool, 245 Incubation temperature, effect of on magnesium content of Aerobacter aerogenes, 235 Induction of enzyme synthesis, use of differential rate in study of, 255 Induced encystment in amoebae, 119 Inducer concentration, control of, 303 Inducer entry, 305 Inducer specificity for amidase, 184-186 Induction kinetics of serotype transformation, 166-170

Induction lag with acetamide, 186 Inhibitors of amidase, 193-195 Inhibitors of energy transfer in bacteria, 81 Inhibitors of the respiratory chain in bacteria., 81 Inophores, 64 5’-Inosinic acid, 26 Inositol dehydrogenase, catabolite repression in synthesis of, 259 Interaction of heavy metals with microbial membranes, 90 Intermediary metabolism of oarbohydrates, 294 Internal membranous organelles in bacteria, 47 Invertase, catabolite repression of, 258 Iodoacetate, effect of on encystment in amoebae, 123 Ion transport and ATPase, 82 Ionic requirements for encystment in amoebae, 119 Iron, and secondary metabolism, 25, 26, 28, 29, 30, 31, 32, 34, 35, 39 and secondary metabolites, 17, 20 Irehdiamine, effect of on bacterial membranes, 57 Isocitrate dehydrogenase, catabolite repression in synthesis of, 263 Isocitrate lyase, catabolite repression in synthesis of, 263 Isoleucine, cataboliterepressionin degradation of, 262 Itoic acid, 17

K Kinetics, of secondary-metabolite synthesis, 5-13 of serotype transformation, 166-170 Kojic acid, 4, 8

L Laboratory artefacts in microbiology, 225 L a c operon, enzymes of, 288 Lactamide and amidase, 184, 192, 195, 209 Lactate oxidase, catabolite repression in synthesis of, 259 Lactobacillus plantarum, catabolite repression of arabinose isomerase in, 258 Lactose, catabolite repression in utilization of, 258 Lactose-glucose diauxie in Escherichia coli, 310 Lactose utilization by Escherichia eoli, catabolite inhibition of, 299 Lag phase in batch culture, 225

348

SUBJECT INDEX

Lanthanum ions, effect of on microbial membranes, 91 Lecithinase, clostridial, 27 Leucine biosynthesis, enzymes involved in, 287 Leucine, catabolite repression in degradation of, 262 Leucocidin, 21 Levallorphan, effect of on RNA synthesis in Escherichia coli, 58 Lipid as a wall component in cysts, 110 Lipopolysaccharide, release of from Gramnegative bacteria, 52 Lipoprotein membranes, compounds which disorganise, 53 Locus for cataholite repression, 290 Longer term adaptation in microorganisms, 304 Lysergic acid, 28 Lysine, catabolite repression in degradation of, 262

M Macrolides, 2, 3 Macromolecular composition of bacteria grown in a chemostat, 246 Macronucleus of Paramecium, 147, 148 Macrotetralides, action of, 76 Magnesium and secondary metabolism, 25, 31, 35 Magnesium ions, effect of on encystment in amoebac, 120 Magnesium-limited cultures of Aerobacter aerogenes, 233 Magnesium metabolism in bacteria, use of chemostats in study of, 232 Magno-constitutive mutants of amidase, 196,197,198, 199, 205,206,217,218 Malate dehydrogenase, catabolite repression in synthesis of, 263 Malate synthase, catabolite repression in synthesis of, 263 Malformins, 2, 3, 4, 28 biosynthesis, 36, 37 Malouetine, effect of on bacterial membranes, 57 Maltose a n a n inducer of a-glucosidase synthesis in yeast, 284 Maltose utilization, catabolite repression of, 258 Manganese and secondary metabolite production, 23, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 Mannitol, catabolite repression in utilization of, 259 Mature cyst of Acanthamoeba, micrograph of, 112

Mayorella palestinensis, encystment in, 108 Measurement of encystment in amoebae, 108 Mechanism, of catabolite inhibition, 302 of transient repression, 281 Medium composition, effect of on encystment in amoebae, 120 Medium for encystment of amoebae, 108 Megacin, action of, 93 Membrane function and antimicrobial agents, 45 Membrane sterols, 61 Membrane, bacterial, integration with wall and nucleus, 51 lipoprotein, compounds which disorganise, 53 Mercury compounds, effect of on microbial membranes, 90 Mesosomes, 47 as source of respiratory enzymes in bacteria, 50 Messenger-RNA and serotype transformation, 170-174 Messenger synthesis and catabolite repression, 283 Metabolic concentrations in regulation of carbohydrate utilization, 256 Metabolic inhibitors, relation t o catabolite repression, 274 N-Methyl acetamide and amidase, 184, 185, 186, 188 6-Methylsalicyclic acid, 5 , 6, 7 Michaelis constants for microbial growth, 228 Microbial enzyme synthesis, use of chemostats in study of, 241 Microbial growth, in a closed system, 224 in a n open system, 228 Microbiological research, place of continuous culture in, 223 Micrococcus lysodeikticus, respiratory enzymes in membranes of, 50 synthesis of cell walls by, 239 Micronucleus of Paramecium, 147 Minerals as wall constituents in cysts, 109 Mitochondria, action of nactins on, 77 effect of oligomycin on, 83 in Acanthamoeba, 110 in cysts in amoebae, I15 Mitochondria formation, catabolite repression of, 266 Mitomycins, 3, 8, 13, 27 Mixed cultures, encystment in, 107 Models of repression, 291 Monactin, formula of, 76 Monazomycin, action of, 80 Monod equation for microbial growth, 228

349

SUBJECT INDEX

Monenoin, 27 action of, 78 M-protein of streptococci, 22 Mucopeptide synthesis, effect of bacitracin on, 5 2 Mucor, catabolite repression in hyphae formation in, 257 Mutants of amidase, 196-217 Mutant organisms, formation of in continuous cultures, 247 Mycobacillin, 26, 29 Mycobacterium amidase, 181, 182, 188 catabolite repression of glycerol kinase in, 258 M . phlei, 17 action of valinomycin on, 71 effect of bacitracin on, 53 Mycobactin, 17, 27 Mycoplasma sp., membranes in, 47 Myxobacter proteases, 21

Organic solvents, effect of on bacterial membranes, 5 4 Ornithine, catabolite repression in degradation of, 262 Orsellinic acid, 36 biosynthesis of, 37 Osmoplasts of Gram-negative bacteria, 5% Osmotic compartments in the microbial cell, 47 Osmotic requirements for encystment in amoebae, 119 Ossamycin, effect of on microbial membranes, 82 Ostioles in Acanthamoeba, 110 Oxidative phosphorylation, uncoupling of in bacterial membranes, 63 Oxygen, effect of on encystment in amoebae, 122 effect of on transient repression, 280

N

P

Nactins, 76 Naegleria, cysts of, 11 1 Narcotics, effect of on bacterial membranes, 57 Neomycin, 8, 27 Nickel ions, effect of on bacterial membranes, 91 Nicotinamidase, 180, 181, 182 Nigericin, 15 action of, 78 Nisin, 17, 18 Nonactin, action of, 76 Non-ionic detergents, effect of on bacterial membranes, 56 Noniset, affect of on bacterial membranes, 56 Nucleus in cysts in amoebae, 115 Nuclear activity in serotype-transformation, 170-172 Nucleolus in cysts in amoebae, 115 Nutrients, effect of on encystment in amoebae, 120 Mystatin, action of on yeast membranes, 62

Paraffin oxygenase, catabolite repression in synthesis of, 259 Paramecium, serotype expression in, 132175 P. aurelia, description of, 133 Patulin, 7, 28, 36 action of on electron transport, 90 Patulin-induced serotype transformation, 170 Peliomycin, effect of on microbial membranes, 82 Pellicle, i-antigen association with, 144,145 Pentachlorophenol, uncoupling action of, 64 Penicillins, 11, 12, 13, 28, 29 Penicillin amidases, 180 Penicillin biosynthesis, 36 Penicillin 13-lactamase, 180 Penicillium,induction of amylase synthesis in, 253 Peptide-analysis of i-antigens, 137-140 Peptide antibiotics, effect of on bacterial membranes, 58 Peptides, as secondary metabolites, 2, 3 Peroxisomes in amoebae, 124 Perylenequinone, 16 Permeability barrier, microbial membranes as a, 47 pH value, effect of on encystment in amoebae, 121 Phagocytic behaviour in relation t o encystment in amoebae, 122 N-Phenylacetamide and amidase, 185, 189 Phenylalanine, catabolite repression in degradation of,262

0 Occurrence of catabolite repression, 256 of transient repression, 276 Ochratoxins, 3 Oligomycin, effect of on bacterial membranes, 82 Onset of secondary-metabolite synthesis, 5-8 Operational problcms in continuous cultures, 246

3 50

SUBJECT INDEX

Phenyl alcohol, effect of on bacterial membranes, 57 Phenethylbiguanidine, effect of on microbial membranes, 86 Phenoxazinone synthase, 6, 15, 21 Philosophy of continuous culture, 223 Phosphatase in autolysosomes in amoebae, 114 Phosphate, effect of on encystment in amoebae, 119 Phosphate limitation, effect of on cell-wall composition of Bacillus subtilis, 240 Phosphoenolpyruvate carboxykinase, catabolite repression in synthesis of, 265 Phosphorylation in mitochondria, effect of oligomycin on, 83 Physical agents and catabolite repression, 275 Physiological heterogeneity in batch cultures, 226 Physiological events in encystment in amoebae, 118 Physiology of encystment in amoebae, 117 Piericidin, action of on microbial membranes, 87 Plasticity of prokaryotic! cells, 225 Polyene antibiotics, 61 Polyenes, 2, 3 Polyethers, action of, 81 Polyglutamate synthase, 35, 36 Polylysine, antimicrobial activity of, 60 Polymyxin, 3, 8, 15 biosynthesis, 36, 37 effect of on bacterial membranes, 60 Polypeptide uncouplers and bacteria, 69 Polyribosomes and i-antigen synthesis, 161, 163 Polytene-like chromosomes of Paramecium, 148 Pool sugars, use of, 256 Popularity of continuous culture, 248 Porphyrin synthesis, 32 catabolite repression in, 271 Positive-feedback control in Paramecium, 152, 153 Potassium, ion movements and valinomycin, 70 limitation in continuous cultures, 236 Potassium metabolism in bacteria, use of chemostats in study of, 232 Prokaryotic cells, plasticity of, 225 Proline, catabolite repression in degradation of, 262 Promotor mutants and repression, 292 Propionamide and amidase, 181, 183, 184, 185, 192, 193, 194, 195, 197, 198, 213,216, 217

Proteinases, bacterial, 179, 180 catabolite repression in synthesis of, 266 of Bacillus, 21 of fungi, 21 of Myxobacter, 21 of Streptomyces, 21, 32 Protamines, antimicrobial activity of, 60 Proteins as wall components in cysts, 109 Proton conduction in bacterial membranes, 63 Proton gradients, role of in oxidative phosphorylation, 51 Protozoa, encystment in, 106 Pseudomonad amidases, 183 Pseudomonas spp., effect of detergents on, 52 Pseudomonas, genetic analysis of, 217, 218 genetic homology of, 218, 219, 220 Ps. aeruginosa, amidase, 183-217 catabolite repression in, 278 Ps. fluoreseem, action of gramicidins on, 15 Ps. ovalis, anaplerotic routes in, 242 effect of carbon source on acetate thiokinase activity of, 244 Ps. putida, magnesium-limited cultures of, 233 Pseudotropine, 37 Puromycin, and catabolite repression, 274 effect on serotype transformation, 172, 173 Pyocyanine, 27, 29 action of on electron transport, 90 Pyrophosphatase, 33 Pyrrolnitrin, 5 Pyruvate oxidase, catabolite repression in synthesis of, 259

Q Quantitative factors in catabolite repression, 254 Quinone function in membranes, effect of piericidin on, 87

R Rates of enzyme synthesis, control of, 254 Regulation of gene-expression in Paramecium, 150-157 Regulator mutants of amidase, 196, 197, 217, 218 Replacement technique, use of in studies of encystment, 107 Repression, of amidase, 188-192 in regulatory mutants, 285 models of, 291

SUBJECT INDEX

Research tool, inadequacies of continuous culture as a, 245 Resistance of cysts in amoebae, 126 Respiratory chain in bacteria, inhibitors of, 86 Respiratory enzymes, catabolite repression of synthesis of, 265 Respiratory, metabolism of encysting amoebae, 123 shock and transient repression, 280 Reversible membrane disorganization, 57 Rhamnose utilization, catabolite repression of, 258 Rhodospirillum rubrum, action of antimycin on photophosphorylation in, 89 offect on Dio 9 on, 86 Itibitol dchydrogenase, catabolite repression in synthesis of, 259 Ribokinase, catabolite repression in synthesis of, 260 Ribonuclease, 21 Ribonucleic acid and trace metals, 35 catabolism in protozoa, 172 Ribonucleic acid composition of Aerobacter nerogenes grown in a chemostat, 246 Ribonucleic acid, influence of magnesium limitation on contents of in bacteria, 234 Ribosomes, and i-antigen, 146 ini-antigen synthesis, 159, 161, 162, 163, 164 Ribosomes, bacterial, effect of magnesium limitation on, 234 Ribulose diphosphate carboxylase, cstabolite repression in synthesis of, 265 Rotenone, action of on microbial membranes, 87 Rutamycin, effect of on bacterial membranes, 82

S Succharomyces, role of catabolite repression in budding in, 257 Saccharomyces carlsbergensis, catabolite repression of or-glucosidase synthesis in, 283 S . cerevisiae, catabolite repression of invertase synthesis in, 258 S. fragilis, catabolite repression of sucrose utilization in, 258 Salicylanilides as uncoupling agents, 64 Salicylic acid, 27 Salmonella typhimzrrium, catabolite renression in. 278 Saramycetin, 18

35 1

Schizophyllum commune, repression of 8-glucosidase synthesis by glucose in, 302 Schizopyrenus, mitochondria in, 115 Secondary i-antigens, 143-144, 157 Secondary metabolites, chemical nature of, 2-5 function of, 13-18 kinetics of synthesis of, 5-13 location of, 12, 13 specialized functions of, 14-18 stability of, 12, 13 yield of, 12, 13 Selection in a chemostat, 245 Semi-constitutive mutants of amidase, 196,197,199,217,218 Sensitivity of Gram-negative bacteria to detergents, 52 Serine deaminase, catabolite repression in synthesis of, 261 repression and synthesis of, 286 Serotype transformation in Paramecium, 165-175 Serotypes of Paramecia, 132-175 Shigella dysenteriae neurotoxin, 2 1 Shigella toxins, 27, 29 Sideramine, 17 Siomycin formation, catabolite inhibition of, 301 Slime-mould cysts, 11 1 Sodium chloride, as an antifoam in continuous cultures, 247 use of in inhibiting uptake of magnesium ions by bacteria, 240 Sodium dodecyl sulphate, effect of on bacterial membranes, 5 5 Sorbitol, catabolite inhibition and utilization of, 301 cataboliterepression in utilization of, 259 diauxie of with glucose in Escherichia coli, 309 Space-filling model of the monactinpotassium complex, 77 Sporulation, and antibiotic production, 15, 16 catabolite repression in, 266 and secondary metabolite production, 23 and toxic production, 20 Stalked particles in bacterial membranes, 50 Staphylococcal, enterotoxin, 18, 19, 20, 21 6-haemolysin, 21 leucocidin, 21 Staphylococcus n.ureus, synthesis of cell walls by, 239 Staphylococcus, enterotoxin, 8, 10 resistance of t o heavv metal ions. 91 Stationary phase in batch culture, 225

352

SUBJECT INDEX

Steady-statc conditions in chcmostats, 231 Steroid diamines, effect on permeability of bacterial membranes, 57 Sterols as polyene receptors in yeast membranes, 62 Stock ofParamecium, definition of, 133 Stratification in cyst wall in amoeba, 111 Streptidine, 6, 16 biosynthesis, 37 Streptococcal M-protein, 22 Streptococcus fuecalis, action of monensin on, 79 effect of dicyclohexylcarbodiimide on, 85 oxidative phosphorylation in, 66 Streptococci, group A, 21 Streptolysin-S, 21 Streptomyces grisems proteinase, 180 Strcptomyces proteases, 21 Streptomycin, 8, 16, 20, 27, 31 -resistant mutants, 15 Structural changes during encystment, in amoebae, 108 time-course of, 116 Structural genes for i-antigens, 148-150 Structural integrity of bacterial membranes, destruction of, 5 3 Structure of microbial membranes, 47 Substrate limitations, effect of on cell-wall composition of Bacillus subtilis, 239 Substrate specificity of amidase, 192, 193 Substrate utilization, catabolite repression, in, 257 Subtilin, 26 Subunits in membrane structure, 4 8 Succinate dehydrogenase, catabolite repression of synthesis of, 264 Succinate, effect of on synthesis of isocitrate lyasc by Pseudomonas ovalis, 242 Succinyl-CoA synthase, catabolite repression in synthesis of, 265 Succinyl perimycin, action of on yeast mcmbranes, 62 Sucrose utilization, catabolite repression of, 258 Sulphydryl compounds, effect on heavy metal action on membranes, 90 Syngen of Paramecium, definition of, 133 Synthesis, of bacterial cell walls, use of chemostats in study of, 238 of effectors in catabolite repression, 306 of i-antigen in vitro, 163, 164 of i-antigen in vivo, 159-162 of microbial enzymes, use of chemostats in study of, 241 of teichuronic acids in walls of Bacillus aubtzlis, 24 1 o f tricarboxylic acid-cycle enzymes, catabolite repression in, 256

T Tartrate permease, catabolite repression in synthesis of, 259 Teichoic acids, in bacterial walls, 239 relation t o potassium contents of bacteria, 238 synthesis of in walls of Bacillus subtilis, 240 Temperature-induced serotype transformation, 166-169 Temperature, effect of on encystment in amoebae, 122 Tetanus toxin, 18, 19, 20, 21 Tetrachlorosalicylanilide, uncoupling action of, 6 4 Tetrachlorotrifluoromethyl benzimidazole, uncoupling action of, 6 4 Tetracycline, 12 effect of on encystment in amoebae, 124 Tetrahymena, and RNA catabolism, 172 regulation of, 151, 155 Thiol reagent as amidase inhibitors, 194, 195 Thioacetamide and amidases, 184, 185, 189, 193 Thiolase, catabolite repression in synthesis of, 260 Threonine deaminase, catabolite repression in synthesis of, 261 Time-course of structural changes during encystment, 116 Toluene, effect of on Escherichia coli, 53 Toluquinol, 36 Torulopsis spp., action of antimycin on, 89 Toxin, botulinus, 18, 19, 2 0 diphtheria, 18, 19, 20, 21 tetanus, 18, 19, 20, 21 Toxins and sporulation, 20 Training for encystment in amoebae, 121 Transient repression, nature of, 276 Transcriptional control in catabolite repression, 281 Transformation of serotype in Paramecium, 165-175 Transient phenomena in continuous culture, 246 Transient repression, and respiratory shock, 280 discovery of, 254 mechanism of, 281 nature of, 254 occurrence of, 276 of P-galactosidase synthesis, 297 of P-galactosidase synthesis in Escherichia coli, 284 relation to catabolite repression, 278 role of, 252

353

SUBJECT INDEX

Translational control in catabolite repression, 281 Transport of i-antigen, 164, 165 Transport systems in bacteria, 48 Trehalose, catabolite repression in utilization of, 259 Tremorgen, 4 Tricarboxylic acid cycle, catabolite repression of enzymes on, 257, 263 Trinactin, action of, 76 Trypanosoma cruzi, cyst formation in, 117 Trypsin-induced serotype transformation, 170 Tryptic digest of i-antigens, 137, 138 Tryptophan, catabolite repression in degradation of, 261 Tryptophanase, catabolitc repression in synthesis of, 261 induction of synthesis of in Escherichia coli, 253 Turbidostat, nature of, 229 Tyrocidine, 3, 4, 13, 15 action of, 68 effect of on bacterial membranes, 58 Tyrosine, catabolite repression in degradation of, 261 Tyrothricin, effect of on bacterial membranes, 58

V Valeramide-utilizing mutants, 21 1-212, 218 Valeramide and amidase, 181, 192, 196, 207, 211, 2 1 2 , 2 1 8 Valine, catabolite repression in degradation of, 262 Valinoniycin, 2, 3, 15 action of, 69 action of on bacterial membranes, 63 antibacterial action of, 71 formula of, 69 Vancomycin, action of, 53 Venturicidin, effect of on microbial membranes, 82 V'ibrio cholerae vascular permeability factor, 21 Viramin A, effect of on bacterial membranes, 5 4

W Wall, cyst, in amoebae, 109 Wall growth as a hazard in continuous cultures, 247 Water-expulsion vacuole in Acanthamoeba, 110

U UDP-glucose epimerase, catabolite repression of, 258 Uncoupling agents and bacteria, 81 Uranyl ions, effect of on bacterial membranes, 91 Urea and amidase inhibition, 193 Urease, catabolite repression in synthesis of, 366 specificity, 180 Urocanase, catabolite repression in synthesis of, 260 Uronic acid contents of bacterial walls, 239 Usnic acid, action of on electron transport, 90 Utilization of carbohydrates, control of, 252 Utilization of gluconate, enzymes involved in, 253 UV-ray induced serotype transformation, 170

X X-ray induced serotype transformation, 170 Xylose utilization, catabolite repression of, 258

Y Yeast, galactozymase induction in, 253 Yeast membranes, action of polyenes on, 62

Z Zinc and secondary metabolism, 25, 27, 28, 29, 30, 31, 34, 35, 36, 39 Zinc ions, effect of on ion accumulation, 91

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