Advances in Microbial Physiology, Volume 10

Advances in

MICROBIAL PHYSIOLOGY

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Advances in

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

and

D. W. TEMPEST Laboratorium voor Microbiologie, Universiteit van Amsterdam, Amsterdam-C The Netherlands

VOLUME 10

1973

ACADEMIC PRESS LONDON and NEW YORK A Subsidiary of Harcourt Brace Jovanovich,Publishers

ACADEMIC PRESS INC. (LONDON) LTD. 24/28 Oval Road London NW1

United States Edition published by ACADEMIC PRESS INC. 111 Fifth Avenue New York, New York 10003

Copyright 01973 by ACADEMIC PRESS INC. (LONDON) LTD.

All Rights Reserved

No part of this bookmay be reproducedin any form by photostat, microfilm, or any other means, without written permission from the publishers

Library of Congress Catalog Card Number: 67-19850 ISBN: 0 12-027710-7

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Contributors t o Volume 10

E. A. DAWES, Department of Biochemistry, University of Hull, Kingston upon Hull, England J. LE GALL,University of Georgia, Athens, Georgia, U.X.A. and C.N.R.X., Marseilles, France J. R. POSTGATE, University of Xussex, Brighton, England P. J. SENIOR,Department of Biochemistry, University of Hull, Kingston upon Hull, England S . RAZIN,Department of Clinical Microbiology, The Hebrew UniversityHadassah Medical Xchool, Jerusalem, Israel

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T

Physiology of Mycoplasmas

SHMUEL RAZlN

I . Introduction . 11. Ecology . 111. Morphology and Mode of Reproduction . A. Size of the Minimal Reproductive Unit . B. Morphology . C. Ultrastructural Features . D. Special Organelles and Structures . E. Mode of Reproduction . F. Motility . IV. The Genome and Mycoplasma Genetics . A. Nature and Mode of Replication of the Genome . B. Genome Size and Attachment to Membrane . C. Base Composition of Mycoplasma DNA . D. Ultraviolet Irradiation Damage and Repair . E. Mycoplasma Genetics . F. Mycoplasma Viruses . V. Ribosomes, Transfer-RNA and Protein Synthesis . A. Properties of Mycoplasma Ribosomes . B. Ribosomal Helices . C. Transfer-RNA . D. Protein Synthesis . E. Co-ordination of Macromolecular Synthesis . VI. The Cell Membrane . A. IsolgLtion of Membranes . B. Chemical Composition . C. Membrane Proteins . D. MembraneLipids . E. Organization of Protein and Lipid in the Membrane . F. Reconstitution of Membranes . G. Transport Mechanisms . VII. Nutrition and Metabolism . A. Nutritional Requirements and Synthetic Capabilities . B. Respiratory Pathways and Energy-Yielding Mechanisms VIII. Acknowledgments . References vii

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viii

CONTENTS

The Physiology of Sulphate-Reducing Bacteria JEAN LE GALL AND JOHN

I. Introduction

.

R. POSTGATE

Culture and Estimation . Inhibition . Classification . Control Processes . Carbon Metabolism . . A. Lactate Oxidation to Acetate via Pyruvate B. Fumarate and Malate Dismutation . C. Formate Oxidation. D. Citrate Synthase . E. Carbon Dioxide Fixation and Mixotrophy . F. Hydrocarbon Oxidation and Formation; Methane Formation G. Glucose Metabolism in Desulfotomaculum . VII. Nitrogen Metabolism . A. Fixation of Nitrogen . B. General Nitrogen Metabolism VIII. Hydrogen Metabolism . . IX. Electron Transport and Phosphorylation X. Chemistry . A. Cytochromesc, . B. Other c-Type Cytochrornes C. Other Cytochromes . D. Non-Haem Iron Electron Carriers . XI, Sulphur Metabolism . A. Reduction of Sulphate to Sulphite . B. Reduction of Sulphite to Sulphide . XII. Primitive Character . XIII. Ecology . XIV. Economic Activities . A. Corrosion of Metals. B. Storage of Town Gas . C. OiITechnology . D. Formation of Minerals . References . 11. 111. IV. V. VI.

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82 83 84 84 87 88 88 89 90 91 91 92 93 93 93 94 94 97 100 101 105 107 107 109 110 111 116 117 119 119 121 121 122 125

CONTENTS

ix

The Role and Regulation of Energy Reserve Polymers in Micro-organisms EDWIN A. DAWES AND PETER J. SENIOR I. Introduction . . A. Criteria for Energy Storage Function . . B. Energy Storage Compounds . . C. Adenylate Energy Charge and Energy Storage . . D. Mutants Defective in Storage Polymer synthesis. . . 11. Glycogen and Glycogen-like Reserves . . A. General Considerations . . B. Occurrence of Glycogen and Glycogen-likeReserves . . C. Structure of Microbial Polyglucans . . D. Biosynthesis of Glycogen-like Reserves . . E. Glycogen Biosynthesis by Prokaryotes . . F. Glycogen Biosynthesis by Eukaryotes . . G. Glycogen Degradation . . H. Conclusions . . 111. Polyphosphate . , A. Status of Polyphosphate as a Reserve Material . . B. Occurrence of Polyphosphate in Micro-organisms . . C. Chemical Structure of Polyphosphates . . D. Accumulation and Utilization of Polyphosphate . . . E. Polyphosphate Metabolism: Enzymology . . F. Regulation of Polyphosphate Metabolism . . G. Physiological Functions of Polyphosphates . . IV. Poly-8-hydroxybutyrate . . A. History. . . B. Occurrence of Poly-p-hydroxybutyrate . . C. The Nature of Poly-,%hydroxybutyrate . . D. Poly-8-hydroxybutyrate Metabolism . . E. The Enzymology of Poly-/3-hydroxybutyrate Biosynthesis . F. The Enzymology of Poly-8-hydroxybutyrate Degradation . G. Regulation of Poly-8-hydroxybutyrate Metabolism and its . . Physiological Significance H. Functions of Poly-&hydroxybutyrate . . V. Conclusions . . VI. Acknowledgments . . References. . Author Index . . Subject Index . . Cumulative Index . .

136 137 137 138 139 140 140 142 142 144 146 163 170 176 178 178 179 179 183 192 197 201 203 203 204 206 214 228 236 244 249 254 256 257 267 279 297

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Physiology of My coplas mas SHMUEL RAZIN Department of Clinical Microbiology The Hebrew University-Hadussuh Medical School Jerusalem, Israel I. Introduction . 11. Ecology 111. Morphology and Mode of Reproduction A. Size of the Minimal Reproductive Unit B. Morphology. . C. Ultrastructural Features . D. Special Organelles and Structures . E. Mode of Reproduction F. Motility . IV. The Genome and Mycoplasma Genetics . A. Nature and Mode of Replication of the Genome B. Genome Size and Attachment to Membrane . C. Base Composition of Mycoplasma DNA . . D. Ultraviolet Irradiation Damage and Repair E. Mycoplasma Genetics . F. Mycoplasma Viruses V. Ribosomes, Transfer-RNA and Protein Synthesis A. Properties of Mycoplasma Ribosomes B. Ribosomal Helices C. Transfer-RNA D. Protein Synthesis . . E. Co-ordination of Macromolecular Synthesis VI. The Cell Membrane A. Isolation of Membranes. B. Chemical Composition . C. Membrane Proteins D. MembraneLipids E. Organization of Protein and Lipid in the Membrane F. Reconstitution of Membranes. G. Transport Mechanisms VII. Nutrition and Metabolism . A. Nutritional Requirements and Synthetic Capabilities B. Respiratory Pathways and Energy-Yielding Mechanisms V I I I . Acknowledgments . References .

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I. Introduction The physiology of mycoplasmas, the smallest organisms capable of autonomous growth, is of special interest in view of their extremely simple structure and limited biochemical activities. I n recent years they have been used quite extensively in biochemical studies, particularly those concerned with the cell membrane, and it seems worthwhile, a t this juncture, to survey the new insights that have been gained since the publication of the last reviews on the subject (Razin, 1969a; Smith, 1971a).

II. Ecology Thanks to recent improvements in cultivation and identification techniques, quite a number of further mycoplasma species could be established. By now over 40 species occurring in primates, farm and laboratory animals, and a variety of wild animals have been named (Razin, 1973; Freundt, 1973). Many more no doubt remain to be cultivated and identified as further biological materials are examined and better culture media become available. From the highly exacting nature of the mycoplasmas, strict host specificity was initially inferred, but recent findings seem to contradict this assumption. Thus, monkeys were shown to harbour human mycoplasmas (Del Giudice et al., 1969), while Mycoplasma canis, a dog mycoplasma, was isolated from man (Armstrong et al., 1971) and M . arginini from a wide range of animals (Barile et al., 1968). Of special interest is the isolation of Acholeplasma laidlawii from a variety of hosts (see Tully and Razin, 1968), casting doubt on the saprophytic nature ascribed to them because they were originally isolated from sewage and soil which they may well have reached via animal excreta. Considering also their strict nutritional requirements and osmotic sensitivity, it seems unlikely that they should be able to lead a truly saprophytic life under the variable ecological conditions of soil or sewage. Nevertheless the recent discovery by Brock and his associates (Darland et al., 1970; Belly and Brock, 1973) of mycoplasma-like organisms in self-heated coal-refuse piles seems to indicate that wall-less prokaryotes can live as true saprophytes. The thermophilic, acidophilic prokaryotes without cell walls, growing best at 55OC and pH 2.0, are somewhat larger than most animal mycoplasmas, but are like them in ultrastructure (Fig. I), DNA base composition, and probably in mode of reproduction. Though the unusual physiological properties of the new Thermoplasma acidophilum suggest a rather distant relationship to the animal mycoplasmas, its inclusion in the Mollicutes class seems warranted, and its very existence considerably broadens the range of habitats in which mycoplasma-like organisms are found.

PHYSIOLOGY O F MYCOPLASMAS

3

The flood of papers describing mycoplasma-like organisms in diseased plants and their insect vectors released during the past four years has been thoroughly reviewed by other authors (Alaramorosch et al., 1970; Davis and Whitcomb, 1971 ; Hull, 1971). The agents of the very large group of yellows plant diseases, long thought to be viruses, have recently been identified as mycoplasmas or, more cautiously, as mycoplasma-like organisms (MLO). Thin sections of diseased plant phloem and of tissues of infected insect vectors show numerous bodies indistinguishable from sectioned animal mycoplasmas (Fig. 1 ) . Phasecontrast microscopy and freeze etching studies accentuate their morphological resemblance to animal mycoplasmas (Davis et al., 1972)) further borne out by the successful treatment of the diseased plants and vectors with tetracyclines, and the ineffectiveness of antibiotics like penicillin that specifically inhibit bacterial cell-wall synthesis (Davis and Whitcomb, 1970). Marked heat sensitivity and rapid death in buffer solutions are further points of similarity (Chen and Granados, 1970). A major obstacle to the biochemical and serological characterization and consequent classification of the plant MLOs is the lack of adequate growth media. Sometimes viability could be sustained for a considerable period in vitro (Chen and Granados, 1 9 7 0 ) ; in other instances the organisms isolated appeared to be laboratory contaminants (Hampton et al., 1969; Lin et al., 1970). Saglio et al. (1971) and Gianotti etal. (1971), however, managed to isolate MLOs from diseased plants which seem to differ from the known animal mycoplasmas, the ones cultured by Gianotti et al. (1971) being capable of infecting the insect vector and the plant. Several laboratories are now comparing their biochemical and serological properties with those of animal mycoplasmas to ascertain whether they really are plant mycoplasmas. Why is it so difficult to cultivate plant lclLOs? One reason may be their intracellular location in the plant and insect as distinct from animal mycoplasmas, which are rarely intracellular. This may indicate a stricter adaptation of the parasite to conditions which may be difficult to simulate in a cell-free medium. Morphology and ultrastructural features alone may be doubtful criteria for the identification of an organism as a mycoplasma, but not for its exclusion from this group. Thus, upon careful examination of electron micrographs of thin sections, the allegedly mycoplasma-like bodies claimed to cause male sterility in Drosophila (Williamson et al., 1971) are seen to be bounded by two rather than by one membrane (Fig. 1). Hence they seem not to be mycoplasmas, but probably belong to the rickettsia or the chlamydia group. The “Greening” agent of citrus also does not seem to qualify for membership since its membrane is about twice as thick as that of mycoplasmas and the distance between

4

SHMUEL RAZIR’

FIG.1. Electron micrographs showing thin sections through : (A)an animal mycoplasma; taken from Anderson arid Uarilo (1965): (B) the therinophilic Thermoplasma acidophilum; taken from Darland et al. (1970): (C) mycoplasma-like organisms in the phloem of diseased plants ; an unpublished electron micrograph of Dr. H. Hirumi: (D) mycoplasma-like organisms in tho testes of a sterile Drosophila male ; taken from Williamson et al. ( 1 97 1 ) . The structural resemblance of all organisms is striking. The Drosophila micro-organism differs, however, from the others in being bounded by two unit mernbrancs.

PHYSIOLOGY OF MYCOPLASMAS

5

6

SHMUEL RAZIN

the two electron-dense lines is not even, as in the typical “unit membrane” of mycoplasmas (Saglio et al., 1971).

Ill. Morphology and Mode of Reproduction A. SIZE OF

THE

MINIMALREPRODUCTIVE UNIT

Though bigger than originally thought, the mycoplasmas may still be regarded as the smallest organisms capable of autonomous growth. Recent data indicate that the diameter of the smallest mycoplasma cell is 0-2-0.3 pm and not, as claimed previously, 0.15 pm which is close to the calculated theoretical minimum cell size (Morowitz, 1967). The earlier estimates were based on filtration experiments (Klieneberger-Nobel, 1962 ; Morowitz et al., 1963) showing that some viable mycoplasma cells can pass through membrane filters of 0.22 pm pore diameter. However, when high negative or positive pressures are employed, the flexible mycoplasma cells may be squeezed through pores much smaller than their diameter (Razin, 1969a; Lemcke, 1971). When pressure is decreased, almost all mycoplasma cells are retained even on a 0.45 pm pore-diameter filter (Razin et al., 1968; Rottem and Razin, 1969; Cho and Morowitz, 1969) so that filtration can be used for the fast separation of mycoplasma ceIls in transport studies. The effect of cell plasticity on filtrability was demonstrated by Lemcke (197 1). Cells of A . laidlawii, prefixed with glutaraldehyde, were retained on membrane filters that allowed the passage of unfixed cells, fixation presumably enhancing the rigidity of the normally soft cells. Thus filtrability through 0.45 pm pore-diameter filters, one of the standard tests recommended for the identification of an organism as a mycoplasma (Edward et al., 1972),cannot be taken as an indication of size but merely of the plasticity characteristic of these organisms. Membrane-bound bodies filled with ribosomes and having a diameter of 0.1-0-2 pm frequently observed in thin sections cannot be taken as the minimal reproductive units as they may represent a section through the tip of a spherical cell, a thin filament, or a thin thread connecting two adjacent cells (Razin, 1969a; Boatman and Kenny, 1970). The small round 0.1-0-2 pm diameter particles observed in electron micrographs of shadowed or negatively-stained mycoplasma preparations (Anderson et al., 1965) seem incapable of reproduction, and may well be fragments of the fragile mycoplasma cells. To be viable, they should contain the entire cell genome, but their sections seldom show the characteristic DNA fibrils. Moreover, the minimum diameter of the sphere required to accommodate the smallest known mycoplasma

PHYSIOLOGY O F MYCOPLASMAS

7

genome together with a single ribosome has been calculated a t 0.13 pm (Morowitz et al., 1967) so that the diameter of the minimal reproductive unit cannot be much less than 0-2 pm. B. MORPHOLOGY I n the absence of a cell wall, the mycoplasmas are so fragile and pliable that their morphology was long disputed. I n preparing them for microscopy, it is obviously difficult to prevent the formation of artifacts, especially when unfixed cells are negatively stained with phosphotungstic acid. The thin filament-like protrusions often formed under these conditions as a result of osmotic damage or dessication are easily distinguishable from true filaments by their smaller calibre-less than 0.1 pm as compared with 0.3-0.4 pm (Razin et al., 1967; Bredt, 1970). Structures resembling the distorted mycoplasma cells may also be obtained by the negative staining of extracts of plant or animal cells not infected with mycoplasmas, so that this is hardly the method of choice for detecting mycoplasmas in tissues (Wolansky and Maramorosch, 1970). The tonicity of the medium also has a marked influence on mycoplasma morphology. Thin, filament-like protrusions were observed in cells of H . gallisepticum exposed t o hypotonic conditions (BernsteinZiv, 1971); and fixation under grossly hypertonic conditions caused cells of A . laidlazuii to become vacuolated or invaginated (Lemcke, 1972). To minimize artifact formation, attention should therefore be paid to the tonicity of the medium before, during and after fixation. The mechanical stress of centrifugation alone may be enough to affect morphology and cause unfixed M . gallisepticum, for instance, t o assume a spherical, swollen shape in the electron microscope unlike their tear-shaped form-presumably their true morphology (Maniloff and Morowitz, 1967)-when prefixed with glutaraldehyde in the growth medium. As is t o be expected from plastic organisms, the coccus is the basic, though by no means the only, form in all mycoplasma cultures. I n most, and under certain conditions, perhaps in all, mycoplasma cultures elongated or filamentous forms may also be discovered. The controversy about their being artifacts or not has been finally resolved upon the production of convincing evidence for filamentous growth in most species. Such evidence has been furnished by phase-contrast microscopy (Razin et al., 1967; Bredt, 1970; Hubbard and Kite, 1971), negative staining (Brunner et al., 1971 ; Rottem and Razin, 1972a),thin sectioning of mycoplasmas grown in broth (Maniloff, 1970; Metz and Bredt, 1971) or in agar (Knudson and MacLeod, 1970), scanning-beam electron

8

SHMUEL RAZIN

microscopy (Biberfeld and Biberfeld, 1970; Kammer et al., 1970; Boatman and Kenny, 1971), and freeze etching (Davis et al., 1972). While there is no longer any doubt that, given the appropriate conditions (Razin et al., 1967), mycoplasma can grow in filaments, the different strains vary in their ability to do so. This may be due to differences in their ability to synthesize certain membrane components when the supply of precursors in the growth medium is limited.

C. ULTRASTRUCTURAL FEATURES The extremely simple ultrastructure found in numerous electronmicroscope studies of thin mycoplasma sections supports the view that they are the simplest and most primitive organisms extant. Essentially the mycoplasma cell is built of only three organelles : the cell membrane, the ribosomes, and the characteristic prokaryotic chromosome. I n several species, specialized organelles or structures have, however, recently been observed (see Section 111, D, p. 9). I n section, the cell membrane shows the characteristic trilaminar “unit membrane” structure, about 8.0-1 1-0nm thick (Domermuth et al., 1964s; Carstensen et al., 1971). The frequently observed fuzziness of its outer surface (Morowitz and Terry, 1969; Maniloff, 1970 ; Bernstein-Ziv, 1971) disappears once the membrane is isolated and washed, and therefore probably represents material adsorbed from the growth medium, or highly polymerized material excreted from the cells, like the galactan of $1.m y c o i d e s (Gourlay and Thrower, 1968). There is no evidence of any intracellular membranous structures, That the membrane-bound vacuoles observed in some sectioned cells represent a section passing through a deep cup-like invagination of the cell membrane (Hirth et al., 1970)has been conclusively proved by means of serial cell sections (Boatman and Kenny, 1970). Serial sections have, moreover, shown that some cells may have a hole in their centre, so that it is doubtful whether any real vacuoles are present. I n old mycoplasma cultures or in cultures growing in nutritionally inadequate media, large bodies are often seen, sometimes containing granules (Anderson and Barile, 1965) which a t one time were commonly regarded as minimal reproductive units liberated into the medium after lysis of the large bodies to start a new life cycle (Klieneberger-Nobel, 1962). Very few workers, if any, still support this notion. The large bodies apparently represent damaged swollen cells of low viability, and the intracellular granules cytoplasmic degeneration products, though the possibility of some of them being viruses should also be considered (Home, 1972).

PHYSIOLOGY OF MYCOPLASMAS

9

D. SPECIALORGANELLESAND STRUCTURES

1. The bleb of Mycoplasma gallisepticum The bleb a t the tip of the worm-like cells of M . gallisepticum has attracted much attention since it was first discovered in the middle sixties (Maniloff et al., 1965;Domermuth et al., 1964b). It is not an artifact since it is observable throughout in organisms that have been negatively stained (Maniloffetal., 1965;Bernstein-Ziv, 1969),sectioned(Domermuth et ul., 196413; Maniloff et al., 1965, Allen et al., 1970; Bernstein-Ziv, 1969) or freeze-etched (Bernstein-Ziv, 1969; Maniloff, 1972). More or less like an oblate ellipsoid in shape, measuring about 80 by 130 nm without the bounding membrane which appears to be part of the plasma membrane (Fig. 2), it probably consists of protein and lipid. Nucleic acids have not been detected histochemically (Maniloff et al., 1965) but, as long as this organelle has not been isolated and purified, its chemical composition remains uncertain. The function of the bleb is still unknown. Onc suggestion is that it is somehow associated with the organism’s reproduction (Maniloff and Morowitz, 1967), but a more convincing hypothesis is that it plays a role in the adsorption of M . gallisepticum to cell surfaces. The excellent electron micrographs of Zucker-Franklin et al. (1966) show cells of M . gallisepticum clustered around leukocytes like iron filings around a magnetic pole or flukes attached to their host, the bleb constituting the site of contact more frequently than would be accounted for by chance alone.

2. Terminal structure of Mycoplasma pneumoniae A special structure a t the tip of filaments of N . pneumoniae was first reported by Biberfeld and Biberfeld (1970) and later confirmed by Collier and Clyde (1971). It consists of a dense central rod-like core surrounded by a lucent space enveloped by the cell membrane (Fig. 2), and measures 80 to 100 nm by 250 to 300 nni (Collier and Clyde, 1971). The electron density, fibrillar nature and affinity of the rod-like structure for uranyl and lead stains suggest that it may contain nucleic acid. Coupled with morphological evidence of filaments with two parallel terminal structures or of partly split terminal structures, this was taken t o indicate participation in the initiation of reproduction by binary fission (Collier, 1972 ; Biberfeld, 1972). If so, however, one would expect the filament to split longitudinally along its axis, whereas all the available data point to transversal division to bead-like structures (Razin and Cosenza, 1966). It seems more probable that, like the bleb of M . gallisepticum, the terminal structure is instrumental in the

FIG.2(A). (B) (For legend see facing page)

PHYSIOLOGY OF MYCOPLASMAS

12

SHMUEL RAZIN

adsorption of M . pneumoniae to cell surfaces (Collier, 1972) or to glass and plastic (Biberfeld, 1972). The terminal structure was always in close proximity t o the host-cell membrane. On the other hand, the retention of the terminal structure by an avirulent M . pneumotaiae strain that had lost its ability to cytadsorb (Collier, 1972) casts some doubt on the surface-attachment hypothesis. Another alternative, that the terminal structure plays a role in the motility of ill. pneumoniae, is discussed on page 13.

3. Striated rods in Mycoplasma sp. strain Y The rigid pleomorphic, elongated forms with beaded swellings that predominate in cultures of a goat mycoplasma strain Y (related to M . mycoides) adapted to grow in a fatty acid-poor medium (Rodwell et nl., 1972) were found to have a striated, filamentous structure, visible not only in sections but also in negatively stained organisms. The rod extended throughout the entire organisms up to a length of 5 pm. The periodicity of the striation varied between 12.0 to 14.5 nm (Fig. 2 ) . Nothing is so far known about the chemical nature and function of these skeleton-like rods but most probably they account for the rigidity of the long and beaded cells.

E. MODE OF REPRODUCTION From the outset, the mode of reproduction of mycoplasmas has been a matter of dispute. Do the mycoplasmas reproduce by the liberation of elementary particles from large bodies (Klieneberger-Nobel, 1962), by the fragmentation of filaments (Freundt, 1969), by budding (Liebermeister, 1960) or by binary fission (Maniloff and Morowitz, 1967 ; Furness et al., 1968)?It seems that the time has come t o end this controversy. Recent data (summarized by Morowitz, 1969) clearly show that replication of the prokaryotic mycoplasma genome, which must precede cell division, follows the same pattern as with other prokaryotes dividing by binary fission. However, for binary fission to occur, cytoplasmic division must be fully synchronized with genome replication, which is not always the case with mycoplasmas. The lack of a cell wall may well be responsible for the poor co-ordination of the two processes, especially considering that in bacteria their co-ordination is also adversely affected by cell-wall removal, when peculiar bud-like forms, as in L-phase cultures, are produced. In many mycoplasmas, cytoplasmic division lags behind genome replication, resulting in the formation of multinucleate filaments. The subsequent division of the cytoplasm leads to the formation of the characteristic chains of beads, which later

PHYSIOLOGY O F MYCOPLASMAS

13

fragment to give single cells (Freundt, 1969; Razin and Cosenza, 1966). Budding, frequently seen in mycoplasma cultures (Anderson and Barile, 1965; Nelson and Lyons, 1965; Bredt, 1970; Furness, 1970), may also be regarded as a form of binary fission in which the cytoplasm is not equally divided between the daughter cells. Perhaps the best experimental evidence for this mode of reproduction was provided by Bredt (1 970), by continuous phase-contrast microscopy of the growth and reproduction of single cells of Jf.hominis in a slightly viscous medium. Classical binary fission was clearly seen to occur side by side with budding and fragmentation of filaments. I n my view, therefore, the mode of reproduction of mycoplasmas is essentially not different from that of other prokaryotes, and the proposed establishment of a new genus Xchizoplasma (Furness et al., 1968) for mycoplasmas which reproduce by binary fission seems superfluous.

F. MOTILITY Although locomotion organelles such as flagella have not been detected in any of the known mycoplasmas, motility has been described in three species, namely M . pulmonis, M . pneumoniae and M . gallisepticum. The early first report on motility of M . pulmonis (Andrewes and Welch, 1946) was later confirmed by Nelson and Lyons (1965), but)little attention was paid to it until recently, when Bredt (1968, 1972) demonstrated motility in $1.pneumoniae and M . gallisepticum. The original description of motility in M . pulmonis also appears to fit the recent representation of motility in $I. pneumoniae. Strings of globules connected by short filaments were seen moving across the field; or else a globule with a short linear stalk would go forward, the stalk in advance, bending slightly from side to side as if feeling its way; or a sphere with a projection on its circumference would keep rotating in one direction. I n both cases, motility is hardly ever observed except where the organisms are in contact with the slide. I n broth only Brownian movement can be seen (Andrewes and Welch, 1946; Bredt, 1968). The mechanism of motility is still obscure. The resemblance to the gliding movement of myxobacteria was already noted by Andrewes and Welch (1946) but, while myxobacterial motility seems to be due to the secretion of viscous material and contraction of fibrils, no similar mechanism has been found in mycoplasmas. Alternate contraction and expansion might possibly account for the gliding motion of the bacilliform elements in the cultures, but not for the spinning of the coccoid forms (Nelson and Lyons, 1966). I n view of association of motility with adherence to glass surfaces, it may well, in the case of M . pneumoniae, be caused by alternate release and re-attachment of the polar tip

14

SHMUEL RAZIN

of the filamer_l to the glass surface (Biberfeld and Riberfeld, 1970). The bleb of M . gallisepticum may play a similar role. As yet there is no evidence for a similar attachment structurc in M . pulmonis, which may, however, be discovered upon careful examination of this organism.

IV. The Genome and Mycoplasma Genetics A. NATUREAND MODE OF REPLICATION OF THE GENOME Early electron micrographs of sectioned mycoplasma cells (Van Iterson and Ruys, 1960; Domermuth et aZ., 1964a) indicated the prokaryotic nature of the mycoplasma genome. The precise nature of the genome has, however, been elucidated only recently. Its observation and measurement were made possible by the radioautographic method of Cairns (1963) and the spread-film technique of Kleinschmidt and Zahn (1959). All of the mycoplasmas examined so far appear to have circular genomes built of double-stranded DNA. Density labelling of the DNA of A . laidlawii with bromouridine according to Meselson and Stahl (1 958) has shown (Smith, 1969) that DNA replication is semi-conservative, proceeding uni-directiona,lly, probably from a single growing point, as in bacteria. This corroborates the electron-microscope findings that the growing mycoplasma chromosomes have replicating Ys or double forks (Bode andnilorowitz, 1967; Morowitz, 1969). B. GENO~VE SIZEAND ATTACHMENT TO MEMBRANE The assumption that the minute and structurally simple mycoplasmas have smaller genomes than ordinary bacteria seems to be borne out by several recent findings. Estimates of genome size arrived a t by measurements of the circular DNA strands in electron micrographs, using 1.95 x lo6 daltoris per micron as a conversion factor (Bode and Morowitz, 1967), have been corroborated by the use of a less cumbersome technique based on comparing the renaturation kinetics of DNA (Bak et al., 1969; Table 1).It appears that the genome of Acholeplasma species that do not require sterols is about twice as big as that of the sterol-requiring Mycoplasma species, supporting their recent classification in a separate family, the Acholeplasmataceae (Edward and Freundt, 1970). The Mycoplasma species appear to have a smaller genome than any kacteria investigated so far, about one-fifth of the size of the genome of Escherichia coli (Bak et al., 1970). On the other hand, the mycoplasma genome is far bigger than the genome of animal viruses (Smith, 1965), but seems to be of the same order of magnitude as that of the chlamydiae and rickettsiae (Kingsbury, 1969).

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PHYSIOLOGY OF MYGOPLASMAS

TABLE1. Size and Base Composition of Mycoplasrna Genornes Genome size (daltons x 10")

Organism

Mycoplasma arthvitidis Mycoplasma gallisepticum Mycoplasma pneumoniae Mycoplasma meleagridis T-strain 27 Acholeplasma laidlawii A Acholeplasma granularum Acholeplasma axansthum Mycoplasma neurolyticum

Determined by Determined by electron DNA renaturation microscopy or autoradiography" kineticsb 4.4-5.1 10.0-12.0 -

-

5.3 -

4.4 4.9 4.8 4.2 0.5 4.7 11.0 9.5 10.0

- ..

-

DNA base cornpositionC ( O h guanine + cytosine) 31-3 34.7 39.6 27.9 28.0 33.0 32-4 31.3 23.0

From Morowitz (1069). * F r o m Bak et ul. (1969), Allen (1971), Freundt (1972). From Neimark (1970).

The average cistron may be assumed to encode a protein of 40,000 daltons, and the coding ratio of DNA to protein is approximately 20: 1, so that 800,000 daltons of DNA constitute an average cistron. On this basis, Morowitz (1967) calculated that the genome of $1. arthritidis contains 637 cistrons, only one order of magnitude more than the theoretical minimal self-replicating cell. The idea that the initiation of replication in the prokaryotic chromosome occurs a t the point where it is attached t o the cell membrane has been supported by experiments showing newly synthesized DNA to be associated with a given portion of the cell membrane (Tremblay et al., 1969). Similarly, by using the technique of Tremblay et al. ( 1 969), Rodwell et al. (1972) isolated a DNA-membrane complex from the goat mycoplasma strain Y. On lysis of the mycoplasmas by the detergent sodium lauryl sarcosinate, and centrifugation of the lysate on a sucrose density-gradient containing Mg2+, a complex was separated containing most of the cellular DKA, part of the ribosomes and a portion of the cell membrane. Maiiiloff (1972) isolated a DNA-membrane complex from &I.gallisepticum by a somewhat different technique. The organisms were grown overnight with T-thymidine, then pulse labelled with 3H-thymidine for 1-3 min, and gently lysed by alternate freezing and thawing followed by a very short sonication. The suspension was centrifuged for 5 min a t 4000 g to remove unbroken cells and large debris. Centrifusation of the supernatant liquid for 20 min at 40,000 g separated

16

SHMUEL RAZIN

a pellet containing membrane fragments and blebs, and highly enriched

with the newly synthesized 3H-labelled DNA. The finding of blebs in this fraction may support the association of this peculiar organelle with DNA replication and cell division of M . gallisepticum (Maniloff and Morowitz, 1967). C. BASE COMPOSITIONOF MYCOPLASMA DNA Apart from its small size, the mycoplasma genome is characterized by its low guanosine + cytosine (GC) content, which in some instances is 23%, less than of any known aerobic bacteria (Table 1 , p. 15). This may further restrict the amount of genetic information available, as it does not allow for much degeneracy in the code. Moreover, however low the GC content of the DNA, the GC content of mycoplasma ribosomal a i d transfer RNAs (rRNA and tRNA) never drops below 43-45% (Ryan and Morowitz, 1969). Hence, in all low-GC genomes, the composition of the regions coding for rRNA and tRNA may be expected to differ considerably from that of the major portion of the DNA which codes for messenger-RNA. Ryan and Morowitz (1969), making use of the different melting temperatures of the low- and high-GC regions of the genome, have in the most elegant fashion separated a small portion of a high-GC DNA from the low-GC (230/) genome of Mycoplasma sp. strain kid. Sonicated DNA of the kid strain was put on a hydroxyapatite column and heated to 86°C to melt all of the low-GC DNA. The denatured DNA was then eluted with 0-17 M-phosphate buffer. Subsequent elution with 0-27 M-phosphate buffer eluted the remaining native high-GC DNA, which had a melting point of over 89°C and constituted only I.+% of the total cell DNA. The high-GC DNA showed a saturation hybridization value of 15.6% with a mixture of the organism’s rRNA and tRNA, as against 0.26% for the bulk of the DNA with rRNA and 0.16% with tRKA. Hence, the high-GC DNA fraction seems t o be highly enriched with the regions coding for rRNA and tRNA. Considering that the size of the kid strain genome, as elicited by electron microscopy, is 6-85 x 10* daltons, it appears to contain enough ribosomal-DNA to code for only one set of 23 S plus 16 S rRNA and enough DNA complementary to tRNA to code for only 44 different tRNA molecules (Ryan and Morowitz, 1969). D. ULTRAVIOLET IRRADIATION DAMAGE AND REPAIR The mycoplasmas are very sensitive to ultraviolet irradiation. When the ultraviolet-sensitivities of A . laidlawii and E . coli are normalized per unit of DNA, A . laidlazuii seems to be 13 times as sensitive

PHYSIOLOGY O F MYCOPLASMAS

17

(Folsome, 1968). Although a variety of mycoplasma species were shown to be inactivated by ultraviolet irradiation (Furness, I969), the mechanism involved has been studied only in A . laidlawii. Here, as in bacterial systems, ultraviolet irradiation causes a drastic reduction in DNA synthesis, leading to DNA degradation. The ensuing cell lysis (Smith and Hanawalt, 1969) may, in view of the frequent virus infestation of A . laidlawii (see page 19), be a t least in some instances due to the activation of latent viruses by ultraviolet irradiation. A strong photoreactivation effect has been reported by all three groups working on ultraviolet inactivation of A . laidlawii (Folsome, 1968; Smith and Hanawalt, 1969; Das et al., 1972). However, Folsome ( I 068) denied the presence of a dark-repair system because the dark inactivation curves remained the same in the presence and absence of caffeine. Das et al. ( 1972) ascribed Folsome’s failure to show dark repair to the gradual loss of viability of mycoplasmas held in buffer for over 90 min a t 37°C or a t room temperature, in the light or in the dark. From the evidence produced by Smith and Hanawalt (1969), it appears that the repair replication consists of small single-strand regions in the parental DNA strands, and that the newly synthesized DNA is capable of normal semiconservative DNA replication. This is supported by the findings of Das ct nl. (1972) that 60% of the DNA strand breaks noted in ultraviolet-irradiated A . laidlawii are repaired within 2 h. It therefore appears that these small micro-organisms do possess an enzymic system capable of repair replication of ultraviolet-damaged DNA, like that found in bacteria.

E. MYCOPLASMAGENETICS Although, theoretically, the small genome size in mycoplasmas should facilitate mapping, several unexpected failures have tended to discourage genetic research so that not much information is available so far. Mutants are required for mapping studies; and it is not particularly difficult to obtain mycoplasma mutants. Transfer of mycoplasmas through increasing concentrations of antibiotics results in antibiotic-resistant mutants (Schwartz and Perlman, 1971 ) . By treatment of cells of llf.pnezirnoniae with nitrosoguanidine, the frequency of temperature sensitive (ts) mutation was increased almost proportionately to the concentration of the mutagen (Steinberg et al., 1969). Similar ts mutants were obtained from cells of A . laidlawii treated with 2-aminopurine or 5-bromodeoxyuridine as inducers (Folsome and Folsome, 1966).It is, however, not so easy to obtain the usual type of biochemicallydeficient mutants, because there are no defined growth media for most mycoplasma species. The goat mycoplasma strain Y, which grows very

18

SHMUEL RAZIN

well in a completely defined medium (Rodwell, 1969),may perhaps serve the purpose. All attempts to locate genetic markers on the chromosomes of the various mutants available have so far failed in the absence of an appropriate recombination system. Of the three methods generally used in genetic studies of micro-organisms, namely transduction, conjugation and transformation, the first has not been reported so far for the simple reason that mycoplasma viruses are a recent discovery (see Section IV, F, p. 18). Likewise, there are no descriptions of transfer of genetic material by conjugation. Several mixed-growth experiments in which two antibiotic-resistant strains were grown in the absence of antibiotic and then tested for recombinants in the presence of both antibiotics have failed (Morowitz, 1969). The absence of a cell wall would seem to favour genetic exchange by transformation, but most of the extensive experiments carried out in various laboratories have given inconsistent results and consequently remained unpublished. Folsome ( 196 8) reported that external homologous DNA, amounting to about 0.1-0.2 of the organism’s total DNA content, was bound t o cells of A . laidlawii by a temperature-dependent process. Despite binding of high molecular-weight DNA, the transformation of streptomycin-sensitive recipients to streptomycin-resistant cells under conditions of maximum DNA uptake was erratic, unstable, and often no higher than spontaneous mutation. From these results and the ultraviolet irradiation data available then, Polsome (1968) concluded that the external DNA is not genetically integrated int80the chromosome for lack of recombination enzyme(s), but the subsequent finding of a dark-repair mechanism by Smith and Hanawalt (1969) and Das et al. (1972) speaks against this interpretation. Slater and Folsome’s (1971) report on a-glucosidase formation in A . laidlawii being induced by maltose is the first indication of a mechaiiism regulating gene expression in mycoplasmas, whose existence seems t o be conclusively demonstrated despite the low level of enzyme induction recorded.

F. MYCOPLASMA VIRUSES That even the smallest prokaryotes in existence might be parasitized by viruses has been an intriguing object of speculation for quite some time. The first to suggest that the small particles within the large swollen mycoplasma cells are viruses rather than minimal reproductive units were Edwards and Fogh (1960). The presence of phage-like particles in an unidentified mycoplasma was also mooted by Swartzendruber et al. (1967). These ideas were put forward on the grounds of otherwise

PHYSIOLOGY O F MYCOPLASMAS

19

unsupported morphological evidence. Stronger, though still indirect, support for the mycoplasma virus theory was provided by genome studies. Morowitz (1969), in his review on the mycoplasma genome, suggested that the small circles of about 20 x l o 6 daltons each observed in the DNA of M . arthritidis, prepared according to Kleinschmidt and Zahn (1959), might represent an episome, a contaminating virus or an artifact of preparation. Haller and Lynn (1969) likewise reported the presence of satellite DNA in M . arthritidis as was later also shown by Dugle and Dugle (1971) on A . laidlawii. Density-gradient analysis of DNA of A . laidlawii showed two peaks, the major peak consisting of the cell genome, having a molecular weight of 99 5 x lo7 daltons and a GC content of 35%, and the minor peak having a molecular weight of 38 & 5 x lo6 daltons and a GC content of 25%. The first direct evidence for mycoplasma viruses was the plaque formation on a lawn of A . laidlawii by an ultrafiltrate of a mycoplasma culture reported by Gourlay in 1970. This set the wheels turning and, in 1971, the first electron micrographs of A . laidlawii viruses were produced (Gourlay et al., 1971). As was to be expected from a virus infecting a wall-less organism, it lacked a tail and injection mechanism (Fig. 3 ) . Soon afterwards came another report (Gourlay, 1971) describing a totally different virus, also infecting A . laidlawii, which was spherical and enveloped while the first one was rod-shaped (Fig. 3 ) . Intensive studies showed the two viruses, designated by Gourlay as MV-L1 and MV-L2 (MV = Mycoplasmatales Virus; L = Laidlawii), to be biologically very different from each other (Table 2 ) . The higher sensitivity of MV-L2 to ether, detergents, and perhaps also to heat may be due t o its lipid-containing envelope. The incorporation of labelled thymidine but not uridine into the virus when the labelled nucleosides were included in the mycoplasma growth medium indicates the presence of DNA as the nucleic acid of MV-Ll. Further chemical studies of MV-L1 and MV-L2 have been hampered by the scarcity of material for analysis. Gourlay also has been unable to collect enough of the, MV-L1 DNA for base composition and sedimentation analyses. If the satellite A . laidlawii DNA characterized by Dugle and Dugie (1971) is MV-L1 DNA, then the MV-L1 DNA has a molecular weight of 38 5 x 106 daltons and a base composition of 25% GC. On the other hand Das et al. (1972), on the basis of sedimentation and electron microscope data, claim that its molecular weight is only 13-14 x 106 daltons. The question can be cleared up only after purified virus DNA becomes available for analysis. From recent detailed electron microscope and optical diffractometry studies (Bruce et al., 1972) i t may be concluded that MV-L1 is an unenveloped helically symmetrical DNA virus, probably the first of this

20

SHI'IUEL RAZIN

FIG. 3. Electron micrographs of negatively-stained viruses of Acholeplasma Zaidlawii. (A) MV-LI. Unpublished electron micrograph of Dr. R. N. Gourlay. (B) MV-L2. Taken from Gourlay (1971).

21

PHYSIOLOGY O F MYCOPLASMAS

TABLE 2. Properties of Acholeplasma laidlawii viruses MV-L1 and MV-LZ“ PIopclrty

MV-L1

Shape Enveloped Size

Hod shaped

Nucleic acid type Sensitivity t o ether and detergents Sensitivity to heat (tiO”C, 30 min) Plaque size Plaque production at 22°C Inhibition by MV-L1 antiserum

DNA

a

-

15 x 90 nm -

< ti mm diameter

+ +

MV-L2 Spherical

+

About 50-100 nm in diameter not determined

+ +

1-2 min diameter -

Nrom Gouriay (1972b)

type to have been isolated. The tubular portion of the particles appears to consist of subunits arranged in hexagons t o form a helix. The two prefemed lengths of virus particles either have both ends rounded (90 nm long) or only one end rounded (80 nm long). Virus particles appear to attach to host membranes a t one end only. As the unattached end was always rounded, degradation would seem to occur a t the end which is normally involved in attachment. Adsorption is rapid and, as well over 300 MV-L1 viruses can adsorb to one cell, there probably are no specific adsorption sites but as in animal viruses each cell can “take up” viruses a t many sites on its membrane surface (Liss and Maniloff, 1 9 7 1 ) . It is still not clear how the MV-L1 DNA penetrates the host membrane. Presumably it is released through the broken end of the cylinder since hollow centres could be seen in negatively stained preparations of MV-L1 (Gourlay et al., 1971). On the mode of infection with MV-L2 as yet practically nothing is known. What happens after the viral nucleic acid has penetrated into the mycoplasma cell? I n one-step growth experiments, Gourlay and Wyld (1972) found the latent period to be between 30 and 60 min and the rise period between 1 and 2 h, while burst size varied from 4 to 213. From this they inferred that the colony-count technique may not be appropriate for this type of experiment since, with mycoplasmas, the colony-forming unit may consist of many clumped cells. This consideration also seems pertinent to the findings of Liss and Maniloff ( 1 971) who isolated two rod-shaped viruses which infected A. laidlawii and resembled Gourlay’s MV-LI in size, sedimentation behaviour and resistance to ether and detergents. Clones of A . laidlawii resistant to

22

SHMUEL RAZIN

infection with MV-L1 were, moreover, also resistant to infection by the two new viruses and vice versa. Nevertheless, Liss and Maniloff (197 1) claimed that, owing to their dissimilar ultraviolet inactivation curves, burst time and burst size, the two viruses are different enough from RIVL1 to warrant designation by new names, MVL62 and MVG5l. I n light of the results of Gourlay and Wyld, however, these differences may not weigh too heavily, and it seems, perhaps, premature to assign new names to the viruses isolated by Liss and Maniloff (1971). The extensive studies of Gourlay’s group (Gourlay, 1972a) and of Liss and Maniloff (1971) show that MV-L1 and MV-L2 can infect only A . laidlawii strains. Viruses infecting A . laidlawii and resembling MV-L1 in their biological properties were, however, also isolated from cultures ofA. granularum (Gourlay, 1972a), a goat mycoplasma (Liss and Maniloff, 1971) and a variety of other mycoplasma species (Maniloff, 1972). Since they were not able to infect the mycoplasma species from which they were isolated, but only A . laidlawii, it seems probable that the cultures were contaminated with this strain. Nevertheless it seems highly likely that other mycoplasmas also harbour specific viruses. Morphological evidence of virus-like particles in sterol-requiring mycoplasmas (Home, 1972) and the finding of satellite DNA in M . arthritidis (Morowitz, 1969; Haller and Lynn, 1969) both seem to point in this direction.

V. Ribosomes, Transfer-RNAand Protein Synthesis A. PROPERTIES OF MYCOPLASMA RIBOSOMES Apart from the DNA strand, ribosomes are the only structures found in the mycoplasma cytoplasm. Though most reports relate to ribosomes of M . gallisepticum and M . hominis (Kirk and Morowitz, 1969 ; Johnson and Horowitz, 1971), we may venture the generalization that mycoplasma ribosomes are much closer to bacterial than to animal ribosomes, both in structure and in function. Their sedimentation coefficients resemble those of ribosomes from E . coli. They have an RNA:protein ratio of about 60:40 compared with about 40:60 in eukaryotic cell ribosomes. The protein mixture they contain is no less complex than that of E . coli ribosomes. Their ribosomal RNAs have sedimentation coefficients of 22 S and 16 S, the bigger component sedimenting slightly slower than the 23 S rRNA of E . coli. The GC content of the ribosomal-RNA is lower than of the corresponding E. coli RNA, but significantly higher than of the DNA of M . gallisepticum and M . hominis (Reich, 1967; Kirk and Morowitz, 1969; Johnson and Horowitz, 1971).

PHYSIOLOGY O F MYCOPLASMAS

23

B. RIBOSOMAL HELICES Under certain conditions, especially when the cells are harvested by centrifugation without prior fixation, the ribosomes of M . gallisepticum, but not of any other mycoplasma examined so far, form helical corncob-like structures of over 50 ribosomes each (Fig. 4;Domermuth et al., 1964b; Maniloff etal., 1965; Bernstein-Ziv, 1969; Allen et al., 1970). Recent optical diffraction and rotational symmetry analyses of electron micrographs (Maniloff, 1971) have shown the structures to consist of a helix repeat of 10 ribosomes in three turns. The interaction of the 50 S ribosomal subunits seems to stabilize the helix since chloramphenicol and lincomycin, which are known t o bind to these subunits, inhibited helix formation, which remained unaffected by streptomycin and tetracycline, which in turn are known to bind to the 30 S subunits.

FIG.4. Ribosomal helices in sectioned cells of Mycoplasma gallisepticum. Taken from Bernstein-Ziv (1969).

24

SHMUEL RAZIN

As inhibition of RNA or protein synthesis by specific inhibitors did not affect helix formation, the helices cannot be active polyribosomes. This view is re-inforced by the finding that ribosomal helices are also formed in E . coli under conditions where protein synthesis is negligible (Nauman et al., 1971).They rather seem to be the result of some environmental perturbation-low pH value in the case of E . coli, centrifugation in the case of M . gallisepticum-but why they appear in M . gallisepticum and not in any other mycoplasma treated in the same way still remains a mystery. C. TRANSFER-RNA Contrary to Kirk and Morowitz’s earlier contention (1969) that the soluble transfer-RNA (tRNA) of M . gallisepticum is much smaller than that of E . coli ( 2 . 5 S as against 5.0 S), Hayashi et al. (1969)found that the two tRNAs cosediment. A sedimentation coefficient of 5.0 S was also found for the tRNA of A . laidlawii, Mycoplasma sp. kid (Hayashi et al., 1969) and M . hominis (Johnson and Horowitz, 1971). The low molecular weight RNA found by Kirk and Morowitz (1969) probably was an artifact produced during isolation by the potent mycoplasma ribonuclease (see Johnson and Horowitz, 1971). Several fundamental questions have been raised in the last few years in connection with the mycoplasma tRNAs. These include : how many tRNA species can a mycoplasma make with its limited genetic information? Can mycoplasma tRNA function in heterologous systems of prokaryotic or eukaryotic origin? Is the low GC content of the mycoplasma genome reflected in the base composition of the tRNAs? Do the mycoplasma tRNAs contain the modified or minor nucleosides characteristic of the tRNAs of other organisms? No definite answer has as yet been given to the first question. The findings of Ryan and Morowitz (1969) suggest that Mycoplasma sp. kid has enough genetic information for only 44 tRNA species, which may be the minimal set (see page 16). Direct experimental data are, however, available only for A. laidlawii, where Feldman and Falter (197 1) found one or more tRNA species with acceptor activity for each of a series of amino acids. More is known about the second question. Transfer-RNA from A . laidlawii was examined for activity in heterologous systems of E . coli and yeast (Feldman and Falter, 1971). Amino-acylation was possible in all combinations of methionine-specific tRNAs and enzymes. However, though capable of formylating tRNA from E . coli, the transformylase of A . laidlawii failed to formylate the methionine-specific tRNAs from yeast. Mycoplasma tRNAs also stimulated polypeptide formation in

PHYSIOLOGY O F MYCOPLASMAS

25

cell-free amino acid-incorporating systems from E. coli (Hayashi et al., 1 9 6 9 ; Johnson et al., 1970). All three mycoplasma strains studied by Hayashi et al. (1969) contained N-formylmethionyl-tRNA which could be formed with formyltetrahydrofolate as formyl donor by both homologous and E. coli enzymes. The-tRNA of M . hominis could also be charged by amino-acyl tRNA ligases of E . coli (Johnson and Horowitz, 1971).A purified isoleucyl tRMA of M y c q l a s m a sp. kid resembled E. coli tRNA in its thermal denaturation profile and in its sedimentation behaviour, and could be equally charged with isoleucine by homologous and by E . coli amino acid-tRNA synthetases. All these findings point to the marked similarity of mycoplasma and bacterial tRNAs. The answer t o the third question seems to be in the affirmative. The GC content of i l l . hominis 5 S RNA is only 43.1% compared with 63.1% GC in E. coli 5 S RNA (Johnson and Horowitz, 1971). The kid strain tRNA, however, has only 4.4% less GC than E . coli tRNA (Hayashi et al., 1969) and, with A . laidlawii tRNA, the difference is a mere 2% (Feldman and Falter, 1971). The low GC content of the mycoplasma DNA accordingly seems to be reflected in the tRNA, but not to the same extent in all species. Perhaps the most intriguing issue is the presence or absence of modified or minor nucleosides. Among cellular RNA species, tRNA is unique in containing numerous modified nucleosides. On the whole one might expect that the less genetic information an organism has the lower the variety and amount of its modified nucleosides, since the enzymes required for this synthesis might be missing. The failure of Hall et al. (1 967) to detect modified nucleosides in RNA from M . pulmonis strain 880 was, therefore, not unexpected. If there really are none it would be necessary t o conclude that modified nucleosides are not essential t o tXNA function. On the other hand, subsequent studies have shown that the tRNAs of A . laidlawii, $1.gallisepticum, M . hominis, M . mycoides var. Capri and the kid strain do contain modified nucleosides though much less than E . coli (Hayashi et al., 1 9 6 9 ; Johnson and Horowitz, 1971; Walker, 1971). The low ribothymidine content of the kid strain tRNA was utilized by Johnson et al. (1970) t o test whether it is essential for tRNA function. They purified a tRNA specific for isoleucine which contained no ribothymidine. Nevertheless it could be charged with isoleucine by E . coli amino acid-tRNA synthetase, and mediated polyisoleucine formation in a cell-free system from E. coli. Hence, ribothymidine is not required for the recognition of tRNA by amino acidtRNA synthetases, nor does it appear to be required for the binding of tRNA to ribosomes. This study is an excellent example of the way in which the special properties of mycoplasmas can be utilized to tackle basic biological problems.

26

SHMUEL RAZIN

D. PROTEIN SYNTHESIS The biophysical similarity of mycoplasma and prokaryotic cell ribosomes is also evidenced by the sensitivity of the protein-synthesizing system of the mycoplasmas to specific inhibitors. Chloramphenicol and erythromycin, which specifically inhibit protein synthesis on the 70 S microbial ribosomes, inhibited protein synthesis in seven mycoplasma strains, while cycloheximide, which inhibits protein synthesis on the 80 S eukaryotic cell ribosomes, had no effect (Tourtellotte, 1969). From the few mycoplasma species investigated, it may be safely concluded that their mechanisms of protein synthesis correspond to the general scheme of free-living organisms. Ribonucleic acid is synthesized by a DNAdependent RNA polymerase, and its synthesis is inhibited by actinomycin D. As in bacteria, the calculated half-life of the messenger-RNA formed is less than four minutes (Kirk, 1966; Tourtellotte et al., 1967). There also is evidence of amino-acid activation by soluble RNAs specific for each amino acid (Tourtellotte, 1969; Feldman and Falter, 1971 ; Johnson and Horowitz, 1971; Johnson et al., 1970). The presence of tRNAfMetin all three mycoplasma strains examined by Hayashi et al. (1969) suggests that, as in other prokaryotes, formylmethionine is involved in peptide-chain initiation. A cell-free protein-synthesizing system according t o Matthaei and Nirenberg's model has been constructed from A . Haidlawii, where ribonuclease, puromycin, and chloramphenicol, but not cycloheximide, acted as inhibitors (Tourtellotte, 1969).

E. GO-ORDINATION

OF

MACROMOLECULAR SYNTHESIS

Since regulation and control mechanisms in mycoplasmas have received little attention, it is not clear whether they are affected by the limited genetic information available, but two recent studies offer some useful indications. The effects of thymine and glycerol deprivation on growth, lipid, protein and DNA synthesis have been investigated in the goat mycoplasma strain Y, which is capable of growth in a defined medium (Rodwell et al., 1972). Because of the nutritionally exacting nature of mycoplasmas, synthesis of each of these constituents can be specifically controlled by withdrawing the appropriate precursors from the growth medium. The products of macromolecular synthesis can moreover be specifically labelled by adding the appropriate radioactive precursors to the growth medium. This is one of the greatest advantages of mycoplasmas in biochemical studies. Omission of glycerol from the growth medium stopped lipid synthesis immediately (Fig. 5 ) while, for 5 h,

PHYSIOLOGY O F MYCOPLASMAS

27

cell mass increased a t the same rate as the control culture. Synthesis of DNA also continued in the absence of net lipid synthesis. Hence it seems that net lipid synthesis is not essential for DNA synthesis, as was also found by Mindich (1970) for Bacillus subtilis. On omission of thymine from the medium (Pig. 6), the number of colony-forming units went up only slightly, but cell mass and lipid synthesis continued to increase and glycine continued to be incorporated into the protein (these data are not shown in Figure 5 ) . Highly peculiar morphological changes took

Glycerol deprivation

DNA synthesis

f-q Thymine deprivation

2

0"' Incubation time (h)

FIG.5 . Effects of glycerol deprivation or thymine deprivation on macromolecular synthesis and growth of Mycoplasma sp. strain Y in a defined growth medium. Lipid synthesis was measured by 14C-palmitateincorporation and DNA synthesis by 3H-t2iymidineincorporation. Adapted from Rodwoll et al. (1972).

place in the thymine-deprived organisms. After 12 to 24 h, the cultures contained numerous minute particles, thin, flexible, threadlike filaments and other minute, often bizarre, forms like the minicells of E . coli, devoid of DNA. Although the effects of inhibition of protein and RNA synthesis on the synthesis of the other macromolecules have not yet been demonstrated, it may be tentatively concluded that synthesis of lipids, proteins and DNA in the mycoplasma strain Y are not stringently co-ordinated, and that a t least this mycoplasma has no stringent control mechanisms for co-ordinating macromolecular synthesis. According t o Rodwell et al. (1972) this may be explained by the fact that, in nature, the parasitic mycoplasmas grow and multiply in the controlled environment of

28

SHMUEL RAZIN

the host, so that the need to evolve control mechanisms is not as acute as in saprophytic micro-organisms. A somewhat different picture was obtained by Smith and Hanawalt (1968),who found that, in A . laidlawii as in other bacteria, deprivation of thymidine results in thymine-less death except that i t occurs after an initial lag of 10 to 15 h, much longer than in E . coli. Conditions preventing RNA synt,hesis also inhibited thymine-less death, which therefore appears to be associated with RNA synthesis. The idea that it might be caused by induction of a defective episome or a prophage, raised as a remote possibility by Smith and HanawaIt (1968), should now be reconsidered in light of the finding that many A . laidlawii cultures are infected with specific viruses. Smith and Hanawalt (1968) also examined the effect of inhibition of protein synthesis on DNA synthesis in A . laidlawii. Removal of tryptophan from the growth medium and addition of 15 pg of chloramphenicol/ ml stopped protein synthesis most effectively. Synthesis of DNA proceeded for only about one generation time (3-5h) and only 40% of the normal amount of DNA was produced. This is the value to be expected i f nearly all of the cells were synthesizing DNA a t the time protein synthesis was stopped, and all of the rounds of DNA replication begun were completed while no replication rounds were initiated. The much larger genome of A . laidlawii (Table 1, p. 15) may account for the fact that its macromolecular synthesis is better co-ordinated than in the Y strain.

VI. The Cell Membrane A. ISOLATION OF MEMBRANES Devoid of a cell wall and intracytoplasmic membranes, the mycoplasmas have only one type of membrane, namely the plasma membrane. This is what makes them particularly suitable as models for membrane study; once isolated, it is certain that the plasma membrane is uncontaminated with other membranes. Osmotic lysis, the gentlest method so far devised for the isolation of mycoplasma membranes, does not always work because the organisms’ sensitivity t o osmotic shock varies with the age of the culture (Razin, 1964) and the species (Razin, 1963). While Acholeplasma strains are usually highly sensitive, the sterol-requiring Mycoplasma strains frequently resist osmotic lysis. Other techniques have therefore been resorted to, such as alternate freezing and thawing (Hollingdale and Lemcke, 1969 ; Williams and Taylor-Robinson, 1967) or ultrasonic treatment (Argaman and Razin, 1969; Hollingdale and Lemcke, 1969).

PHYSIOLOGY O F MYCOPLASMAS

29

By the first, however, the bulk of the cells often remains unlysed (Hollingdale and Lemcke, 1969) while, by the second, the membranes are fragmented into minute particles (Pollack et al., 1965b; Kahane and Razin, 1969a). Recent experiments in our laboratory indicate that osmotic lysis of sterol-requiring mycoplasmas can be most effective when the organisms are harvested a t the right time, a t the end of the logarithmic phase of growth. Perfect control of the growth rate is absolutely essential and can only be achieved when the strain is well adapted to the growth medium. Under these conditions, very pure membrane preparations were obtained from M . hominis, M . mycoides var. mycoides, M . mycoides var. capri, M . arginini and M . anatis (Razin et al., 1972 and unpublished data). Although with the slowly growing mycoplasmas it is difficult to determine the correct harvesting time, attempts to obtain membranes from M . pneumoniae by osmotic lysis have met with some success (Pollack et al., 1970). It is not yet clear how the wall-less mycoplasmas resist osmotic lysis during most of their growth cycle, and why it is least effective in the cold. One explanation is that, because of the high surface to volume ratio of the minute cells, rapid liberation of the internal solutes may occur upon transfer to a hypotonic solution, quickly lowering the internal osmotic pressure so as to cushion the osmotic shock. The marked leakiness of mycoplasmas in non-nutrient solutions is well-known. Filamentous growth is likely to augment these effects, since the surface to volume ratio is much higher than in spherical cells, and filaments are able to absorb enormous amounts of water and turn into large spherical bodies without any stretching of the membrane. Special properties of the membrane itself may, however, also enhance resistance to osmotic lysis. The mycoplasma membrane is, in fact, far more resistant to fragmentation by sonication than the bacterial protoplast membrane. Whether the high cholesterol content has anything to do with the marked tensile strength of the mycoplasma membrane is still a matter of conjecture (Razin, 1967). A new approach to the isolation of mycoplasma membranes, circumventing many of the difficulties encountered with osmotic lysis, has recently been proposed by Rottem and Razin (1972a). It is based on the marked sensitivity of the sterol-requiring mycoplasmas to lysis by digitonin. Membranes of 31. hominis so obtained had the same ultrastructure, density, gross chemical composition, detailed lipid composition and protein composition as membranes obtained by osmotic lysis (Table 3). The advantage of digitonin lysis is that it takes place in the presence of divalent cations (e.g. Mgz') and is less dependent on the age of the culture, so that it may be useful for isolating membranes from the slow-growing mycoplasmas and from M . gallisepticum.

30

SHMUEL RAZIN

TABLE 3. Composition of Mycoplasrna Mernbrnrios

Membranos

Acholeplasma laidlawii B Mycoplasma bovigenitalium Mycoplasma mycoides var. capri Mycoplasma mycoides var. mycoides Mycoplasma hominis Mycoplasma pneumoniae

a

Protein

Lipida

___

Percent dry weight of Carbohydrate RNA DNA

Rrference

~-

~~

57

32 (4)

0.5

2.0

1.0

Razin (1967)

59

37 (24)

2.2

2.5

0.8

Raziri (1967)

50

40 ( 3 0 )

2.0

4.5

1.1

Razin (1967)

51

39

3.2

5-6

0.8

Hazin et al. (1969)

57

41 (37)

--

0.8

0.6

Rottem and Razin (1972a)

37

58

3.2

2.1

-

Pollack et al. (1970)

Figures in parentheses represent the percentage of cholesterol in lipid.

B. CHEMICAL COMPOSITION Mycoplasma membranes resemble other biological membranes in being essentially built of protein and lipid. Unless they are extensively washed with dilute buffer or sodium chloride solution and treated with nucleolytic enzymes, small amounts of RNA and DNA can be detected (Table 3 ) . The amount of protein usually exceeds that of the lipid, but considerable variations in the protein-to-lipid ratio may occur when the growth conditions are changed (Kahane and Razin, 1970). Endogenous peptidases and phospholipases may also affect the composition of isolated membranes (Choules and Gray, 1971 ; Van Golde et al., 1971). The isolated membranes should therefore be kept under conditions that minimize the activities of these enzymes (preferably a t -70°C). The long protracted incubation of isolated membranes of 1M. pneumoniae with nucleolytic enzymes (90 min a t 37°C) employed by Pollack et al. (1970) may have stimulated the activity of endogenous peptidases and of peptidases contaminating the nucleolytic enzyme, so as to account for the unusually low content of protein (Table 3 ) . Carbohydrate, a minor component of the mycoplasma membranes analysed so far, may include the carbohydrate moiety of glycolipids if it is determined before the membrane lipids have been removed.

PHYSIOLOGY O F MYCOPLASMAS

31

There is no chemical evidence as yet for the membranes containing glycoproteins, major components of eukaryotic plasma membranes, whose presence in bacterial membranes, however, is poorly documented (Weinbaum et al., 1970). On the other hand, negatively stained cells of M . gallisepticum (Chu and Horne, 1967) and M . pulmonis (Hummeler et al., 1965; Reuss et al., 1967) have spikes on the outer membrane surface, resembling those of the myxoviruses in length and spacing; and some of the viral spikes are known to consist of glycoprotein. Membranes from A . laidlawii contain considerable amounts of glucosamine and galactosamine (Naide, 1963; Engelman and Morowitz, 1968a; Morowitz and Terry, 1969; Ne’eman et al., 1972).The hexosamine does not appear to be part of a glycolipid since it is not extracted with aqueous acetone and moves independently of the lipid in polyacrylamidegel electrophoresis. It does not appear t o be bound to the protein because it is not released when membrane protein is digested by pronase. It might thus constitute a polymer by itself (Morowitz and Terry, 1969).

C. MEMBRANE PROTEINS Proteins are the major component of the mycoplasma membrane. Apart from their structural and catalytic role they also have a major share in the immunological activities of mycoplasma cells (for a review, see Razin et al., 1972).

I. Localization of Enzymic Activities Although the relative ease with which mycoplasma membranes can be separated from the cytoplasmic constituents is a boon to the study of enzyme localization, their enzymic build-up is still largely unknown. The list of enzymic activities detected so far in the A . laidlawii membrane (Table 4) shows that they have not been systematically surveyed even in the most extensively investigated membranes. Since localization studies have been confined to a few enzymic activities and mycoplasma species, the only general conclusion warranted a t this stage is that ATPase activity is exclusive to the membrane (Pollack et al., 1965a; Rottem and Razin, 1966) while NADH, oxidase activity is associated with membranes from A . laidlawii and with the soluble fraction of all the other mycoplasma strains examined (Pollack et al., 1965a, 1970; Rodwell, 1969). The inability to detect NADH, oxidase activity in membranes from M . mycoides var. mycoides isolated by digitonin in the presence of Mg2+, conditions minimizing release of loosely associated membrane proteins (Z. Ne’eman, unpublished data) argues against its being loosely bound to the membranes of the sterol-

32

SHMUEL RAZIN

TABLE 4. Enzyrnc Activities Localized in Membranes from ilclkoleplasma laidlatuuii Enzyme activity Adenosine triphosphatase Para-Nitrophenyl phosphatase ii’-Nucleotidasc Ribonnclease Deoxyribonueleasc NADH, oxidase P-Clucosidase Phosphoglucolipid syrithetesc Aminoacylphosphatidylglyoerol syrithctasc synthetases Mono- and di~lucosyldi~lyceride Lysophospholipase Peptidase

Reference Pollack et al. (1965a) Nr’ernan et al. (1971) IZottcm and Razin (1966) Pollack et al. (1965a) Pollack et al. (lQ65a) Pollack et al. (1965a) Heririkson and Smith (1964) Smith (1971b) Koostra and Smith (1969) Smith (196Qa) Van Golde et nl. (1971) Chotdes and Gray ( 1971 )

requiring mycoplasnias. I n M . arthritidis stra.in 07, on the other hand, quinone-like material and cytochromes seem to be associated with the membrane fraction (VanDemark, 1967) while flavins have been extracted and spectrophotometrically identified in membranes from A . Zaidlawii (Morowitz and Terry, 1969). Hence the location of the respiratory enzyme system is not necessarily in the membrane as is generally the case with bacteria, but seems to vary with the species. That a t least some of the enzymes involved in membrane lipid synthesis are membrane-bound is borne out by recent studies on the biosynthesis of membrane phospholipids and glycolipids in A . Zaidlawii (Table 4).

2. Xolubilixation of Membrane Proteins Solubilization is a prerequisite for the fractionation and characterization of membrane proteins. Under mild non-denaturing conditions, only a minor fraction can be solubilized. These are the proteins held to the membrane mainly by ionic bonds which are broken by changes in the ionic strength or the pH value of the suspending medium, solubilization usually being improved by the addition of a chelating agent such as EDTA (Razin, 1972b). Upon prolonged treatment of membranes of A . laidlawii with EDTA in media of low ionic strength, about 11% of the total membrane protein was released in a water-soluble, almost lipid-free, form (Ne’eman et al., 1971), but no ATPase, p-nitrophenyl phosphatase and NADH, oxidase activities and none of the important protein antigens of the membrane were present. This gentle procedure may, however, prove more successful in releasing enzymically active proteins from membranes of other mycoplasmas.

PHYSIOLOGY O F MYCOPLASMAS

33

Most membrane proteins are tightly bound to membrane lipids, apparently by a combination of hydrophobic and ionic bonds. To break up these composite bonds, more drastic procedures have to be applied. The agents most commonly employed for this purpose are detergents and organic solvents, but they are liable to denature the solubilized protein molecules (Razin, 1972b) and, since the solvent must be water soluble, the choice is limited. Although aqueous 90% acetone removes over 90Y0 of the membrane lipids of A . laidlawii, it does not solubilize the protein. I n electron micrographs of thin sections, acetone-extracted membranes from A . laidlawii were found to retain their trilaminar structure (Morowitz and Terry, 1969). Cold n-butanol, under conditions of low-ionic strength (Maddy, 1966), effectively solubilizes membranes from A . laidlawii. Some 80-90% of the proteins with very little lipid are separated out in the aqueous phase, but apparently not in the monomeric form, since they are excluded in the void volume of a Sephadex G-200 column (S. Rottem, M. Hasin and S. Razin, unpublished data). I n solubilizing membrane proteins from A . laidlawii, detergents have proved more effective (Ne’eman et ul., 1971), the strongly ionic ones (sodium dodecyl sulphate and cetyltrimethylammonium bromide) not unexpectedly more so than the non-ionic ones (Triton X-100, Lubrol W or Brij !%), sodium deoxycholate occupying an intermediate position. The ionic detergents also caused more intensive denaturation of membrane proteins, as indicated by enzyme inactivation (Ne’eman et ul., 1971). Not only the membrane proteins and lipids but also the different membrane protein species were solubilized at different rates, so that there was no random disaggregation of the membrane (Ne’eman et al., 1971 ; Morowitz and Terry, 1969). It appears that, during membrane solubilization by detergents, two antagonistic processes take place simultaneously, namely the activation or unmasking of some enzymic activities and the denaturation and consequent inactivation of the solubilized enzymes. The activation effect has been described for a variety of membranes and detergents (e.g. Pollack et al., 1965a; Eisenberg et al., 1970) and may be ascribed t,o the exposure of sterically hindered catalytic sites in the course of membrane solubilization.

3. Fractionation of Membrane Proteins Solubilization of the hydrophobic membrane proteins is only the first step in their characterization. Next comes their fractionation and separation which are usually more difficult. Most of the techniques available so far are designed for soluble hydrophilic proteins and are not easily adaptable to hydrophobic proteins.

34

SHMUEL RAZIN

Attempts to fractionate mycoplasma membrane material, solubilized by detergents on Sephadex or Sepharose columns devoid of detergents, have failed, apparently because the proteins and lipids re-aggregated upon dilution or removal of the detergent during filtration (Ne’eman et al., 1972; see page 57). Detergents, therefore, had to be included throughout the fractionation procedure. When the detergent used for membrane solubilization was also incorporated into the column and the 0.3

-

25

E

0

0.2

c

20

s

L_ %

-

0

e

15

:: D

-

01

0

c

E8

g

0

0.1

10 I

-e n .-c

9

W

2

05

0

I I Void VOI.

Fraction number

FIG.6. Fractionation of solubilized membrane proteins and lipids from Acholeplasma laidlawii on a Sephadex G-200 column containing 16 mg sodium deoxycholate/ml. 0 - 0 , indicates membrane protein ; 0-0, membrane lipid labelled during growth with 3H-oleicacid; A-A, NADH, oxidase activity. From Ne’eman et al. (1972).

elution buffer, a considerable portion of the enzymic and antigenic activities of the membrane proteins was preserved despite the prolonged exposure to detergents (Ne’eman et al., 1972; Razin et al., 1972).Sodium deoxycholate appears to be best suited for this purpose. Its inclusion in the Sephadex G-200 coIumn resulted in the separation of membrane proteins into several reproducible peaks most of which were devoid of membrane lipid (Fig. 6 ) . Their fractionation was a t least as good as with sodium dodecyl sulphate while the fractions retained more of their biological activity. Sodium deoxycholate also does not seem to cause gross conformational changes in membrane proteins (Crumpton, 197 1) or form complexes with them (Philippot, 1971 ; Allan and Crumpton,

PHYSIOLOGY OF MYCOPLASMAS 35 1971), whereas sodium dodecyl sulphate binds to them in considerable quantities and unfolds the polypeptide chains (Reynolds and Tanford, 1970).

4 . Purijication and Characterization of Membrane Enzymes Separation of membrane-bound enzymes of mycoplasmas without denaturation and loss of activity is so difficult that so far none has been purified and characterized. The marked sensitivity of ATPase from A . laidlawii to detergents has failed all attempts to purify it by detergent solubilization. Ultrasonic treatment liberated about 50% of this enzymic activity into a fraction not sedimentable a t 100,000 q for 45 min, but it apparently remained bound to minute membrane particles so that it could not be purified by the conventional protein purification techniques (Rottem and Razin, 1966). Adenosine triphosphatase from A . laidlawii seems to be more tightly bound to the membrane than the ATPases of Micrococcus lysodeikticus, Xtreptococcus faecalis and Bacillus meqaterium, which can be easily detached by repeated washings with dilute buffers without Mg2+ (Munoz et al., 1969; Abrams and Baron, 1968; Ishida and Mizushima, 1969). Nevertheless it is like the microbial and mitochondria1 ATPases in that it is not stimulated by sodium and potassium and is insensitive to ouabain (Rottem and Razin, 1966; Cho and Morowitz, 1969). It is still not clear whether the ATPase andp-nitrophenyl phosphatase activities of membranes from A . laidlawii are the expression of a single enzyme. Their different sensitivities to detergents (Ne’eman et al., 1971), organic solvents (L. Qottfried and S. Razin, unpublished data) and to pronase digestion (I.Kahane, Z. Ne’eman and S. Razin, unpublished data) seem to indicate that they are not. The NADH, oxidase of membranes from A . laidlawii may be a better candidate for purification than the ATPase since it is less sensitive to detergents. Resistant to brief exposure to sodium dodecyl sulphate, it could even be detected in reconstituted membranes from A . laidlawii (Razin et al., 1965). The NADH, oxidase activity was eluted from a deoxycholate-containing Sephadex G-200 column in the first lipid-free protein peak (Fig. 6) and accordingly, unlike many membrane enzymes (Razin, 1972b), it does not depend on membrane lipids. Solubilization of membranes from A . laidlawii with anionic or non-ionic detergents, followed by extensive dialysis with a view to solubilize the enzymes that synthesize the glycolipids, resulted in inactive preparations. Acetone powders of the membranes were completely devoid of activity even when the acetone-soluble lipids were again added (Smith, 1969a). 3

36

SHMUEL RAZIN

5 . Molecular Properties of Membrane Proteins Amino-acid analysis of membrane proteins from A . laidlawii revealed a high content of alanine, glycine, valine, leucine, isoleucine, phenyl-

alanine, and tyrosine but not enough to account for the hydrophobic character of the protein fraction (Engelman and Morowitz, 1968a; Morowitz and Terry, 1969; Choules and Bjorklund, 1970).This bears out the view of Wallach and Gordon (1968) that hydrophobicity of membrane proteins may depend on certain specialized amino-acid sequences or specific regional adaptations of the polypeptide structure. As in many other biological membranes (Wallach and Zahler, 1968) only minute amounts of cysteine or cystine have been found in membranes from A . laidlawii (Engelman and Morowitz, 1968a; Choules and Bjorklund, 1970; Kahane, 1971). The absence of disulphide bridges appears to be a characteristic of proteins associated with lipids either in membranes or in soluble lipoprotein complexes. The polypeptide chains are consequently more flexible so that the hydrocarbon chains of membrane lipids can penetrate more easily into the corresponding hydrophobic regions of the proteins (Camejo et al., 1968). The marked sensitivity of ATPase (Rottem and Razin, 1966) and the active transport systems of mycoplasmas (Razin et al., 1968; Rottem and Razin, 1969; Cho and Morowitz, 1969; VanDemark and Plackett, 1972)to sulphydrylblocking reagents indicate, however, that sulphydryl groups are present, and titration for these groups in membranes from A . laidlawii was, in fact, positive (Kahane, 1971). Electrophoresis of mycoplasma membrane proteins in polyacrylamide gels usually yields 15-30 different protein bands, depending on the species and on the method used (Rottem and Razin, 1967a; Morowitz and Terry, 1969). There undoubtedly are more proteins than are discernible in this way, since the electrophoretic patterns reveal only the major ones and some may migrate together in the same band. The electrophoretic patterns of the mycoplasma membrane proteins are highly reproducible and species-specificand, as they are usually not affected by variations in growth conditions, they may be used for mycoplasma identification and classification (Rottem and Razin, 1967a). The molecular weight of the various membrane proteins could be assessed by electrophoretic analysis in sodium dodecyl sulphate-containing polyacrylamide gels (Morowitz and Terry, 1969) or in the acidic gel system of Takayama (Ne’eman et al., 1972). The molecular weight of membrane proteins from A . laidlawii varied from 9,000 t o over 70,000 (Ne’eman et al., 1972), well within the range of other biological membrane proteins. Do mycoplasma membranes contain structural proteins that have

PHYSIOLOGY OF MYCOPLASMAS

37

no catalytic role? No definite answer can be given a t the present. What seems certain, however, is that the hydrophobic protein fraction isolated from detergent-solubilized mycoplasma membranes by salting out with ammonium sulphate a t 12% saturation (Rodwell et al., 1967; Kahane and Razin, 1969a) is not a structural protein fraction. The finding that it contains almost all of the protein species of the membrane (Rodwell et al., 1967) was one of the first disclaimers of the hypothesis that biological membranes contain a major structural protein common to all (see Senior and MacLennan, 1970).

D. MEMBRANELIPIDS One of the main objects of this review is to point out the usefulness of the mycoplasma membrane in investigating the role of lipids in biological membranes. Their limited biosynthetic capacity makes mycoplasmas dependent on the supply of many nutrients from the growth medium, including lipid precursors. All of the mycoplasmas tested so far require long-chain fatty acids for growth and most strains require substantial amounts of cholesterol. Several strains, like M . mycoides var. mycoides, also require glycerol (Rodwell and Abbot, 1961), and are thus entirely dependent on the external supply of lipid precursors. This enables us, within limits, to regulate the amount of a particular lipid component in the membrane and to analyse the effects of such variations on membrane properties. Virtually all mycoplasma lipids are located in the cell membrane (Razin and Cleverdon, 1965; Razin, 1967) and, as in other biological membranes, consist mostly of polar lipids (phospholipids and glycolipids) with smaller amounts of cholesterol and sometimes also carotenoids.

1. Phospholipids Phosphatidylglycerol has been found in all of the mycoplasmas examined so far and appears to be the major phospholipid synthesized by them (Shaw et al., 1968; Plackett, 196%; Smith and Koostra, 1967; Plackett et al., 1969; Prescott et al., 1970; Romano et al., 1972). Diphosphatidylglycerol is usually found in smaller quantities or not a t all (Smith and Koostra, 1967; Romano et al., 1972). Phosphatidic acid, recently identified as a major lipid of a T-strain mycoplasma (Romano et al., 1972), has not been reported in any other species. Lyso compounds, like monoacylglycerophosphoryl glycerophosphate, phosphatidylglycerophosphate, or pliosphatidylmonoglyceride that have been detected in mycoplasma lipids, may be either breakdown products or biosynthetic precursors (Smith and Koostra, 1967 ; Plackett et al., 1969).

38

SHMUEL RAZEN

Presumably the mechanism of biosynthesis of phosphatidylglycerol and diphosphatidylglycerol is the same as in bacteria, although it has not been examined in any detail. Evidence presented by Plackett and Rodwell (1970) suggests that phosphatidylglycerol is synthesized from cytidine diphosphodiglyceride and sn-glycerol-3-phosphate. Phosphatidylglycerol may then be the precursor of diphosphatidylglycerol. The mono- and diglycerides often found in mycoplasma lipids (Pollack et al., 1970; Romano et ul., 1972; S. Rottem, unpublished data) may be either ,degradation products of the polar lipids following the action of endogenous lipases or, what is more likely in the light of their rapid turnover, precursors of the polar lipids (Plackett and Rodwell, 1970). A somewhat unusual lipid was discovered by Smith and Henrikson (1965) in A . laidlawii. Containing glycerol, glucose, fatty acids and phosphate, it was tentatively identified as “phosphatidyl glucose”. Since chemically prepared phosphatidylglucose (Verheij et al., 1970) was, however, found to have different properties, this identification is untenable. Shaw et ul. (1970) have accordingly proposed an alternative structure, namely glycerylphosphoryldiglucosyl diglyceride. The tentative structure of this phosphoglycolipid is : CH,OH

I

CHOH

I

OH

I

CHZ-0-P-OH

l

OH

I

CH,-O-CO-I%

Since this phospholipid is completely resistant to hydrolysis by phospholipases A, C, and D, the generic name “phosphatidyl” is inapplicable and phosphoglycolipid seems to be more appropriate. From recent findings it appears that phosphoglycolipids are quite common in lactic acid bacteria (Stead et al., 1972). As phosphoglycolipids are always structurally related to the diglucosyl diglycerides found in micro-organisms, transfer of glycerophosphate from CDP-glycerol to diglucosyl diglyceride is regarded by Stead et al. (1 972) as the most likely biosynthetic route. Alternatively, phosphatidic

39

PHYSIOLOGY OF MYCOPLASMAS

acid may be transferred from CDP-diglyceride, initially producing a diacylglycerophosphoryl diglucosyl, and then losing two acyl residues. Conversion of cliglucosyl diglycerides to phosphoglycolipids has been demonstrated using a particulate enzyme preparation of Streptococcus faecalis (Ambron and Pieringer, 1971). A third version, according to which the phosphoglycolipid of A . laidlawii is synthesized by a membrane-associated enzymic system from phosphatidic acid and uridine-5'diphosphoglucose, was put forward by Smith (196933) before it became clear that the lipid in question is not phosphatidylglucose. Mycoplasmas grown in serum-containing media may contain appreciable quantities of lecithin and sphingomyelin (Smith and Henrikson, 1965; Plackett et al., 1969; Razin et al., 1970b) but labelling experiments have shown conclusively that they are adsorbed from the medium and are not synthesized by the organisms. The common microbial lipid, phosphatidylethanolamine, has been reported only once, in small quantities, in a T-strain mycoplasma (Romano et al., 1972). An interesting finding of Plackett et al. (1970) is that the recently discovered A . axanthum, which cannot synthesize carotenoids and does not require cholesterol for growth (Tully and Razin, 1969, 1970), synthesizes a sphingolipid as its major phospholipid, a feature rarely noted in bacteria. The tentative structure of this phosphosphingolipid is : 0

cH3,+cHz-o-P-o-cH, OR, NH

"

I

OH

I

+

CH,OH

OH

CH, R,

= H; HZ= fatty

acid

R, = fatty acid; R,

=H

Every labelled fatty acid added to the medium, except erucate (C22:1),led to the formation of a labelled long-chain base with two more carbon atoms, so that the fatty acids in the medium evidently account for synthesis of the long-chain base. It is doubtful whether the 0-amino acyl esters of phosphatidylglycerol isolated from acidified cultures of A . laidlawii and M . arthritidis strain 07 occur naturally, since the organisms do not grow a t the pH values necessary for their formation (Smith and Koostra, 1967; Koostra and Smith, 1969).

40

SHMUEL RAZIN

Little or no turnover of glycerophospholipids takes place during growth of A . laidlawii (McElhaney and Tourtellotte, 1970a; Smith, 1969c), M . arthritidis strain 07 and M . gallinarum strain J (Smith and Koostra, 1967). Transacylation also does not seem to occur (Smith, 197l a ) . Smith’s contrary claim for the phosphoglycolipid (“phosphatidyl glucose”) of A . laidlawii (Smith, 1969c) was not corroborated by McElhaney and Tourtellotte (1970a) who tend to ascribe the discrepancy to differences in experimental techniques and in the age of the culture. Kahane and Razin (196913) were unable to show turnover of polar lipids except in young cultures of A . laidlawii. While it would be precipitate to assume that polar lipids from A . Zaidlawii are metabolically stable under all conditions, it may be safely concluded from McElhaney and Tourtellotte’s findings that turnover of polar lipid is not obligatory for proper membrane function.

2. Glycolipids The glycolipids that form a major constituent of the poIar lipids of fermentative mycoplasmas are glycosyl diglycerides, acylated sugars and cholesteryl or carotenyl glycosides. I n A . laidlawii, monoglucosyl- and diglucosyldiglycerides sometimes represent almost half of the lipid (Plackett and Shaw, 1967; Shaw et al., 1968; Rottem and Panos, 1969) whereas, in Gram-positive bacteria, though widely distributed, they constitute only a minor fraction (Shaw, 1970). The monoglucosyl diglyceride from A . laidlawii has the following structure : 0

II

R-C-0-CHZ CH2OH

I

HC-0-C-R,

0

I1

OH

Both M . mycoides var. rnycoides and M . pneumoniae synthesize galactolipids which apparently are responsible for the serological cross-reactions between the two mycoplasmas. I n M . mycoides, the galactolipids consist mostly of a monogalactosyldiglyceride (Plackett, 1967a, b ) and, in M . pneumoniae, of digalactosyl- and trigalactosyldiglycerides, a dihexosyldiglyceride containing both glucose and galactose, and a trihexosyl diglyceride fraction containing both hexoses, probably representing a mixture of two or more compounds (Plackett et al., 1969). The

PHYSIOLOGY O F MYCOPLASMAS

41

galactose-containing glycolipids of M . pneumoniae are its major antigenic determinants (Plackett et al., 1969; Beckman and Kenny, 1968; Razin et al., 1970b). The enzymic system that synthesizes the glycosyldiglycerides in A . laidlawii has been localized in the cell membrane (Smith, 1969a). Monoglucosyldiglyceride is synthesized from 1,2-diglyceride and UDP-glucose, and diglucosyldiglyceride from monoglucosyldiglyceride and UDP-glucose. Essentially this system is the same as in other microorganisms. Biosynthesis of the tri- and tetraglycosyldiglycerides has not been elucidated, but their structural features suggest that they are formed by successive transfers of hexose residues from the appropriate nucleotide precursor. Mycoplasrna gallinarum strain J contains two major glycolipids : cholesteryl- 8 - D -glucopyranose and 3,4,6-triacyl-p-D-glucopyranose. Each of them accounts for about half of the total glycolipids or about 10% of the total lipid (Smith, 1971a). The acylated sugar from Af. gallinarum strain J has the following structure : 0

II

CHZOCR

OH

Acylated sugar derivatives have been isolated from many organisms, and may be more widely distributed than glycosyldiglycerides, although their quantity is usually very small. Their biosynthesis has not been established experimentally, but the most likely route seems to be the direct acylation of the appropriate hexose or a phosphorylated derivative (Smith, 1971a). Biosynthesis of cholesterylglucoside by M . gallinarum strain J was shown (Smith, 1971b) to proceed by transfer of glucose from UDPglucose to membrane-bound cholesterol. The enzymic activity, again associated with the membrane, was expunged when the endogenous cholesterol was removed from the membrane. Since only 5% of exogenously added cholesterol became glycosylated, the cholesterol bound during growth seems to be oriented or sited differently from that bound by resting cells or membrane suspensions (see also page 47). The function of glycolipids in mycoplasma membranes is still not clear. The apparent absence of turnover speaks in favour of a, structural role (Smith, 1 9 6 9 ~ McElhaney ; and Tourtellotte, 1970a) and against the

42

SHMUEL RAZIN

attractive hypothesis that glycosyldiglycerides are intermediates in polysaccharide biosynthesis (Plackett, 1967b).

3. Fatty Acids Fatty acids with their long hydrocarbon chains constitute the major portion of the hydrophobic region in the bioIogica1 membrane. The physical properties of this region are consequently largely determined by their composition. Since they are dependent on the external supply of fatty acids, mycoplasmas have proved most useful in elucidating the effect of the fatty acid composition on membrane structure and function. Usually the fatty acids added to the growth medium are quickly incorporated into the membrane polar lipids (Razin et al., 1966b; Kahane and Razin, 1969b), but Romano et al. (1972) found large quantities of free fatty acids in the lipids of a T-strain mycoplasma. Since lyso compounds could not be detected, they do not seem to derive from the breakdown of phospholipids. Also, in M . hominis (S. Rottem, unpublished data), 30-40% of the labelled palmitic or oleic acid added to the growth medium was found as free fatty acid in the membrane, without indication of any enzymic breakdown of membrane lipids that might account for them. Of the mycoplasmas tested, M . mycoides var. mpoides, the related Y strain (Rottem and Razin, 196713; Rodwell, 1968), M . arthritidis, M . gallisepticum, M . gallinarum (Smith, 1971a) and probably M . pneumoniae (Pollack et aH., 1970) seem totally unable to synthesize de novo, or even elongate, unsaturate or interconvert any long-chain fatty acid. Acholeplasma laidlawii, on the other hand is capable of synthesizing saturated long-chain fatty acids from acetate (Pollack and Tourtellotte, 1967; Rottem and Razin, 1967b) which does not hinder it from incorporating them from the medium, when synthesis de novo is arrested, apparently by end-product inhibition (Rottem and Razin, 1967b). The soluble fatty-acid synthetase system of A. laidlawii strain A has been characterized by Rottem and Panos (1970). Depending on ATB, Mg2+,and NADPH, for activity, its need for malonyl-CoA is absolute, possibly because the acetyl-CoA carboxylase activity of this organism is very low. The sole end-products were saturated fatty acids (stearic, palmitic and arachidic acids) of which, unlike in whole-cell growth experiments, stearic acid was the major one, so that the mechanism regulating the type of fatty acid synthesized may possibly alter once cellular integrity is destroyed. The work of Rottem and Panos (1970) also explains the inability of A . laidlawii t o synthesize unsaturated fatty acids by the absence ofthe

PHYSIOLOGY OF MYCOPLASMAS

43

dehydrase acting on /3-hydroxydecanoicacid, the normal precursor at the stage in bacteria where the pathway for formation of unsaturated fatty acids branches off. Addition of a purified /3-decanoylthioesterdehydrase from E . coli to the synthetase system of A . laidlawii enabled it to form unsaturated fatty acids. Like other synthetases, the A . laidlawii system contains an acyl carrier protein (ACP) which resembles the E. coli acyl carrier protein in molecular weight, but is more sensitive to heat (Rottem and Panos, 1970; S. Rottem, 0. Muhsam and S. Razin, unpublished data). Some mycoplasmas are able to elongate fatty acids. Thus Mycoplasma sp. strain KHS has been shown to elongate palmitoleic acid to cisvaccenic acid, and cis-5,6-tetradecenoic acid to cis-7,8-hexadecenoic acid (Panos and Henrikson, 1969). The cis-7,8-hexadecenoic acid, an oleic acid precursor, did not, however, elongate further to oleic acid. While oleic acid is the predominating octadecenoic acid of higher forms of life, its positional isomer, cis-vaccenic acid, prevails in bacteria (Panos and Henrikson, 1969). Whereas the KHS strain seems to be capable of forming cis-vaccenic acid by elongation of the appropriate hexadecenoic acid, A . laidlawii strain A could not elongate externally supplied fatty acids with a chain length of 16 carbon atoms, though it was capable of elongating dodecenoic and tetradecenoic acids (Panos and Rottem, 1970). Owing to the limited ability of A . laidlawii to synthesize fatty acids, the fatty-acid composition of its membrane lipids can be controlled by regulating the fatty-acid supply in the growth medium (Razin et al., 196613;Henrikson and Panos, 1969;Rottem and Panos, 1969;McElhaney and Tourtellotte, 1969). As seen in Table 5 the exogenous fatty acid is incorporated in high amounts, reaching up to 80 mole percent of the total fatty acids. Laurie, palmitic and stearic acids are present throughout as a biosynthetic background over which fatty-acid incorporation is superimposed. The range of acceptable fatty acids does, however, have its limits. Increasing numbers of double bonds or methyl branches on a fatty acid lowers and eventually eliminates its incorporation into membrane lipids. Likewise long-chain saturated fatty acids (22 casbons and more) are not incorporated in significant quantities. By manipulation of the fatty-acid composition of the growth medium, McElhaney and Tourtellotte (1970b) determined the distribution of the different fatty acids between the 1- and 2-positions of phosphatidylglycerol from A . laidlawii. The strength of the apolar cohesional force developed by the fatty acid was found to be correlated with its positional specificity. The strength of the apolar attractive forces decreases in the order: stearic > isostearic > elaidic > oleic > linoleic, the same as the decreasing order of affinity of these fatty acids for the 1-position. The

44

SHMUEL RAZIN

Table 5. Fatty-Acid Composition of Acholeplasma laidlawii strain B Grown with Various Fatty Acids Iiicorporation of f a t t y acids i n polar lipids (moles per 100 moles) ~~

Added F a t t y Acids

~

12:O.

14:O

16:O

18:O

18:l

None Palmitic ( 16 :0) Stearic (18 :0) Oleic (18:l cis) Elaidic ( 18 : 1 trans)

7.5 13.5 5.0 3.5 3.9

18:2 __

___-_____.

24.8 3.9 5.0 6.4 4.7

53.5 68.3 8.0 20.2 16.5

3.0 2.0 65.0 0.9 0.7

6.6 6.8 10.3 68.9 Y3.Y

4.4 5.4 6.7 Trace

0.5

The organisms were grown in a lipid-extracted tryptose-containing medium supplemented with 0.4% lipid-depleted bovine albumin and 50 pg of the fatty acid/ml. Taken from McElhaney and Tourtellotte (1969).

acylating enzymes of A . laidlawii thus tend to minimize the formation o f phosphatidylglycerol molecules containing two fatty acyl groups characterized by either very strong or very weak apolar interactions, and instead produce a more physiochemically homogeneous class of phospholipid molecules than would result from the random incorporation of fatty acids into the 1- and %positions. Since M . mycoides and the related Y strain are entirely unable to synthesize or transform fatty acids, their composition can be still more stringently controlled than in A . laidlawii. The Y strain could be grown in a defined medium supplemented with one saturated and one unsaturated fatty acid (Rodwell, 1967). As the chain length of the unsaturated acid increased from C,, to C,,, saturated acids of progressively shorter chain length were required. Further study (Rodwell, 1968) showed that the pair of fatty acids could be replaced by elaidic acid, the trans isomer of oleic acid, which became the only fatty acid o f the membrane lipids, a vivid illustration of the high flexibility of the membrane-lipid composition. Controlled changes in the fatty-acid composition of membrane lipids in A . laidlawii have a marked influence on the growth of the organism. Thus, an increase in oleic acid content was accompanied by a substantial rise in cell yield and osmotic stability, and a tendency to grow in very long and branching filaments. On the other hand, an increase in the palmitic or stearic acid content inhibited growth; the cells were swollen and very fragile and filaments were not found (Razin et al., 1966a, b). These effects may be attributed t o physical changes in the membrane which affect its permeability and elasticity. The chain length and degree of unsaturation of the fatty acids ma,y alter the packing of the polar lipids

PHYSIOLOGY O F MYCOPLASMAS

45

in the membrane (Razin, 196910). Experiments with phospholipid films (Van Deenen, 1965) have shown that unsaturated or short-chain fatty acids prevent the lipid layer from becoming over-condensed and enhance membrane permeability. The cohesive forces between the long hydrocarbon chains are mostly of the London-van der Waals type, which act between CH, groups on adjacent fatty acids and, since they are additive and depend on the number of CH, pairs, apolar attraction increases with chain length. When unsaturated bonds are introduced into the chain, steric hindrance causes the film to become more loosely packed (Van Deenen, 1965). Thus, in A . laidlawii, oleic acid could be partly replaced

Temperature (“C)

FIG.7. (a)Initial swelling rates in isotonic glycerol of intact celIs of Acholeplasmu laidlawii grown in the presence of various combinations of fatty acids as a function of temperature. (b)Initial swelling rates in isotonic glycerol of liposomes, prepared from total membrane lipids of A . laidlawii, as a function of temperature. The fatty-acid combinations are indicated on the graphs. Taken from McElhaney et al. (1970).

by the short-chain lauric acid (Razin and Rottem, 1963; Razin et al., 1966a). Similarly, the fatty-acid pairs required for growth of the goat mycoplasma strain Y (p. 44) may be needed to regulate the cohesion forces between the lipid molecules in the membrane, so that it should not become either too fluid or too condensed. Elaidic acid, being a trans fatty acid, may physically simulate a mixture of saturated and unsaturated acids and provide the right fluidity for the lipid region (Rodwell, 1968). These ideas, advanced several years ago (Razin, 1969a, b), have since received experimental support by McElhaney et al. (1970) who grew A . laidlawii in a lipid-poor medium supplemented with palmitate together with either oleate, elaidate or linoleate, so that the membrane lipids contained approximately equimolar proportions of saturated and unsaturated acids. Permeability to glycerol of membranes of intact cells and of liposomes prepared from their lipid was determined by the rate of swelling at different temperatures. The intact cells were somewhat

46

SHMUEL RAZIN

more permeable than the liposomes, but the rates for both increased in the order elaidate < oleate < linoleate (Fig. 7), in agreement with permeability studies through films of synthetic phospholipids. Acholeplasma laidlawii apparently has some regulatory mechanism by which it can control the fluidity of the lipid region of the membrane under different physiological conditions. Thus, the drastic lowering of the growth temperature resulted in a marked increase in oleic acid and lowered the amount of cholesterol incorporated into the cell membrane. Increased membrane-lipid fluidity was evident from the much higher freedom of motion of a spin-labelled fatty acid in these membranes than in membranes of cells grown a t 37°C (Rottem et al., 1970).

4 . Cholesterol and Carotenoids A requirement for cholesterol for growth is a major criterion for distinguishing the Mycoplasmataceae from bacteria (Edward and Freundt, 1969). Cholesterol, or other sterols, while being important constituents of membranes of eukaryotic organisms, are not normally found in prokaryote cell membranes. Cholesterol is incorporated into membranes of growing or washed mycoplasma cells by a process which does not require energy (Smith and Rothblat, 1960; Razin and Shafer, 1969). When esterified cholesterol as well as free cholesterol is present in the growth medium, the mycoplasmas selectively incorporate the free cholesterol (Argaman and Razin, 1965; Razin and Shafer, 1969).Animal cells in culture have similar preference (Rothblat et al., 1966) and animal cell membranes were also shown to be practically free of esterified cholesterol (Kleinig, 1970).Several cholesteryl esters have indeed proved unsuitable for mycoplasma growth (Edward and Fitzgerald, 1951), but some could nevertheless be traced in the membranes of sterolrequiring species (Smith, 1971a; Romano et al., 1972; S . Rottem, unpublished data). According to Smith (1971a) some mycoplasmas are capable of esterifying cholesterol with short-chain fatty acids, like butyrate. Mycoplasma gallinarum strain J is also capable of glycosylating cholesterol to cholesteryl glucoside (Smith, 1971b). Rodwell (1963),Argaman and Razin (1965) and Razin and Shafer (1969), however, found that labelled cholesterol incorporated by growing mycoplasmas of different species was neither esterified nor changed in any other way. When examining the binding of cholesterol to isolated mycoplasma. membranes in a buffer system containing cholesterol and Tween $0 (8. Razin, N. Gershfeld and M. Wormser, unpublished data), cholesterol appeared to be incorporated into the lipid region. Lipid-depleted membranes bind much less cholesterol than native membranes, and the

47

PHYSIOLOGY O F MYCOPLASMAS

kinetics of cholesterol uptake and washout are comparable with those obtained using polystyrene particles (Pig. 8), so that here most of the cholesterol is adsorbed to the surface rather than incorporated into the interior of the membrane. The fact that mycoplasma cells are lysed by digitonin (Rottem and Razin, 1972a) and filipin (Weber and Kinsky, 1965), compounds that are known to complex with cholesterol, also indicates that cholesterol forms an integral part of the membrane lipid region. This does not mean, however, that all the cholesterol is necessarily bound to the membrane 700 600

500 400

300

ted membranes

200

epleted membranes

I00 OO

I

2

3

4

5 0

I

2

3

4

5

lncubotion time ( h )

FIG.8. Uptake and washout of cholesterol from polystyrene particles and from native or lipid-depleted membranes of Acholeplasma laidlawii. Membranes (50 pg protein/ml) or polystyrene particles (0.02% suspension of 0.3 pm diameter particles) were incubated with labelled cholesterol (pM)and 0.01% Tween 80 in 50 mM phosphate buffer, pH 7.0 a t 37°C. Aliquots were withdrawn, centrifuged and the sediments were washed once in buffer. Washout was tested by suspending the membranes or particles which had bound cholesterol in the same reaction mixture as above but with unlabelled cholesterol. Unpublished data of S. Razin, N. Gershfeld and M. Wormser.

in the same way. Our experiments (S. Razin, N. Gershfeld and M. Wormser, unpublished data) show that the cholesterol bound to isolated A . laidlawii membranes in buffer is washed away faster than the cholesterol bound to them during growth. Smith (1971b) also noted that only 5% of exogenously added cholesterol was glycosylated by membranes from M . gallinarum, compared with a much higher proportion of endogenous cholesterol bound during growth. The idea that biological membranes have two cholesterol pools, advanced by Bell and Schwartz (1971) on the basis of their work with erythrocyte membranes, therefore deserves serious consideration. The role of cholesterol in biological membranes has been the subject of much investigation. Prom recent studies it appears to control the

48

SHMUEL RAZIN

fluidity of the hydrocarbon chains of the membrane polar lipids by disrupting the crystalline chain lattice of the gel phase and inhibiting the flexing of the chains in the dispersed liquid crystalline phase. There is strong evidence of a dual effect on phospholipids, depending on their fatty-acid constituents (De Gier et al., 1969). Thus, cholesterol increased the mobility of the hydrocarbon chains of dipalmitoyl lecithin, but decreased their mobility in egg lecithin, which contains oleic acid. Its condensing effect on phospholipids containing mono-enoic fatty acids and interference with crystallization in phospholipids containing saturated fatty acids have been confirmed by measurements of the surface area and permeability of phospholipid monolayers (Demel, 1968; Chapman et al., 1969), bilayers (Rand and Luzzati, 1968) and liposomes (Demel et al., 1972; McElhaney et al., 1970; De Kruiff et al., 1972); by X-ray diffraction studies of fatty-acid mobility in bilayers and biological membranes (Levine and Wilkins, 1971) and by electron paramagnetic resonance spectra of spin-labelled fatty acids (Hubbell et al., 1970; Butler et al., 1970). All of these studies point to the biological function of cholesterol in membranes being related t o its ability to produce the preferred orientation of the lipid in a bilayer structure so that the long axes of the lipids are perpendicular to the lamellar plane. The recent work of Butler et al. (1970) indicates that, for the sterols to exercise their ordering effect on membrane lipids, they must have a planar steroid nucleus and single hydroxyl group in the 3P-position (see also Oldfield and Chapman, I 971 ; Demel et al., 1972; De Kruiff et al., 1972). The ordering effect is enhanced by the presence of a hydrocarbon chain a t C-17 of the steroid nucleus. A planar steroid nucleus, a free hydroxyl group a t the 3P-position, and a hydrocarbon side chain are precisely the properties common to all sterols capable of supporting mycoplasma growth (Smith, 1971a). Why are sterols required for growth of Mycoplasma and not for growth of Acholeplasma and bacteria? A possible answer may be derived from a paper by Oldfield and Chapman (1971). They claim that the mechanism controlling the fluidity of the lipid region in the membrane must be linked to the turnover of the lipid. Thus in myelin, where the turnover is low, long-term stability of lipid fluidity is required so that the relatively saturated lipids are kept fluid by the presence of cholesterol. Following this line of thought, it is quite conceivable that the sterolrequiring mycoplasmas, whose dependence on fatty acids provided by the host and slow, if not non-existent, turnover of polar lipid (see pp. 40 and 42) restricts their ability to change and regulate the fluidity of the lipid region in their membrane, overcome this obstacle by the incorporation of varying amounts of cholesterol into the lipid region. Bacteria which are able to synthesize fatty acids and have a

49

PHYSIOLOGY O F MYCOPLASMAS

rapid lipid turnover can presumably regulate fluidity without cholesterol. The ability of Acholeplasma strains t o grow without cholesterol may likewise be associated with their capacity to synthesize saturated longchain fatty acids. Acholeplasma laidlawii and A. granularum strains synthesize carotenoids which resemble cholesterol in their planar hydrocarbon structure (Smith, 1971a) and may consequently simulate cholesterol in their effect on the fluidity of lipid bilayers. Acholeplasma carotenoids were indeed able to replace cholesterol in the growth of a couple of sterol-requiring mycoplasmas (Henrikson and Smith, I966 ; Smith, 1988). However, as there seems to be a far smaller amount of carotenoids in Acholeplasma membranes than of cholesterol in the membranes of sterol-requiring mycoplasmas (Razin, 1969a), it has been suggested that they play a catalytic rather than a structural role, such as the protection of Acholeplasma membrane enzymes against photodynamic destruction (Rottem et al., 1968a). Acholeplasma axanthum does not require cholesterol for growth and also does not synthesize carotenoids (Tully and Razin, 1969). Here the unique sphingolipid synthesized by this organism may be the regulator of membrme fluidity. The composition of the hydrocarbon chains in this lipid has in fact been shown t o be under some type of control, which appears to be associated with maintaining the right degree of membrane fluidity (Plackett et al., 1970). If cholesterol functions only in the regulation of the physical state of the lipid region in the membrane, then one should be able to replace the need for it by an appropriate mixture of fatty acids which provides the right degree of fluidity to the membrane. This has not been accomplished so far, but preliminary experiments (8. Rottem, unpublished data) show that N . mycoides var. Capri can be adapted t o grow in the presence of very low concentrations of cholesterol. Analysis of the fatty acids of the adapted strain showed a most significant increase in the percentage of the saturated palmitic acid, indicating that the adaptation to grow without cholesterol was associated in some way with the development of a mechanism by which this organism is able to incorporate higher amounts of palmitic acid from the medium and thus regulate the fluidity in its membrane.

E. ORGANIZATION OF PROTEIN AND LIPID IN

THE

MEMBRANE

1. Evidence for a Lipid Bilayer Carstensen et al. (1971) have shown the membranes of A. laidlawii and M . gallisepticum to have the same capacitance value (0.9 pF/cm3) although, in thin sections, the M . gallisepticum membrane was about 20-40 A thicker. The similar capacitance values of the membranes suggest

50

SHMUEL RAZIN

that both organisms are surrounded by an insulation barrier of about the same thickness, and that the difference in membrane thickness probably reflects a membrane ultrastructure other than the insulating dielectric layer. This is consistent with the insulation barrier in both membranes consisting of a lipid bilayer, and protein or other material bound to the surfaces of the lipid bilayer being responsible for the difference in membrane thickness. Substantiation of a bilayer configuration of the lipid in mycoplasma membranes has been forthcoming from differential scanning calorimetry, X-ray diffraction, and electron paramagnetic resonance spectra of spinlabelled fatty acids incorporated into the membranes. Steim et al. (1969), Reinert and Steim (1970) and Melchior et al. (1970) have shown, by differential calorimetry of viable cells of A . laaidlawii, and of isolated membranes, and aqueous dispersions of membrane lipids, that the endothermic phase transition of the lipid from a crystalline to a liquidcrystalline state occurred over the same temperature range in all three materials. The transition temperatures were lowered upon increased unsaturation of the fatty-acid residues but were throughout the same for membranes and isolated lipids (Pig. 9). The identical transition temperatures of the lipid in membranes and water suggest that, in the membrane, the lipid forms a bilayer in which the hydrocarbon chains associate with each other rather than with protein (Steim et ul., 1969).The ratio of the transition enthalpy per gram of lipid in membranes to that of the isolated lipid was found to be 0.9 & 0.1, suggesting that 90 5 10% of the lipid in the membrane is in the bilayer conformation (Reinert and Steim, 1970). The constancy of the lipid transition temperature upon removal of membrane proteins by pronase digestion (Melchior et ul., 1970) also indicates that the bulk of the lipid in the membrane is free rather than associated with protein. Apolar binding of lipids to protein via fatty-acid hydrocarbon chains perturbs or abolishes transition as will cholesterol in phospholipid dispersions (Steim et al., 1969). Chapman and Urbina (1971) call for caution in the interpretation of calorimetry data. They showed that the different lipid classes of membranes from A . luid&~,wii differ considerably in their heats of transition, glycolipids having almost four times the transition enthalpy value of phospholipids. They contend that, if all glycolipids were arranged together in the membrane, their thermal transition alone would account for the heat of transition measured by Reinert and Steim (1970)for whole membranes. This would leave the remaining over 60% of the membrane lipids organized differently, so as to allow lipid-lipid and lipid-protein interactions. They also noted that the transition temperature of phospholipids shifted to about 10°C below that of the lipids alone upon

PHYSIOLOGY OF MYCOPLASMAS

51

electrostatic interaction with the basic cytochrome c. Since electrostatic interactions between lipid and protein can thus affect the gel-liquid crystal transition of membrane lipids (see also p. 52) the similarity in the transition temperature and enthalpy change of total lipids and intact membranes is not conclusive proof that all of the (i.e. 9 0 & 10%) lipids in the membrane are in a n extended bilayer configuration nor is it

f

,:I

4

, , ,

, ,

1

I

I

necessarily consistent with electrostatic linkage of lipid and protein, i.e. the Danielli-Davson membrane model. Engelman's (1971) X-ray diffraction data again tend to confirm the lipid bilayer structure. I n membranes from A. laidlawii enriched to the extent of 60-70% with palmitate, oleate or erucate, he found almost the same phase transition temperature as was demonstrated by differential scanning calorimetry. If close hexagonal packing of the fatty acids is responsible for the sharp 4-15 A diffraction band seen below the phase transition, a change in average chain length ought to be observable in the peak-to-peak separation, since the fatty acids seem to lie practically perpendicular to the plane of the membrane. And indeed, in membranes enriched with erucate (Czo)the peak-to-peak distance was 5 A 4

52

SHMUEL RAZIN

greater than in palmitate (C,,)-enriched membranes, 2 A less than the theoretical value. Fatty acids into which a nitroxide radical is introduced at various positions of the hydrocarbon chain have paramagnetic properties. The following is the general formula for a spin-labelled fatty acid:

’

CH, (CHJm-C-(CH,),COOH

O

W

N

I

‘0

I

The electron paramagnetic resonance spectra of such spin-labelledfattyacid derivatives introduced into membrane lipids provide further insights into the physical state of the lipid region. They may be administered in vitro as free fatty acids by exchange from bovine serum albumin (Rottem et al., 1970), or enzymically incorporated into membrane polar lipids by incubating them with the organisms (Tourtellotte et al., 1970). The preferred orientation of the spin-labelled fatty acid is perpendicular to the plane of the membrane (Hubbell and McConnell, 1969) when the freedom of motion of its hydrocarbon chain can be estimated by measuring the hyperfine splitting (2Tm) of the electron paramagnetic resonance spectrum caused by the swinging motion of the nitroxide radical. Both Rottem et al. (1970) and Tourtellotte et al. (1970) showed that the freedom of motion of the spin label was higher in membranes from A . laidlawii enriched with oleic acid than when enriched with stearic, palmitic or elaidic acid, indicating that most of the label was in a semiviscous hydrocarbon environment, most probably a lipid bilayer (Fig. 10). Heat denaturation and glutaraldehyde fixation of the membranes did not affect the mobility of the spin label, again indicating that it is largely determined by lipid-lipid and not by lipid-protein interactions. Nevertheless, the spin label was throughout slightly but significantly less mobile in the intact membrane than in a dispersion of lipids from A . laidlawii. Tourtellotte et al. (1970)thought that this might be due to the weak interaction of the hydrocarbon chains with the particulate components which freeze-etching has shown to be immersed in the central region of the membrane (see P. 60). Rottem et al. (1970) however showed a similar restriction in mobility in reconstituted membranes from A . laidlawii which do not have particles (Tillack et al., 1970a). More recently Rottem and Samuni (1973)have found that the mobility of the spin label in membranes from A . laidlawii and M . hominis increased significantlywhen membrane protein was digested by pronase. The binding of cytochrome c or lysozyme to the pronase-digested membranes caused spin-label mobility to decrease to the value of native

PHYSIOLOGY OF MYCOPLASMAS

53

untreated membranes. The changes were almost completely reversed when the soluble proteins were removed by M-NaCI. It accordingly seems that proteins bound electrostatically to the polar heads of the lipids on the membrane surface still decrease the mobility of the lipid hydrocarbon chains. This, however, does not rule out the possibility that the positively charged proteins have some hydrophobic regions in their molecules which do not participate in their binding to the membrane, but do affect the mobility of the hydrocarbon chains.

-

FIG.10. Electron paramagnetic resonance spectra of N-oxyl-4’,4’-dimethyloxazolidine derivative of 5-ketostearic acid (spin label I (12, 3)) in membranes from Acholeplasma Eaidlawii derived from cells grown in medium containing tryptose with added oleate (A) or palmitate (B). The smaller hyperfine splitting (2Tm) in the oleate-enriched membranes indicates a higher freedom of motion of the spinlabelled fatty acid. From unpublished data of s. Rottem.

It should be noted that, since the distribution of the spin-labelled fatty acids in the membrane is unknown, the nitroxide probe may be systematically excluded from other environments in which unlabelled lipids participate. This applies particularly to the introduction of the probe as a free fatty acid (Rottem et al., 1970), but even when it is incorporated into the polar lipids, there is no proof of its random distribution in all polar lipid types. Therefore, the extent of the assumed bilayer structure in the membrane cannot be definitely assessed from the available spin-label data. 2. Localization and Conformation of Membrane Proteins While the membrane lipids appear to be largely organized in a bilayer, much less is known about the organization of the proteins, although they make up a far bigger proportion of the membrane. There are some good

54

SHMUEL RAZIN

indications that, in mycoplasma membranes, most of them occupy a surface position. Thus, membranes from A . laidlawii from which over 95% of the lipid was removed with acetone preserved their typical trilaminar structure. Removal of over SO% of the membrane proteins by pronase digestion was, however, accompanied by a decrease in thickness and contrast and the disappearance of most of the trilaminar structure (Morowitz and Terry, 1969). From the release of only about 80% of the protein from membranes of A. laidlawii following pronase digestion (Morowitz and Terry, 1969; I. Kahane, Z . Ne’eman and S. Razin, unpublished data), it appears that Some protein species are wholly or partly buried within the lipophilic interior of the membrane so as t o be inaccessible to proteolytic attack. This is supported by the finding that almost all of the proteins in lipiddepleted mycoplasma membranes are sensitive to pronase (Morowitz and Terry, 1969;Rottemand Samuni, 1973).Furthermore, when mycoplasma by a lactoperoxidasemembrane proteins were iodinated with 1251 dependent process which labels only surface proteins, the pronaseresistant protein residue had a much lower specific label than the total membrane protein (Rottem and Razin, 1972b). The amino-acid composition of the pronase-resistant protein residue is similar to that of the total membrane protein, and there is no enrichment in hydrophobic amino acids as there should be if it contained hydrophobic “tails” of proteins buried in a lipid interior. It may accordingly constitute, or form part of, the particles that cover 7-20% of the convex fracture faces of mycoplasma membranes, which are apparently immersed within the lipid bilayer (Tourtellotte et al., 1970).The presence of particles on the fracture faces of pronase-digested membranes from A . laidlawii seems to support this idea (I. Kahane, Z. Ne’eman and S. Razin, unpublished data). The selective disappearance of the higher molecular-weight proteins when membranes from A . laidlawii were digested with very low concentrations of pronase was recorded by Morowitz and Terry (1969). The membrane ATPase was inactivated by pronase much faster than the membrane p-nitrophenyl phosphatase (I. Kahaiie, Z. Ne’eman and S. Razin, unpublished data). This speaks in favour of the differential location of the protein species within the membrane, some high molecularweight proteins like ATPase lying more towards the exterior. Although the bulk of the protein appears to be on the mernbrarip surface, recent data obtained with mycoplasma and other biological membranes indicate that it does not form a continuous layer, leaving some of the polar heads of the lipids exposed on the membrane surface. This is borne out by the agglutination of cells of 111.p,neurnonicrPtoa high titre by a specific antiserum to membrane glycolipids (Razin rt nl..

PHYSIOLOGY OF MYCOPLASMAS

55

1970a). The electrostatic binding of considerable quantities of cytochrome c , lysozyme (S. Rottem, M. Hasin and S. Razin, unpublished data) and divalent cations (I. Kahane, Z. Ne’eman and S. Razin, unpublished data) to the lipid component of membranes from A . laidlawii likewise points to a considerable portion of the polar groups of the lipids being exposed. A highly schematic and tentative representation of the protein and lipid organization in the mycoplasma membrane is given in Fig. 11. The use of optical techniques based on determining the circular dichroism and optical-rotatory dispersion spectra of membranes in investigating the conformation of their proteins has gained considerable popularity. The circular dichroism and optical-rotatory dispersion spectra of membranes from A. laidlawii closely resemble those of other biological membranes (Choules and Bjorklund, 1970; Rottem and

LIPID{ POLAR HEAD-

FIG.11. A highly schematic representation of the possible organization of proteins and lipids in the mycoplasma membrane. Taken from Razin (197213).

Hayflick, 1973). Computer analysis of the circular dichroism spectrum of membranes from A. laidlawii has led Choules and Bjorklund (1970) to suggest that the proteins have 56% of /3-structure, 30.7% a-helix and 13.2% random coil. Whereas this a-helix value roughly corresponds to the vaIues obtained with other membranes (Wallach and Zahler, 1966)the value for the /3-structure is much higher and would lead one to expect a high proportion of fibrous proteins. The circular dichroism and optical-rotatory dispersion spectra of various biological membranes grossly resemble those of an a-helix, but with characteristic distortions, a low amplitude of molar ellipticities and molar rotations and red shifts of the negative extrema (Wallach and Zahler, 1966). Whether the distortions are due to the interaction of membrane protein with lipid or to an artifact caused by light scattering of the membrane suspension is still a controversial issue (Urry et aZ., 1971). A recent study by Rottem and Hayflick (1973) seems to weight the scale in favour of light scattering. With clear solutions of membranes of A. laidlawii solubilized with sodium dodecyl sulphate, undistorted

56

SHMUEL RAZIN

a-helix spectra were obtained. The characteristic red-shift distortion re-appeared on re-aggregation of the solubilized membrane material. Their finding that the circular dichroism spectrum of membranes from A . laidlawii is unaffected by the removal of over 90% of membrane lipids is a further indication that lipid-protein interactions have n o or little effect on the secondary structure of membrane proteins, and are thus not responsible for the spectral distortions.

3. Co-ordination of Nembrane Protein and Lipid Xynthesis Although the presence of only one membrane makes the mycoplasmas convenient objects for the study of membrane biosynthesis, not much seems to have been done in this direction. Kahane and Razin (1969b) concerned themselves with the question whether membrane lipid synthesis is synchronized or co-orclinated with membrane protein synthesis. Membrane lipid synthesis in A . laidlawii was found to continue for over 4 h after protein synthesis was stopped by chloramphenicol. The membranes of clilorampheiiicol-treated cells had a significantly lower buoyant density than membranes of untreated cells, indicating a higher lipid-to-protein ratio, but were still physiologically active, the cells bounded by them being alive and multiplying on removal of the drug. Further studies (Kahane and Razin, 2970) have shown that considerable variations occur in the membrane lipidto-protein ratio also under physiological conditions, without inhibitors. Thus membranes of A . laidlawii grown a t a high pH value had a much lower lipid-to-protein ratio than membranes of cells grown a t a low pH value. Membrane lipid synthesis may accordingly proceed independently of membrane protein synthesis, and the rates of synthesis may vary considerably under different growth conditions. Hence it seems that membrane composition is not very stringently controlled, and that a stoicheiometric relationship between protein and lipid is not necessary for membrane function. These findings also argue against proteins being incorporated into the membrane as lipoprotein subunits. Are membrane lipids synthesized throughout the membrane during growth and replication or a t the presumed point of attachment of the chromosome to the membrane? To answer this question, Rodwell et al. (1972) grew goat strain Y with its membrane lipids labelled with 3H-glycerol. Aliquots of the culture were withdrawn in the exponential phase of growth and mixed with unlabelled glycerol and 14C-palmitate, when the newly synthesized lipids should be recognizable by their high I4C : 3H ratio. There was no significant difference between the 3H:I4C ratio of the lipids of the membrane-DNA complex isolated by the sodium laurylsarcosinate technique (Tremblay et al., 1969) and the rest

PHYSIOLOGY O F MYCOPLASMAS

57

of the membrane lipids. These negative results may be explained by the rapid lateral diffusion of the newly synthesized lipids in the membrane, a phenomenon recently described for bilayers of phospholipids (Kornberg and McConnell, 1971).

F. RECONSTITUTION OF MEMBRANES Membrane reconstitution offers a most promising approach to elucidating the molecular organization of the protein and lipid in biological membranes, the nature of the building blocks participating in membrane assembly, and the type of bonds holding them together. Since a review on membrane reconstitution has just appeared elsewhere (Razin, 1972b), I shall confine myself to the fairly extensive use of mycoplasma membranes for this purpose.

1. Membrane Xolubilixation and Solubilization Products For reconstitution studies t o be meaningful, the membranes must first be properly solubilized. As long as any fragments retaining the basic membrane structure remain in the solubilized preparation, neither disaggregation nor reconstitution experiments can be meaningful. This rules out sonication, because the minute fragments into which the membrane is broken up by this technique still have the typical trilaminar structure (Rosenberg and McIntosh, 1968). Organic solvents, or detergents, are therefore used, particularly sodium dodecyl sulphate which solubilizes mycoplasma membranes more effectively than bile salts and non-ionic detergents, though it also does more harm to their enzyme activities (p. 33). The initial claim, based on analytical ultraeentrifugation, that a solution of mycoplasma membranes in sodium dodecyl sulphate consists of homogeneous lipoprotein subunits was withdrawn when the protein proved to be separable from the lipid by prolonged centrifugation on sucrose density gradients (Engelman et al., 1967; Rodwell et al., 1967 ; Razin and Barash, 1969), electrophoresis in polyacrylamide gels (Rottem et al., 1968b) or filtration through Sepharose or Sephadex columns equilibrated with the detergent (Ne’eman et al., 1972). Hence the solution appears to consist of complexes of protein and sodium dodecyl sulphate and micelles of lipid with this detergent which are inseparable in the analytical ultracentrifuge. 2. Re-aggregation of Solubilized Membrane Components

It seems obvious that the solubilizing agent has t o be removed for the solubilized membrane components to re-associate. The complete removal of detergents, which is usually extremely difficult, is not

58

SHMUEL RAZIN

necessary. Reconstitution may be induced by merely lowering the detergent concentration below a certain value by simple dilution, by filtration through Sephadex columns, or by dialysis, provided divalent cations are also supplied (Pig. 12). Dialysis against a buffer containing a divalent cation can pIay the double role of diluting the detergent and slowly adding the required cation (Razin et al., 1965). The requirement for a divalent cation for the reconstitution of mycoplasma membranes solubilized by sodium dodecyl sulphate, first noted by Razin et al. (1965), has since been not only confirmed (Terry I

Concentrotion of magnesium chloride in diolysis buffer ( m M )

FIG.12. Effect of Mg2+ concentration on the re-aggregation of protein and lipid of solubilized membranes from Mycoplasrna mycoides. The membranes containing labelled lipid were solubilized by 10 m M sodium dodecyl sulphate and dialysed against dilute buffer containing various concentrations of Mg2+ for 3 days a t 4°C. The ratio of labelled lipid to protein (expressed as radioactivity per mg protein) decreases as the Mg2+ concentration is increased. Taken from Razin et al. (1969).

et al., 1967; Rottem et al., 1968b; Engelman and Morowitz, 1968a, b ; Razin et al., 1969; Smith et al., 1969) but also extended to a variety of other biomembranes (Razin, 1972b). Of the divalent and trivalent cations effective in reconstitution, Mg2+ appears to be the best as, unlike most of the others particularly Ca2+, it does not precipitate sodium dodecyl sulphate in the dialysis bag. Moreover, although with trivalent cations and Ca2+faster re-aggregation is achieved at a lower concentration, the resultant aggregates are frequently amorphous (Butler et al., 1967). Is the Mg2+incorporated into the reconstituted membranes? According to Engelman and Morowitz (1968b), native and reconstituted membranes from A . laidlawii have about the same Mg2+ concentration (1.2-1.4 pg Mg2+ per mg protein). A more recent study (I.Kahane,

PHYSIOLOGY O F MYCOPLASMAS

59

Z. Ne’eman and S. Razin, unpublished data) shows, however, that the Mg2+ concentration in the reconstituted membranes is much higher (about 11 pg Mg2+ per mg protein) though, in native membranes, it comes close to the value reported by Engelman and Morowitz (1968b). High values for Mg2+were also obtained for native membranes dialysed against the Mg2i--containing buffer used for reconstitution. Hence, isolated niembranes seem to contain sites for Mg2+binding that are not saturated during growth. Prolonged dialysis against 5mM-EDTA brought the Mg2+ content of the reconstituted membranes down to that of native EDTA-treated membranes (about 0.4 pg Mg2+ per mg protein). Thus, a fairly constant and small amount is unavailable for chelation and is apparently buried within the native and reconstituted membranes. This may be associated with the failure of EDTA to disaggregate reconstituted membranes (Rottem et al., 1968b). Why are divalent cations essential for reconstitution? It appears that they neutralize the negatively charged groups on membrane lipids and proteins which interfere by electrostatic repulsion with membrane re-assembly. This is supported by the smaller Mg2+requirement when the pH value of the system is low (Rottem et al., 1968b). The divalent cations may also contribute to the stability of the reconstituted membranes by inter- or intra-chain cross linking through protein carboxyl or lipid phosphate groups, possibly accounting for the relative stability of membranes reconstituted in the presence of divalent cations whereas membranes reconstituted at a high Na+ concentration disaggregate on washing in de-ionized water (Rottem et al., 1968b). The concentration of Mg2+ in the dialysis buffer has a marked effect on the lipid-to-protein ratio of the reconstituted membranes (Terry et al., 1967; Rottem et al., 1968b; Razin et al., 1969). At a low concentration of Mg2+ (5 mM), density-gradient analysis of the reconstituted material shows a “light” lipid-rich band (density = 1.140 g/cm3)which, a t zt higher concentration (10-20 mM-Mg2+),is usually replaced by one or two heavier bands with densities approximating those of the native membranes (about 1.170 g/cm3). Hence, the solubilized membrane lipid tends to re-aggregate at a lower Mg2+ concentration than the solubilized protein.

3. Ultrastructure of Reconstituted Membranes Only under fairly restricted conditions, both of initial detergent Concentration and of subsequent exposure to divalent cations, do the solubilized membrane components interact to produce material which resembles the original membrane in morphology and density. Globules of concentric lamellae and amorphous clumps frequently accompany

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the trilaminar membranous structures (Terry et al., 1967; Cole et al., 1971). The myelin-like globules consist of re-aggregated membrane lipid and the amorphous clumps of re-aggregated membrane proteins (Cole et al., 1971). The typical trilaminar membranes, having the same thickness as the native membrane, contain both protein and lipid. Vesicle formation is obviously essential for the reconstitution of transport mechanisms or for oxidative phosphorylation (see Razin, 1972b) but so far the vesicularization of reconstituted membranes cannot be fully controlled nor are the factors governing it properly understood. Time seems to be one of them. Reconstituted membrane profiles of A . luidluwii appearing after short dialysis periods varied in length and had free ends. Only after several hours did the membranous sheets fold to vesicles with almost quantitative vesicularization being obtained after 7 2 h (Razin et ul., 1969). Do the reconstituted membranes have the same ultrastructure as native membranes? The results of thin sectioning tend to be in the affirmative, but freeze-etching has revealed some differences (Fig. 13). Both the convex and concave fracture faces of reconstituted membranes from A . laidluwii were relatively smooth, like those of myelin (Branton, 1967) or lipid bilayers (Staehelin, 1968), lacking the characteristic

FIG.13 A

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FIG.13 B FIG.13. Freeze-etched replicas of native (A) and reconstituted (B)membranes of Acholeplasma lazdlawii. The convex fracture face of the native membrane (IF) contains globular particles, while the corresponding fracture face of the recoiistituted membrane is relatively smooth. Taken from Tillack et al. (1970a).

particles of the native membranes (Tillack et al., 1970a; Tourtellotte et al., 1970). The chemical nature of the particles is still unknown, but there is strong evidence that they contain protein (Branton, 1969; Tillack et al., 1970b). Probe techniques and X-ray diffraction, recently applied to comparative studies of native and reconstituted membranes, provide more stringent tests of structural organization than electron microscopy analysis. Electron paramagnetic resonance spectra of spin-labelled fatty acid derivatives incorporated into native and reconstituted membranes from A . laidlawii corroborate the bilayer structure of the lipids in both

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(Rottem et ul., 1970).From X-ray diffraction data it also appears that the lipid bilayer is restored (Metcalfe et al., 1971a) but nuclear magnetic relaxation measurements with benzyl alcohol as the probe, and fluorescence measurements with 1-anilinonaphthalene-8-sulphonateas the probe, indicate that the original organization of the membrane proteins is not fully reconstituted (Metcalfe et ul., 1971a, b). The retention of a considerable number of abnormal protein-binding sites in reconstituted membranes prepared from material solubilized with sodium dodecyl sulphate indicates that either the conformation of the proteins is not restored to the native state or that some specific interaction with the lipids is missing. Treatment with proteolytic and lipolytic enzymes may also offer some clue to the organization of the proteins and lipids in the membmne. Reconstituted membrane proteins from A . laidluwii were more sensitive to pronase digestion than proteins of the native membrane. When treated with 200 pg pronase/ml for 20 h a t 50"C, 94% of the total protein of reconstituted membranes was digested as against only 83% of native membrane proteins (I. Kahane, Z. Ne'eman and S. Razin, unpublished data). This may be taken as an indication that more protein is exposed on the surfaces of reconstituted than of native membranes.

4 . Specificity of the Reconstitution Phenomenon Can proteins or lipids of one membrane be incorporated into a membrane of a different type'! I n vivo the incorporation of membrane proteins and lipids is specific to each membrane type, as evidenced by its characteristic chemical composition. Is this specificity controlled by factors that operate only during membrane synthesis or is there some control mechanism inherent in the membrane components? Incorporation of the different membrane proteins and lipids into reconstituted membranes from A . laidluwii membranes was shown to be non-selective (Kahane and Razin, 1971) so that there appears to be no strict specificity. But, as the material was solubilized by sodium dodecyl sulphate, which may cause radical conformational changes in membrane proteins (Reynolds and Tanford, 1970), the results are not conclusive. The non-specific reconstitution of sodium dodecyl sulphate-solubilized membrane proteins and lipids is amply demonstrated by the formation of hybrid membranes from a mixture of membrane proteins and lipids of different organisms. Dialysis of a mixture of sodium dodecyl sulphatesolubilized membranes from A . laidlawii and M . gallisepticum against Mg" produced an aggregate with a density intermediate between that of reconstituted membranes from A . laidluwii and M . gallisepticum. It consisted of trilaminar membranes as well as amorphous material, and

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contained the antigens of both membrane species (Razin and Kahane, 1969). The finding that reconstituted mycoplasma membranes retain their immunogenicity (Kahane and Razin, 1969a) has led to the use of hybrid membranes for the preparation of highly specific and potent antisera t o serologically-active membrane lipids (Razin et al., 1970a, 1971a, b). By themselves the lipids are unable to elicit an antibody response; hybrid membranes prepared from purified glycolipids of M . pneumoniae or cytolipin H from bovine spleen and membrane proteins of A . laidlawii were most effective in eliciting the production, in rabbits, of antibodies t o the lipid component. The high immunogenicity of the lipid hapteii when bound t o membrane proteins by reconstitution is apparently due to intimate binding, probably by the same kind of bonds as are responsible for the association of proteins and lipids in native membranes. The membranous nature of a considerable part of the hybrid material supports this assumption (Coleet al., 1971).The main advantage of using reconstituted hybrid material for the production of antibodies t o membrane lipids is that the lipid and protein components can be selected so that the type of antibodies produced is controllable. An intriguing question is whether non-membranous proteins, such as albumin, can be incorporated into reconstituted membranes. From the still scanty data it appears that soluble non-membranous proteins are either not incorporated a t all or in much smaller amounts than membrane proteins. Bovine serum albumin added t o sodium dodecyl sulphate-solubilized membranes from A . laidlawii bound t o the reconstituted membranes only up t o 0.5% of the total protein in the reconstituted membrane (Rottem et al., 1971a).On the other hand, appreciable quantities of the highly basic proteins lysozyme and cytochrome c did bind electrostatically to membrane phospholipids (S. Rottem, M. Hasin and S. Razin, unpublished data). The reconstitution technique has proved useful in studying the effects of environmental changes on the properties of the soluble penicillinase of Bacillus cerew.9 (Rottem et al., 1971a). When added to the reconstitution mixture of solubilized membranes from A . laidlawii, appreciable amounts of the enzyme were bound to the reconstituted material. About two-thirds were obviously bound electrostatically as they could be removed by washing with M-NaC1. As the rest (X--10 pg penicillinase per mg membrane protein) could not so be released, it was apparently bound by hydrophobic, or by a combination of hydrophobic and electrostatic, bonds. That the tightly bound penicillinase could be eluted with M-NaC1 after extraction of the lipid from the reconstituted membranes shows that i t was embedded in a largely hydrophobic environment, a constraint likely to modify its conformation.

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I n effect, the bound penicillinase was much more resistant to iodination in the presence of methicillin and more sensitive to thermal inactivation than the soluble native enzyme (Rottem et aZ., 1971a).

5 . Possible Mechanisms for Membrane Reconstitution The findings leading to the rejection of the lipoprotein subunit hypothesis (see pp. 57 and 59) support the alternative hypothesis (Razin et al., 1969; Kahane and Razin, 1971) that the reconstituted mycoplasma membranes are built by a multi-step assembly process. First, a lipid-rich membrane is formed on which more protein is successively bound, depending on the Mg2+ concentration. The primary membrane is accordingly built of a bimolecular lipid leaflet coated on both sides with protein, according to the classical model of Danielli and Davson. Corroborating evidence is the absence of particles on the fracture faces of the reconstituted membranes from A . laidlawii (Fig. 13) which implies a resemblance to the myelin membrane whose ultrastructure seems to conform to the Danielli-Davson model (Eylar, 1970). Moreover, the solubilized membrane proteins, lacking S-S bonds (Auborn et al., 1971; Kahane, 197I ), lose most of their tertiary and quaternary structure on solubilization by sodium dodecyl sulphate (Reynolds and Tanford, 1970),so that the polypeptide chains unfold. Spread tangentially over the surfaces of the lipid bilayer, like the A, protein of myelin (Eylar, 1970), the open polypeptide chains fit in well with the role assigned to the proteins in the leaflet model. The exposure of the protein on the membrane surface may also explain its higher sensitivity to pronase digestion (p. 62). Mycoplasma membrane proteins have a net negative charge a t the neutral p H value a t which reconstitution takes place. Since the unfolding of the polypeptide chains by the detergent presumably exposes further ionizable groups, electrostatic repulsion is likely to interfere with the attachment of proteins to the negatively charged phosphate groups. Hence the need for Mg2+ or some other cation to neutralize the charge repulsions. Another mechanism for the reconstitution of sodium dodecyl sulphatesolubilized mycoplasma membranes was suggested by Engelman and Morowitz (1968a, b). They claim that when detergent-solubilized components of A . la~dlawiimembrane are dialysed against a buffer lacking divalent cations, small lipoprotein particles are formed which sediment in the analytical ultracentrifuge as a single peak of about 4.8 S and that, upon dialysis against a buffer containing Mg2+, they re-aggregate further into trilaminar membranes. The molecular weight of the particles was estimated a t 160,000 f 40,000, and their diameter at 80 & 8 d, approximating the thickness of the native membrane. No

PHYSIOLOGY OF MYCOPLASMAS

65

such particles could, however, be traced by electron microscopy and they are difficult t o reconcile with the varying lipid-to-protein ratio of reconstituted membranes formed with different Mg2+concentrations, especially as the Mg2+effect remained the same when sodium dodecyl sulphate was removed before reconstitution (Rottem et al., 1968b; Razin et al., 1969). Polyacrylamide-gel electrophoresis moreover separated the lipid from the protein after removal of sodium dodecyl sulphate by Sephadex G-25, so that it is unlikely that the material should consist of homogeneous lipoprotein particles (Rottem et al., 1968b). I n conclusion, it seems that there is no difficulty in restoring the bilayer configuration of membrane lipids because, in aqueous solution, it is energetically the most favourable. The problem is how to restore the original conformation of the proteins which is often irreversibly altered by solubilization, with inevitable effects on their molecular properties and biological activities. Thus membrane proteins from A . laidlawii, when solubilized by sodium dodecyl sulphate, lose most of their biological activities and reconstitute with membrane lipids to membranes that, unlike the native membranes, seem to have all, or almost all, of their protein on the surface. The gentler non-ionic detergents and bile salts, however, seem to enable many solubilized membrane proteins and lipoprotein complexes to retain their original conformation. By using these detergents it has proved possible to restore the activity of multi-enzyme systems in mitochondria1 membranes (Racker, 1970), and their substitution for sodium dodecyl sulphate may eventually lead to the reconstitution of biologically active mycoplasma membranes.

G. TRANSPORT MECHANISMS Thanks to the single cell membrane of mycoplasma and the ability of the experimenter to control its lipid composition, they are most suitable micro-organisms for transport studies. Active transport systems for sugars and amino acids have been described in several species (Razin et al., 1968; Rottem and Razin, 1969). Some of the mycoplasmas, including M . gallisepticum, M . mycoides var. mycoides and var. capri and the related goat strain Y, have the highly efficient phosphoenolpyruvate (PEP)-dependent phosphotransferase system found in bacteria (VanDemark and Plackett, 1972; V. P. Cirillo and S. Razin, unpublished data). As in E. coli, the PEP-phosphotransferase system of M . rnycoides var. capri depends on both cytoplasmic and membrane proteins for activity (V. P. Cirillo and S. Razin, unpublished data). Although the Acholeplasma species are fermentative and require a metabolizable sugar for growth (Razin and Cohen, 1963), they have

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no PEP-phosphotransferase system. Their sugar-transport mechanism, which is far less efficient than that of M . gallisepticurn or M . mycoides, is still obscure. L. F. Guymon and G. L. Choules (private communication) claim that A . laidlawii cells have a sugar-transport system which is highly specific for D-glucose but, as only small amounts of the nonmetabolizable glucose analogues were taken up by the cells, transport could not be uncoupled from assimilation, so that the interpretation of the data is difficult. The evidence for carrier-mediated transport and the kinetic data provided by Guymon and Choules might perhaps indicate that, in A . laidlawii, glucose is transported by facilitated diffusion. The systems responsible for the active transport of amino acids in M . fermentans and M . hominis generally behave like typical microbial permease systems, as evidenced by the transport kinetics and the marked accumulation of metabolites in the intracellular pool. They require energy for activity and are strongly inhibited by sulphydryl-blocking reagents (Razin et al., 1968). The discovery of these transport systems has shaken Smith’s hypothesis (1969c, 1971a) that cholesterol in mycoplasmas and carotenoids in acholeplasmas act as carriers of glucose and short-chain fatty acids through the cell membrane. Even if they do perform this function, and in many strains and under various growth conditions apparently they do not (Razin, 1969a),they are likely to be less effective considering the low biosynthetic efficiency of the cholesterol and carotenol glycosides and esters. Mycoplasmas also have transport systems for inorganic ions. The presence of an active transport system responsible for potassium accumulation in A. laidlawii was shown by Rottem and Razin (1966) and confirmed by Cho and Morowitz (1969). Potassium uptake is energydependent, and appears to be mediated by sulphydryl-containing proteins. As in other prokaryotes, but in contrast to animal cells, it is not aswciated with a (Na+-K+)-activated ATPase and is therefore not inhibited by ouabain (Rottem and Razin, 1966). The ability to control the composition of membrane lipids has been used to find out whether their physical state affects active transport. I n E . coli mutants with different fatty-acid compositions, a marked change in the rate of galactoside transport was noted at a temperature corresponding t o that of the phase transition of membrane lipids. Below the phase transition, when the lipids lose their liquid-crystalline state, transport was drastically decreased (Schairer and Overath, 1969). Attempts to show phase-transition effects on potassium uptake by A. laidlawii were less successful (Cho and Morowitz, 197%).No direct attempt was, however, made to alter the cornposition of the membrane lipids and the cells were merely grown a t different temperatures: nor

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were data given on lipid composition and thermal transition temperatures.

VII. Nutrition and Metabolism A. NUTRITIONAL REQUIREMENTS AND SYNTHETIC CAPABILITIES Definition of the nutritional requirements of a micro-organism may help in elucidating its biosynthetic pathways. The development of defined media for the growth of mycoplasmas has proved particularly difficult, not only because of their exacting nature but also because of their sensitivity to lysis by agents to which cell wall-enveloped bacteria are much more resistant. It was not until recently that two completely defined media were evolved, one for the goat Mycoplasma sp. strain Y (Rodwell, 1969) and the other for A . Euidlawii strain B (Tourtellotte, 1969). That so little research has been done in the past few years on mycoplasma nutrition may well be due to the tedious work involved in compounding complex synthetic media, and the recognition that many biosynthetic pathways can be elucidated in undefined media by the use of labelled precursors. 1. Lipid Requirements Perhaps the most distinctive nutritional requirements of mycoplasmas are lipids and lipid precursors for membrane synthesis. Here the main difficulty is to provide the lipid materials in an aqueous medium and in an assimilable,non-toxic form. Thus cholesterol,which is essential for the growth of most mycoplasmas (Razin and TuIly, 1970; Rottem et ul., 1971b; Edward, 1971), must either be supplied together with a carrier-a thermostable defatted serum fraction C (Rodwell, 1967) or a similar serum lipoprotein (Smith and Boughton, 1960)-or may be dispersed in Tween 80 and serum albumin (Razin and Tully, 1970). Since small quantities of unesterified fatty acids cause rapid lysis of mycoplasmas, i t is still more difficult to furnish an adequate supply of the long-chain fatty acids. They are included in various forms in the synthetic media devised, as listed by Rodwell (1969) or Smith (1971a). Unesterified fatty acids may be added together with serum albumin which acts as a buffer, binding them so as to liberate minute non-toxic quantities (Razin and Cohen, 1963; Rodwell, 1967). Serum albumin is, however, contaminated with considerable amounts of fatty acids, The commercial “fatty-acid poor” preparations still contain appreciable quantities. Mild alkaline methanolysis probably is the best way of removing these contaminants (Rodwell and Peterson, 1971). Tween 80 5

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may serve as a water-soluble less-toxic supply of oleic acid since it is slowly hydrolysed by mycoplasma lipase (Razin and Rottem, 1963 ; Rottem and Razin, 1964). Perhaps better still is TEM-4T (diacetyl tartaric acid ester of tallow monoglycerides) which contains a balanced mixture of saturated and unsaturated fatty acids in a non-toxic form found suitable for growth of the M . gallinarum strain J (Lund and Shorb, 1966) and the goat strain Y (Rodwell, 1969). TEM-4T has in its turn been replaced by synthetic homologues which, in addition to supplying the required fatty acids, also solubilize cholesterol, thus eliminating the need for a carrier protein and making the medium completely defined (Rodwell, 1969). As stated (p. 44) it is not enough to supply fatty acids in a nontoxic form; it is perhaps still more important to provide them in a balanced mixture (e.g. Rodwell, 1967, 1971 ; Rodwell and Peterson, 1971; Rottem and Panos, 1969). Since the correct fluidity of the lipid region in the membrane depends on the interplay between fatty acids and cholesterol (p. 48), this balance is evidently affected by the amount of cholesterol present in the medium. Though the replacement of cholesterol by a balanced mixture of fatty acids has not yet proved completely successful, the goat mycoplasma strain Y (Rodwell et al., 1972) and the related ill. mycoides var. Capri ( S . Rottem, unpublished data) could be adapted to grow with very little cholesterol when provided with a certain combination of fatty acids.

2. Amino -Acid Requirements Once a completely defined medium for the goat strain Y became available, its amino-acid requirements could be established. They were found to be absolute for all except glutamic and aspartic acids and cystine (glutamine, asparagine and cysteine were present in the medium; Rodwell, 1969), indicating an unusually low biosynthetic capability. Acholeplasma laidlawii, which also depends on an external supply of most amino acids (Razin and Cohen, 1963; Tourtellotte et al., 1964), was unable to incorporate aspartic and glutamic acids but only their amides (Tourtellotte, 1969). A limited capacity to synthesize amino acids has, however, been found in several mycoplasmas. In A. laidlawii, for instance, the shikimic acid pathway is active in the synthesis of aromatic acids (Tourtellotte, 1969), and the recent finding of glutamate dehydrogenase (Yarrison et al., 1972), an enzyme which serves as a link between nitrogen metabolism and carbohydrate metabolism, points to the ability to synthesize glutamate and probably also other amino acids from keto acids.

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3. Nucleic Acid Precursor Requirements Thanks to their nucleolytic activity, many mycoplasmas can use RNA and DNA as a source of nucleic acid precursors (Razin et ul., 1964; Stock and Gentry, 1969). The nucleic-acid precursor requirements of A. luidlawii (Razin, 1962; Smith and Hanawalt, 1968) indicate that this mycoplasma has no functional orotic acid pathway for pyrimidine synthesis and apparently lacks thymidylate synthetase. Unlike E. coli, it cannot convert thymine to thymidine monophosphate, but like E . coli it can produce uridine monophosphate, the direct precursor of all four pyrimidine triphosphates, from uridine and from cytosine (Smith and Hanawalt, 1968). By the enzymic methylation of deoxycytidine or deoxyuridine, with folinic acid as a cofactor, it is also capable of producing thymidine (Razin, 1962). Production of extracellular deoxyribonuclease by M . hominis suggested that a scavenger pathway may be used to supply thymidine nucleosides for DNA synthesis (Stock and Gentry, 1971). To verify this idea, the activity of several enzymes involved in the synthesis of thymidylate and its incorporation into DNA were assayed in fractions of M . hominis when both de novo and scavenger pathways for thymidylate synthesis were found. As with A . luidlawii, the inability to satisfy the thymidine requirement of M . hominis with cytidine points to a defect in folate metabolism.

4 . Vitumin Requirements Vitamin requirements have been determined for only a few strains. Acholeplaasmu ZuidZawii (Razin and Cohen, 1963 ; Tourtellotte et al., 1964), M . mycoides, and the goat strain Y (Rodwell, 1969) require nicotinic acid, thiamine, coenzyme A, and riboflavin, the last probably in connection with their flavin-terminated respiratory chain. Coenzyme A is required to activate and incorporate fatty acids into their membrane lipids and, in A . luidlawii,also to synthesize saturated fatty acid and carotenoids (Razin and Rottem, 1967; Rottem and Razin, 1967b). The coenzyme A requirement of A . luidlawii could be replaced by pantetheine but not by /I-alanine (S. Rottem, 0. Muhsam and S. Razin, unpublished data).

5. Urea Requirement A requirement for urea for growth and urease activity may be the main distinction between T-mycoplasmas and other sterol-requiring mycoplasmas. The role of urea in the growth of T-mycoplasmas is not yet clear. It seems rather doubtful that the ammonia liberated during urea hydrolysis has a beneficial effect since exogenous ammonia added to

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cultures of T-mycoplasmas proved highly toxic even when the pH value was kept constant (Ford and MacDonald, 1967; Rottem et al., 1971b). Since an increase in carbon dioxide tension did not replace the requirement for urea, and the use of I4C-urea showed no significant incorporation of labelled carbon by the cells (Ford et al., 1970),the carbon dioxide liberated during urea degradation a190 is an unlikely adjuvant. To find out whether the ammonia liberated during urea hydrolysis is incorporated into cell material, it may be necessary to use urea in which the nitrogen is labelled. Since the energy source of T-mycoplasmas is still unknown, there also is a remote possibility that the urea serves this purpose, remote because urea hydrolysis by known metabolic pathways does not yield energy. This enigma notwithstanding, further evidence has been forthcoming of the need for urea hydrolysis for growth of T-mycoplasmas (Ford and McCandlish, 1972).

B. RESPIRATORY PATHWAYS

AND

ENERGY-YIELDING MECEANTSMS

On the basis of their ability to produce acids from carbohydrates, the mycoplasmas may be classified into fermentative and non-fermentative strains. Sugars are fermented by the glycolytic pathway whose associated enzymic activities were detected in several fermentative strains (Castrejon-Diez et al., 1963; Tourtellotte and Jacobs, 1960; Rodwell, 1960).There is still very little evidence for the presence of the tricarboxylic acid cycle, except in M . arthritidis strain 07 (VanDemark and Smith, 1964a).More seems to be known about the electron-transport pathway. It appears that, in most mycoplasmas,the respiratory pathway is flavin-terminated, as cytochromes and catalase are absent (Weibull and Hammerberg, 1962; Rodwell, 1967 ; Low et al., 1968) and hydrogen peroxide accumulates in cultures of almost all known mycoplasmas (Cole et al., 196s). Spectrophotometric evidence for the presence of flavin in membranes from A . laidlawii was provided by Morowitz and Terry (1969). The respiratory pathway in these mycoplasmas may perhaps be represented by the following scheme : AH,

--f

NAD -+ Flavoprotein + 0,

‘The NADH, oxidase activity of M . mycoides (Rodwell, 1967) and A . laidlawii (Z. Ne’eman and S. Razin, unpublished data) decreases considerably on storage. It may be restored by sulphydryl-containing compounds, like cysteine or P-mercaptoethanol, and by the addition of flavin adenine dinucleotide, indicating the flavin requirement of the oxidase system. Ferricyanide, dichlorophenolindophenoland menadione, but not cytochrome G, can replace oxygen as hydrogen acceptors in M . mycoidees and A . laidlawii (Rodwell, 1967; Z. Ne’eman and S. Razin, unpublished data).

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PHYSIOLOGY OF MYCOPLASMAS

A more complex respiratory chain containing quinones and cytochromes as well as flavoproteins has been demonstrated in M . arthritidis strain 07 (VanDemark and Smith, 1964b): AH, + NAD + Flavoprotein -+ Quinones + Cytochrome b + Cytochrome a + a3 -+ 0,

According to unpublished data cited by VanDemark (1969),catalase, quinones, and cytochromes are also found in several, mostly unclassified, avian and bovine strains. These findings have not been followed up further, and it seems that the more fashionable molecular biology problems have put the respiratory systems in the shade so as to leave enormous gaps in our knowledge of this basic aspect of mycoplasma physiology. It is to be expected that mycoplasmas with a flavin-terminated respiratory pathway derive their energy from substrate-level phosphorylation, while those equipped with a complete respiratory chain derive theirs from oxidative phosphorylation. Again, only preliminary reports are available (VanDemark, 1969). It appears that oxidative phosphorylation, as measured by ATP formation during NADH, oxidation, takes place in M . arthritidis strain 07 and an unclassified bovine strain. The low P/O ratios indicate a relatively labile system, not unlike that of most bacteria. Oxidative phosphoryIation was blocked or diminished by known uncouplers. The type of oxidizablesubstrates utilized as an energy source obviously depends on whether or not the mycoplasmas possess the glycolytic cycle. The fermentive strains prefer a carbohydrate, and some were actually shown to depend on a metabolizable sugar for growth (Razin and Cohen, 1963; Rodwell, 1969).The nature of the substrates used by non-fermentative mycoplasmas is less clear. Mycoplasma arthritidis strain 07, the most extensively studied mycoplasma, is capable of reducing succinate, lactate and butryl-CoAby reactions not linked to nicotinamide nucleotides (VanDemark and Smith, 1965).There also is good evidence for the presence of the tricarboxylic acid cycle (VanDemark and Smith, 1964a) and another metabolic cycle for the oxidation of butyric acid to acetyl-CoA,which proceeds via the tricarboxylic acid cycle (VanDemark and Smith, 1965).Like many other mycoplasrnas, M . arthritidis also has the arginine dihydrolase pathway (Barile et al., 1966): arginine

arginine deiminase

citrulline +Pi

citrirllinc f NH3

P

ornithinc carbnmoyltrnnsferase ~

carbamoyl phosphate

~

+ ADP

ornithine

carbamoyl yhosphokinase ~

N@+

f

carbamoyl phosphate

ATP

+ NH, + CO,.

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SHMUEL RAZIN

This pathway is generally present in the non-fermentative species. Schimke et al. (1966) claimed that the conversion of arginine to ornithine with the concomitant formation of an equimolar amount of ATP is sufficient to account for all of the energy required for macromolecular synthesis in growing cells of M . arthritidis. The marked growth stimulation, by arginine, of mycoplasmas having this metabolic pathway argues in favour of this suggestion (Barile et al., 1966; Schimke et al., 1966; Razin et al., 1968). Schimke et al. (1966)further suggested that the arginine dihydrolase pathway helps the mycoplasmas to economize in genetic information, and the information for synthesizing the three enzymes that form this metabolic pathway is sufficient for the synthesis of the organism's entire ATP requirement. It is hard to accept this suggestion in view of the enzymic complexity of M . arthritidis, which is undoubtedly capable of using a variety of substrates as energy sources by a variety of metabolic pathways. VIII. Acknowledgements The preparation of this review was supported through the Special Foreign Currency Program of the National Library of Medicine, National Institutes of Health, Public Health Service, U.S. Department of Health, Education and VC'elfare, Bethesda, Maryland, under an agreement with the Israel Journal of Nedical Sciences, Jerusalem, Israel. Investigations originating from this laboratory were supported by grants from the U.S. Department of Agriculture (FG-Is-174 ; FG-Is-286) under Public Law 480, and from the Ford Foundation (5/B-5; 6/D-IV). The review was written between February and May 1972. I thank the authors who generously supplied illustrations and the results of unpublished work. My thanks are also due to Dr. S. Rottem for critical reading of the manuscript,

REFERENCES Abrams, A. and Baron, C. (1968). Biochemistry, N.Y. 7,501. Allan, D. andCrumpton, M. J. (1971). Biochem. J. 123,967. Allen, T.C. (1971). J. gen. Microbiol. 69, 285. Allen, T. C., Stevens, J. O., Florancc, E. R. andHampton, R. 0. (1970). J. Ultrastruct. Res. 33, 318. Ambron, R. T. and Pieringer, A. (1971). J. biol. Chem. 246,4216. Anderson, D.R. and Barile, M. F. (1965). J. Bact. 90, 180. Anderson, D. L., Pollock, M. E. and Brower, L. F. (1965).J. Bact. 90, 1764. Andrewes, C. H. and Welch, F. V. (1946). J. Path. Bact. 58, 578. Argaman, M.and Razin, S. (1965).J. gen. Microbiol. 38, 153.

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Ryan, J. L. and Morowitz, H. J. (1969). Proc. natn. Acad. Sci. U.S.A. 63,1282. Saglio, M. P., Laflhhe, D., Bonissol, C. and B o d , J. M. (1971).C. r. hebd. Sianc. Acad. Sci., Paris, 272, 1387. Schairer, H. and Overath, P. (1969).J. molec. Biol. 44, 209. Schimke, R . T., Berlin, C. M., Sweeney, E. W. and Carroll, W. R. (1966). J. biol. Chem. 241,2228. Schwartz, J. L. and Perlman, D. (1971).J. Antibiot. 24, 575. Senior, A. E. and MacLennan, D. H. (1970).J. biol. Chem. 245,5086. Shaw, N. (1970). Bact. Rev. 34, 365. Shaw, N., Smith, P. F. andKoostra, W. L. (1968). Biochem. J. 107, 329. Shaw, N., Smith, P. F. and Verheij, H. M. (1970). Biochem. J. 120,439. Slater, M. L. and Folsome, C. E. (1971).Nature, Lond. 229, 117. Smith, D. W. (1969).Biochim. biophys. Acta 179, 408. Smith, D. W. and Hanawalt, P. C. (1968).J. Bact. 96, 2066. Smith, D. W. and Hanawalt, P. C. (1969).J. mobec. Biol. 46, 57. Smith, K. 0. (1965).Science, N . Y . 148, 100. Smith,P. F. (1968).J. Bact. 95, 1718. Smith, P. F. (1969a).J. Bact. 99, 480. Smith, P. F. (196913).Bact. Proc. 121. Smith, P. F. (1969~). Lipids,4, 331. Smith, P. F. (1971a).“The Biology of Mycoplasmas”. New York: Academic Press. Smith, P. F. (1971b).J. Bact. 108, 986. Smith, P. F. and Boughton, J. E. (1960).J. Bact. 80, 851. Smith, P. F. and Henrikson, C. V. (1965).J. Lipid Res. 6, 106. Smith, P. F. andKoostra, W. L. (1967).J. Bact. 93, 1853. Smith, P. F., Koostra, W. L. andMayberry, W. R. (1969).J. Bact. 100, 1166. Smith, P. F. and Rothblat, G. H. (1960).J. Bact. 80,842. Staehelin, L. A. (1968).J. Ultrastruct. Res. 22, 326. Stead, A., Freeman, R. and Shaw, N. (1972).J.gen. Microbiol. 68. Steim, J. M., Tourtellotte, M. E., Reinert, J. C., McElhaney, R. N. and Rader, R. L. (1969).Proc. natn. Acad. Sci. U.S.A. 63, 104. Steinberg, P., Horswood, R. L. and Chanock, R . M. (1969). J. inject. Dis. 120, 217. Stock, D. A. and Gentry, G. A. (1969).J. Virol. 3, 313. Stock, D. A. and Gentry, G. A. (1971).J. gen. Microbiol. 65, 105. Swartzendruber, D. C., Clark, J. andMurphy, W. H. (1967). Bact. Proc. 151. Terry, T. M., Engelman, D. M. and Morowitz, H. J. (1967). Biochim. biophys. Acta 135, 391. Tillack, T. W., Carter, R. and Razin, S. (1970a). Biochim. biophys. Acta 219, 123. Tillack, T. W., Scott, R. E. and Marchesi, V. T. (1970b).J.Cell Biol.47,213a. Tourtellotte, M. E. (1969).I n “The Mycoplasmatales and the L-Phase of Bacteria” (L. Hayflick, ed.), pp. 451-468. New York: Appleton Century Crofts. Tourtellotte, M. E., Branton, D. and Keith, A. (1970). Proc. natn. Acad. Sci. U.S.A. 66, 909. Tourtellotte, M. E. and Jacobs, R . E. (1960).Ann. N.Y. Acad. Sci. 79, 521. Tourtellotte, M. E., Moromitz, H. J. and Kasimer, P. (1964).J. Bact. 8 8 , l l . Tourtellotte, M. E., Pollack, M. E. and Nalewaik, R. P. (1967). Ann. N . Y . Acacl. Sci. 143, 130. Tremblay, G. Y . ,Daniels, M. J. and Schaechter, M. (1969).J. molec. Biol. 40, 65. Tully, 5. G. and Razin, S. (1968).J. Bact. 95, 1504. Tully, J. G. andRazin, S. (1969).J. Bact. 98, 970. Tully, J. G. and Razin, S. (1970).J. Bact. 103, 751.

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The Physiology of Sulphate-Reducing Bacteria JEANLE GALL University of Georgia, Athens, Georgia, U.S.A. and C.N.R.S., Marseilles, France AND

JOHN R. POSTGATE University of Sussex, Brighton, England I. 11. 111. IV. V. VI.

.

Introduction Culture and Estimation Inhibition . Classification Control Processes Carbon Metabolism . A. Lactate Oxidation to Acetate via Pyruvate . B. Fumarate and Malate Dismutation. C. Formate Oxidation . D. Citrate Synthase E. Carbon Dioxide Fixation and Mixotrophy F. Hydrocarbon Oxidation and Formation ;Methane Formation . G. Glucose Metabolism in Desulfotomaculum VII. Nitrogen Metabolism. . A. Fixation of Nitrogen B. General Nitrogen Metabolism . VIII. Hydrogen Metabolism . IX. Electron Transport and Phosphorylation X. Chemistry A. Cytochromesc, B. Other c-Type Cytochromes . C. Other Cytochromes D. Non-Haem Iron Electron Carriers . . XI. Sulphur Metabolism A. Reduction of Sulphate to Sulphite . . B. Reduction of Sulphite to Sulphide . XII. Primitive Character . XIII. EcoIogy XIV. Economic Activities A. Corrosion of Metals . B. Storage of Town Gas . C. OilTechnology D. Formation of Minerals References

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.

.

.

. .

.

.

.

.

.

81

.

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

. . . .

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

82 83 84 84 87 88 88 89 90 91 91 92 93 93 93 94 94 97 100 101 105 107 107 109 110 111 116 117 119 119 121 121 122 125

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JEAN L E GALL AKD JOHN R. POSTGATE

I. Introduction The sulphate-reducing bacteria form a physiologically distinctive group of anaerobic bacteria, their oxidative metabolism being based, not on fermentation, but on the reduction of sulphate or certain other inorganic sulphur compounds. Their physiology has broad analogies with that of the nitrate-reducing bacteria (denitrifying bacteria), but they are all exacting anaerobes and no examples of facultative aerobes are known (though an artefactual aerobic metabolism can be obtained in special conditions). Some representatives of the group are capable of growth by non-respiratory processes involving dismutation of substrates such as pyruvate, fumarate or choline. Even when reducing sulphate, these organisms are unable completely to oxidize their carbon compounds, so fatty acids, usually acetic acid, plus carbon dioxide are the normal end products of carbon metabolism. Though once thought to be facultative autotrophs, they are now known not to be capable of normal autotrophic growth; they can conduct assimilatory reactions at the expense of a lithotrophic energy-yielding process (oxidation of molecular hydrogen with sulphate) and in certain conditions a considerable proportion of the assimilated carbon can arise from carbon dioxide (see p. 91). Their wide distribution on this planet, coupled with the fact that the group includes representatives of thermophilic, psychrophilic and halophilic organisms, means that they are likely to grow and multiply in a wide variety of relatively extreme natural environments. Their multiplication can have considerable ecological and economic consequences and, since these are due to their special physiology, in particular the production of hydrogen sulphide, we have regarded a brief account of their ecology and economic activities as relevant to the subject of this review. The present review is a continuation of a series initiated by one of us (Postgate, 1959, 1960,1965) and brings up to date all those publications on this group of bacteria which had come to the authors’ attention by early 1972. Reviews of related subjects which have been published during this period, and which make reference to these bacteria, are by Silverman and Ehrlich (1964) discussing their role in the formation of sulphide ores; La Riviere (1965)on their importance in connection with the sulphur cycle in geology; Peck (1968) in the general context of energy coupling in lithotrophic bacteria; Rittenberg (1969) in a discussion of mixotrophy and coupled assimilatory reactions in bacteria; and Trudinger (1969) reviewing the general metabolism of sulphur bacteria. Two books (Roy and Trudinger, 1970; Nickless, 1968) include chapters which discuss the chemical and biochemical activities of these bacteria.

THE PHYSIOLOGY OF SULPHATE-REDUCING BACTERIA

83

11. Culture and Estimation Recipes for media specific for sulphate-reducing bacteria, together with some details of their cultivation and estimation, were given by Pankhurst (1971) whose article incorporates most of the material available in a briefer account by Postgate (1966; corrigenda 1969~). The problem of growing these bacteria anaerobically on the surface of plates of agar-containing medium appears to be largely one of obtaining long-term anaerobic conditions ; claims for “good growth” on trgptic soy-agar plates were made by Iverson (1966a) ; Pankhurst (1966)

FIG. 1. A Pankhurst tube set up for growing sulphate-reducing bacteria. See text for details.

mentioned plate counts on a more specific thioglycollate medium. Quantitative evaluations of plate counts on these bacteria have not been published but one of us (J.R.P.)has checked Pankhurst’s and Iverson’s techniques against the more conventional deep agar procedures, and the results are statistically comparable. The problem of estimating members of the genus Desulfotomaculum remains ; a “versatile” medium was claimed by Lin and Lin (1970) but comparative data with other techniques were not provided. I n an admirable paper, including appropriate controls, Mara and Williams (1970) observed that a deep 6

84

JEAN LE GALL AND JOHN R . POSTGATE

iron-sulphite agar was satisfactory for counts of Desulfovibrio desulfuricans, D. vulgaris, D . salexigens, but less quantitatively satisfactory for D. africanus and D. gigas. They could find no really satisfactory medium for Desulfotomaculum species. Pankhurst (1967) described special tubes for the cultivation of nongasogenic anaerobes with special reference to sulphate-reducing bacteria. Figure 1 illustrates such a tube, set up with two rubber seals rather than with the single seal and rubber bung which Pankhurst recommended. The advantage of the system illustrated is that an appropriate volume of an inert gas such as molecular nitrogen or argon can be injected with a hypodermic syringe into the side arm, to compensate for the oxygen absorbed by the pyrogallol, and thus the necessity of relying on the tubesnotleakingatsub-atmosphericpressuresisavoided. The familiar effects of organic matter, pH value and salinity on growth have been noted once more by Leban et al. (1966); a rather complex continuous culture system for a halotolerant Desulfovibrio desulfuricans was described by Hallberg (1970). Alico and Liegey (1966) obtained satisfactory correlation between viable and total counts during growth of cultures, and confirmed that a redox potential lower than zero was necessary for the initiation of growth of Desulfovibrio species. Toerien et al. (1968) counted sulphate-reducing bacteria in sewage in atmospheres containing 1%1 H, + 99% CO, and found populations in the region of 4 % 1 x 104/ml. Their claim to have purified seven strains must be viewed with a little scepticism since their only check for purity was apparently microscopic examination, a procedure which is notoriously unreliable with so pleomorphic a group as D.desulfuricans.

111. Inhibition Little has been published specifically concerning the inhibition of sulphate-reducing bacteria since the last general review. Plessis and Gattellier (1965) confirmed earlier reports that the presence of contaminant organisms can influence the apparent resistance of sulphatereducing bacteria to inhibitors in petroleum waters. Costello et al. (1970) reported that the concentration of sodium chloride influenced the sensitivity of Desulfovibrio species to a quaternary ammonium. Though chromate has been widely used and recommended as a specific inhibitor in natural environments, Krassowski et al. (1966) reported that an apparently chromate-resistant strain of Desulfovibrio was responsible for spoilage of pig leather in tanning by the “one and half bath” process.

IV. Classification A proposal to classify the spore-forming sulphate-reducing bacteria in a genus Desulfotomaculum (Campbell and Postgate, 1965) was mentioned

THE PHYSIOLOGY OF SULPHATE-REDUCING BACTERIA

85

in the review by Postgate (1965). A further proposal to divide the genus Desulfovibrio into five species has also been published (Postgate and Campbell, 1966). The essential features of their classifications are indicated in Table 1. A formal description of the new species Desulfovibrio africanus was given by Campbell (1966). I n conflict with the original description of this species, Jones (1971) has claimed that the flagellation of D. africanus is bipolar. The new classification has required the revision of the type strains of the various species; those quoted by Sneath and Skerman (1966) must be regarded as obsolete and new type strains are included in Table 1 . A confusion of records led to a mis-assignment of the type strain of Desulfotomaculum ruminis which has been formally corrected (Campbell and Postgate, 1969). An international committee on nomenclature of the sulphate-reducing bacteria has provisionally accepted the classification in Table 1 (Postgate, 1967), and recommended that it should stand the test of time. I n view of the rarity of reports of authentic natural halophilic thermophiles (Ochynski and Postgate, 1963) a precise identification of Roemer and Schwarz’s (1965) isolate PM, which grew a t 56”C, would be of considerable interest. Booth et al. (1966a) reported that infrared spectra of sulphatereducing bacteria enabled them to distinguish Desulfovibrio from Desulfotomaculum, but that salt relations, rather than species, influenced these spectra within the genus Desulfovibrio. Doubts have been expressed by Miller et al. (1968) about the value of a high resistance to “hibitane” as a criterion for the species of D. salexigens; Trueper et al. (1969) also found hibitane resistance of relatively little value in an examination of marine isolates of Desulfovibrio. As mentioned later, the report of a h-type cytochrome in D. africanus makes the presence of cytochrome c3 the critical taxonomic criterion for the genus Desulfovibrio, not the absence of cytochrome b. Pace and Campbell (1971) found that the homology of rRNA from various species towards DNA obtained from D. vulgaris was consistent with the proposed classification. Saunders and Campbell (1966) surveyed twenty strains of thermophilic Desulfotomaculum nigri$cans and reported that the G+C content of their DNA fell within the range quoted in Table 1. Oulette et al. (1969) measured the G+ C content of three sub-strains of the type species of Desulfovibrio vulgaris and found them identical, despite apparent laboratory-tolaboratory differences in superficial properties. Iizuka et al. (1969) have proposed a new species, Desulfotomaculum antarcticum, for an organism isolated from antarctic soil which differs from other mesophilic Desulfotomaculum strains in forming acid from glucose and possessing gelatinase. Sefer and Pozsgi (1968) examined the serology of many isolates of Desulfovibrio and observed a degree of heterogeneity which did not

00

TABLE1. Simplified K e y t o t h e Classification of Sulphate-Reducing Bacteria Desulfotomaculum

Character

nigrijicuns

Desulfoaibrio africanus

d wu l-

orientis

43

ruminis

juricans

vulguris

salexigens

gigas

Form

rod

curved rod

rod

vibrio

vibrio

fat vibrio

Flagella

peritrichous

peritrichous

peritrichous

single, polar

single, polar

single, polar

+

+

+

-

-

-

-

c3

+

c3 c

c3

55.3 f I

61.2 + 1

+

cj,

46.1 i 1

61.2 _t 1

t60.2

2.5

1,000

2.5

2.5

Spores Type of cytochrome b Desulfoviridin 44.7 Percentage of G + C in DNA Growth in medium containing : pyruvate but not sulphate + choline but not sulphate malate and sulphate formate and sulphato acetate and sulphate Requirement for sodium chloride Hibitaiie resistance 0.25 (mg/litre) Capacity for thermophily + ” Hilo/neotype Delft 74T ( N U B number) (8395)

b

-

41.7

b 45.6

sigmoid vibrio lophotrichous, bipolar?

+

spirillum lophotrichous -

b

CJ

+ + -

-

0.25

1

10-25

-

-

-

Singapore 1 Coleman DL Essex 6 (8382) (8452) (8307)

-

-

-

-

Hildenborough (8303)

British Guiana (8403)

Benghazi (8401)

-

Hibitane or “chlorohexidinc acetate” is bis-p-chlorophenyldlguanidohexane diacetate, a. general microbicide.

(9332)

THE PHYSIOLOGY O F SULPHATE-REDUCING BACTERIA

87

necessarily correspond to Postgate and Campbell’s (1966) classification; Petrovici et al. (1968) discussed their morphology and expressed the view that sprilloid forms are chains of vibrios. The observation that many more strains than hitherto suspected of Desulfovibrio fix nitrogen (see Section on “Nitrogen Metabolism”, p. 93) suggests that the proposal for a variety of “azotovorans” within the species D. desulfuricans was mistaken. The ability to fix nitrogen appears to be rather randomly distributed among the species of Desulfovibrio but, as far as is a t present known, is absent from D. salexigens and D . africanus. It is probable that taxonomic understanding of these organisms will not now advance substantially until large numbers of isolates are available for study by a numerical taxonomy and until reproducible tests, particularly for carbon metabolism, can be devised. It is curious, however, that the percentage of G + C within the genus Desulfovibrio seems to cluster within three narrowly defined values rather than, as with most other bacterial genera, to spread over a moderate range.

V. Control Processes Hespell et al. (1969) observed that coenzyme-A relieved inhibition of pyruvate phosphoroclasm by desulpho-coenzyme-A in several anaerobes including D . vulgaris. Coenzyme-A also stimulated carboxylation of pyruvate and the formation of acetoin from pyruvate (the first report of the lattter reaction in this genus). They proposed that coenzyme-A might have a regulatory function on pyruvate metabolism ; Postgate (1969b) attributed the large coenzyme-A requirement for the “minimethane system” (Wolfe, 1971) of Desulfovibrio to distortion of a control process. Ware and Postgate (1970, 1971) studied the pyrophosphatase of D. desuljuricans which, in common with the pyrophosphatases of other Desuljovibrio species and certain clostridia, was usually activated by reducing agents. They observed that, if the population had been exposed to air, the pyrophosphatase was inactive when extracted but became active on treatment with reductants such as sodium dithionite; if the organisms were grown anaerobically, the extracted enzyme was active and was not influenced by reductants. They purified the pyrophosphatase, a protein of molecular weight 43,000 daltons, requiring Mg2+,Co2+ or Mn2+for activity, which existed in two forms distinguishable by their electrophoretic mobility a t p H 10; an inactive form with three free sulphydryl groups and a form, active after treatment with reductants, withnine free sulphydryl groups per molecule. Because activity appeared to be regulated intracellularly in response to environment, they proposed that the regulation had the physiological function of conserving ATP

88

J E A N LE GALL AND J O H N R . POSTGATE

when the organism was in an unfavourable environment. I n the scheme below, “switch off”of the pyrophosphatase (step 2 ) would conserve ATP by preventing formation of adenosine phosphosulphate (APS-step 1 ) : ATP+S04Z-

______7

APS+PPi

I

-

2Pi

(2)

(3)

AMP

+ S0,Z-

Crude extracts of D. desulfuricans gave some support for this view in that reductant-activated formation of AMP (via step 3) could sometimes be detected. Control via pyrophosphatase of reactions involving pyrophosphate formation, including many biosynthetic processes, may well be widespread in anaerobes, since reductant activation of pyrophosphatase was first observed in clostridia (D’Eustachio et al., 1965). Control of biosynthesis via pyrophosphatase in Rhodospirillum rubrum was proposed by Klemme and Gest (197 1) on grounds of inhibition by reduced nicotinamide nucleotides ; reductant activation was not sought.

VI. Carbon Metabolism A. LACTATE OXIDATION TO ACETATE VIA PYRUVATE Lactate is the most common carbon and energy source used in media for sulphate-reducing bacteria. It becomes oxidized to acetate, yet the nature of the lactate dehydrogenase of sulphate-reducing bacteria has never been described so far, largely because of its instability. Barton and Peck (1971) observed that the L(+) lactate dehydrogenase of D.gigas is active only when bound t o membrane preparations; this report is the first demonstration of an active lactate dehydrogenase in extracts of sulphate-reducing bacteria. The pyruvate phosphoroclastic reaction of Dm. nigrificans was studied by Akagi (1964). The crude extract required pyruvate, coenzyme-A, phosphate buffer and methyl viologen for the reaction. Thiamine diphosphate (TPP) could not be completely dissociated from extracts, but precipitation by ammonium sulphate gave extracts stimulated 33% by addition of TPP. The reaction was inhibited 96% by EDTA, but the activity could be restored by Co2+, Be2+ or Mg2+ chlorides. This reaction was studied further by Akagi (1965). A ferredoxin was isolated which could couple between pyruvate dehydrogenase and hydrogenase, both from Clostridium pasteurianum. It could couple also the dehydrogenation of pyruvate with sulphite as electron acceptor, but not from pyruvate to hydrogenase in extracts of Dm. nigrijcans. Suh and Akagi (1966) showed that the pyruvate-carbon dioxide

THE PHYSIOLOGY O F SULPHATE-REDUCING BACTERIA

89

exchange reaction in D. vulgaris (NCIB 8303) required the presence of phosphate and coenzyme-A, but that the requirement for phosphate disappeared if the concentration of coenzyme-A was increased. Passing the extract through DEAE and Amberlite to remove ferredoxin and cytochroine c3 resulted in a diminution of exchange activity. Full activity was restored by addition of the ferredoxin preparation or cytochrome c3. The reaction was stimulated by either Fe2+or Co2+. Akagi and Verna (1966), then Akagi (1967), reported that, in the phosphoroclastic reaction of D. vulgaris, ferredoxin alone was sufficient t o couple pyruvate dehydrogenase to hydrogenase to a certain extent, but that addition of cytochrome c3 was necessary to recover activity fully. A discussion on the effect of these carriers will be found in the section on electron transport (p. 97). Acetokinase has been purified by Brown and Akagi (1966). The enzyme, with a p H optimum of 7.4, required Mg2+,Mn2+,Co2+or Fe2+. It was specific for acetate and inactive with propionate, formate, butyrate and succinate. Adenosine triphosphate could be replaced by ITP ; GTP or UTP gave slight activities ; p-chloromercuribenzoate inhibited the reaction. That ATP stimulates the pyruvate phosphoroclasm of sulphate-reducing bacteria has been shown by Pates (1967). B. FUMARATE AND MALATEDISMUTATION Resting sulphate-reducing bacteria can dismute fumarate or malate without growth (see Postgate, 1959). Miller and WakerIey (1966) discovered that D.gigas and several strains of D. desulfuricans were able to grow while dismuting fumarate. During growth, malate, succinate and acetate were formed. Sulphate reduction by D. desulfuricans, but not by D. gigas, was inhibited by fumarate. The fact that D . gigas had a much higher -QH2 value in the presence of sulphate than with fumarate as electron acceptor can account for its abnormal behaviour. Similarly, growth by malate dismutation was reported by Miller and Neumann (1970) in nine strains of Desulfovibrio belonging to four species. Succinate, fumarate and acetate were end products. Two dismuting strains of D . desulfuricans and two dismuting strains of D. vulgaris, all grown in lactate-containing medium, showed fumarate hydratase activities of the same order of ma,gnitude. The disrnuting strains showed high succinate dehydrogenase activity but, in another non-dismuting strain, the activity was low and, in D. vulgaris strain Hildenborough, its presence could not be conclusively proved. The enzymes of the fumarate dismutation pathway in D. gigas were studied by Hatchikian aiid Le Gall (1970, a, b). A fumarate hydratase was detected, and malate was decarboxylated to pyruvate by a malate

90

J E A N L E GALL AND JOHN R. POSTGATE

enzyme ; direct formation of lactate from malate could not be demonstrated. The malate enzyme was partly purified; it was specific for NADP and was stimulated by both Mn2+and K+ ions. It did not possess an oxaloacetate decarboxylase activity. A pyruvate phosphorocIastic reaction also occurred with production of acetyl phosphate, carbon dioxide and molecular hydrogen. Since a membrane-linked fumarate reductase was also present, the authors proposed the following scheme for fumarate dismutation : HZ

T

- - -

fumarate

SO4*-

succinate

S2-

I

fumarate ---+

malate

NADP

I

PYruvate

KADPH, +H+

2 H+

+ 2e

carriers

acetyl phosphate + CO,

I

acetate

The conclusions concerning the electron-transfer chain related t o this metabolism are reported in the corresponding section (p. 100).

C . FORMATE OXIDATION A formate dehydrogenase from a strain of D. vulgaris was purified 40-fold by Yagi (1969). The enzyme was inhibited by potassium cyanide but not p-chloromercuribenzoate or o-phenanthroline. The acceptors NAD, NADP, FAD and FMN were not reduced by formate in the presence of an enzyme preparation. Since cytochrome c3 was not reduced and the enzyme seemed to be specific for a low molecular-weight cytochrome, c S s 3 (molecular weight 6500 daltons), a new name was proposed for Desulfovibrio formate dehydrogenase : formate ferricytochrome c S s 3(molecular weight 6500 daltons) oxido-reductase. I n contrast, a formate dehydrogenase from D . gigas, purified 50-fold by RiedererHenderson and Peck (1970), was still able to reduce cytochromes c3 either from D . gigas or D. vulgaris strain Hildenborough. Benzyl viologen or methylene blue were also reduced. Material similar to Yagi’s preparation was used by Ambler et al. (1971b): the enzyme preparation reduced both cytochrome c3 and cytochrome c s s (molecular weight 9000 daltons) from D . vulgaris strain Hildenborough. Furthermore, it contained hydrogenase activity. It seems then premature to link Desulfovibrio formate dehydrogenase in name to a

THE PHYSIOLOGY OF SULPHATE-REDUCING BACTERIA

91

cytochrome that has been found so far in only one particular strain of D. vulgaris.

D. CITRATESYNTHASE Gottschalk and Barker (1966, 1967) found that some strict anaerobes, such as Clostridium kluyveri or D. vulgaris, contained an atypical citrate synthase: it formed [1-14C] citrate from oxaloacetate and [1-'4C] acetyl-CoA when the usual citrate synthase (S-citrate synthase) forms [5-I4C] citrate from these substrates. The atypical enzyme was referred to as (R)-citratesynthase. Gottschalk (1965) later found that both types of enzyme were present in sulphate-reducing bacteria, the (S)-type in D. salexigens, D. gigas and Dm. ruminis, and the (R)-typein two strains of D. desulfuricans and two strains of D. vulgaris. When grown on pyruvate and radioactive carbon dioxide, the protein of the two latter species contained [5-14C] glutamate. The author reasoned that the presence of a (R)-citrate synthase in some anaerobic bacteria could be explained by the fact that this enzyme appeared historically when micro-organisms became prototrophic with respect to glutamate, and that only a few organisms remained which had been able t o defend their ecological niche successfully. O'Brien and Stern (1969) claimed that the stereospecificity of citrate synthase from Cl. kluyveri could not be changed from the (R)-typeto the (S)-type by p-chloromercuribenzoate. These authors proposed that sulphydryl groups are essentials for the (R)-type and that their incomplete oxidation could account for mixed labelling patterns of citrate. Dittbrenner et al. (1969) were unable to confirm these observations, and proposed as possible explanations that Cl. kluyveri contains genetic information for both synthases or that some cultures may have been contaminated with microbes that synthesize p-chloromercuribenzoateinsensitive (S) synthase.

E. CARBONDIOXIDE FIXATION AND MIXOTROPHY I n a series of articles, Sorokin (1966a, b, c, d) demonstrated that a strain of D . desulfuricans isolated from the stratum water of an oil deposit was able to grow by oxidation of molecular hydrogen or formate. Biosynthesis occurred only in the presense of small amounts of acetate which, together with carbon dioxide, fully provided the carbon requirement of the bacteria. Sorokin noted the very interesting fact that acetate and carbon dioxide formed during the oxidation of formic acid and ethanol had to be excreted from the cell prior to their assimilation. He then postulated that the processes of oxidation and biosynthesis are spatially separated in the cell and proposed the pathway shown in Fig. 2 .

92

JEAN LE GALL AND JOHN R. POSTGATE

so:CH,--CH,OH

CH ,-CO

0-

FIG.2 . Pathways involved in mixotrophy in Desulfovibrio desulfuricans ; after Sorokin (1966 a , b, c , d).

F. HYDROCARBON OXIDATIONAND FORMATION ; METHANEPORMATION The details of the importance of sulphate reduction in oil microbiology cannot be included in this review; the book by Davis (1967) provides further information. Postgate (1969b) could not obtain evidence for methane utilization by D.desulfuricuns strain Berre-sol, but Davis and Yarbrough (1966) have published results indicating that 14C was sIowly incorporated into their strains of D.desuilfuricuns when radioactive hydrocarbons were added to growth media. They also show that oxidation of ethane was accelerated by addition of sulphate. Enzymological data have not been reported yet. The presence of NAD : rubredoxin oxido-reductase in D. gigus

THE PHYSIOLOGY OF SULPHATE-REDUCING BACTERIA

93

(Le Gall, 1968)) similar to the enzyme reported by Coon’s group (see Peterson et al., 1966) in Pseudomonas oleovorans, could be an indication of a carbon oxidation system (see section on electron transport, p. 99, for discussion). I n an interesting article, Oppenheimer (1965) demonstrated the presence of “hydrocarbon-like” compounds in mixed bacterial cultures. When a pure culture of sulphate reducers was used, the organisms contained 0.8% of hydrocarbon-like material and, after heat treatment, 4% of the same material. Postgate (1969a, b) reported that methane was a minor product of pyruvate metabolism in D.desulfuricans strain Berre-sol and other sulphate-reducing bacteria as well as Clostridium pasteurianum. Production of methane by extracts required pyruvate, adenine nucleotides, acetyl phosphate, coenzyme-A, thiamine pyrophosphate, vitamin B and Mg2+ions. In the optimum conditions, 0.1 to 0.02 mol % of pyruvate was transformed into methane. I n contrast to normal methane bacteria, the methane came from the methyl-carbon of pyruvate and, correspondingly, ethane was formed from a-ketobutyrate. Methane was not produced in sufficient quantities to account for ATP stimulation of the pyruvate phosphoroclasm noted by Yates (1967). The author reasoned that methane formation in Desulfovibrio species and other non-methane bacteria may be a “vestigial” biochemical process of the kind proposed by Kelly (1968) for Thiobacillus neapolitanus and Gottschalk (1968) for Desulfovibrio and certain clostridia.

G. GLUCOSEMETABOLISM IN DESULFOTOMACULUM Degradation of glucose by proliferating Dm. nigrificans was examined by Akagi and Jackson (1967). Glucose was degraded to acetate, ethanoI and carbon dioxide, The Embden-Meyerhof and Entner-Doudoroff pathways were shown to be operative. Amino acids supplied by yeast extract or peptone in the fermentative medium also contributed to the formation of acetate and carbon dioxide.

VII. Nitrogen Metabolism A. FIXATION OF NITROGEN Recognition of nitrogen fixation among sulphate-reducing bacteria has a chequered history. An early report by Sisler and ZoBell(1951),based on changes in argon :nitrogen ratios over cultures of sulphate-reducing bacteria, suggested that many strains could fix nitrogen. Nitrogen fixation could not be confirmed by other techniques in other laboratories

94

J E A N LE CALL AND J O H N R . POSTGATE

including that of one of us (J.R.P.) and in the Department of Biochemistry in the University of Wisconsin. A report of unequivocal fixation by the “Berre” strains (Le Gall et al., 1959) was confirmed in those laboratories and, for some time, these appeared to be the only authenticated nitrogen-fixing strains of Desulfovibrio. Riederer (1966) re-examined several strains and isolates, including some hitherto believed negative, and obtained fixation with all of them. Publication of her data was delayed for several years because of failure to repeat these observations, in other laboratories, under conditions in which the Berre strains fixed nitrogen readily and and reproducibly. With the advent of the sensitive acetylene test for nitrogen fixation (see Postgate, 197la), Riederer’s findings proved to be correct (Riederer-Henderson and Wilson, 1970) and her findings were confirmed by Postgate (1970a). The Berre strains appear to be particularly good a t fixing nitrogen, and one strain has been grown for long periods in continuous culture in a medium free of fixed nitrogen. Cell-free extracts have been obtained showing some of the properties of nitrogenase (see Silver, 1967) but detaiIs have not been published. Nitrogen fixation by both mesophilic species of Desulfotomaculum has been reported (Postgate, 1970a) but fixation by the thermophilic Dm. nigrijicans was not obtained.

METABOLISM B. GENERAL NITROGEN An adenine nucleotide deaminase showing wide specificity was isolated and purified from D. desulfuricans strain Berre-sol (Yates, 1969). Its metabolic function is obscure, though it was specific to the Berre strain, and absent from four other strains of Desulfovibrio; there was no correlation between its presence and whether the organism had fixed nitrogen during growth. Germano and Anderson (1967, 1968) purified the L-alanine dehydrogenase of D. desulfuricans 56-fold by conventional protein-purification techniques. They reported on the substrate specificity, products and sensitivity to mercurial inhibitors of this partially purified enzyme. Germano and Anderson (1969) studied the pathway of biosynthesis of serine by the same strain, and reported that it followed a conventional pathway, namely from 3-phosphoglycerate by way of phosphohydroxypyruvate and phosphoserine. A brief report on biosynthesis of threonine has been made (Daly and Anderson, 1966).

VIII. Hydrogen Metabolism A classification of micro-organisms that evolve molecular hydrogen was given by Gray and Gest (1965) according to the nature of hydrogen donor and electron donor used in the reaction (see Table 2).

THE PIIYSIOLOCY O F SULPHATE-REDUCING BACTERIA

95

TABLE 2. Gray and Gcst’s (1965) Classification of Micro-Organisms Evolving Molecular Hydrogen. The classification is based upon tho different electron donors which arc effective towards hydrogenasc in the classes of organism possessing that enzyme. EIectron donor(s) fur hydrogen formation .

~

I. Hoterotrophic strict rtnacrobos Clostridia Peptmtreptococcus Micrococcus lactilyticus 11. Heterotrophic facultative anaerobes Escherichia coli and related bacteria 111. Hctcrotrophic strict anaerobrr (cytochrornrs) Sulpliate reducing bacteria

IV. Photosynthetic Non-sulphur purple Sulphur purple Anaerobically “adapted” algae

pyruvato pyruvate pyruvate formate pyruvate formate organic compounds organic compounds t l i iusulphatc reduced nicotinamide nucleotidcs

Another type of classification, based on the general properties of hydrogenases (molecular weight, solubility and susceptibility to aggregation), was elaborated by Kidman et al. (1969). They proposed that Uesulfovibrio hydrogenase had a molecular weight of 56,000 daltons and was composed of several distinct molecular species. Yu and Wolin (1969) compared the activity of several hydrogenases by following methylene-blue reduction or photochemically-reduced methyl viologeri oxidation. A partially purified hydrogenase preparation from D.desulfuricans was inactive in the methylene-blue assay, although it re-oxidized reduced methyl viologen. Yagi et al. (1968), using a highly purified hydrogenase from D. desulfuricans, showed that molecular hydrogen was evolved from dithionite in the presence of cytochrome c3 but not of ferredoxin from Cl. pasteurinnurn. Since the possible reaction of Desulfovibrio ferredoxin (in which the active centre appears to be different from the clostridial type, see Section X, p. 107) with Ltesulfovibrio hydrogenase was not tested, the specificity of hydrogenase for cytochrome c3remains to be demonstrated. Furthermore, Akagi (1967) showed that D.vulgaris hydrogenase was able to evolve molecular hydrogen from pyruvate dehydrogenase in the presence of ferredoxin only. Using dry hydrogenase previously reduced by dithionite, Yagi et al. (1969) demonstrated that it can catatyse the para-hydrogen, ortho -hydrogen conversion.

96

JEAN LE GALL AND JOHN R . POSTCATE

Iron has been shown to be necessary in the oxidation of hydrogen (Sadana and Rittenberg, 1964). Haschke and Campbell (1971a) purified the hydrogenase from D.vulgaris and presented visible and ultraviolet absorption spectra of the enzyme. The main characteristic features were an absorption peak a t 280 nm and a much smaller one in the 400 nm region, reminiscent of non-haem iron absorption. The purified enzyme was active towards cytochrome c, and methyl viologen. Nakos and Mortenson (1971a, b) reported the properties of a very highly purified preparation of hydrogenase from Cl. pasteurianum W5. The enzyme (molecular weight 60,000 daltons) could be dissociated into two sub-units (molecular weight 30,000) by treatment with urea. It contained four iron atoms and four “acid-labile” sulphide groups per molecule. Twelve thiol equivalents could be titrated by mercurials, and half-cysteine residues were present in the molecule. Treatment with o-phenanthroline removed two iron atoms, but its hydrogenevolution and hydrogen-uptake activities were not affected. The enzyme was isolated in its reduced form and exhibited a strong 1.94 electron paramagnetic resonance signal. The signal was lost on oxidation and re-appeared after incubation with hydrogen. The absorption spectrum exhibited a peak a t 280 nm and a broad absorption in the 400 nm region, probably due to non-haem iron. The authors also demonstrated that the multiple forms of hydrogenase from Cl. pasteurianum, previously reported by several authors, were probably artefacts due to the tendency of hydrogenase to form complexes with other proteins. This explanation could also apply to the multiple forms of sulphate-reducing bacteria hydrogenases reported by Kidman et al. (1969). Le Gall et al. (1971) purified a hydrogenase from D.vulgaris strain Hildenborough. Its properties were essentially similar to the clostridial type, with 3.5 non-haem iron and 3.2 “labile sulphur” atoms per molecule of 60,000 daltons molecular weight. It could exist as a dimer of molecular weight 60,000 daltons and a monomer of molecular weight 30,000 daltons. The dimer-monomer dissociation was much easier than with the clostridial type and was concentration-dependent. This property may account for the well known fact that diluted hydrogenase preparations rapidly lose their activities. The spectrum exhibited a peak a t 280 nm and a maximum around 400 nm. The protein was isolated in the oxidized form (argon was used instead of hydrogen during the purification) ; upon reduction with hydrogen, a g + 1.86 electron paramagnetic resonance (EPR) signal appeared, equivalent to the g 1.9 type of E P R absorption observed in other iron-sulphur proteins. The protein contained traces of molybdenum and was not activated

+

THE PHYSIOLOGY OF SULPHATE-REDUCING BACTERIA

97

by permolybdate. Molecular hydrogen did not reduce the added molybdenum ; that reaction occurred only upon addition of dithionite. The purified protein catalysed reduction of both methyl- and benzyl viologen. Although the methyl viologen assay was used by Nakos and Mortenson in the reverse reaction (formation of molecular hydrogen), the specific activities of the two purified hydrogenases are strikingly similar ; 53 pmol molecular hydrogen oxidized per min per mg protein for D. vulgaris hydrogenase, 50 pmol molecular hydrogen evolved per min per mg protein for hydrogenase from Cl. pasteurianum. No experiments were reported concerning the reactivity of hydrogenase from D. vulgaris toward natural electron carriers. The purified hydrogenase still contained traces of flavin and haem iron. This is due t o the tendency of hydrogenase to bind to other proteins as reported by Nakos and Mortenson ( l 9 7 l b ) . It renders difficult the studies of hydrogenase specificity for natural electron carriers.

IX. Electron Transport and Phosphorylation Since many observations indicate that sulphate-reducing bacteria are present-day representatives of very primitive organisms, it has been thought that this “primitivity” should be reflected by the presence in these organisms of a rather truncated electron-transfer chain which could not compete with the complexity of the highly sophisticated mitochondria1 system. Indeed, following the discovery of cytochrome c3 by Postgate (1954) and Ishimoto et al. (1954) in two strains of D. vulgaris (respectively Hildenborough and Miyazaki) and the detection of b-type cytochromes in Desulfotomaculum (Adams and Postgate, 1959), several years elapsed before indications of other electron carriers appeared in the literature. However, recent discoveries, including the extreme intragenus and even intraspecies heterogeneity, have completely changed the picture and it is rapidly becoming confusing even to the specialist. Multiple c-type cytochromes, b-type, d-type, non-haem iron proteins, flavoproteins and quinones have been shown to participate in the electron-transfer system of sulphate-reducing bacteria. Information is now available on the chemistry of these constituents, though very little is known on the electron-transport chain itself. I n fact, most of the activities reported for a given electron carrier are “stimulating” activities. Such activities are not necessarily a demonstration for a direct participation of the carrier in the reaction. The best example to illustrate this point is certainly cytochrome c 3 .Since this protein is extremely auto-oxidizable (Postgate, 1956) it acts as a very efficient oxygen scavenger. It is then possible that the major effect of its addition to a reaction mixture is to

98

JEAN LE GALL AND JOHN R . POYTGATE

remove traces of oxygen (a very powerful hydrogenase inhibitor) thus allowing the reaction to proceed without direct participation of the haem-protein. This could explain why, in many reported stimulations, the rate of the reaction is not proportional t o the amount of added carrier. Another difficulty lies in the fact that, in contrast to higher organisms, sulphate-reducing bacteria possess several terminal oxidases, including those for sulphate, trithionate, thiosulphate, sulphite and fumarate. With the elegant demonstration by Sorokin (see Section on Carbon Metabolism, p. 91) that the sulphate-reduction system is spacially separated from the biosynthetic processes, one might wonder if it is not the case for the different terminal oxidases, each having its own specialized chain of electron carriers. Disruption of the cells would then lead to misleading conclusions concerning the reconstruction of a given activity. For example, Guarraia et al. (1968) have reported that both ferredoxin and flavodoxin can couple reduction of thiosulphate by molecular hydrogen in extracts of D. gigas. Bruschi et al. (1969) reported that, in addition, cytochrome cc3' from the same organism had the same coupling activity. Three proteins, all present a t the same time in D. gigas, seem to share the same activity, a fact that does not appear to be very useful to the cell economy. The first indications of electron exchange between two electroncarrier proteins, ferredoxin and cytochrome c,, have been given by Akagi (1967) and Suh and Akagi (1969) in the pyruvate phosphoroclastic reaction and reduction of thiosulphate by D. vulgaris. The proposed electron-transfer chains were as follows : pyruvate dehydrogenase

pyruvate dehydrogenase

thiosulphate reduetaae

thiosulphate reductase

forredoxin ox.

1(

ferrodoxin red.

1(

'

cyt. r j Pel+

cyt

cj

Pe3 +

ferredoxin ox

cyt c 3 Fe*+

ferredoxin red

L y t c3

Fe3+

1I:

H,

hydrttgenme ox.

1

hydrogrnaae red

( h ydrogenase ox

YH2

hgdrogenaar

Perredoxin has been isolated from Dm. nigr{ficuns (Akagi, 1965). It was active in the reduction of sulphite with pyruvate as an electron donor, but its coupling activity between pyruvate dehydrogenase and hydrogenase from the same organism was poor. Le Gall and Dragoni (1966) reported the crystallization of ferredoxin from D.gigus, and its capability of stimulating hydrogen oxidation in the presence of sulphite. The product of sulphite reduction was not indicated. Flavodoxin was isolated by Le Gall and Hatchikian (1967) and shown to be able to replace ferredoxin in sulphite reduction by molecular

T H E PHYSIOLOGY O F SULPHATE-REDUCING BACTERIA

99

hydrogen. Guarraia et al. (1968) confirmed these results and, in addition to the effect of ferredoxin or flavodoxin on sulphite reduction already reported, indicated that they are also efficient in reduction of thiosulphate and sulphate plus ATP. Hatchikian and Le Gall (1970b) showed that the two proteins were capable of stimulating evolution of molecular hydrogen from pyruvate in a pyruvate phosphoroclastic reaction in extracts of D. gigas. The electron carriers between hydrogenase and fumarate reductase (an enzyme found to be present in a particulate fraction from D.gigas by the same authors) are still unknown; the reaction was not stimulated by any of the above carriers nor by soluble cytochromes ; the particle contained all necessary carriers. The role of rubredoxin, isolated from D.gigas by Le Gall and Dragoni (1966)) by Haschke and Campbell (1967) from D. vulgaris, and by Newman and Postgate (1968) from D. desulfuricans, remains obscure. It was not active in reduction of sulphite (Le Gall and Dragoni, 1966) by extracts of D. gigas nor in reduction of thiosulphate by extracts of D. vulgaris (Suh and Akagi, 1969). Le Gall (1968) showed that an enzyme that catalyses reduction of rubredoxin from NADH, was present in D.gigas, and that the reduced rubredoxin could give its electrons to mitochondria1 cytochrome c, but not to cytochrome c 3 . The physiological electron acceptor for reduced rubredoxin is still unknown. However, this system is reminiscent of the hydroxylation of hydrocarbons by Pseudomonas oleovoruns (Peterson et al., 1966). I n this organism, hydrocarbons are oxidized according to the following overall reaction : R-CH,

+ NADHZ + 0,

-P

R-CHZOH

+ NAD + HZO

Three enzymes are required and have been purified and identified as rubredoxin (Peterson and Coon, 1968)) rubredoxin :NAD oxidoreductase (Ueda, 1971) and an w-hydroxylase (McKenna and Coon, 1970). Rubredoxin from D. gigas has been tested in the P. oleovoruns octylhydroperoxide NADH,-dependent disappearance reaction, and was unable to replace rubredoxin from P. oleovorans for the reaction (Boyer et al., 1971). Nevertheless, because of the similarities between the two systems, the possibility remains that a different w-hydroxylation system exists in sulphate-reducing bacteria or that, by analogy with hydroxylation, a sulphydration system would be present in cultures of sulphate-reducing bacteria. The first report that oxidative phosphorylation occurs in cell-free extracts of sulphate-reducing bacteria was published by Peck in 1966. I n a particulate extract from D.gigas, ATP was formed during reduction of sulphite by molecular hydrogen. Some acidic, soluble proteins were

100

JEAN LE GALL AND JOHN R . POSTGATE

necessary for the reaction, and this observation led to the discovery of ferredoxin and flavodoxin in this organism. The phosphorylation was uncoupled by 2,4-dinitrophenol, pentachlorophenol and gramicidin, but was insensit)ive to oligomycin. Curiously, although flavodoxin replaced ferredoxin for reduction of sulphite, it did not replace i t for ATP formation (Barton and Peck, 1970). Oxidative phosphorylation also takes place during fumarate reduction by molecular hydrogen in D. gigas. The reaction does not require soluble proteins (Barton et al., 1970). The observation (Barton and Peck, 1971) that the same particulate preparation can form ATP when fumarate is reduced by L(+) lactate constitutes (see Section on Carbon Metabolism, p. 88) the first demonstration of an active L-lactate dehydrogenase in extracts of a sulphate-reducing bacterium. An ATPase activity has been found in soluble and particulate fractions of B. gigas (Guarraia and Peck, 1971) which is probably associated with anaerobic oxidative phosphorylation. A NAD(P)H, menadione oxidoreductase was present in D . gigas (Hatchikian, 1970). The function of this enzyme is not yet understood since menaquinone MK-6 present in the cells could not replace menadione for reduction. I n a more physiological approach to the question of oxidative phosphorylation in Desulfovibrio, Vosjan (1970) estimated a phosphorylation ratio (P:2e) of less than 0.5 from the ratios of growth yields with pyruvate and lactate a t various concentrations of their substrates. Catalase activity was reported in extracts of D. vulgaris and D. gigas, but not in those from D. desulfuricans strain El Agheila Z by Bell and Le Gall (1971). The enzyme from D. vulgaris was purified 620-fold, and was a haem-protein with protohaem IX as prosthetic group.

X. Chemistry The discovery of multiple low molecular-weight electron-carrier proteins in sulphate-reducing bacteria will certainly prove to be an extremely interesting tool in the future for studies on structure-function relationships as well as in the field of comparative biochemistry. During the course of this review, the fact that sulphate-reducing bacteria appear to be living relics of very primitive organisms has been pointed out. It is then extremely tempting t o utilize the new data provided in particular by amino-acid sequence determination to try to understand relationships between sulphate-reducing bacteria and between these organisms and the other groups of micro-organisms such as photosynthetic bacteria, clostridia and pseudomonads. It is our belief, however, that one must not jump teo easily to conclusions and that much more data are needed before a clear view of the evolution

THE PHYSIOLOGY O F SULPHATE-REDUCING BACTERIA

101

pattern in bacteria can be given. I n particular, the function of all electron carriers, and their mechanism of action, are still poorly understood. It is obvious that any phylogenetic tree built only on the amino-acid sequences is not sufficient in itself and should be duplicated by a similar tree based on reactivity. The general survey of c-type mitochondrial cytochromes and their reactivity toward cytochrome oxidase is certainly a model in this research field (see Fitch and Margoliash, 1970). Before reaching any conclusion, one should a t least bear in mind that the genera1 shape and aquatic habits of the porpoise are not sufficient to classify it (or him) as a fish.

A. CYTOCHROMES c3 “Cytochrome c3” was described by Postgate (1956) as a dihaem cytochrome. Drucker et aE. (1970b) proposed that it contains three haems per molecule. From amino-acid sequence analysis (Ambler, 1968; Ambler et al., 1969, 1971a, b) it became clear that cytochromes c3 possess attachment sites for a maximum of four haems, two with the classical arrangement : -C-a-b-C-Hand two others (excepted in D.desulfuricans strain El Agheila Z) with the sequence : -C-a-b-c-d-C-H (In the above code, C represents cysteine, H represents histidine and a to d are amino-acid residues whose character may vary. For complete

sequences, see Fig. 3. Amino-acid compositions are listed in Table 3). I n a paper dealing with the chemical properties of cytochrome c3, DerVartanian and Le Gall (1971) pointed out that, during reduction of hydroxylamine with ferrocytochrome c3, a stable half-reduced intermediate with an original electron paramagnetic resonance absorption was detectable. That intermediate resisted further reduction and was thought to represent the catalytic unit. With four haems per molecule, this would then correspond to a two-electron reduction. Yagi and Maruyama (1971) purified cytochrome c3 from D. vulgaris strain Migazaki, and estimated the haem content to be equal to four; Mayer et al. (1971) found the same value with a preparation of cytochrome c3 fsom D.vulgaris strain Hildenborough from Le Gall’s laboratory. It seems probable that four is the correct number of haems in cytochrome c3 and that earlier quoted values are under-estimated. No direct relationship can be found between the amino-acid sequences of mitochondrial cytochrome c and cytochromes c 3 .The high number of mutational events that separate one cytochrome c3 from the other

I.

v.d.a.P.A.d.m.v.

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

-

-

__

- -

t.k.a.p.-.-.V. a.F.s.6.k.g.H.a.s.m.d.C.k. t.C.H.?.? . ? .d.g.a(a,g) i.z(p,c,z)a. s.G.C.H.a.b.t.2.

11. :i .k.a.P.A.-.G.a.K.v.-.-.-.-.

--_

a.p.k.a.P.A.d.G.1.K.m.e.-.-.a.t.k.e.p.-.-.~.v.F.n.~.s.t.~.k.s.v.k.~.g.d,~.~.~.p.v.n.g.k.e.d.y.r.k.~.g.t.a.G.~.~.d,s.m.d.

111. v.d.v.P.A.d.G.a.K.

i .d.f. i .a.g.g.e.k . n . l ( V , v , F)n.E(s ,t,h)k.d.v.k.c.b.b.c.B.s.z .p.g. b.k.q.-.y.a.g.c.t.t.d.G.c.E.n.i.l.d.

P Y

~

I.

.s.k.-.K.s.d.d.s.f.Y.m.a.f.H.e.r.k.s.e.-.K.

-

_ -

.s.C.v.g.C.H.k.-.-.-.-.

-

- _

.-.-.s.m.K.K.g.p.T.k.C.-.-.-.t.e.~.~.p.k.n

11. .k.k.d.K. s . a . k . g . y . Y . ~ . v . m . ~ . d . k . n . t . k . f . K . - . s . ~ . v . g . ~ . ~ . v . s . v . a . g . a . d . a . a . k . K . K . d . l . T . ~ . ~ . k . k . s . k , ~ . ~ . e

111. .k.a.d.K.s.v.n.s.w.Y.k.v.v.~.d.a.k.g,g,a.K.~.t.~,i.s.~.~.k.d.k.a.g.d.d.k.e.l.K.K.k.l.~.g.~.k.~. c .a.C.i.p.s 1 2 3 4 5 6 7 6 9 0 1 2 3 4 5 6 7 6 9 0 i 2 3 4 ~ 6 7 6 9 0 1 2 3 4 S 6 7 8 9 0 1 2 3 4 5 6 7 ~ ~ 0 1 ~ ~ 7 6 9 0 1 i 1

FIG.3. Amino-Acid Sequences from Cytochromes c3 of Desulfovibrio Species. From Ambler el nl. (1971~1). I , indicates Desulfovibrio desuZfuricans (“El Agheila Z”); 11,Desulfovibrio vulgaris (“Hildenborough”) NCIB 8303 ; 111, Desulfovibrio gigan NCIB 9332. The following abbreviations are used for amino acids: A, alanine: C, cysteine; E, glutamic acid; F, phenylalanine; G, glycine; H, histicline; I , isoleucine; K , lysine; L, leucine; M, methionine; N, aspa,ragine; P, proline; Q, glutamine; R, arginine; S, serine; T, threoninc : V, valine; W, tryptophan; Y , tyrosine.

&

bU

THE PHYSIOLOGY O F SULPHATE-REDUCING BACTERIA

103

indicate that Desulfovibrio species diverged a t a very remote time; but, again, much more information is needed. Amino-acid compositions of six cytochromes c3 are known so far (Table 3) including two strains of D.vulgaris and two strains of D. desulfuricans. Although the two cytochromes cg from D.vulgaris seem to be quite related, important differences appear in the two cytochromes c3 from D.desulfuricans indicating a high degree of heterogeneity within these species (see also Drucker et al., 1970b). The main characteristics of all cytochromes c3 are a high number of cysteine residues (eight) of histidine residues (6-10) and of lysine residues (at least 14). Methionine appears to be ligated a t the sixth position of haem iron in mitochondrial as well as some bacterial cytochromes c (Fanger et al., 1967; McDonald et al., 1969a). Evidence has been presented that the 695 nm band in the spectrum of these cytochromes is due to an interaction between a methionine residue and haem iron (Eaton and Hochstrasser, 1967 ;Vinogradov, 1970).This band is absent in all cytochromes c3 described so far, and nuclear magnetic resonance studies (McDonald et al., 1969b)with cytochrome c3 from D.vulgaris indicate that methionine is not the ligand a t the sixth position. This ligand is not known with certainty. By analogy with mitochondrial cytochrome c, histidine could be ligated a t the fifth position and, as already noticed, there is enough histidine or lysine to accommodate the sixth position of the four haem irons. Furthermore, the g values from electron paramagnetic resonance studies of cytochrome c3 from D. vulgaris (DerVartanian and Le Gall, 1971)are in favour of histidine being ligated a t the sixth position. Although electrophoretic and immunological properties of the three cytochromes c3 were different (Drucker and Campbell, 1966b, 1969), circular dichroism and optical rotatory dispersion spectral studies (Drucker et al., 1970a) showed that the haem group environment seems to be identical in all three proteins. A splitting of the circular dichroism Soret-band spectrum was interpreted as resulting from exciton coupling due to the interaction of two or more haems, similar to cytochrome oxidase (Urry et al., 1967). The electron paramagnetic resonance spectrum of cytochrome c3 from D.vulgaris (Le Gall et al., 1971a) is similar to the autoxidizable aggregate cytochrome c haem undecapeptide (DerVartanian, 1970), also an indication of haem-haem interaction. One of the most interesting features of cytochrome c j is undoubtedly its low redox potential : a value of -210 mV was reported by Postgate in 1956 using dithionite titration. Yagi and Maruyama (1971), using molecular hydrogen and hydrogenase to reduce cytochrome c j from D. vulgaris strain Migazaki, obtained the value of -290 mV. BruschiHeriaud and Le Gall (1968) obtained lower redox potential values (close to -260 mV) when using the same system, than by dithionite

TABLE3. Amino-Acid Compositions of c,-Type Cytochromes From Sulphate-Reducing Bacteria

Desulfovibrio

Amino acid Lysine Histidine Arginine Tryptophan Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cystine (half) Valine Methionine lsoleucine Leucine Tyrosine Phenylalanine ~

vulgaris Hilden borough

vulgaris Migazaki

22 9 1 0 12 5

17 9 0 0 12-13 6-7 5 5 4 8 9 8 9 1 1 6 2 3

6

5 4

9 10 8 8 3 0 2 3 2 ~~~

For references, see text.

desulfuricans El Agheila Z

desulfuricans cholinicus

salexigens

gigas ci

14 8 1 1 8 5 8 6 8

8 13 8 5 4 2 0 1 3

17 9 0 0 7 5

6 11 6 9 9 8 9 1 1 6 2 3

22 6 0 0 9 6 12 5

17 8 0 1 18 5 6 4

5

4

11 13 6 8 2 2 4 1 3

11

9 6 9 0 4 4 2 1

6

r M

0

kr

b

U 0

z4

m d

8 0

2Ml

THE PHYSIOLOGY OF SULPHATE-REDUCING BACTERIA

105

titration (-2 15 mV). The low redox potential of cytochrome c3 is probably responsible for its direct chemical reactivity towards both nitrite and hydroxylamine (Senez and Pichinoty, 1958). A re-investigation of this property by DerVartanian and Le Gall (1971) indicated that, when hydroxylamine was converted into ammonia by reduced cytochrome c 3 , a 14Ntriplet appeared on half reduction, with a 52-gauss shift upfield of the signal. A similar shift was seen by reaction of half-reduced cytochrome with ammonia. This was an indication of the appearance of an enzyme-substrate complex in which nitrogen is probably ligated a t the sixth position of the haem-iron. It was proposed that, upon reduction of the protein, a conformational change occurs freeing that position. The different g values obtained with the half-reduced state also indicated the formation of a stable half-reduced intermediate that could also be detected by nuclear magnetic resonance spectroscopy (McDonald et al., 1969a, b). It is interesting that the product of nitrite reduction was not ammonia but nitric oxide, which became firmly bound to the haemiron. Since, with crude extracts of D. vulgaris, NO,- is stoicheiometrically reduced to ammonia (Senez and Pichinoty, 1958), this property should be re-investigated. Cytochromes c and d appear to have nitrite reductase activity in other micro-organisms ; the recent discovery of a d-type cytochrome in Desulfovibrio (Jones, 1971) may be relevant to that problem. Since the haem environments of all cytochromes c3 appear to be quite similar, differences should be pointed out. The most striking are certainly between cytochromes c3 from D. vulgaris and D . gigas; their isoelectric points are extremely different, 10.5 and 5 . 2 respectively (Postgate, 1956 ; Le Gall et al., 1965). Cytochrome cg from D. vulgaris is immediately soluble, D.gigas cytochrome c3 is not. It is interesting t o note that soluble cytochrome c3 from D. gigas can be obtained simply by washing the cells with a slightly alkaline buffer without disrupting the cells (Le Gall et al., 1965). A similar procedure has been used by Scholes et al. (1971) t o purify a cytochrome c from Mic~ococcusdenitrijicans. This phenomenon is probably an indication of the membrane location of the cytochromes. B. OTHERC-TYPECYTOCHROMES The first indication that cytochromes c other than c j were present in Desulfovibrio was given by Le Gall and Bruschi-Heriaud (1968). This cytochrome, called cJS3,was extracted from D . vulgaris strain Hildeiiborough; it had a higher redox potential than cytochrome c3 (between 0 and -100 mV), a molecular weight closed t o 9000 daltons and an amino-acid composition showing that it could not be a dimer of cyto-

106

J E A N L E GALL A N D J O H N R . POSTGATE

chrome c3;see Table 4 (Bruschi et al., 1970). It contains only two cysteine and one histidine residues per mole, representing one haem-binding site. It possesses the 695 nm band characteristic of methionine ligated a t the sixth iron co-ordination position (Le Gall et al., 197lb). Yagi (1969) extracted three different cytochromes c from D.vulgaris Migazaki : c3 (molecular weight 13,000 daltons), c 5 5 3 (molecular weight 6500 daltons) and another cytochrome with a molecular weight of 70,000 daltons. TABLE4. Amino-Acid Composition of Cytochromes cSs3, c3 and cc3 from Desulfovibrio vulgaris strain Hildenborough

Lysine Histidine Arginine Tryptophan Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cystine (half) Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Total

12 1 1 0 6-7 1 5-6 6 2 11 13 2 2 5 1 5 6 0 79-81

22 9 1 0 12 5 6 5 4

9 10 8 8 3 0 2 3 2 107

29 16 11-12 N.D. 23 10-1 1 8 22 16 18 30 16 13 0 9 13 1 6 242

N.D. indicates not determined.

With a molecular weight of G500 daltons, the cytochrome cSs3 from D. vulgaris Migazaki, absent from D.vulgaris Hildenborough, is, to our knowledge, the smallest cytochrome ever reported. Bruschi et al. (1969) and Ambler et al. (1971b) described another type of cytochrome provisionally called cytochrome cc3 from D.gigas and D. vulgaris Hildenborough. The main characteristics of these cytochromes are their molecular weight of 26,000 daltons, their cysteine and histidine contents (18 residues of each), the high number of arginine residues (1112),their low redox potential close t o the cytochromes c3 redox potential, and the absence of an absorption a t 695 nm. Their amino-acid compositions show that they cannot be dimers of a cytochrome cj (Table 4).

THE PHYSIOLOGY O F SULPHATE-REDUCING BACTERIA

107

Electron paramagnetic resonance data (Le Gall et al., 1971a) justify the classification of Desuvovibrio cytochromes into three species, namely : cSs3,c3 and cc3.

C. OTHERCYTOCHROMES A recent publication (Jones, 1971) indicates that both b- and d-type cytochromes are present in D.africanus. The absence of cytochrome c 3 , and not the presence of a b-type cytochrome, should therefore be taken as a criterion to differentiate Desuvotomaculum species from Desuvowibrio species (see the Section on Classification, p. 85).

D. NON-HAEM IRON ELECTRON CARRIERS 1. Ferredoxin The first indication of the presence of a ferredoxin in sulphatereducing bacteria was given by Tagawa and Arnon (1962). The ferredoxin from Dm. nigriJicans was isolated by Alragi (1965) and that from D. vulgaris by Akagi (1965). The first crystalline preparation of a ferredoxin from sulphate-reducing bacteria was reported by Le Gall and Dragoni (1966).For a study of their function, see the sections on Carbon Metabolism (p. 89) and Electron Transport (p. 91). The amino-acid composition of ferredoxin from D.gigas was given by Laishley et al. (1969; see Table 5 ) and some of its properties described by Newman et al. (1969). The protein has a low content of iron (four atoms per mol) and four acid-labile sulphurs. Its amino-acid sequence (Travis et al., 1971) indicates six cysteine residues per molecule. Four cysteine residues are contained in the first half of the molecule (molecular weight 6000 daltons) and the two other in the second half. Comparison with other bacterial ferredoxins indicates strong homologies in the first half of the molecule, but not in the second, which seems to bear homologies with ferredoxins from green plants. It is then tempting to place the ferredoxin from D. gigas between the bacterial and the green-plant type but, as pointed out before, numerous sequences are needed from other sulphate-reducing bacteria before answering this question. Gene duplication is thought to have been a major event in evolution (Eclr and Dayhoff, 1966; Matsubara et al., 1968) and, because of the strong similarities between the two halves of bacterial ferredoxins, the hypothesis of a proto-ferredoxin with four iron and half of the residues of the normal ferredoxin has been formulated. The example of ferredoxin from L). gigas could mean that it is not always so, since such a symmetry does not exist in the molecule.

108

J E A N LE GALL AND J O H N R . POSTGATE

TABLE5. Amino-Acid Composition o f Some Non-Haern Electron Carrier Protcirrs from Desulfovabrao species

Desulfovibrio gigas gigas desulfuricans vulgaris ferredoxin rubredoxin rubredoxin flavodoxin

gigas flavodoxin

~-

Lysine Histidine Arginine Tryptophan Aspartic acid Tlireonine Serine Glutamic acid Proline Glycine Alanine Cystine (half) Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Total

2 0 1 0 11 0 3 10 4

2 6 6 6

2 6

2 0 1 61

5 0 0 1 8 2 4 5 5 6 5 4 4 1 3 2 3 3 61

4 0 0 1 7 2 2 8 6-7 G

6 4 5 1 2 0 3 3 60

4 1 6 1-2 20 6 7 17 4 19 18 5 10 0 9 13 5 6 151-152

8 0 3 1 17 9 8 18 6 4- 1 15 5 16 2 5 14 5 3 149-150

For references, see text. Clear homologies exist within the two examples of rubrodoxin and o f flavodoxin.

2. Rubredoxin Rubredoxin from sulphate-reducing bacteria was first reported in D. gigas by Le Gall and Dragoni (1966). It has also been isolated from D. vulgaris (Hascke and Campbell, 1967) and D. desu7j'uricans strain Berre-sol (Newman and Postgate, 1968). These authors reported several of its properties : optical rotatory, circular dichroism and ultraviolet spectra all indicated it to be similar to other bacterial rubredoxins. It contains four thiol groups and one Fe per molecule of 6000 molecular weight daltons. The amino-acid compositions of rubredoxins from Desulfovibrio are similar (see Table 5).

3. Flavodoxin Flavodoxin was first reported from D.gigas by Le Gall and Hatchikian (1967). Several of its properties have been given by Dubourdieu et al. (1968) and Dubourdieu and Le Gall (1970).It has also been isolated from

THE PHYSIOLOGY O F SULPHATE-REDUCING BACTERIA

109

D. vulgaris. The two flavodoxins (molecular weight 16,000 daltons) bear several similarities with flavodoxins from other microbes : they form a stable semiquinone intermediate, contain one FMN per mol, and can replace ferredoxin in several reactions. Recombination of apoflavodoxin with FMN is quite effective and gives a reconstituted flavodoxin active in sulphite reduction. Amino-acid compositions of the two flavodoxins are given in Table 5 (p. 108); the X-ray structure to 2.5 resolution of flavodoxin from D. vulgaris has been published (Watenpaugh et al., 1972). 4 . Other Compounds Menaquinone MK-6 has been detected in D.vulgaris Hildenborough and in D. gigas by Weber et al. (1970). It has also been shown t o be present in D. gigas and D. desulfuricans El agheila Z by Maroc et al. (1970). Its physiological function is still unknown. Cinquina (1968) has reported the presence of a a-tocopherol in sulphate-reducing bacteria.

XI. Sulphur Metabolism Assimilatory and dissimilatory sulphate reduction take place by two different pathways. Although they appear to bear little similarities, the enzymes involved in the two sets of reactions might prove to have several properties in common. As an introduction to this section, we outline the principal characteristics of the two processes. Synthesis of the enzymes of the assimilatory pathway is repressed by cysteine (Dreyfus and Monty, 1963) and reduction of sulphate to sulphite is preceded by the formation of 3’-phosphoadenylyl sulphate (Asahi et al., 1962). Sulphite is reduced by a complex molecule, sulphite reductase (EC 1.8.1.2))involving several proteins and cofactors. The literature on assimilatory sulphite reductases is abundant and its discussion is out of the limits of this review. The following publications provide further information : sulphite reductase from E. coli Mager (1960), Kemp et al. (1963), Siegel et al. (1971) ; yeast sulphite reductase, Yoshimoto and Sato (1965, 1968a, b), Naiki (1965), Wainwright (1961), Prabhakararao and Nicholas (1969) ; Salmonella typhimurium, Dreyfus and Monty ( 1963), Siegel and Kamin (1968); Aspergillus nidulans, Yoshimoto et al. (1961); Neurospora crassa, Siegel et al. (1965) ; Clostridium pasteurianum, Laishley et al. ( 197 1); higher plants, Asada et al. (1968), Tsmura ( 1965). An important feature of most of these enzymes is the presence of a haem-like component with an absorption peak in the 585-590 nm region. The product of the reaction is hydrogen sulphide, and no intermediate can be detected (Siegel and Kamin, 1968). The reduced form of sulphur

110

J E A N L E GALL A N D J O H N R . POSTGATE

obtained from this pathway is utilized for amino-acid biosynthesis, and hydrogen sulphide does not accumulate. I n contrast, during reduction of sulphate via the dissimilatory pathway, large amounts of sulphide accumulate (see Peck, 1962).This process is a specific property of sulphate-reducing bacteria and involves the formation of adenylyl sulphate (APS) and its subsequent reduction to sulphite and AMP (Ishimoto, 1959; Peck, 1959, 1960). The pathway for reduction of sulphite to hydrogen sulphide is still unclear, but significant progress has been made during the last few years. It now appears that, a t least in some sulphate reducers, stable intermediates are involved in the reductive process.

A. REDUCTION OF SULPHATE TO SULPHITE The process invokes three enzymes, according t o the following steps : SO,*-

+ ATP

H,O

+ PPi

APS

+ 2e

2 APS

+

+ PPi

2 Pi

AMP + SO,z-

(ATP sulphurylase)

(1)

(pyrophosphatase)

(11)

(APS reductase)

(111)

Purification and properties of a pyrophosphatase from a strain of D. desulfuricans (Ware and Postgate, 1971) have been reported, and its role in control processes discussed (see Control Processes p. 87). Some progress has been made on the mechanism of action of APS reductase. This fascinating enzyme catalyses the reduction of APS to sulphite and AMP in the presence of methyl viologen, and also the oxidation of sulphite in the presence of AMP and ferricyanide (Ishimoto and Fujimoto, 1961; Peck et al., 1965) according to reaction I V :

+ AMP + 2 Fe(CN),3-

2 APS + 2 Fe(CN),4-

(IV)

The mechanism of the reaction is of extreme interest since oxidation of sulphite in the presence of AMP results in the formation of a phosphosulphate bound with a AF" equal to 18-19 Kcal (Robbins and Lipmann, 1958). The purified enzyme from D.vulgaris incorporates 35Sfrom AP35S (Peck and Davidson, 1967). It contains one mol of FAD and 6-8 atoms of non-haem iron per mol of protein (estimated molecular weight, 220,000 daltons) and forms a flavin-sulphite adduct, probably on N-5 of the iso-alloxazin ring of the flavin (Michaels et al., 1970). Because of its weak reactivity with molecular oxygen, APS reductase appears to be a true reductase; as noted by Michaels et al. (1971), formation of a sulphite adduct does not necessarily contradict the classification of Massey et al. (1969) which correlates molecular oxygen and sulphite

THE PHYSIOLOGY O F SULPHATE-REDUCING BACTERIA

111

reactivities in oxidases, since the latter form a catalytically inactive sulphite adduct. Michaels et al. (1971) proposed that, due to electronic configuration change, enough energy is acquired by the nitrogen-sulphur adduct for the formation of a phosphosulphate linkage (reactions V, VI, VII) : /

X(IW

\

FAD

I”,

E

/x

‘

’

XW,)

+so;- 2===

FAD-SO,-

(W

/ XHZ

- E

\ FAD

FADH,

I n the first step (reaction V), the 5-N-S03- adduct is formed (X represents a chromophore, which could be non-haem iron); during the second step (reaction VI), the sulphur moiety is transferred from FAD to AMP, or some other mononucleotide acceptor ; and finally (reaction VII), because of equilibrium conditions, the chromophore is reduced. More detailed information is awaited with great interest because of possible relations between oxidative phosphorylation mechanisms and the mode of action of APS reductase.

B. REDUCTION OF SULPHITE TO SULPHIDE The direct reduction of sulphite to sulphide, as it occurs in the assimilatory pathway, is a six-electron transfer process, which can be written as follows : 3 H,

+ 80,’-

+

S2- + 3 H,O

(VIII)

The ratio between oxidized molecular hydrogen and reduced suIphur is three. It is well known to workers in the field that this ratio is rarely obtained with more or less purified cell-free extracts from sulphatereducing bacteria. A difficulty in studying the sulphite pathway lies in the fact that these bacteria might reasonably be expected to contain enzymes that would allow them to utilize as many as possible of the free sulphur compounds usually available in nature. This is indeed the case, and the problem is then to determine whether a given reducible sulphur compound is an intermediate in sulphite reduction or is reduced by an independent pathway. The stable intermediate most likely to be included in the pathway is thiosulphate. As early as 1957, Ishimoto and Koyama reported the

112

J E A N L E GALL AND J O H N R. POSTGATE

partial separation of hydrogenase and thiosulphate reductase activities in Desulfovibrio extracts. Suh et al. (1968) have shown that, in cell-free extracts from Dm. nigri$cans or D.vulgaris, the ratio 3 : 1 (hydrogen oxidized :H,S formed) was not consistently observed and that, during the reduction of sulphite, dithionite and thiosulphate were detectable. With extracts of D. vulgaris, these authors were able to separate a fraction containing sulphite and thiosulphate activities from a thiosulphate-forming fraction. This fraction was able to utilize either sulphite, bisulphite or metabisulphite ions and did not produce hydrogen sulphide. Nakatsukasa and Akagi (1969) reported the isolation of a thiosulphate reductase from Dm. nigri$cans without activity towards sulphate, sulphite, tetrathionate or dithionate. Using 3SS-labelledthiosulphate, the authors concluded that the outer atom of sulphur is reduced to hydrogen sulphide when the inner atom accumulates as sulphite. These observations are consistent with Ishimoto and Koyama’s (1957)evidence that sulphite is generated during thiosulphate reduction : H,

+ S-SO,*-

SO,’-

+ 3H,

+ -+

H,S

+ SO,,-

S2- + 3H,O

(thiosulphate reductase) (sulphite reductase)

(1X)

(XI

Findley and Akagi (1969) and Suh and Akagi (1969) confirmed that thiosulphate is formed during sulphite reduction by extracts of D. vulgaris. The optimal p H value of 6.0 led to their proposal that the bisulphite ion (HS03-), which is dominant in sulphite solutions a t that p H value, was the actual species being reduced. They did not do kinetic experiments comparable t o those of Woolfolk ( 1962) which provided evidence that metabisulphite (S202-)was the true substrate for Micrococcus lactyliticus but they adopted his suggestion that dithionite (S,Oa-) might be the first intermediate. They did not publish evidence for its formation, though they mentioned unpublished work suggesting that it could appear by non-enzymic reactions. Dithionite is reduced by sulphate-reducing bacteria in hydrogen (Postgate, 1956) despite the fact that its standard potential is more reducing than the hydrogen electrode. The idea of a recycling sulphite pool was clearly established by Findley and Akagi (1970) with the observations that, in extracts of D. vulgaris, the rate of 3sS2-formation is smaller when inner labelled thiosulphate (S-3sS0,2-) is used instead of outer labelled thiosulphate ( 3sS-S032-),and that time studies showed that, with inner labelled thiosulphate, there is an increase of 3 5 S 2 - with time and an increasing ratio of double-labelled to inner-labelled thiosulphate remaining in the reaction mixture. They also showed that double-labelled thiosulphate is formed when 35S032-is used by the thiosulphate-forming fraction in

113

THE PHYSIOLOGY O F SULPHATE-REDUCING BACTERIA

contrast to results obtained from other organisms (Schiff and Hodson, 1970). A scheme for sulphate reduction based on these views is : 2u

2e

--? >

sCb2-

--

s,o,2-

_ j

s,o,2-

_3

s,o,z-

s2-

so;-

which depicts a cyclic process for sulphite reduction, modified from the report of Akagi and his colleagues, who prefer to regard the primary substrate as HSO,’ (see text, p. 112). The purification of a thiosulphate reductase from D. vulgaris has been reported by Haschke and Campbell (1971b). I n contrast to the thiosulphate reductase from Dm.nigri$cans (Nakatsukasa and Akagi, 1969), the purified enzyme, homogeneous by ultracentrifugation, was not specific for thiosulphate and also reduced sulphite and bisulphite. Interestingly, the protein did not seem to contain a prosthetic group ; neither visible or ultraviolet absorption was observed. Rhodaiiese (thiosulphate : cyanide sulphur transferase ; EC 2.8.1.1) activity has been found in Dm.nigri$cans by Burton and Akagi (1971). The authors postulate that such an activity might be necessary for the cleavage of the thiosulphate molecule during the reductive process. Kobayashi et al. (1969) published a very interesting paper in which they observed that, with crude extracts from D.vulgaris strain Migazaki, the ratio of molecular hydrogen oxidized to sulphite reduced is numerically equal to three. Chromatography of the crude extract on hydroxyapatite separated two fractions, A and B. The B fraction gave a H, :SO?- ratio less than one, and the products of the reaction were trithionate and thiosulphate. Fraction A contained both trithionate and thiosulphate reductase activities. The authors proposed, then, that the steps leading to formation of hydrogen sulphide from sulphite would be as in the scheme below :

so32-

- - _ _ _+_

5,0,2-

s,062-

S032I

- j L s,z-

so32I

This is a pathway consistent with the idea of a recycling sulphite pool. At least three enzymes would then be included in such a pathway, namely a trithionate-forming enzyme, a trithionate reductase or thiosulphate-forming enzyme, and a thiosulphate reductase. Jin-Po Lee and Peck (1971a, b) demonstrated that, in D.gigas, the trithionate-forming enzyme system, which they called “bisulphite reductase”, and desulfoviridin, the green protein, described in detail by Postgate in 1956, was purified a t the same time. Desulfoviridin from

114

J E A N LE CALL AND J O H N R. POSTGATE

D.gigas, purified to homogeneity, still formed trithionate from sulphite ; trithionate was the only reaction product. Almost all of the trithionate-forming activity present in the crude extract could be accounted for as desulfoviridin a t p H 6.0. This result, together with the fact that desulfoviridin is present in large quantities in most Desuvovibrio species, indicates the importance of the trithionate pathway in this genus. However, there are indications that sulphite can be reduced through different pathways by sulphate-reducing bacteria. Two different sulphite reductases have been reported in D. vulgaris (presumably the Hildenborough strain) by Haschke and Campbell (1967, 1968). The first enzyme, with a molecular weight of 14,000 daltons, is obviously different from desulfoviridin (molecular weight 200,000 daltons) as well as the second (molecular weight 40,000 daltons) which was not specific for sulphite but reduced also other sulphurcontaining compounds with an oxidation state between sulphite and sulphide; it exhibited a strong absorption a t 260 nm. More recently, Jin-Po Lee et al. (1971) described an enzyme from D. vulgaris, strain Hildenborough, termed assimilatory-type sulphite reductase, because its spectrum, with a peak a t 590 nm, was reminiscent of other assimilatory enzymes. The protein (molecular weight 26,800 daltons) separated from desulfoviridin, and exhibited other resemblances with the assimilatory type enzyme: its optimum p H value was not acidic; it was specific for sulphite and hydrogen sulphide was the only reaction product detected. However, the authors did not present proof that the protein had an exclusive assimilatory function. Desulfotomaculum species are devoid of desulfoviridin, so another type of enzyme has t o be involved in reduction of sulphite. Trudinger ( 1970)reported purification of a carbon monoxide-binding pigment from Dm. nigri.cans which contains a haem-like component with an absorption peak a t 582 nm, and seems to be involved in sulphite reduction. The bacteria contain an enormous amount of the protein, namely 10-15% of their dry weight. A highly purified preparation of the enzyme reduced sulphite to hydrogen sulphide with a maximum activity a t p H 6.4. However, with reduced methyl viologen as electron donor, only half of the expected sulphide was recovered. This fact might indicate either a partial denaturation of the enzyme, or that hydrogen sulphide is not the only product of the reaction. The protein also reduced nitrite and hydroxylamine, as is the case for assimilatory sulphite reductases, but not sulphate or thiosixlphate. Its molecular weight was estimated to be 145,000 daltons and it contained Fe, Zn and “labile” sulphide. Another indication that desulfoviridin is not the only sulphite reductase in Desulfovibrio species is the fact that D. desulfuricccns strain Norway 4 (Miller and Saleh, 1964) does not contain that protein. The

4

TABLE6. Properties of Sulphite Reductases from Sulphate-Reducing Bacteria

Organism Desulfovibrio vulgaris Hildenborough

Substrates siilphite

Products

NR

sulphite hydrogen other sulphur sulphide compounds bisulphite trithionate (metabisulphite?)

sulphite Desulfovibrio gigas

Desulfotomaculum nigr@cans

bisulphite (metabisulphite?)

hydrogen sulphide trithionate

bisulphite hydrogen (metabisulphite?) sulphide nitrite NR NR hydroxylamine

Molecular weight

Absorption maxima (nm)

Provisional name

14,000

NR

sulphite reductase (14,000)

40,000

260

sulphite reductase (40,000)

200,000

26,800 220,000

145,000

280, 408, 508 630

275, 405, 545, 590 390, 408, 580, 628 280, 392, 582, 700

Reference

li M

Haschke and Campbell (1967) Haschke and Campbell (1968) Postgate (1965)

2

bisulphite reductase* or trithionate-forming enzyme ; desulfoviridin “assimilatory” sul- Jin-Po Lee phite reductase et al. (1971) bisulphite reductase Jin-Po Lee or trithionatee t a l . (1971) forming enzyme ; desulfoviridin sulphite reducTrudinger tase carbon (1970) monoxide-binding pigment

0 F 0 0

Ic 0

w

38

F $

2

2 td

? i e M

$

NR indicates that the value was not reported. * indicates that the name is assigned by analogy with desulfoviridin from Destrlfovibrio gigus. k-

c.’

u1

116

JEAN LE GALL AND JOHN R . POSTGATE

spectrum ofa crude extract from that organism showed only the presence of a c,-type cytochrome, and the high absorption in the 590 nm region did not allow detection of a protein similar to the Dm. nigri$cans carbon monoxide-binding pigment. The claim that disulphur dioxide and elemental sulphur appeared in cultures of D.desulfuricans might also indicate another pathway for sulphate reduction (Iverson, 1967). Table 6 shows the principal properties of the different sulphite reductases that have been detected in sulphate-reducing bacteria. It will probably indicate mainly that the pathway of sulphite reduction is still far from being clearly understood, Because of the heterogeneity found in sulphite-reducing bacteria, still other proteins with sulphite (or other sulphur compound)-reductive properties will probably be discovered. As already noted, little work has been done concerning the natural electron carriers and donors ; with the separation and characterization of the different reductases, the electron-carrier chain of the sulphate-reducing bacteria will become better understood.

XII. Primitive Character Earlier reviews have drawn attention to evidence, from the extent of fractionation of the sulphur isotopes in geological strata of known age, that sulphate reduction was a widespread biological process on this planet between 0.8 x lo9 and 2 x lo9 years ago, a period well before fossil records of metazoa are found. Peck (1966/7) discussed the evolutionary significance of biological sulphate reduction, and pointed out that present-day sulphate-reducing bacteria had many properties which, from the point of view of general microbiology, could legitimately be regarded as primitive. These included possession of hydrogenase, ferredoxin and nitrogenase, as well as ability to conduct the reductive carboxylation of acetate and the pyruvate phosphoroclastic reaction. To these may be added the partial autotrophy shown by Sorokin (see Section VI, p. 91) whereby carbon dioxide or acetate are not assimilated lithotrophically unless both are present together. Peck pointed out that many of the these properties overlapped with those of the coloured sulphur bacteria, which are also regarded as primitive, and that the two groups share the use of energy-rich sulphate, as adenosine phosphosulphate, as part of their dissimilatory metabolism (Trueper and Peck, 1970). Klein and Cronquist (1967), in a monumental survey of the early evolution of living things, came to the conclusion from a very wide variety of physiological and morphological data that sulphate-reducing bacteria are present-day representatives of organisms which were only one evolutionary stage removed from those which represent the most primitive microbes of all, namely the fermentative clostridia. Postgate

THE PHYSIOLOGY O F SULPHATE-REDUCING BACTERIA

117

(1968) pointed out that the sulphuretum was probably an important primitive primary-producing ecosystem which would be dominant on a planet whose atmosphere was not oxidizing. Such ideas do, of course, depend on acceptance of the view stemming from Oparin (Bernal, 1967) that life developed on this planet when the atmosphere was essentially anaerobic. Some authorities (Broda, 1970) have felt that such an atmosphere excludes the possibility of abundant sulphate in terrestrial soils and water at that period and have proposed placing the photosynthetic sulphide-oxidizing bacteria as more primitive than the sulphate-reducing bacteria. Such arguments are not entirely convincing because, as the photochemical trend to an oxidizing atmosphere on this planet proceeded, free oxygen would tend to be scavenged, by purely chemical reactions, until most of the free sulphide was converted to sulphate. Hence free sulphate probably antedated free oxygen during the transitional phase of this planet’s existence. The idea that sulphate-reducing bacteria have many primitive characteristics has led to proposals that aspects of their physiology which seem to have no clear function may be vestiges of an ancestral biochemistry; Gottschalk (1968) took such a view of the variable stereospecificity of the citrate synthetase in Desulfovibrio (see Section VI, p. 91); Postgate (1969b) suggested that the “mini-methane system” (Wolfe, 1971) of Desulfovibrio might be an evolutionary vestige; Han and Calvin (1969) reported that the hydrocarbon components of Desulfovibrio fell into the “pristine” class. At present, the evolutionary status of sulphate-reducing bacteria is very much a matter for speculation. The structures of both haem and non-haem proteins which are now emerging (see Section X, p. 100) make it likely that the evolutionary position of these bacteria can be discussed in more concrete biochemical terms within a few years.

XIII. Ecology The specialized physiology of sulphate-reducing bacteria leads to specialized ecological requirements and associations, as well as highly specific effects in ecosystems where these bacteria become established. For this and subsequent sections, we have arbitrarily distinguished between “natural” ecological studies and those situations in which the specific physiology of these bacteria influences man’s economy. Kadota and his colleagues (Hata et al., 1964) have given a detailed account of their studies on the ecology of sulphate-reducing bacteria in Hiroshima Bay. The organisms are present in relatively small numbers, and are probably inactive, in the superficial waters and are much

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more abundant, and more variable in number, in the sediments. Novozhilova and Berezina (1968) reported the presence of these bacteria in sediments of a turbulent, highly aerated lake in a Russian semi-desert (Lake Balkash) ;in such aerobic circumstances, winds and other bacteria removed hydrogen sulphide rapidly. Ivanov and Gorlenko (1966) used radioactive sulphate to obtain an estimate of their activity in oil-well waters. Numerical data for numbers of sulphate-reducing bacteria in sewage were quoted by Toerien et al. (1968); Holm and Vennes (1970) mentioned their prescnce a t low concentrations of sulphate in a sewage lamgoon.The importance of a low redox potential for multiplication of these bacteria is evident from a number of ecological studies, and was emphasized by Genovese and Rigano (1964). They are present in large numbers in the mud of Lake Ganzirri (Genovese et al., 1964) but are few in the superficial waters ; they are present in the sediment, but absent from the superficial waters, of a marine lagoon near Marsala (Genovese, 1969). They were absent from aerobic sediments from the Mediterranean examined by Genovese (1970) during various marine excursions. Connell and Patrick (1968) emphasized the need for a low redox potential for development of these organisms in Louisiana soil. A high p H value is customarily regarded as favouring multiplication of these bacteria, but Ivanov and Karavaiko (1966) detected sulphate-reducing bacteria a t p H values in the region of three in volcanic lake systems, and Kiister (1969) mentioned their presence in peat with a p H of 3-4. Whether localized micro-environments of high p H value or specific acid-tolerant strains account for these reports is not clear. Sulphate-reducing bacteria show a rather limited substrate specificity, so syntrophy with other bacteria is of considerable ecological importance. I n a painstaking study of the ecology of the Sumida river near Tokyo, Tezuka showed that a lactic-acetic fermentation of carbohydrate-like material provided carbon sources for bacterial sulphate reduction. Among the responsible organisms were Escherichia coli and Aerobacter aerogenes : crude enrichment cultures could utilize a wider variety of amino acids and other materials than could pure cultures of sulphatereducing bacteria from the same source. Artificial mixtures of these pure cultures with coliform bacteria showed a widened range of substrate specificity (Tezuka, 1963, 1964, 1965, 1966; Tezuka et al., 1964). Syntrophism enabled sulphate-reducing bacteria t o grow on the breakdown products of alginic acid derivatives (Billy, 1963); Cahet (1965, 1966) emphasized the importance of syntrophy in providing carbon nutrients in natural waters. Another form of syntrophy occurs where these bacteria provide sulphide for photosynthetic sulphur bacteria in a sewage lagoon. The natural ecosystem based on sulphate reduction has been called a sulphuretum, and has been studied by Schwartz and his

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colleagues, noting associations with chitin-decomposing bacteria (Durner et al., 1965),during a study of the metal tolerance of these bacteria. The failure of Schwartz and Schwartz (1965) to find many sulphate-reducing bacteria in volcanic environments may have been due to use of a medium notpoisedforredox potential. Though their description of the appropriate ecosystem as “new” is an overstatement, it is interesting that Fenchel and Riedl (1970) find that the sulphuretum can form the basis of a biotic complex involving multicellular organisms. Marine or naturally salt-resistant strains of Desulfotomaculum remain elusive ; the organism isolated by Fernandez et al. (1968)from flooded Venezuelan soils appears to be a freshwater type. Alexander (1965) discussed the ecological role of these bacteria in the persistence of petroleum deposits; even if their still uncertain ability to metabolize petroleum is accepted, the amounts of sulphate needed for them t o affect the World’s petroleum deposits would be vast. Postgate (1970b) mentioned a possible extraterrestrial function in conceptual ecosystems based on oxidation of carbon monoxide generated by photodissociation of carbon dioxide.

XIV. Economic Activities A. CORROSION OF METALS Metallic corrosion by microbes features extensively in an elementary textbook on microbes in metallurgy (Miller, 1971). Corrosion due t o sulphide originating from non-sulphate-reducing bacteria has been reported again by Brisou and De Rautlan de la Roy (1965)but, since the major economic source of bacterial corrosion is bacterial sulphate reduction, emphasis has continued t o be placed on the dissimilatory sulphate-reducing bacteria. The British research group headed by Dr. G. A. Booth has now been dispersed despite the impressive contributions it made over the last decade, and its demise has been accompanied by a considerable number of papers and a relatively elementary review (Booth, 1968). The theory of cathodic depolarization proposed that a major part of the corrosive effect of sulphate-reducing bacteria was attributable to their possession of the enzyme hydrogenase, with which they “depolarized” the surface of a wet metal such as iron by removing the protective film of cathodic hydrogen. The normal anodic attack by hydrogen sulphide was considered t o be a minor process. Electrochemical studies and a correlation between corrosiveness and hydrogenase content among diverse strains of bacteria, discussed in earlier reviews, tended to support this theory, though clearly it would never be universally applicable in natural systems and other effects, such as anodic attack by hydrogen sulphide, effects of intermittent

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aeration, protection by chelating agents such as tannates, variations in salinity and redox potential, would augment or mask cathodic depolarization in various circumstances. Booth et al. (1965) found that the correlation between hydrogenase content and corrosiveness towards iron among various bacterial strains was less pronounced in semi-continuous culture than in batch cultures, though hydrogenase-positive strains were always the most corrosive. Booth et al. (1966b) showed that, if free ferrous ions were present in sufficient amounts to precipitate sulphide as it was formed continuously, corrosion rates became independent of hydrogenase content. Booth et al. (1968)grew a strain of D. desulfuricans with fumarate and without sulphate, so as to obtain cathodic depolarization uncomplicated by anodic attack by sulphide ; they then observed that added ferrous sulphide accelerated corrosion. These observations suggested that the principal mode of attack, in their conditions, was anodic. Enhancement of iron corrosion by ferrous sulphide has long been known, and King and Miller (1971) proposed that both anodic attack by iron sulphide and cathodic depolarization occurred in their experiments. Tests with dyestuffs as electron acceptors (Chemistry Research, 1952; Iverson, 1966b) provide unequivocal evidence that cathodic depolarization does in fact take place in Desulfovibrio as well as with hydrogenase-containing strains of Desulfotomaculum (Booth and Tiller, 1968). The rapid growth which can be sustained in continuous culture leads to remarkably high corrosion rates (Booth et al., 1967b) and it is possible that corrosion mechanisms may depend on whether the population is multiplying (and rich in hydrogenase) or dormant (when hydrogenase activity normally declines). A strange mechanism for bacterial corrosion was proposed by Iverson (1965a, b) on a basis of experiment’swith cell-free extracts of Desulfovibrio : he claimed t o detect iron phosphide (Fe,P) by X-ray and Mossbauer studies of the corrosion products, and suggested that hydrogenase reduced phosphate to phosphine, which then attacked the metal. There is no evidence for the formation of phosphine from phosphate by hydrogenase, and the reaction is thermodynamically implausible. I n more practical studies, Booth et al. (1967a, d, e) carefully reexamined the criteria of agressivity to be used with soils and waters, and concluded that the traditional “bottle test”, or more exact enumeration of populations of sulphate-reducing bacteria, were relatively valueless, presumably because they did not reflect the actual physiological activity of such organisms as were present. Measurements of redox potential and conductivity correlated more satisfactorily with field aggressivities, a conclusion consistent with the beliefs of earlier workers and of Connell and Patrick (1968). Simplistic views of the ecology of microbial corrosion are not encouraged by the report of Booth et al.

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(1967a) on a field trial in which corrosion rates were faster in a relatively unpolluted river in Wales than in the seriously polluted River Thames. That bacterial corrosion is accelerated in conditions of partial or intermittent aeration has long been known; Rozanova et al. (1969) documented cases of corrosion in a flooded petroleum stratum and showed that it was fastest a t moderate concentrations of hydrogen sulphide ; high concentrations of hydrogen sulphide were protective. A rather unusual site for bacterial corrosion has been reported (Anon., 1971); the organisms attacked bolts beneath an algal film in an industrial cooling tower. Tiller and Booth (1968) studied corrosion of aluminium by sulphate-reducing bacteria, and concluded that the process was largely, but not entirely, cathodic.

B. STORAGE OF TOWNGAS Spoilage of stored town gas by sulphate-reducing bacteria can present a serious economic problem, since the gas becomes corrosive, smells offensive and generates sulphur oxides when burnt. Pankhurst (1968a) reviewed the considerable literature on the subject, and the only additional relevant material which has come to the notice of the present authors is her own later contribution (Pankhurst, 1968b) on their potential importance in underground storage of natural gas. These bacteria were present in all samples from potential storage sites and, though some may have been introduced in the drilling muds, most were probably indigenous. Hence they are likely to cause a serious economic problem as underground storage becomes widespread ; the outlook for the effective use of microbicides in this context is not promising.

C. OIL TECHNOLOGY Despite the acknowledged role of sulphate-reducing bacteria in many aspects of oil technology, relatively few publications bearing directly on this aspect of their economic activities have come t o our attention during the period under review. I n an important contribution on the ecology of these bacteria in oil deposits, samples and enrichment cultures from the Romashkino oil field were studied by Kuznetzova and Gorlenko (1965), who observed maximum production of hydrogen sulphide in samples at 25-3OoC, though the optimum temperature for sulphate-reducing bacteria isolated from such samples was 40°C.Evidently a temperaturesensitive syntroph was contributing t o sulphate reduction from natural substrates ; they demonstrated the presence of a temperature-sensitive pseudomonad capable of generating substrates for sulphate reduction.

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Murzaev (1965a) has again mentioned a possible role of these bacteria in the desulphurization of petroleum hydrocarbons, though their general metabolism would incline them to contribute sulphur t o oil rather than t o remove it. Reference was made to sulphate-reducing bacteria in the context of oil technology in certain earlier sections of this review; inhibition (Plessis and Gattellier, 1965), ecology (Alexander, 1956) and corrosion (Rozanova et al., 1969).

D. FORMATION OF MINERALS I . Sulphur Deposits Ivanov’s (1964) book on the microbiology of the formation of natural sulphur deposits has been translated into English. The basic role of sulphate-reducing bacteria in the natural genesis of reduced sulphur seems we11 established, but the mechanism of oxidation of S2- t o So seems t o have differed from place t o place. Detailed discussion of this step is outside the scope of this review, but attention should be drawn t o the report of Nissenbaum and Kaplan (1966) of an Israeli deposit in which the So fraction contained more of the heavy sulphur isotope than the sulphate. This is the converse of what is usually found, and implies that the sulphur cycle had been initiated in this area from materials which were already enriched. Research and speculation on potential industrial processes continue t o be published. Burgess and Wood (1961) gave an account of pilot-scale use of K. R. Butlin’s process in a London sewage works, and Sadana and Morey (1962) reported an evaluation of a comparable process in India, including confirmation of the need to “adapt” the microbial population to sulphate-enriched sewage. Barta (1964) gave a review of the Czech process making use of Desulfovibrio to produce sulphur from industrial wastes. Murzaev (1965b) calculated that the sewage of Moscow could make a useful contribution to the sulphur supplies of the U.S.S.R. if treated appropriately, and a process has been proposed based on the premise that sulphate-reduction can be coupled to hydrocarbon oxidation (Anon., 1967). Since the major industrial need for sulphur is for the manufacture of sulphuric acid, microbiological procedures seem unlikely to become economic, despite an accelerating shortage of natural sulphur (Gittinger, 1966), because processes making use of pyrites, anhydrite or sour gas from oil fields are becoming increasingly used. Microbiological processes can, however, be valuable as waste disposal and water purification procedures, and will probably become competitive when, in a few decades, the World’s resources of native sulphur are acutely strained.

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2. Metal Xulphide Ores The view that sulphate-reducing bacteria play a part in the formation and deposition of non-magmatic sulphide ores is now widely accepted. The known toxicity of copper to Desulfovibrio has been held against a role for these bacteria in the formation of copper-sulphide minerals, but Temple and Le Roux (1964a) showed that production of hydrogen sulphide by active Desulfovibrio was sufficient to detoxify copper in any normal copper-enriched water, thus supporting an independent criticism of earlier arguments (Postgate, 1965). I n a laboratory model system, Temple and Le Roux (1964b) observed that active sulphate reduction in a culture led to desorbtion and banded precipitation, as sulphides, of iron, lead, copper or zinc adsorbed on a matrix which was separated from the culture by a barrier through which the metal ions could diffuse. The conclusion, that environments where sulphate reduction was proceeding tended t o concentrate and precipitate metal ions from the neighbourhood, was discussed in relation t o the natural deposition of mineral ores by Temple (1964). Rickard (1969) has shown that five types of sulphide of iron (greigite, mackinawite, marcasite, pyrite and pyrrhotite) occur in cultures of a halotolerant strain of D. desulfuricans. Hallberg (1970) reported that the hydrated iron sulphide found in his continuous-culture system was non-magnetic, so the precise conditions for formation of Freke and Tate’s (1961) magnetic material remain elusive. Lyalikova and Sokolova (1965), studying the microbiology of natural copper, iron and molybdenum sulphides, obtained evidence for the presence of D. desulfuricans in a copper deposit, and believed that the ore was biogenic. Accretion of pyrites around centres of active sulphate reduction is a probable mechanism for formation of pyritic fossils, and Kato (1970) has illustrated many instances of replacement of diatomaceous tests by pyrites of biogenic origin. But few microbiologists would agree with his proposal that the extent of isotope fractionation in biogenic deposits could be an index of the type of microbe involved.

3. Mineral Carbonates Alkaline earth and alkali metals do not form sulphide deposits ; their sulphides hydrolyse and the final product in nature is a carbonate, as discussed in earlier reviews. Roemer and Schwartz (1965) reported formation of carbonates of calcium, strontium and barium in laboratory cultures subjected to prolonged incubation which partly simulated natural mineral-forming processes. 4 . Mortality of Higher Organisms i. Plants. Damage to rice crops caused by sulphate-reduction consequent on anaerobiosis in rice paddies has been recognized for many years. A

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review by Takai and Kamura (1966) cites Shiori (1943)) Mitsui (1955) and Aomine (1962) as key references on pedological aspects; earlier reviews mentioned the microbiological side (e.g. Postgate, 1960). Autumn wilting of rice crops, called “aki-ochi” in Japan and “Brusone disease” in Europe (Vamos, 1959))can lead to considerable crop losses, and Takai and Kamura (1966) report that yields in Japan have increased by a third since the Second World War as a result of control of redox potential in rice paddies. The presence of hydrogen sulphide in the paddy soil causes root rot and accumulation of indolylacetic acid (Vamos, 196613, 1968). Comparable problems with other crops have now been reported. Ford (1965) reported death of citrus plants grown in waterlogged soils in Florida, which was partly attributable to production of hydrogen sulphide by bacteria. Dommergues and his colleagues take the view that the rhizosphere of plants and the spermosphere of seeds can become sites of localized sulphate reduction, which will have toxic effects on growing plants or seedlings. Dommergues et al. (1969a, b) attributed the sudden death of lucerne in saline Tunisian soils t o sulphate reduction in the rhizosphere of maize, a process which was enhanced by strong sunlight, and led to death of the plants in 25 days (Jacq and Dommergues, 1970a).Jacq and Dommergues (1970b)reported sulphate reduction in the spermosphere of maize seeds which was most pronounced deep in dense soils. Death of seeds from this cause was favoured by a high content of sulphate and a low rate of diffusion of oxygen ; they observed no correlation with bacterial numbers but their medium for counting sulphate-reducing bacteria lacked a redox-poising agent (Jacq and Dommergues, 1971). ii. Animals. Toxicity to fish, which can reach catastrophic proportions, was mentioned in earlier reviews; an instance in Tunisia (Cabasso and Roussel, 1942) associated with “red water” was inadvertently omitted. Vamos and Tasnadi (1971) attributed fish mortality less t o poisoning by hydrogen sulphide rather than to acid formed by oxidation of biogenic iron sulphide. Suspended lime in the water can protect, and accounted for a relatively low mortality of fish in the Danube. I n inland ponds, sulphate reduction a t the expense of decaying algal blooms may produce the “poisonous dawn fog” of Italian and Hungarian peasants (Vamos, 1959) in appropriate climatic conditions (Vamos, 1966a) and cause death of fish a t rates as high as 25 tons in 3 h (Vamos, 1964). Folklore maintains that the “poisonous dawn fog” can kill water birds. According to Vamos (1971) the first deaths of man attributable t o sulphate-reducing bacteria have now been recorded : workmen died of poisoning due to biogenic hydrogen sulphide while cleaning out waste traps in town sewers in Hungary; death was characterized by a very rapid onset of rigor mortis.

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5. Miscellaneous Econom,ic Activities Blackening of leather by sulphate-reducing bacteria during tanning (Krassowski et al., 1966) was mentioned on p. 84. Lin and Lin (1970) reported involvement of Desuvotomaculum species in spoilage of canned mushrooms. A possible role of nitrogen-fixing mesophilic Desulfotomaculum in the nitrogen nutrition of ruminants was mentioned by Postgate (1971b) but is unlikely to be serious in view of the low numbers present. Sefer and Calinescu (1969) demonstrated Desulfovibrio, as well as oral streptococci, in seven specimens of human dental caries, a hitherto unsuspected habitat for these bacteria. Whether they contribute to the progress of dental infection is not yet clear. Jones-Mortimer (1968) used D. desulfuricans to make radioactive hydrogen sulphide from radioactive sulphate. Lewis (1966) mentioned experiments on the use of cultures of sulphate-reducing bacteria as the cathodic halves of biochemical fuel cells. Vamos and Ando (1969), in a discussion of the origins of black alkaline soils, have again emphasized the role of sulphatereducing bacteria. Their alkalizing effect in nature has been put to use by Tuttle and his colleagues for the control of acid effluents : acid mine waters, rich in sulphuric acid, contain acid-tolerant heterotrophs as well as the autotrophs responsible for the processes leading t o acid formation (Tuttle et al., 1968). Where such water accumulated behind a porous dam of residues from handling timber, sulphate reduction leading to partial neutralization of the mine water took place (Tuttle et al., 1969a). Tuttle et al. (1969b) discussed the practical use of this property in pollution control, and defined optimal conditions. Clearly, the variegated economic effects which result from the peculiar physiology of the sulphate-reducing bacteria are economically very costly; the human nose is still probably a cheaper device for detecting their activities than the 10 cent piece recommended by Keup and Ballinger (1969).

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The Role and Regulation of Energy Reserve Polymers in Micro-organ isms EDWINA. DAWES AND PETER J. SENIOR Department of Biochemistry, University of Hull, Kingston upon Hull, H U 6 7 R X , England I. Introduction . . A. Criteria for Energy Storage Function . . B. Energy Storage Compounds . . C. Adenylate Energy Charge and Energy Storage . . D. Mutants Defective in Storage Polymer Synthesis . . 11. Glycogen and Glycogen-likeReserves . . A. General Considerations . . B. Occurrence of Glycogen and Glycogen-likeReserves . . C. Structure of Microbial Polyglucans , . D. Biosynthesis of Glycogen-likeReserves . . E. Glycogen Biosynthesis by Prokaryotes . . F. Glycogen Biosynthesis by Eukaryotes . . G. Glycogen Degradation . H. Conclusions . . 111. Polyphosphate . . A. Status of Polyphosphate as a Reserve Material . . B. Occurrence of Polyphosphate in Micro-organisms . . C. Chemical Structure of Polyphosphates . . D. Accumulation and Utilization of Polyphosphate . E. Polyphosphate Metabolism: Enzymology . . F. Regulation of Polyphosphate Metabolism . . G. Physiological Functions of Polyphosphates . . IV. Poly-8-hydroxybutyrate . . A. History . B. Occurrence of Poly-a-hydroxybutyrate . . C. The Nature of Poly-B-hydroxybutyrate . . D. Poly-/?-hydroxybutyrate Metabolism. . . E. The Enzymology of Poly-P-hydroxybutyrateBiosynthesis . . F. The Enzymology of Poly-P-hydroxybutyrateDegradation . . G. Regulation of Poly-a-hydroxybutyrateMetabolism and its Physio. logical Significance H. Functions of Poly-a-hydroxybutyrate . . V. Conclusions . . VI. Acknowledgments . . References . . 135

136 137 137 135 139 140 140 142 142 144 146 163 170 176 178 178 179 179 183 192 197 201 203 203 204 206 214 228 236 244 249 264 256 257

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I. Introduction Energy-storage compounds in higher plants and animals have been recognized for many years. Their function in providing both carbon and energy, as in the case of starch, glycogen and lipids, or only energy, as with phosphagens such as creatine phosphate and arginine phosphate, led to speculation on the possible role of analogous compounds in microorganisms. The fact that polysaccharides resembling starch and glycogen occur in some microbes is not, of course, evidence for their function as energy-storage compounds and, indeed, when Wilkinson first reviewed this topic in 1959 he was initially concerned with dispelling the scepticism which a t that time surrounded the existence of such compounds in bacteria. It is, for example, evident that a simple undifferentiated unicellular organism will not require energy for many purposes that demand it in a higher form of life, such as homeiostasis, secretion and muscular contraction. However, the concept of an energy of maintenance for micro-organisms is now well-established and supported by experimental evidence, as witnessed by the work of Mallette (1963), Marr et al. (1963) and Pirt (1965). Thus, in the absence of growth, energy is required for osmotic regulation, maintenance of intracellular pH, for motility, for the turnover of proteins and nucleic acids, and for the specialized phenomena of sporulation, encystment and luminescence. In the absence of an exogenous source of energy these requirements must be met from endogenous sources and Dawes and Ribbons (1962, 1964), in reviewing various aspects of the endogenous metabolism of microorganisms, focused attention on those intracellular compounds which could fulfil these demands. A clear distinction can be made between compounds which serve as specialized reserves of carbon and of energy and those that are essentially basal components of the cell, e.g. proteins and RNA, but which, under conditions of starvation, may be degraded to provide energy. There is evidence that some micro-organisms, of which Pseudomonas aeruginosu is a good example (Campbell et al., 1963), do not synthesize any specific reserve compounds and they are therefore entirely dependent on protein and RNA degradation for their endogenous metabolism and energy of maintenance. Certain organisms are able to accumulate more than one type of reserve material and, in these instances, the environmental conditions and the regulatory mechanisms involved will determine the principal reserve deposited. Examples of such dual reserves include glycogen and lipid (Mycobacterium phlei, Antoine and Tepper, 1969c), glycogen and poly-P-hydroxybutyrate (Rhodospirillum rubrum, Stanier et al., 1959 ; Bacillus megaterium, Wilkinson and Munro, 1967), and glycogen and polyphosphate (Aerobacter aerogenes, Duguid et al., 1954 ; Herbert,

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1961). It may be noted that B. megaterium can, in addition, synthesize polyphosphate under the appropriate environmental conditions (Wilkinson and Munro, 1967).

A. CRITERIAFOR ENERGY-STORAGE FUNCTION Wilkinson (1959) advanced three criteria for the establishment of the energy-storage function of a compound, namely (i) that the compound is accumulated under conditions when the supply of energy from exogenous sources is in excess of that required by the cell for growth and related processes a t that particular moment in time; (ii) that the compound is utilized when the supply of energy from exogenous sources is no longer sufficient for the optimal maintenance of the cell, either for growth and division or for maintenance of viability and other processes ; and (iii) that the compound is degraded to produce energy in a form utilizable by the cell and that it is, in fact, utilized for some purpose which gives the cell a biological advantage in the struggle for existence over those cells which do not have a comparable compound. Wilkinson stressed the undesirability of relying on the first two criteria for the evaluation of a storage function since clearly some compounds might be produced by a cell in an attempt to detoxicate end-products of metabolism which could otherwise accumulate at too rapid a rate and prove toxic. However, if Wilkinson’s first criterion is qualified as the intracellular accumulation of a compound then much of his original concern to distinguish between genuine storage compounds and shunt or overflow products of metabolism, as discussed for fungi by Foster ( 1947), becomes unnecessary. These latter compounds accumulate in the medium under certain conditions of growth, usually where a large excess of carbohydrate has been provided.

B. ENERGY-STORAGE COMPOUNDS Three main classes of compound have usually been considered as possible storage compounds in micro-organisms, namely polysaccharides, lipids (including poly-p-hydroxybutyrate) and polyphosphates. The cellular content of each of these may vary widely depending on environmental conditions (Herbert, 1961; Harold, 1966) but since they are of high molecular weight they affect the internal osmotic pressure of the cell only slightly when they are synthesized. I n the present review we shall confine our attention t o polymeric reserve materials and thus exclude consideration of lipids other than poly-P-hydroxybutyrate, and the disaccharide trehalose, which plays an important role in the economy of the yeast cell but displays certain features which are at variance with the normally accepted behaviour of storage compounds. We shall thus consider the evidence for the energy-storage roles of

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glycogen, polyphosphates and poly-p-hydroxybutyrate and the current state of knowledge concerning the regulation of their biosynthesis and degradation in the microbial cell. Various aspects of the regulation of the biosynthesis of a- 1,4-glucans in bacteria and plants was the subject of an excellent, comprehensive review by Preiss (1969) and the structure and biosynthesis of storage carbohydrates in yeast were surveyed by Manners (1971). Energy reserves in yeast and their mobilization were considered by Sols et al. (197 1) as part of a wider review of energy-yielding metabolism in yeasts, and the structure, metabolism and function of polyphosphates were last reviewed by Harold (1966). There has been no general review of poly-/3hydroxybutyrate, a lipid peculiar to micro-organisms and the centre of much research activity in recent years, since those of Dawes and Ribbons (1964) and Doudoroff (1966).

C. ADENYLATE ENERGY CHARGE AND ENERGY STORAGE Acceptance of the criteria for establishing the role of a compound as an energy-storage material leads to the general prediction that under conditions where microbial growth is limited by some factor other than the carbon and energy source, accumulation of the reserve material should occur. Such conditions should lead t o a favourable intracellular energetic state which would presumably act as a signal to stimulate biosynthesis of the appropriate storage compound. Since some microorganisms are able t o accumulate more than one type of reserve material the regulatory processes are of considerable significance. The last decade has witnessed the discovery of the allosteric control of enzymes and recognition of the importance of adenine and nicotinamide nucleotides in many of these regulatory processes. These features of metabolism have received a unifying treatment with the adenylate energy charge concept of Atkinson (1968, 1971). The energy charge is defined as [ATP] + O*5[ADP] [ATP] [ADPI [AMP]

+

+

and its value is an indication of the energetic state of a cell; a value of unity means that all the adenine nucleotide is present as ATP and the cell is in its maximum energetic state. Atkinson suggested that it is the energy charge which regulates the pathways that produce and utilize high energy compounds, thereby ensuring that these processes are maintained in a steady state for optimum cellular economy; an energy charge of about 0.85 was considered to represent the equilibrium point for the bala,nce of the energy-producing and utilizing reactions. Chapman et al. (197 1) have shown that the energy charge of Escherichia

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cobi growing in glucose-ammonium salts medium approximated to 0.8, that when growth ceased in the presence of an excess of the carbon and energy source (glucose) it then declined slowly to 0.6 without significant loss of viability, but that when the energy charge had fallen below 0.5 the cells began t o die rapidly. Their results suggested that growth could occur only a t energy charge values above or equal to 0.8, that viability was maintained a t values between 0.8 and 0.5 and that death occurred when the energy charge fell below 0.5. They did not measure the glycogen content ofthe bacteria so that it is not possible to correlate energy charge and survival with the presence of this storage material which would certainly be expected t o be present in nitrogen-limited cells. As energy-storage compounds must necessarily be involved in the overall economy of the microbial cell it would seem reasonable t o suppose that their biosynthesis and subsequent degradation might be in some way controlled by the energy charge. At the time of writing there is, unfortunately, relatively little information available on this particular aspect of reserve materials but it is worth noting that the biosynthesis of glycogen and polyphosphate both involve the direct utilization of ATP whereas that of poly-/3-hydroxybutyrate does not, a feature unique amongst such compounds.

D. MUTANTSDEFECTIVE IN STORAGE POLYMER SYNTHESIS While the isolation of mutants that are unable to accumulate storage compounds is obviously of considerable significance in relation to the light they may throw on the metabolic pathways involved in the biosynthesis of these materials, and their regulation, there are also important applied aspects. Current interest in the production of single cell protein focuses attention on means for securing the maximum possible protein yield. However, optimum conditions for biomass synthesis often produce cells which have accumulated reserve materials and the products may therefore be undesirable. If stable mutants which did not synthesize reserve materials could be obtained then this particular problem would be overcome. I n this latter connection Schlegel et al. (1970) and Schlegel and Oeding (1971) have isolated PHB-less mutants of Hydrogenomonas H16, a hydrogen-utilizing organism which can be used to achieve the transformation of electrical energy into foodstuff by deriving the necessary hydrogen and oxygen for its growth from the electrolysis of the nutrient medium in the culture vessel (Schlegel, 1969). Relatively few mutants lacking reserve materials have, in fact, so far been isolated but valuable information concerning polyphosphate metabolism in Aerobacter aerogenes (Harold, 1964) and glycogen metabolism in Escherichia coli (Damotte et ab., 1968; Govons et al., 1969) and

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Saccharomyces cerevisiae (Chester, 1967a, b ; Chester and Byrne, 1968) has stemmed from such mutants and is discussed subsequently in the appropriate sections. 11. Glycogen and Glycogen-like Reserves A. GENERALCONSIDERATIONS

It is generally true that micro-organisms which accumulate glycogen and glycogen-like materials do so under conditions where growth is limited by the supply of utilizable nitrogen and there is a plentiful supply of exogenous carbon, but exceptions are known (see, for example, Kuenzi and Fiechter, 1972; Section IIF 1, p. 165). Wilkinson and Munro (1967) showed that limitation of the sulphur, potassium or carbon and energy source of Bacillus megaterium in a chemostat did not result in significant glycogen deposition whereas nitrogen limitation did so. Some organisms, such as Rhodospirillum rubrum and B. megaterium, can accumulate reserves additional to glycogen and the nature of the carbon source may then play an important role in determining the actual storage compound synthesized (Stanier et al., 1959;Wilkinson and Munro, 1967). However, a report that the carbohydrate content of Escherichia coli decreased and the lipid content increased if the carbon source in an ammonium salts medium was progressively changed from glucose to acetate (Dagley and Johnson, 1953) could not subsequently be substantiated by Damoglou and Dawes (1968) who found that the lipid content was unaffected by the carbon source and remained constant a t 8-9% (w/w) irrespective of the conditions of growth. The rate of growth affects the quantity of glycogen accumulated and Holme (1957) demonstrated an inverse relationship between growth rate and the glycogen content with nitrogen-limited, glucose-grown cultures of E. coli B (Fig. 1).He and his colleagues went on to show that there are two main fractions of glycogen in this organism, one with a high molecular weight (40 to 90 x l o 6 daltons) and the other with a low molecular weight (4x l o 6 dsltons) and that the major portion of the glycogen that accumulates in slow-growing cells is the high molecular weight fraction while a t fast growth rates (when the cells contain less glycogen) the low molecular weight fraction comprises u p t o half of the total glycogen content (Holme et al., 1958; Holme, 1958). Herbert (1961) reported that under nitrogen-limiting conditions the glycogen content of Torulopsis utilis varied inversely with the extracellular NH,+ concentration and he suggested that glycogen synthesis might be inhibited by NH,+ ions. Dicks and Tempest (1967)subsequently found that the synthesis of glycogen by washed suspensions of

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A . aerogenes [which had been grown under potassium-limited conditions in a chemostat with excess of the carbon source (glycerol) and Mg2+] depended on the relative concentrations of extracellular K+ and NH,+ ; polysaccharide deposition was promoted by K+ and inhibited by NH,+. The mechanism of the effect was not elucidated but competition for a common transport pathway seemed unlikely. However, under other growth conditions the effect of potassium concentration could be different, for when K+ was increased from 5 to 200 mM in a phosphateI

1

1

0:

02

I

0.4

I

06 Flow rate ( I / h )

I

I

08

10

FIG.1. Output of bacteria and glycogen as a function of the flow rate in a continuous culture of Escherichia coli B growing with nitrogen (NH4+)limitation. From Holme (1957).

limited chemostat culture (dilution rate 0.1 h-I), the cellular carbohydrate content decreased from 18 to 8% of the dry weight. Dicks and Tempest (1967)therefore suggested that differences in media composition might well account for their finding that phosphate- and sulphatelimited organisms contained significant amounts of glycogen in contrast to the results of Holme and Palmstierna (1956) with E. coli. The accumulated polysaccharides are subsequently degraded during starvation and in several instances, such as A . aerogenes (Strange et al., 1961),E . coli (Dawes and Ribbons, 1963;Strange, 1968)andfltreptococcus mitis (van Houte and Jansen, 1970), the possession of glycogen reserves has been correlated with a prolonged viability of the organism. However, in the case in A . aerogenes it is possible that magnesium ions, associated with the glycogen, may have an important role in the extended period of survival (Strange, 1968; Tempest and Strange, 1966). On the other hand, polyglucose-rich Sarcina &tea died at a faster rate than cells without the polymer when starved in phosphate buffer (Burleigh and Dawes, 1967). The maximum synthesis of granulose in Clostridium pasteurianum and in Clostridium botulinum coincided with the onset of sporulation (Mackey and Morris, 1971; Strasdine, 1972) suggesting a possible role

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for glycogen in the formation of spores and the subsequent survival of the organism. Ribbons and Dawes (1963) showed that during the period of rapid catabolism of glycogen by starved suspensions of E . coli the formation of ammonia by endogenous metabolism was prevented, although protein turnover was occurring (Dawes and Ribbons, 1965). Further, Strange and Hunter (1967) suggested that ammonia (nitrogen)-limited cells of A . aerogenes were less susceptible t o substrate-accelerated death (Postgate and Hunter, 1963) by ammonia. On the basis of these findings Wilkinson and Munro (1967) have suggested that glycogen reserves could be used by the cell as a method for the detoxication of ammonia under certain conditions. I n our discussion of the synthesis of microbial glycogen the differences between synthesis by prokaryotes and eukaryotes will be emphasized, noting that the former organisms utilize ADP-glucose as the glucose donor to the polysaccharide primer, and that the rate-limiting step, which is subject to regulation during synthesis, is the formation of ADP-glucose. By contrast, UDP-glucose is the glucose donor of eukaryotes, and glycogen synthetase (UDP-glucose : glycogen a-4-glucosyltransferase) is the regulated enzymic step in biosynthesis. Activation of glycogen synthesis is achieved by many effectors but it is a general rule that effector specificity is dictated in some measure by the pathways used for carbohydrate metabolism. I n most systems that have been studied the balance of synthesis and degradation is maintained by a combination of precursor activation for synthesis and end-product inhibition for degradation, further, high adenylate energy charge, as defined by Atkinson (1968), facilitates synthesis and inhibits degradation, while low energy charge promotes degradation and inhibits synthesis.

B. THEOCCTJRRENCE OF GLYCOGEN AND GLYCOGEN-LIKE RESERVES Glycogen-like polyglucans are synthesized by a wide variety of both prokaryotic and eukaryotic organisms. Table 1 is not intended to be a complete survey of the occurrence of glycogen in micro-organisms, but it clearly indicates the widespread distribution of the polymer. It must be borne in mind that although many micro-organisms synthesize both intracellular and extracellular polysaccharides, in this review we are concerned only with intracellular materials which serve as reserves.

C. STRUCTURE OF MICROBIAL POLYGLUCANS Microbial glycogen-like materials differ widely in their structural characteristics. But a factor common to both microbial and mammalian

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TABLE1. Occurrence of Glycogen and Glycogen-like Reserves in Microorganisms Organism

Aerobacter aerogenes Agrobacterium tumefaciens Arthrobacter viscosus Bacillus cereus Bacillus megaterium Clostridium pasteurianum Escherichia coli Mycobacteriurm phtei Mycobacterium smegmatis Mycobacterium tuberculosis Rhodopseudomonas capsulatus Rhodospirillum rubrum Streptococcus mitis

Reference (a) PROKARYOTES Strange et al. (1961); Gahan and Conrad (1968) Madsen (1961a, b, c) ; Eidels et al. (1970) Mulder et al. (1962): Shen and Preiss (1966) Mulder et al. (1962) Barry et at. (1952) Gavard and Milhaud (1952); Robson et al. (1972) Holme and Palmstierna (1956); Preiss et al. (1966); Strange (1968) German et al. (1961); Antoine and Tepper (1969th) Antoine and Tepper (1969a) Chargaff and Moore (1944); Antoine and Tepper (1969a and b) Eidels et al. (1970) Stanier et al. (1959); Furlong and Preiss, (1969a); Paule and Preiss (1971) Gibbons and Kapsimalis (1963); van Houte and Jansen (1970); Builder and Walker (1970) (b) EUKARYOTES

Blastocladiella emersonii Dictyostelium discoideum Neurospora crassa Nostoc muscarum Saccharomyces cerevisiae Tetrahymena pyrvormis

Cantino and Goldstein (1961) ; Camargo et al. (1969) Ashworth and Watts (1970) ; Weeks and Ashworth (1972); Hames et al. (1972) Traut and Lipmann (1963); Shepherd and Segel (1969); Shepherd et al. (1969) Chao and Bowen (1971) Harden and Rowland (1901) ; Chester ( 1 9 6 7 4 ; Chester and Byrne (1968) Blum (1967) ; Blum (1970)

This list is not intended to be exhaustive. Generally the first reference records the initial report of the presence of glycogen in the organism and the other references cover recent developments.

glycogen is the possession of a-(1 + 4) glycosyl linkages and occasional a-(1 +-6) branch linkages. Streptococcus mitis glycogen is a highly branched polymer (Builder and Walker, 1970) as is the polymer from Nostoc muscarurn (Chao and Bowen, 1971). The glycogen of N . muscarum is in granule form with a n average chain length of 1 3 units, and it is

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highly branched. Distinct structures inside the glycogen granule have been revealed by electron microscopy. The N . muscarum glycogeniodine complex had a similar absorption spectrum to that obtained with glycogen from either oyster or rabbit liver. During the course of glycogen accumulation in Mycobacterium smegmatis, the degree of branching decreases and the sedimentation coefficient increases as the glycogen granules increase in size. Mycobacterium phlei possesses a glycogen of molecular weight 1-2 x lo8 (Antoine and Tepper, 1969s) compared with 8.2 x lo7 for E. coli (Holme et al., 1957) and 9.2 x lo6 for A . aerogenes (Levine et al., 1953). The butyric clostridia synthesize granulose, an intracellular glucan, whose structure resembles that of amylopectin (Gavard & Milhaud, 1952). The glycogen of Arthrobacter is a highly branched, short outer chain length, polyglucan with mean chain length of seven to nine units (Zevenhuizen, 1966) compared with 12-13 units for other bacterial glycogens. A comprehensive account of bacterial glycogen structure, including structural analysis and the results of enzymic degradation has been given by Zevenhuizen (1966), and yeast glycogen has been reviewed by Manners (1971). OF GLYCOGEN-LIKE RESERVES D. BIOSYNTHESIS

During recent years there has been tremendous activity in this field arising from the discovery that ADP-glucose was the glucose donor for the glycogen synthetase of bacteria. I n particular Preiss and his associates have made substantial contributions to our knowledge, and the regulation of biosynthesis of a-(l + 4) glucans by bacteria and plants ha,s been excellently reviewed by Preiss (1969). Microbial glycogen biosynthesis can conveniently be divided into two classes, one effected by prokaryotes and the other by eukaryotes. The common precursor in all glycogen synthesizing organisms is glucose 1-phosphate. I n prokaryotes, glucose 1-phosphate is adenylated to ADPglucose in a reaction requiring ATP and eliminating inorganic pyrophosphate. ATP + glucose 1-phosphate

+ ADP-glucose + pyrophosphate

(PP,)

This reaction is catalysed by ADP-glucose pyrophosphorylase, an enzyme found in many micro-organisms (Table 2a). The corresponding reaction in eukaryotes involves the conversion of glucose 1-phosphate t o UDP-glucose, utilizing UTP with the concomitant formation of pyrophosphate UTP

+ glucose 1-phosphate $ UDP-glucose + pyrophosphate (PPi)

The UDP-glucose pyrophosphorylase is found in several eukaryotes (Table 2b).

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The microbial synthesis of glycogen de novo is rare, a primer usually being required for the addition of a glucose unit in the reaction catalysed by glycogen synthetase (Glucosyl),+

ADP-glucose UDP-glucose

-

ADP (G1ucosyl)~+l + UDP

The presence of glucosyl transferase-branching enzyme activity TABLE2. The Occurrence of Nucleotide Diphosphate Pyrophosphorylases in Micro-organisms Organism

Reference

(a) Prokaryotes (ADP-GlucosePyrophosphorylases) Aerobacter aerogenes RibBreau-Gayon et al. (1971) Aerobacter cloacae RibBreau-Gayon et al. (1971) Agrobacterium tumefaciens Eidels et al. (1970) Arthrobacter viscosus Shen and Preiss (1966) Citrobacter freundii RibBreau-Gayon et al. (1971) Clostridium pasteurianum Robson et al. (1972) Escherichia aurescens RibBreau-Gayon et al. (1971) Escherichia coli B Preiss et al. (1966); Govons et al. (1969); Preiss et al. (1971) Rhodopseudomonas capsulatus Eidels et al. (1970) Rhodospirillum rubrum Paule and Preiss ( 1971) RibBreau-Gayon et al. (1971) Salmonella typhimurium RibBreau-Gayon et al. (1971) Serratia marcescens Builder and Walker (1970) Streptococcus mitis (b) Eukaryotes (UDP-Glucose Pyrophosphorylases) Blastocladiella emersonii Camargo et al. (1969) Dictyostelium discoideum Wright and Anderson (1959); Edmundson and Ashworth (1972) Neurospora crassa Shepherd et al. (1969); Tillez-Inon and Torres (1970) Saccharom yces cerevisiae Rothman and Cabib (1966, 1967a, b, 1970, 1971); Schlender et al. (1969); Chester and Byrne (1968) Tetrahymena pyriformis Blum (1970)

catalysing the formation of cr-l,B-linkages has been reported in Arthrobacter viscosus (Zevenhuizen, 1964) and E. coli (Sigal et al., 1965), while experiments with enzymes from A . aerogenes, which apparently can carry out de novo synthesis of glycogen in the absence of primer, suggest that synthesis may occur from the reducing end of the molecule by a mechanism not involving branching enzyme (Ga,hanand Conrad, 1968).

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E. A . DAWES AND P. J. SENIOR Glucose 6-phosphate

Glucose I-phosphate-

4

ATP or UTP Pyrophosphate ADP-Glucose (Proliaryotos) UDP-Glucose (Eukaryotes)

Glycogen primer

ADP or UDP

(glucosy1),

FIG.2. The pathway of microbial glycogen synthesis and degradation.

A general scheme for the biosynthesis and degradation of glycogen is shown in Fig. 2.

E. GLYCOGEN BIOSYNTHESIS BY PROKARVOTES Most glycogen-synthesizing bacteria possess, along with ADPglucose pyrophosphorylase, UDP-glucose pyrophosphorylase. Indeed, in the early work on glycogen synthesis in Agrobacterium tumefaciens it was believed that the UDP-glucose pathway operated (Madsen, 1961a, b, c). However, it has subsequently been clearly shown that only ADPglucose is significantly active in glycogen synthesis and therefore presumably UDP-glucose is utilized for the biosynthesis of other polysaccharides.

1. Enterobacteriaceue Many of the earlier studies (from 1966) were carried out by Preiss and Segel, and their collaborators, with Escherichiu coli ; in this work the

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enzymic reactions and their regulation were charted. More recently comparative aspects of the ADP-glucose-pyrophosphorylases of enteric organisms have been investigated by Ribkreau-Gayon et al. (1971) who studied Citrobacterfreundii, Salmonella typhimurium, Serratia marcescens, Escherichia aurescens, Aerobacter cloacae, Aerobacter aerogenes, Proteus vulgaris and Erwinia carotovora. With the exception of P. vulgaris and E . carotozjora, all these organisms possessed ADP-glucose pyrophosphorylase and ADP-glucose :crl,4-glucan-4-glucosy1transferase in high activities. I n most cases (except in S. marcescens) fructose 1,6-diphosphate activated the ADP-glucose pyrophosphorylase, and glycogen synthetase required a primer. Glycogen synthetase was not activated by glycolytic intermediates, NADPHz or pyridoxal phosphate. (a) Escherichia coli. The ADP-glucose pyrophosphorylase of E. coli B requires ATP, Mg2+and glucose 1-phosphate for activity (Preiss et al., 1966). Other nucleotide triphosphates, such as GTP, CTP, UTP, dATP, ITP and TTP, were active but a t only 1 to 4% of the rate with ATP; Co2+and Mn2+ could replace Mg2+.The E . coli enzyme was activated some 30- to 40-fold by fructose 1,6-diphosphate but only with respect to ADP-glucose pyrophosphorylase utilizing ATP, and activities with other nucleotide triphosphates were unaffected. Sedoheptulose 1,7diphosphate and u-arabinitol 1,5-diphosphate, like fructose 1,6-diphosphate, produced some 30- to 40-fold activation but other compounds (glucose 1,6-diphosphate, uL-glyceraldehyde 3-phosphate, 2-phosphoglycerate, erythrose 4-phosphate, phosphoenol pyruvate and 3-deoxy2-0x0-6-phosphogluconate)were less effective. At high concentrations 3-phosphoglycerate, acetyl phosphate and acetyl-CoA produced slight activation as did phosphohydroxypyruvate and ribulose 1,5-diphosphate. Compounds which did not activate included fructose 1-phosphate, glucose 6-phosphate, glucosamine 6-phosphate, fructose 6-phosphate, ribose &phosphate, dihydroxyacetone phosphate, S’,B’-cyclic-AMP, lactate, pyruvate, fructose, glucose and tricarboxylic acid cycle intermediates. I n their discussion of these findings Preiss et ab. (1966) suggested that since most of these activators are either substrates or inhibitors of rabbit muscle aldolase, there may be similarities between the aldolase active sitefs) and the activation site(s) for E. coli ADP-glucose pyrophosphorylase. A flaw in their argument, as they pointed out, is the absence of any activation by dihydroxyacetone phosphate. I n the presence of Mg2+, plots of activator concentration versus initial rate of reaction were sigmoidal indicating that, at this stage in glycogen biosynthesis, regulation is effected by allosteric interactions. The enzyme also displayed allosteric kinetics with respect to ATP. Fructose 1,6-diphosphate and other activators acted by decreasing the 8

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K , values for glucose 1-phosphateand ATP (11-and 13-foldrespectively) while at the same time increasing V,,,, for ADP-glucose synthesis seven-fold. Inhibition of ADP-glucose formation was effected by AMP ( K ,= 84 pM). Again, as for activation, inhibition was by allosteric interactions. However, AMP was only effective in the presence of either fructose 1,6-diphosphate or other activators. This latter form of control would ensure that when the energy charge was low but the supply of hexose phosphates in excess, glucose 6-phosphate would be directed towards oxidation and subsequent ATP generation, rather than t o the production of reserve glycogen. Preiss et al. (1971),working with a glycogen-deficient mutant of E . coli (B+)(SG-14))have shown that while the activity of glycogen synthetase was roughly the same in wild-type and mutant, the ADP-glucose pyrophosphorylase activity of the mutant was only 23% of that of the parent strain. The parent and mutant enzymes were stimulated by fructose 1,6-diphosphate but the mutant enzyme required much higher concentrations of ATP, Mg2+and activator. The wild-type enzyme was AMP-sensitive, and NADPH, stimulated, whereas the SG-14 enzyme was unaffected by either AMP or NADPH,. However, even with this decreased activity, the mutant still accumulated large quantities of glycogen (some 55 t o 75% of wild-type content) because even though the activators were less effective, inhibitors such as AMP also had lowered activities. They concluded that in glucose-grown aerobic cultures of E . coli, fructose 1,6-diphosphate is the main activator for glycogen synthesis. Support for their in vitro findings existed in the literature with the results of i n vivo experiments previously carried out by Hempfling et al. (1967), who had shown that E. coli grown under these conditions contained 0.6 to 1.2 mM fructose 1,6-diphosphate. Activation of the ADP-glucose pyrophosphorylase of E . coli B by NADPH, and pyridoxal phosphate, reported by Preiss et al. (1971), may play a significant role in the activation of glycogen synthesis under other growth conditions. Thus NADPH, activation may be of considerable importance for the bacterial cell when there is a temporary imbalance of the NAD(P)H,/NAD(P) ratio caused, for instance by the imposition of oxygen-limitation or sudden anaerobiosis. The significance of pyridoxal phosphate activation is more difficult to assess. While the role of this compound as a prosthetic group for many enzymes, including glycogen phosphorylase, is well documented, the possibility of a physiological role for the free coenzyme seems more remote. A possible role for cyclic-AMP in glycogen biosynthesis in E. coli is suggested by the results of Moses and Sharp (1970) who found that

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cyclic-AMP stimulated the incorporation of 32P0,3-into fructose 1,6-diphosphate. They concluded that phosphofructokinase was, in some way, activated by cyclic-AMP. As we have already described in detail the stimulatory effects of fructose 1,g-diphosphate on the ADPglucose pyrophosphorylase of E. coli, it is tempting to suggest that cyclic-AMP may activate phosphofructokinase, increase the pool size of fructose 1,6-diphosphate (the most potent ADP-glucose pyrophosphorylase activator), stimulate the rate of formation of ADP-glucose and thus increase the glycogen content. Stimulation of phosphofructokinase would, however, decrease the energy charge and this would tend to counteract the activating effects of fructose 1,6-diphosphate. It has been shown that the levels of both ADP-glucose pyrophosphorylase and glycogen synthetase increase markedly towards the end of exponential growth of E . coli in batch culture and preceding the accumulation of glycogen, although the effect is less dramatic with glucose-ammonium salts than with glucose-yeast extract media (Preiss, 1969). Apparently derepression of these enzymes occurs when the organism ceases to grow, and the observation that chloramphenicol inhibits glycogen accumulation in the stationary phase (Cattaneo et al., 1966) suggests that protein synthesis is involved in the mechanism of derepression in E . coli. The increase in the levels of the two enzymes in the stationary phase is independent of the carbon source or composition of the medium. Preiss (1969) has drawn attention t o the similarity between the derepression of the glycogen-synthesizing enzymes and the derepression of the synthesis of various proteins required for sporulation in Bacillus species, noting that both glycogen synthesis and spore formation may be regarded as survival mechanisms and suggesting that the controlling factors for both processes might conceivably possess basic similarities. During the period when the rate of glycogen synthesis in E . coli is maximum, the amounts of the pyrophosphorylase and synthetase present in the cell are 6- to 10-fold greater than would be required for the observed in vivo rate of glycogen accumulation, an observation which indicates that other factors besides genetic regulation control the activities of the glycogen biosynthetic enzymes. Consequently the allosteric control of ADP-glucose pyrophosphorylase is probably of paramount importance in modulating the rate of glycogen synthesis. Although glycogen synthetase has been found in both the soluble and particulate fractions of bacterial cells, in the Enterobacteriaceae the bulk of it (70-95%) is particulate and sedimented between 30,000 and 105,000 g in the same fractions that contain most of the glycogen (Greenberg and Preiss, 1964, 1965; Ghosh and Preiss, 1965; Preiss, 1969). These observations suggest that the synthetase may be bound t o

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the glycogen to form a particulate enzyme complex. Fox [unpublished results quoted by Preiss (1969)l found that a-amylase solubilized the enzyme without loss of activity but the nature of the binding is not yet known. (b) Aerobacter aerogenes. Kindt and Conrad (1967), in the course of investigations of the role of glycogen primer in the biosynthesis of glycogen by A . aerogenes, noticed that while a primer was required for glycogen synthesis in vitro by a cell-free extract of A . aerogenes NCTC 243, de novo biosynthesis could be achieved with in vitro extracts of a different strain, A3 (S,). When grown on nitrogen-limited complex or nitrogen-limited glucose salts media NCTC 243 accumulated glycogen, whereas A3 (S,) only accumulated glycogen in the simple medium. Further work on the A3 (S,) strain by Gahan and Conrad (1968) proved conclusively that de novo biosynthesis could occur, thus challenging the suggestions of Leloir and Cardini (1962), Greenberg and Preiss (1965), Preiss and Greenberg (1965) and RibBreau-Gayon et al. (1971) that all glycogen synthetases, regardless of source, require a glycogen primer molecule. The A3 (S,) system was shown to consist of two fractions, one soluble and the other sedimentable a t 170,000 g (150 min.). The glycogen synthetase in the pellet fraction of a centrifuged crude extract could synthesize glycogen from ADP-glucose. However, activity was greatly stimulated by a supernatant protein fraction : supernatant activator plus glycogen synthetase a,nd glycogen gave full activity. Although glycogen stimulated the synthesis of glycogen, certain lipopolysaccharide fractions from various strains of A . aerogenes spared the effect of glycogen, as did the surfactant Triton X-100, suggesting that glycogen was not stimulating through its action as a glucosyl acceptor molecule. The supernatant activator, like glycogen synthetase, was found to be glycogenfree and during growth the specific activity of the supernatant activator remained constant. Towards the end of exponential growth the activity of glycogen synthetase increased 20-fold suggesting that the rise in glycogen content a t the onset of the stationary phase was not due to changes in specific activity of the supernatant activator, but to regulatory action a t the level of ADP-glucose pyrophosphorylase. (c) Salmonella typhimurium. RibBreau-Gayon et al. (1971) partially purified the ADP-glucose pyrophosphorylase from S. typhimurium and C. freundii and carried out a detailed investigation of the kinetics of regulation. Theeffectsof fructose 1,6-diphosphate,NADPH,, pyridoxal phosphate, glyceraldehyde 3-phosphate, and glycerol 1,3-diphosphate on the rate

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of ADP-glucose formation by the ADP-glucose pyrophosphorylase are shown in Fig. 3. Activation ranges between 16- and 35-fold. The activation curves are sigmoidal and the corresponding Hill plots reveal that the enzyme binds, in all cases, two or more activator molecules. An almost

4

i

g OO 20 I

I

!

!

.

IO-~

IO-~

0 05

I

IO-~

,Activator concentrotion ( M )

0 10

Pyridoxal 5'-phosphate (mM)

Activator concentration (mM 1

FIG.3. Activation of ADP-glucose pyrophosphorylase of Salmonella typhimurium. The inset of the upper graph is a Hill plot of the data. V,,, were estimated from reciprocal plots of rate versus activator concentration ;Av is the increase in velocity due to addition of activator, and n is the interaction coefficientof the Hill equation. Ao.5is the concentration of activator giving 50% of the maximum stimulation. Pyridoxal phosphate, w ; fructose 1,6-diphosphate, 0 ; glycerol 1,3-diphosphate, A ;NADPH,, o ; glyceraldehyde 3-phosphate, A . Values for A and n for each activator were : A n

tw)

Pyridoxal phosphate 9.4 Fructose diphosphate 98 NADPH, 105 Glyceraldehyde 3-phosphate 690 Glycerol 1,3-diphosphate 305 From Rib6reau-Gayon et al. (1971).

3.1 2.3 2.3 2.3 2.2

152

E.A. DAWES AND P. J. SENIOR

identical pattern was observed with C. freundii but activator interaction coefficients were lower than those recorded with S . typhimurium. The ATP-saturation curve for the S. typhimurium enzyme (Fig. 4) reveals that activators function in two ways: (a) by decreasing the

Concentrotion of ATP ( m M1

I

I

0

I

I

10 /%--iO Concentration of ATP [mM)

5

Fig. 4. The effect of ATP on ADP-glucose synthesis by the ADP-glucose pyrophosphorylase of Salmon,ella typhimurium. The values So.5are the concentrations of substrate required for 50% of maximum velocity. No activator, A ; pyridoxal 5-phosphate, 0 ; fructose l,g-diphosphate, o ; NADPH,, A. The upper graph is a Hill plot of the data, and n the interaction coefficient. The and n values for the various activators were: 80.5

(mM) 0.22 Pyridoxal phosphate 0.48 Fructose diphosphate NADPH, 0.37 Control 24 From RibBreau-Gayon et al. (1971).

n

1.0 2.2 1.5 1.5

ENERGY RESERVE POLYMERS I N MICRO-ORGANISMS

153

concentration of ATP required for half maximum velocity (So.,) and (b) increasing the V,,, of ADP-glucose formation. Interactions were allosteric in nature except for pyridoxal phosphate, for which an n value of 1.0 was recorded. The activators employed in the experiment of Fig. 4 also decreased the flo.5for glucose 1-phosphate and ADP-glucose. Similar results were obtained with the C. freundii enzyme. Table 3 summarizes the kinetic parameters of the ADP-glucose pyrophosphorylase from S. typhimurium and, for comparison, the relevant data for the C. freundii enzyme are given. The 8. typhimurium enzyme is inhibited by AMP, ADP and Pi and, of these, AMP is the most effective. However, the presence of an activator modulated these effects. Thus, in the presence of 1.0 mM fructose 1,B-diphosphate, 0.11 to 0.13 mM AMP was required for 50% inhibition ( I o . 5 ) If . the fructose 1,6-diphosphate concentration was decreased to 0.25 mM, then I,,.5 for AMP became 0.028 mM. The AMP interactions were all allosteric. (d) General conclusions concerning the Enterobacteriaceae. The comparative studies of RibBreau-Gayon et al. (1971) suggest that glycogen synthesis in the Enterobacteriaceae is regulated a t the level of the enzymic reaction which forms ADP-glucose. The ADP-glucose pyrophosphorylases from E. coli, S. typhimurium and C. freundii are all very similar in their kinetics and regulatory properties. Table 4 summarizes the activation by various metabolites of ADP-glucose pyrophosphorylases of the enteric organisms studied by these workers. I n the majority of cases fructose 1,6-diphosphate, pyridoxal phosphate and NADPH, are the most potent activators. Activators of the enzyme apparently exert four different effects : namely, (i) they increase the V,,, of ADP-glucose synthesis; (ii) they increase the apparent affinities of the enzyme for the substrates; (iii) AMP, ADP and Pi inhibition is modulated by the presence of an activator; and (iv) the presence of an activator allows the enzyme to be fully inhibited by AMP, ADP and Pi. Energy charge (Atkinson, 1968) is of major importance in regulating glycogen synthesis such that under conditions of high energy charge, i.e. high ATP concentration, and in the presence of excess carbohydrate, glycogen synthesis will be stimulated. Under conditions of low energy charge, i.e. high AMP and low ATP, glycogen synthesis will be inhibited. The activation of ADP-glucose pyrophosphorylases from Enterobacteriaceae by NADPH, is particularly interesting. It has been demonstrated that the availability of NADP is the significant factor governing the operation of the oxidative pentose cycle in E. coli (Model and Ritten-

TABLE3. Activation of RDP-glucose pyrophosphorylases by various metabolites Activity (nmoles ADP-glucose formed in 10 min)

Activator None Pyridoxal 5’-phosphate Fructose 1,6-diphosphate NADPH, NADP Glyceraldehyde 3-phosphate 2-Phosphoglycerate 3-Phosphoglycerate Phosphoenolpyruvate Glucose 1,6-diphosphate 6-Phosphogluconate 3-Deoxy-2-oxogluconate6-phosphate Ribose 5-phosphate Glycerol 1,3-diphosphate Pyridoxal D-Arabinitol 1,5-diphosphate After RibBreau-Gayonet al. (1971).

Concentration (mM)

0.05 1.5 1.0 1.0 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 0.5 1.0

Aerobacter cloacae 1.2 9.6 8.5 7.5 5.3 7.5 8.9 6.2 8.6 3.75 3.5 5.6 3.2 6.1 0.96 -

Aerobacter aerogenes 0.70 21.1 19.3 17.1 15.1 17.8 20.0 12.7 21.9 11.8 7.8 17.9 5.7 15.6 0.74 -

Escherichia aurescens

0.40 20.9 15.8 13.5 2.1 12.0 5.8 1.45 1.60 4.55 0.99 4.32 2.72 7.5 0.40 -

Corynebacterium freundii

Salmonella t yphirnurium

6.7 19.8 18.5 16.0 7.5 15.0 12.5 6.7 17.3 6.0 8.8 6.9 6.7

0.46 22.5 15.6 13.9 4.4 16.6 8.2 1.42 6.2 2.2 1.3 6.8 0.67 10.6 0.50 19.1

TABLE4. Kinetic parameters of the ADP-glucose pyrophosphoryleses from Salmonella t y p h i m u r i u m and Corynebacterium f r e u n d i i ~

Enzyme source Salmonella t yphimurium

Corynebacterium f r e u n d i i

Substrate

Activator (mM)

None FDP, 1.0 PLP, 0.05 NADPH2, 0.5 Glucose 1-phosphate None FDP, 1.0 PLP, 0.05 NADPH,, 0.5 None ADP-glucose FDP, 0.8 PLP, 0.04 NADPH,, 0.8 None ATP FDP, 1.0 PLP, 0.05 Glucose 1-phosphate None FDP, 1.0 PLP, 0.05 NADPH,, 1.0 None MgC1, (synthesis) FDP, 1.0 Pyrophospha,te None FDP, 0.75 None ADP-glucose FDP, 0.75

ATP

vm,,

80.5

(mM)

n

2.4 0.48 0.22 0.37 0.21 0.034 0.042 0.039 0.57 0.10 0.046 0.10 0.72 0.26 0.15 0.13 0.05 0.07 0.09 3.4 1.0 0.5 0.28 0.38 0.12

1.5 2.2 1.0 1.5 0.9 1.06 1.05 1.06 1.8 1.9 1.1 1.7 2.5 1.5 1.0 0.9 0.9 1.0 0.9 3.5 3.4 0.92 0.84 2.0 1.3

(pmoles mg-I 10 min-')

No activator +FDP, 1.0 mM +PLP, 0.05 mM +NADPH,, 1.0 mM

No activator +FDP, 0.8 mM +PLP, 0.04 mM +NADPH,, 0.8 mM

1.2 4.8

6.4 4.3

4.0 9.6 11.2 8.6

3 g

g M 2 d

2

B 8

NOact,ivator +FDP, 1.0 mM +PLP, 0.05 mM +NADPH,, 1.0 mM

No activator +FDP, 0.75 mM

4.0

4.5

is the concentration of substrate or metal ion required + Abbreviations: FDP, fructose 1,6-diphosphate;PLP, pyridoxal 5'-phosphate. 01 for half maximum activity and n. is the interaction constant obtained from Hill plots. After Ribbreau-Gayon et al. (1971). 01

156

E. A. DAWES AND P. J . SENIOR

berg, 1967); when the NADPH, concentration increased, pentose cycle activity diminished. Consequently when growth becomes nitrogenlimited in the presence of excess carbohydrate and the demands for NADPH, for biosynthesis dramatically decrease, the concentration of NADPH, will increase and that of NADP decrease. These conditions will favour glycogen synthesis at the expense of pentose cycle activity. However, a similar situation could presumably be brought about by the imposition of oxygen-limitation on an aerobic culture of E . coli, although in this case ATP production via oxidative phosphorylation would be decreased, and thus the energy charge might not be sufficiently great to bring about activation of glycogen synthesis.

2. Arthrobacter viscosus A somewhat different pattern of regulation of ADP-glucose pyrophosphorylase exists in Arthrobacter viscosus (Shen and Preiss, 1966). Fructose 6-phosphate, deoxyribose 5-phosphate, pyruvate and ribose 5-phosphate, when added separately a t 2.0 mM, caused, respectively, eight-, seven-, six-, and five-fold activation of the ADP-glucose pyrophosphorylase. Over the concentration range of 1 t o 5 mM, glucose 6-phosphate, glucosmmine 6-phosphate, mannose 6-phosphate, galactose 6-phosphate, xylose 5-phosphate, ribulose 5-phosphate, sedoheptulose i’-phosphate, 2-deoxyglucose 6-phosphate, ribose 1-phosphate, deoxyribose 1-phosphate, glyceraldehyde %phosphate, dihydroxyacetone phosphate, 3-phosphoglycerate, fructose, sucrose, acetylphosphate and fructose 1,6-diphosphate were inactive as were various tricarboxylic acid cycle interniediates. 2-Oxobutyrate stimulated the enzyme slightly possibly because of its chemical similarity to the effector pyruvate. As with E . coli the activators function allosterically, decreasing the K , for ATP, but unlike E. coli they do not change the K , for glucose 1-phosphate. Inhibition of the enzyme by AMP, ADP, GMP, GDP, Po4’- and, surprisingly, phosphoenolpyruvate, was overcome completely by fructose G-phosphate and the other activators. Another similarity of the A . viscosus system with that of E. coli was the insensitivity to the activators of ADP-glucose pyrophosphorylase of the UDP-glucose and TDP-glucose pyrophosphorylases. More recent work with the E. coli enzyme by Gentner and Preiss (1968) has shown that AMP inhibits the enzyme by increasing the K , for ATP. The conditions required for the accumulation of glycogen in A. viscosus are either a low p H or nitrogen- or phosphorus-limitation of growth (Shen and Preiss, 1966) and under these circumstances glycogen is accumulated to the extent of 30 to 80% of the cell dry weight. These workers suggest that under conditions of high ATP and low AMP concentrations, and of excess carbon supply, the rate of ADP-glucose

ENERGY RESERVE POLYMERS IN MICRO-ORGANISMS

157

formation would increase and, since ADP-glucose synthesis is considered to be the rate-limiting step in glycogen synthesis in prokaryotes, the overall rate of glycogen synthesis also would increase.

3. Rhodospirillum rubrum Pyruvate was the sole activator of the ADP-glucose pyrophosphorylase from R. rubrum and glycolytic and pentose phosphate pathway intermediates were not active (Furlong and Preiss, 1969a). Pyruvate was shown to increase the V,,, by two and one half- to three-fold while a t the same time decreasing the So.5value (i.e. the substrate concentration a t half maximum velocity of reaction) for ATP lo-fold; the effector action was by allosteric interaction. ATP decreased the binding coefficient ( n )for pyruvate and pyruvate decreased the n value for ATP. The enzyme became totally dependent on the presence of pyruvate when the concentration of ATP was below 1 mM. Thus we have another example of precursor activation of glycogen synthesis. Pyruvate is the product of the light-dependent C0,-fixation reaction involving ferredoxin and acetyl-CoA which has been shown to operate in R. rubrum (Buchanan et al., 1967), thus Ferredoxiqredt+ acetyl-CoA + C 0 2 + ferredoxiq,,)

+ pyruvate

Consequently, the central position occupied by pyruvate in the carbon metabolism of R. rubrum is reflected by the fact that pyruvate is the sole activator for ADP-glucose pyrophosphorylase irrespective of whether the bacteria are grown in the light or dark on a variety of carbon sources (Furlong and Preiss, 196913). Furlong and Preiss (1969a) recorded the effect of various activators on many different microbial ADP-glucose pyrophosphorylases, and concluded that the mode of carbon metabolism dictated, to a large extent, the activator specificity of the enzyme.

4 , Rhodopseudomonas capsulatus and Agrobacterium tumefaciens (a) Comparison of two Entner-Doudoroff pathway-utilizing organisms. An excellent comparative survey of the ADP-glucose pyrophosphorylases of R. capsulatus and A . tumefaciens has been carried out by Eidels et al. (1970). I n crude preparations of the enzyme from R. capsulatus, fructose 6-phosphate, pyruvate, 3-deoxy-2-oxo-6-phosphogIuconate [an intermediate in the Entner and Doudoroff (1952) pathway of glucose metabolism] and ribose &phosphate stimulated the activity of the pyrophosphorylase 5.7-fold, 4.0 to 4.8-fold, 3 to &fold and 1.3 to 1-5-fold, respectively. With the purified enzyme the findings were slightly different;

158

E. A. DAWES AND P. J. SENIOR

3-deoxy-2-oxo-6-phosphogluconate lost a great deal of its stimulatory capacity, presumably because in crude extracts some of it was metabolized to pyruvate and fructose 6-phosphate. The enzyme was inhibited by fructose 1,6-diphosphate, 6-phosphogluconate, sedoheptulose 7phosphate, sedoheptulose 1,7-diphosphate, erythrose 4-phosphate, dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, 3-phosphoglycerate or acetylphosphate (each a t 1 mM). All these compounds inhibited activity some 70 t o 80% but the inhibitory effects could be completely overcome by the addition of 1 mM fructose 6-phosphate. Glucose 6-phosphate, pyruvate, citrate and acetyl-CoA (each a t 1 mM) inhibited 20%. Phosphoenol pyruvate was an extremely potent inhibitor and inhibition was only partly overcome by fructose 6-phosphate. The A . tumefuciens enzyme gave similar results with fructose 6phosphate, and with both organisms it was shown that fructose 6phosphate functions as an activator by lowering the K , values for ATP, glucose 1-phosphate and pyrophosphate. Likewise reversal of the inhibitory effect of ADP, AMP and phosphoenol pyruvate by fructose 6-phosphate was achieved by a decrease of the binding coefficient (n) for these effectors. Eidels et ul. (1970) concluded that in both R. capsulutus and A . tumefuciens regulation of glycogen synthesis occurs a t the level of ADP-glucose synthesis, i.e. a t the rate-limiting reaction. Unlike enteric organisms such as E . coli, where fructose 1,6-diphosphate, pyridoxalphosphate and NADPH, activate ADP-glucose pyrophosphorylase, organisms metabolizing glucose either partly or wholly by the EntnerDoudoroff pathway show different activator specificity. Thus glycogen synthesis in enteric organisms is stimulated by intermediates of the glycolytic sequence whereas in organisms possessing the EntnerDoudoroff pathway pyruvate is the stimulator. 5. Streptococcus mitis Streptococcus mitis, a facultative anaerobe which has been associated with dental caries, can accumulate up t o 37% of its dry weight as a highly branched glycogen-like reserve material when grown anaerobitally on 2% tripticase plus 0.1% glucose (Builder and Walker, 1970). Previous studies by Gibbons and Kapsimalis (1963) showed that growth on 0.1% glucose led to storage of polysaccharide immediately after inoculation, as indicated by an increase in the carbohydrate to bacterial nitrogen ratio (Fig. 5a). When growth ceased due to glucose exhaustion, glycogen degradation was accompanied by a decrease in turbidity without loss of bacterial nitrogen. These results suggested a reserve function for glycogen in this organism.

ENERGY RESERVE POLYMERS I N MICRO-ORGANISMS

159

When these workers repeated the experiment in the presence of excess glucose ( l % ,w/v), they found that the glycogen content of the cells increased rapidly during the exponential growth phase (Fig. 5b), until a point was reached where the carbohydrate to bacterial nitrogen ratio

.-.

5 (b)

.ae

4-

5

...ii

3

e

21

...-~ ? .P

- 1.0 3 - 0.75 :. U

0,

2 "O

- 1.50 - 1.25

.

A-~-~-A

m

c

I.75

-0.50

/A

- 0.25 w

lr/A

u

0

I

I

'

I

I

.

I

'

I

I

I

'

'

N

I

-0

remained constant, indicating that the rate of glycogen synthesis was proportional t o the rate of biosynthesis of bacterial nitrogen-containing compounds. At the end of exponential growth the glycogen content again increased rapidly and remained a t a high level for a further six to eight hours during the stationary phase. Glycogen was not degraded during this period. Nitrogen-limited populations in phosphate buffer plus glucose, accumulated massive quantities of polysaccharide.

160

E.

A. DAWES AND P.J . SENIOR

When organisms containing polysaccharide were incubated anaerobically in phosphate buffer, catabolism of carbohydrate was accompanied by production of an equivalent weight of lactic acid. The acid produced by packed glycogen-rich cells was sufficient to maintain the p H a t between 5.7 and 6.0 for two hours even in the presence of a continuous flow of 67 mM-phosphate buffer, pH 7-0. I n the absence of buffer the pH decreased to 4-7. The high rate of lactic acid formation, derived from glycogen catabolism in the absence of exogenous carbohydrate, may be of etiological significance in the creation of dental caries.

Storage period (h)

FIG.6. Survival of glycogen-positiveand glycogen-negative strains of Streptococcus mitis when starved in phosphate buffer, pII 6.5, a t 37°C. Glycogen-positive ( 0 ) and negative (M)strains were grown for 6 h in 1% glucose trypticase broth to obtain exponential-phase cells. Glycogen-positive ( A )and negative (v) strains were grown in medium containing 0.1% glucose for 16 h to obtain stationary-phase cells. The corresponding open symbols record the glycogen content as the extinction a t 565 nm of the polysaccharide-iodine complex (Eps-I). The authors state that, for suspensions containing lo9 cells /ml, an Eps-I value of 0.4is equivalent to a glycogen content of 50% of the bacterial dry weight. (From van Houte and Jansen, 1970).

Builder and Walker (1970) came to similar conclusions concerning the role of reserve carbohydrate of S. mitis and extended the studies of Gibbons and Kapsimalis (1963) to include the enzymology of glycogen synthesis. As with aerobic prokaryotes the substrate for the constitutive glycogen synthetase was ADP-glucose and the synthetase was unaffected by either 5 mM-glucose,glucose 6-phosphate, ribose 5-phosphate, fructose 6-phosphate or fructose 1,g-diphosphate. The survival characteristics of S. mitis have been investigated by van Houte and Jansen (1970) who showed that a washed suspension of glycogen-rich exponentially-growing cells (in 20 mM-phosphate buffer, pH 6-5, containing 0.13 M-NaCl, a t 37°C) survived well during the period when glycogen was being degraded. Cells which possessed little or no glycogen died rapidly (Fig. 6). The viability of glycogen-rich cells decreased by 40% during a 16-hour storage period, with a concomitant

ENERGY RESERVE POLYMERS I N MICRO-ORGANISMS

161

decrease in the amount of lactic acid produced. However, the survival of glycogen-free cells over the same storage period was only 0.01 %. Thus, although glycogen in S. mitis appears t o obey the criteria laid down by Wilkinson (1959) for a reserve material, the synthesis of glycogen during exponential growth reported by Gibbons and Kapsimalis (1963), is anomalous. The oral environment of S. mitis may have selected an organism which produces glycogen whenever exogenous glucose is in excess. As the presence of oral carbohydrate is intermittent, an organism capable of storing reserve glycogen under these conditions would be selected since degradation of reserve material during starvation would maintain viability.

6 . Clostridium pasteurianum Recent investigations by Morris and his colleagues have revealed that granulose synthesis in Cl. pasteurianum possesses regulatory features unlike any previously recorded in bacteria. Two wild type organisms, which differed in being good and poor spore-formers respectively, also differed in their patterns of granulose accumulation. The former produced little polysaccharide during exponential growth but synthesized this polymer in amounts up to 60% of its dry weight either just prior to, or during, initiation of sporulation (Mackey and Morris, 1971);the latter organism formed granulose throughout exponential growth, but only to some 15% of the dry weight. They have therefore investigated the possibility of a link between the pattern of granulose accumulation and the capacity to sporulate well. Robson et al. (1972) obtained granulosedeficient mutants of C1. pasteurianum and observed that the exponential growth rates of these mutants were the same as that of the parent strain, thus demonstrating that granulose biosynthesis is not a pre-requisite for growth. Granulose biosynthesis in CI. pasteurianum involved the customary two enzymes, ADP-glucose pyrophosphorylase and glycogen (granulose) synthetase. The activity of the former enzyme was not enhanced by glycolytic sequence intermediates, pyruvate, NADPH, or pyridoxal 20 mM) were required phosphate, and high concentrations of ADP (ai, for inhibition; AMP and pyrophosphate were without effect. The glycogen synthetase, while specific for ADP-glucose (K,,,, 25 pM), was not activated by hexose phosphates. However, ADP, AMP and pyridoxalphosphate all inhibited the enzyme. These findings suggest that glycogen biosynthesis in Cl. pasteurianum although probably regulated by the energy charge status of the organism (Shen and Atkinson, 1970), is not affected by the allosteric activators that typify the fine control of glycogen biosynthesis in other bacteria. The clostridial system may possibly represent a cruder, less evolved

162

E. A. DAWES AND P. J. SENIOR

regulatory system in which glycogen biosynthesis is an all-or-nothing phenomenon dictated solely by the availability of carbon as glucose 1-phosphate and resulting in the manufacture of the polymer under all growth conditions. Robson and Morris (unpublished observations) have also investigated the role of granulose in relation to the survival of Cl. pasteurianum. To avoid the complications presented by sporulation they used the poorsporulating wild type and a granulose-negative (granulose synthetase deficient) mutant derived from it, which sporulated t o the same low extent in liquid media. When both organisms were held in carbon-free basal medium for 30 hours neither lost viability, although the granulose of the wild type (22% of the dry weight) was virtually completely utilized in 21 hours; under these conditions possession of the carbohydrate apparently does not confer advantages for maintenance of viability. Further experiments in which the organisms were allowed to remain in the growth medium after growth had ceased and in the presence of exogenous glucose, did, however, reveal some differences between the wild type and mutant. If the p H was allowed to remain a t 4.4,the value attained a t the end of growth, there was little loss of viability with either organism over the initial 8 hours but thereafter the mutant died more rapidly than the wild type. When the p H of the fully grown medium was adjusted to 7, both organisms lost most of their viable population in the first 6 h but subsequently the wild type again survived better than the mutant. There was no gross loss of granulose from the wild type culture during the experiments (46 h) and thus there is clearly no simple relationship between possession of the reserve material and the capacity for survival. Further work is being carried out t o examine the response of the organisms to different stresses and physiological conditions in an effort to elucidate the possibility of more subtle relationships. Strasdine (1968, 1972) demonstrated the accumulation of granulose (amylopectin) in Cl. botulinum type E and subsequently found that it was rapidly synthesized and then lost during growth and spore formation in glucose-trypticase medium. When non-proliferating suspensions of granulose-rich cells were held in carbon-free media the reserves were quickly fermented concurrently with the formation of mature spores. The presence of exogenous glucose did not alter the fermentation rate although subsequent spore formation was either markedly decreased or inhibited. These observations led Strasdine (1972) to conclude that granulose serves as a mobile endogenous source of carbon and/or energy for spore maturation. I n view of the findings of Morris and his associates with GI. pasteurianum, however, it is possible that the role of granulose in Cl. botulinum may also be rather more complex than it appears a t first sight.

ENERGY RESERVE POLYMERS IN MICRO-ORGANISMS

163

7. Summary of Regulation in Prokaryotes It is apparent that glycogen synthesis is regulated a t the ADPglucose level in bacteria, unlike the mammalian or yeast system in which glycogen synthetase is the rate-limiting, and regulated, reaction. This difference may arise because UDP-glucose, the glycosyl donor in eukaryotes, is used for galactose and glucuronic acid synthesis in addition t o its role in the formation of a-(1 + 4)and a-(1 --f 1)(trehalose) glucosides. I n prokaryotes ADP-glucose may not have functions other than that of a glycosyl donor to poly a-(1+ 4) glucans and therefore conservation of the ATP utilized in its synthesis is achieved by control of ADP-glucose pyrophosphorylase, the first enzyme unique to glycogen synthesis. The energy charge regulation of the E. coli ADP-glucose pyrophosphorylase is also in keeping with the observations that phosphofructokinase in this organism in inhibited by ATP and the inhibition is reversed by 5’-AMP (Mansour, 1963;Passonneauand Lowry, 1962 ; Ramaiahet al., 1964;Atkinson and Walton, 1965).The NAD-specific isocitrate dehydrogenase requires ADP or AMP for activity (Chen and Plaut, 1963; Sanwal etal., 1963; Sanwal and Stachow, 1965; Sanwal et at., 1965; Hathaway and Atkinson, 1963), whereas fructose 1,6-diphosphate phosphatase, a key enzyme involved in gluconeogenesis, is inhibited by AMP (Taketa and Pogell, 1965; Krebs et al., 1964; Salas et al., 1964). The outcome of a low energy charge would therefore be the inhibition of glyconeogenesis and a stimulation of glucose catabolism, while a high energy charge would inhibit glucose catabolism and stimulate glycogen biosynthesis.

F. GLYCOGEN BIOSYNTHEYIS IN EUKARYOTES Unlike the bacterial system, yeast, moulds and fungi resemble mammals in utilizing UDP-glucose as the glucosyl donor for glycogen synthesis. Furthermore, the locus of regulation is glycogen synthetase. 1. Yeasts Rothmaii and Cabib ( 1966) demonstrated that NH,+-limitation of growth was responsible for the rapid deposition of glycogen and that the glycogen synthetase of yeasts was inhibited allosterically by C1-, an inhibition reversed by glucose 6-phosphate. They subsequently suggested that the stimulation of the synthetase by glucose 6-phosphate was an example of precursor activation, a phenomenon first recorded by Leloir et al. (1959), and found that the enzyme was inhibited by UDP (competing with UDP-glucose) and high concentrations (0.2 M) of C1-, Br-, I-, NO,-, SO,*- and PO,3-, and also by organic acids such as pyru-

164

E. A. DAWES AND P. J. SENIOR

vate, Iactate, oxalate, malonate, oxaloacetate, fumarate and maleate (Rothman and Cabib, 1967a). Glucose 6-phosphate relieved the inhibition exerted by all these compounds except that caused by UDP. The synthetase was also stimulated by glucosamine 6-phosphate and Mg2+. I

During exponential growth the glycogen content of Xaccharomyces cerevisiae S2SSC was low but when the growth ceased, following exhaustion of NH,+, glycogen accumulated rapidly (Rothman-Denes and Cabib, 1970). The glycogen synthetase isolated from exponentially growing cells was inactive in the absence of glucose 6-phosphate but, as the glycogen content rose, so the enzyme became more active and the dependence on glucose 6-phosphate activation decreased. This observation suggested the conversion of a glucose 6-phosphate-dependent enzyme to a glucose 6-phosphate-independent form of the enzyme (D t)I interconversion'), where the I-form increased 60-fold in activity. At low concentrations of glucose 6-phosphate the D-form was inhibited to a greater extent by ATP than was the I-form. I n the absence of ATP the I-form was activated two-fold by glucose 6-phosphate so that, in vivo, even if all the D-form was converted to the I-form, glycogen would not be accumulated. However, a t intermediate concentrations of glucose 6-phosphate the D -+ I conversion would become decisive in determining glycogen synthesis. Rothman and Cabib (1969)have also studied the regulation of glycogen synthesis in intact yeast. They measured the concentrations of the intracellular pools of ATP, ADP, glucose 6-phosphate and UDPglucose during both glycogen storage and degrading conditions. Glycogen accumulation in a glucose-salts medium proceeded after a short lag and reached 18.2% (w/w) of the dry weight. Synthesis ceased when the intracellular concentration of glucose 6-phosphate fell below 0.4 mM, after which time glycogen was degraded, indicating its function as a reserve material. The addition of 50 mM (NH,),SO, to cultures accumulating glycogen had two major effects, namely (i) glycogen synthesis ceased abruptly without any stimulation of degradation; and (ii) there was a very rapid decline in the glucose 6-phosphate pool concentration. Irrespective of whether (NH,),SO, was present or absent during the accumulation phase, there was an initial increase in ATP and ATP + ADP concentrations, followed by a slow decline. However, in the presence of The D- and I-forms have also been referred to as a and b forms respectively. I n a recent review of mammalian glycogen synthetase, Larner and Villar-Palasi (1971)have proposed a new nomenclature in which represents the active, more active or activated forms (i.e., I and a )while represents the inactive, less activated or non-activated forms (D and b of the enzyme e).

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(NH,),SO, the ATP/AMP ratio decreased. Uridine diphosphateglucose concentrations remained the same in all these experiments. It is thought that the action of NH4+ ions in inhibiting glycogen synthesis is a t the level of phosphofructokinase where stimulation occurs (Sols and Salas, 1966) causing a decrease in the adenylate energy charge and a diminution in the glucose 6-phosphate concentration. Ammonium ions do not change the activity of glycogen synthetase nor do they alter the effector interactions. The intracellular concentrations of ATP and ADP (3-6 mM) were considered to be sufficient to inhibit glycogen synthetase in the absence of glucose 6-phosphate. Rothman and Cabib (1969) suggested that because of the sigmoidal shape of the curve of glucose 6-phosphate concentration versus degree of activation for glycogen synthetase (Le. glucose 6-phosphate functioning as an allosteric effector), there is a critical concentration range of glucose 6-phosphate over which a very small change in the effector concentration produces a wide fluctuation in activation. They concluded that the regulation of glycogen biosynthesis in yeast was primarily by effector antagonism exerted by ATP and glucose 6-phosphate. This argument accords with the general assumption that the regulation of the biosynthesis of storage materials is by precursor activation and energy charge rather than by end-product inhibition. More recently Rothman-Denes and Cabib (1971) described the properties and interconversions of the glucose 6-phosphate-dependent and independent forms of yeast glycogen synthetase in detail and showed that while both D- and I-forms are inhibited by ATP, the D-form is more susceptible and the I-form is not totally glucose 6-phosphateindependent. This finding, they suggest, would mean that the I-form is the only physiologically active enzymic form. Conversion of I-t o D-form was achieved in vitro in the presence of ATP and Mg2+,indicating the interconversion of a phosphorylated enzyme, as occurs in rabbit muscle (Larner, 1966). 1%vivo the D to I conversion took place towards the end of exponential growth and during the stationary phase. The enzyme(s) that catalyse(s) the interconversion did not change in activity during t.his time indicating that they must also be regulated. 3',5'-Cyclic-AMP does riot appear to be an effector for the yeast glycogen synthetase unlike the muscle system (Schlender et al., 1969). A glycogen-deficient strain of Sacch. cerevisiae did not effect the D to I conversion but the systems necessary to catalyse the conversion were shown to be present in the cell extracts. It has generally been assumed that nitrogen-limitation of growth, in the presence of excess carbon source, is the factor responsible for glycogen accumulation in micro-organisms and a great deal of experimental evidence supports this belief. However, recent work by Kuenzi and

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Fiechter (1972) casts some doubt on the universal validity of this assumption. Working with continuous cultures of Sacch. cerevisiue they showed that the glycogen content of slow growing, glucose-limited cultures (specific growth rate = 0.076 h-') was even higher than that of nitrogen-limited cultures. Kuenzi and Fiechter (1969) had found previously that part of the glycogen reserve was mobilized a t the beginning of the budding process and then resynthesized during the single cell phase. I n their later work they demonstrated that a partiallysynchronized, glucose-limited culture growing aerobically and metabolizing glucose purely by respiration (i.e. with RQ = 1 ) displayed wide variations in glycogen (and trehalose content) during the cell cycle. These variations occurred a t all the specific growth rates studied (0.076 t o 0.169 h-l ) but the mean glycogen content did decrease with increasing specific growth rate of glucose-limited cultures. It was also apparent that as the specific growth rate was increased, less glycogen and trehalose were mobilized during the budding cycle. At dilution rates higher than 0.24 h-' the culture grew fermentatively, producing ethanol, and the glycogen content decreased rapidly as the growth rate was increased. During the growth of a synchronous culture in the presence of excess glucose, there was no change in the glycogen content throughout the cell cycle, suggesting that when sufficient exogenous glucose was available glycogen was not used during the budding process, i.e. the glucose exerted a sparing effect. These same workers also examined the variations of specific activity of glycogen phosphorylase and UDP-glucose pyrophosphorylase with specific growth rate of glucose-limited cultures (Fig. 7 ) . They found that as the dilution rate was increased the glycogen content diminished, as did the specific activities of the two enzymes. These results are similar to the findings of Rothman-Denes and Cabib (1970) with batch cultures of Succh. cerevisiae, that glycogen accumulation was reflected by the specific activity of glycogen synthetase ; they, however, used nitrogen-limited cultures. Thus a partial explanation can be offered for the increase in glycogen content of slow growing, nutrient-limited cultures. The slower the growth rate, the longer are the individual cells exposed to limiting conditions, and the higher are the specific activities of the enzymes involved in glycogen biosynthesis ; these factors presumably result in the increased content of glycogen observed. The observations recorded suggest that glycogen in yeasts plays a role additional to that commonly accepted for a reserve material. Consequently, if a rapid flux of glucose catabolism is required at certain periods during the growth cycle, then a glycogen-deficient mutant of yeast might be expected to be a t a disadvantage and not t o grow as well as the wild-type during carbon-limited conditions. If this type of chemostat

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experiment were carried out it would give added weight to the arguments presented by Kiienzi and Fiechter (1972) for the role and regulation of glycogen metabolism in yeasts. Although Chester and Byrne (1968) have obtained a glycogendeficient mutant of Sacch. cerevisiae, it would appear that the lesion is probably in the transport system for glucose since the rate and extent 200)

I

120

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A

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-8

-.-

n a

8 21

20-

3

3

.e

8

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-

Specific growth rate (h-‘)

FIG.7. Comparison of reserve carbohydrate contents and specific activities of UDP-glucose pyrophosphorylase and glycogen phosphorylase as a function of the specific growth rate ofh’accharornyces cerevisiae in a chemostat culture grown under glucose limitation. From Kuenzi and Fiechter (1972).

of growth was about the same as the parent strain if the mutant was grown in media containing 8% glucose, whereas growth was poor on 1% glucose. Under all conditions tested the mutant contained about half the glycogen of the wild type. To answer the problem posed of the role of glycogen it would seem essential to secure a glycogen-less mutant and use the controlled conditions of a chemostat. 2. Blastocladiella emersonii The water mould B1. emersonii forms zoospores towards the end of growth when glycogen is accumulated (Camargo et al., 1969). Glucose 6-phosphate was shown to activate the zoospore glycogen synthetase

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30-fold whereas the enzyme from vegetative cells was only activated four- to five-fold. As in other eukaryotes, UDP-glucose was the glucosyl donor and UDP, GDP-glucose and ADP-glucose competitively inhibited the reaction. Of these inhibitors UDP was the most effective but glucose 6-phosphate did not reverse any of the inhibitory effects. Adenosine triphosphate, GTP and ADP all inhibited the enzyme non-competitively and GTP was the most effective on a molar basis. Thus in this system ATP and glucose 6-phosphate are competitive antagonists. We can conclude from this work that in i:i*uo,a t low concentrations of glucose 6-phosphate and high ATP, the glycogen synthetase would be inhibited. However, a small increase in the pool size of glucose 6-phosphate would stimulate glycogen synthetase and relieve inhibition by ATP giving a two-fold effect rather than a single activation of the enzyme. Thus, as with yeast, it is likely that in Bl. emersonii the intracellular pool size of glucose 6-phosphate directly controls glycogen synthesis. Energy charge probably regulates the pool size of glucose 6-phosphate mediated by effects a t the level of phosphofructokinase. Unlike the yeast system, Carmargo et ul. (1969) could find no evidence for glucose 6-phosphate dependentlindependent forms of glycogen synthetase.

3. Dictyostelium discoideum The cellular slime mould D.discoideum is stimulated to differentiate by starvation conditions. The source of energy during differentiation is derived from the degradation of cellular proteins, and it has been suggested that the saccharide end-products accumulated during the ageing process are derived mainly from glycogen reserves synthesized prior to starvation (Baumann, 1969; Cleland and Coe, 1968, 1969). Rosness et ul. (197 1 ) have shown that this organism possesses a glycogen synthetase similar to that found in mammals in that inter-convertible forms exist which are glucose 6-phosphate-dependent or independent. Cyclic-AMP stimulates the formation of the less active form of the enzyme, i.e. the glucose 6-phosphate-dependent form. More recent work by Weeks and Ashworth (1972) and Hames et al. (1972) with D. discoideum has revealed that there is no correlation between the rate of glycogen synthesis a,nd the amounts of glycogen synthetase present, i.e. regulation is by precursor activation or product inhibition and not by changes in enzymic content. I n contrast to the findings of Rosness et al. (1971), Weeks and Ashworth (1972) were unable to show glucose 6-phosphate-dependence for glycogen synthetase, but they found that inhibition of the enzyme by ATP, ADP and AMP could be overcome by glucose 6-phosphate ; both effects were marked a t physiological concentrations of UDP-glucose.

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Weeks and Ashworth (1972) concluded that the rate-limiting factor for glycogen synthesis in D.discoideum was the supply of UDP-glucose, glucose 6-phosphate and ATP, rather than glycogen synthetase concentration. They based this conclusion on three observations : (i) during growth on axenic medium plus 86 mM-glucose, cells contained seven-fold the quantity of glycogen found in cells grown in the absence of glucose

I -

- 100

E

\

3 $::

E"

L.

50-

7.0 -

L

9"

5

l0-

x

Y)

Time ( h )

FIG.8. Cellular glycogen content of Dictyostelium discoideum at various times during growth. Cell density of cells grown in the absence of glucose, 0 ;cells grown in the presence of 86 mM-glucose, A. Cellular glycogen content of cells grown in the absence of glucose, 0 ; cells grown in the presence of 86 mM-glucose A.From Weeks and Ashworth (1972).

(Fig. 8) ; (ii) glucose 6-phosphate and UDP-glucose concentrations were increased during growth with glucose, and (iii) there was no correlation between the rate of glycogen accumulation and the activity of glycogen synthetase. It appears that while glycogen is degraded and utilized as a carbon source during the starvation-induced differentiation of D.discoideum, it does not fulfil the other criterion for a reserve material in acting as an energy source; its true role in this organism has yet to be established, Undoubtedly glycogen degradation has an important function during differentiation and it may be that its role during this process is similar to that of glycogen degradation during the budding cycle in Sacch, cerevisiae (Kuenzi and Fiechter, 1972). Although 3',5'-cyclic-AMP and the necessary enzymes for its synthesis and breakdown have been found in many micro-organisms, cyclic-AMP has not yet been reported to have any effect on microbial glycogen biosynthesis and degradation. However, this field of study could well

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profit from further investigation in the light of the importance of cyclicAMP in mammalian glycogen metabolism.

4. Tetrahymena pyriformis A recent extremely interesting development in the study of the regulation of glycogen metabolism in the protozoon T . pyriformis has been the observation by Blum (1970) that the drug theophylline increased the glycogen content of the organism. The addition of theophylline to aerobically-grown cells produced an increase in the specific activity of glycogen synthetase with a concomitant decrease in the specific activity of glycogen phosphorylase. 3‘,5‘-Cyclic-AMP and the enzyme adenyl cyclase have not as yet been reported in T . pyriformis, but Blum (1970) observed that this organism does possess a 3’,5’cyclic-AMP phosphodiesterase which is inhibited by caffeine and t heophylline . Reserpine, dichloroisoprenaline hydrochloride and triiodothyronine when added to Tetrahymena cultures decreased the specific activity of glycogen synthetase and isocitrate lyase, while increasing the specific activity of the glycogen phosphorylase. All the drugs investigated were without effect on glycogen synthetase activity i n vitro. These results suggest that glycogen metabolism in Tetrahymena may be regulated by a primitive system of andrenergic and/or seretoninergic control, which would be a unique finding for a unicellular organism. However, this concept, with its implied role for 3’,5’-cyclic-AMP in glycogen synthesis and degradation, is somewhat confused by tfhe finding that the presence of glucose in Tetrahymena cultures stimulated glycogen accumulation and increased the activity of glycogen synthetase. With E . coli Makman and Sutherland (1965) demonstrated that glucose stimulated an efflux of cyclic-AMP from the cell and that the removal of glucose increased the concentration of the intracellular pool of cyclic-AMP. It appears that the function of cyclic-AMP in E. coli is to induce in some fashion those enzymes which are repressed in the presence of glucose. Raised levels of cyclic-AMP reverse the transient and permanent catabolite repression exerted by glucose on the inducible /3-galactosidase system of E. coli. It now seems clear that the synthesis of all inducible enzymes of E. wli which may be repressed by glucose can be stimulated by cyclic-AMP (Pastan and Perlman, 1970). G. GLYCOGENDEGRADATION All organisms that have so far been studied degrade glycogen to glucose 1-phosphate in reactions catalysed by glycogen phosphorylases and which require inorganic phosphate (Glucosyl),+l + Pi

-+

(Glucosyl), + glucose 1-phosphate

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1. Prokaryotes (a) Escherichia coli. Chen and Segel (1968a) found that when E. coli K 12 was grown under conditions of nitrogen-limitation the organism possessed two polyglucan phosphorylases, a dextrin phosphorylase present to high activity in maltose-grown cells and a glycogen phosphorylase of low activity. The activity of the dextrin phosphorylase in maltose-grown cells was some lo-fold that of glucose-grown bacteria. The activities of both phosphorylases were stimulated by 5‘-AMP, the glycogen enzyme to a greater extent than the dextrin phosphorylase.

Incubation time ( h )

FIG.9. Phosphorylase activity and glycogen accumulation and utilization by Escherichia coli K-12 in a glucose-ammonium salts medium. The nitrogen source (one g ammonium chloride per litre) was exhausted a t the point indicated by the arrow. From Chen and Segel (1968a).

Growth studies by Chen and Segel (1968a) revealed that during the early stages of exponential growth the glycogen content fell from 5 to 3% (wlw) of the dry weight. Prior t o the end of exponential growth, caused by nitrogen exhaustion, the glycogen content increased slightly to 4% of the dry weight. However, in the stationary phase following an ammonium limitation there was a rapid rise in glycogen content to 14%. At this time roughly 50% of the original culture glucose remained and during the stationary phase both glycogen and exogenous glucose were being degraded simultaneously (Fig. 9). This latter observation is not in accord with the criteria used to define a reserve material in that the glycogen was degraded in the presence of an exogenous carbon and energy source. I n further work Chen and Segel(1968b)investigated the properties of the two polyglucan phosphorylases from E. coli in detail. The dextrin

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phosphorylase was found to be induced by maltose while the glycogen enzyme was constitutive. Spectrophotometric investigation revealed the presence of pyridoxal-phosphate in the glycogen phosphoi-ylase. The activation of the enzyme by 5'-Ait!tP observed by Chen and Segel (1968a) was quite specific : ADP, ATP, 2'-AMP, 3'-AMP and 3',5'-cyclicAMP and adenosine were without effect, and B'-AMP activated by decreasing the K , for glycogen. The product of the reaction was glucose 1-phosphate which inhibited the enzyme at sub-saturating glycogen concentrations. Glycogen phosphorylase was also inhibited by ADPglucose, TDP-glucose, UDP-glucose and glucose, all of t'hese effectors competitively inhibiting with respect to glucose 1-phosphate ; ADPglucose was, however, the most potent inhibitor. The observed stimulatory and inhibitory effects would therefore provide the organism with an excellent regulatory system in which high intracellular concentrations of ADP-glucose would stimulate glycogen synthesis and inhibit glycogen degradation. Activation of glycogen phosphorylase by 5'-AMP would a t the same time inhibit the activity of ADP-glucose pyrophosphorylase. Consequently the response of the organism to a low energy charge would be the stimulation of glycogenolysis and inhibition of glyconeogenesis. Chen and Segel (1968b) noted that the specific activity of the E. coli glycogen phosphorylase was very low in comparison with that of the muscle system. This observation would accord with the concept of a slow rate of glycogen degradation in E . coli during a period of starvation and stimulation of the rate during conditions of low energy charge. However, Dawes and Ribbons (1965) found that E . coli degraded its glycogen in two t o three hours when starved a t 37OC in phosphate buffer (pH 6.8), and commented on the rapidity with which this reserve was utilized. They also demonstrated isotopically that the effect of glycogen in suppressing the release of ammonia from starved cells was not due to a complete suppression of protein metabolism ; turnover of protein occurred while the bacteria still contained glycogen and thus the glycogen apparently served as both a source of energy and of carbon to permit re-incorporation of the ammonia released by protein breakdown. It may be that its function as a carbon source is more important under these conditions, especially since over longer periods of starvation energy can be provided by the oxidation of ribose derived from RNA breakdown (Dawes and Ribbons, 1961; 1965). Nonetheless, there are still some rather puzzling features concerning the rate of glycogen breakdown during starvation of E. coli. (b) Clostridium pasteurianum. Recent work by Robson and Morris (unpublished observations) on the control of granulose degradation in

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this strict anaerobe has revealed a phosphorylase, which appears to be bound to the granulose. The enzyme can be separated from the polymer and is then only active with added amylopectin or granulose. It has a K , for Pi of 2 to 3 mM but is inhibited by low concentrations of ADPglucose, with Ki of 20 to 50 pM. They observed that exogenous glucose repressed granulose breakdown in the organism, probably because of the maintenance of intracellular ADP-glucose levels.

2. Eulcaryotes Glycogen synthetase in eukaryotes such as yeasts, moulds and fungi is UDP-glucose-specific, and it was believed that the regulation of glycogen metabolism in these organisms was via allosteric control of both glycogen synthetase and glycogen phosphorylase. I n mammals the regulation of glycogen degradation is extremely complex involving hormonal control, S’,B’-cyclic-AMPand phosphorylated/dephosphorylated forms of glycogen phosphorylase. This latter requirement for a phosphorylated/dephosphorylatedenzyme now seeins to be established also for the enzymes from yeast and Neurospora crassa. (a) Neurospora crassa. The glycogen phosphorylase from N . crassa has a molecular weight of 320,000 (Shepherd and Segel, 1969) and incubation of this enzyme with ATP and Mg2+gave a 10-fold increase in activity. After 30 min incubation 3’,5’-cyclic-AMP had an activating effect. Cyclic-AMP was effective in the concentration range of 10W to lo-* M (Tillez-Inon and Torres, 1970). The inactive form of the enzyme was stimulated by 5‘-AMP, but after incubation of the enzyme with ATP and Mg2+,5’-AMP sensitivity disappeared. Adenyl cyclase, the enzyme catalysing the formation of 3’,5’-cyclic-AMPfrom 5‘-ATP, was found in membrane fractions of the mould. Thus striking similarihies exist between the N . crassa and muscle systems both in sensitivity to cyclic-AMP activation and regulation of activity in general. Further evidence of this similarity is provided by the finding that in both systems N a p inhibits the inactivation reaction brought about in the presence of ATP and Mg2+. Earlier work on the N . crassa enzyme by Shepherd et al. (1969) demonstrated that glycogen, dextrin and amylopectin were all equivalent substrates for the enzyme ; 5’-AMP increased the V,,, but did not change the K , values for glycogen or Pi.Glucose 6-phosphate and UDPglucose in the range 2 t o 5 mM competitively inhibited phosphorylase with respect to Pi, as did TDP-glucose and ADP-glucose. However, rabbit muscle glycogen phosphorylase a phosphatase did not convert the enzyme to an AMP-dependent form. While 0.15 mM-AMP activated by 30%, ADP, ATP, UMP, UDP, UTP, TMP, TDP, TTP, CDP, CTP,

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GMP, GDP, IMP, 3’-AMP, 2‘-AMP and 3’,5’-cyclic-AMPhad no effect. I n an attempt to investigate the in vivo significance of their in vitro findings, Shepherd et al. (1969) estimated intracellular Pi and found it to be 20 mM. This concentration was shown to be sub-optimal in terms of in vitro studies ( K , for Pi = 26 to 31 mM) and thus, since ADP-glucose, TDP-glucose, UDP-glucose and glucose 6-phosphate all inhibited competitively with Pi, these inhibition patterns were considered to have importance in vivo. Therefore under conditions of high energy charge the glucose 6phosphate concentration will increase, activating glycogen synthetase (Traut and Lipmann, 1963) and inhibiting glycogen phosphorylase. I n this situation the concentration of UDP-glucose would be expected to follow a similar course to that of glucose 6-phosphate.

(b) Saccharomyces cerevisiae. Sagardia et al. (1971) found the glycogen phosphorylase from Sacch. cerevisiae could be fractionated into several components, each of which was active in glycogen phosphorolysis but unaffected by AMP. Glucose, glucose 6-phosphate and UDP-glucose all inhibited phosphorolysis. Studies have shown that while free intracellular glucose is undetectable in growing yeasts the concentrations of glucose 6-phosphate and UDP-glucose may be as high as 3 and 0-2 mM respectively (Rothman and Cabib, 1969). However, Operti and Panek (1968) have observed free intracellular glucose which undergoes periodic oscillation and decreases during the growth cycle of Sacch. carlsbergensis. Sagardia et al. (1971) showed that the intracellular UDP-glucose concentration in Sacch. cerevisiae 1338 attains a value as high as 2.4 mM in the early stationary phase of a carbon-limited culture. This concentration, they calculated, would in vivo give 50% inhibition of phosphorolysis. But in vivo studies revealed that glycogen is degraded quite rapidly during the stationary phase and it has been suggested that under these conditions, where the intracellular phosphate concentration is 0.02 M (Goodman and Rothstein, 1957), competitive inhibition by phosphorylated effectors may be overcome by inorganic phosphate. The yeast phosphorylase system has been further elucidated by the recent work of Fosset et al. (1971). Two forms of the Sacch. cerevisiae enzyme were found, consisting of sub-units of molecular weight 103,000 daltons. Each sub-unit possessed a pyridoxal phosphate prosthetic group. One enzymic form had one phosphate per sub-unit (specific activity 135 units/mg protein) while the dephosphorylated form had little or no phosphate and displayed a specific activity some five-fold lower. The two forms, designated a and b, were combinations of subunits where the a-form was a slightly associated dimer (molecular weight 250,000 daltons) and the b-form a slightly dissociated tetramer (mole-

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cular weight 390,000 daltons). The K , values for each form for glycogen and glucose 1-phosphate were similar. The major difference between the two forms was in their inhibition by glucose 6-phosphate; K , for glucose 6-phosphate with the a-form was 11 mM whereas it was 1 mM for the b-form. An ATP-dependent kinase which converts the a- to the b-form has been isolated but neither the a- nor the b-form were activated by 5‘-AMP or 3’,5’-cyclic-AMP. (c) Tetrahymena pyriformis. The ciliated protozoon T . pyriformis contains epinephrine and seretonin and is sensitive to a variety of adrenergically reactive drugs. The glycogen formed by this organism is very similar to that of mammals, and it has been suggested that glycogen metabolism in T .pyriformis may be regulated in a similar, albeit cruder, manner to the mammalian system (Blum, 1967; Shepherd and Segel, 1969). The glycogen phosphorylase of T . pyriforrnis has recently been purified and characterized by Kahn and Blum (1971a). The enzyme has a molecular weight of 200,000 daltons and is only active with glycogen ; linear polyglucans were not phosphorylated. On passage through Sephadex G-200 some evidence was obtained to suggest that the enzyme consisted of two sub-units of molecular weight 100,000 daltons each. Although AMP and associated mononucleotides did not activate the enzyme, they did afford protection against thermal inactivation. Glycogen and glucose 1-phosphate could not spare AMP in this latter function. Further studies of the glycogen phosphorylase from T . pyriformis by Kahn and Blum (1971b) revealed that EDTA was a potent inhibitor. This inhibition could not be reversed by the addition of a variety of divalent cations, so apparently EDTA inhibition is not a simple matter of metal chelation. Unlike the liver phosphorylase and mammalian muscle phosphorylase b, glucose 6-phosphate did not inhibit the T . pyriformis enzyme. However, UDP-glucose, the precursor of glycogen in this organism, did inhibit phosphorolysis. I n this respect T .pyriformis is similar to N . crassa and S . cerevisiae. Inhibition of T . pyriformis phosphorylase by ATP was not reversed by AMP but 0.015 mM-AMP largely prevented the inhibitory effects of 15 mM-ATP, and Kahn and Blum (1971b) suggest that AMP caused a structural change in the enzyme. The glycogen phosphorylase of T . pyriformis seems to be regulated by an additional mechanism : Kahn and Blum (1971b) have found that growth conditions alter the properties of the enzyme. Phosphorylase isolated from glucose-grown cells (under conditions leading t o a high accumulation of glycogen) is more stable to heat inactivation and is more resistant to ATP and EDTA inactivation. These workers suggest that

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T. pyriformis glycogen phosphorylase can exist in two forms, one of which is insensitive to ATP and EDTA, and one which is sensitive to these compounds. The authors’ cautionary note may be echoed that ageing of the freshlyisolated extract, containing ATP and EDTA-sensitive enzyme, caused changes in ATP- and EDTA-sensitivity such that EDTA-sensitivity decreased but ATP became more effective. It may be that proteolytic activity brings about these changes and that what appears to be two forms of the enzyme at different stages of growth, may in fact be brought about by differing proteolytic activities during these growth phases. I n conclusion it can be stated that there is still no incontrovertible evidence to suggest that glycogen metabolism in Tetrahyrnena (particularly the glycogen phosphorylase) is regulated by the 3’,5’-cyclicAMP-mediated, complex hormonal system of regulation found in the mammalian system. More likely, regulation of glycogen metabolism in Tetrahymena will fall, in complexity, somewhere between bacteria and mammals and the incorporation of features from both groups might be expected. H. CONCLUSIONS A general pattern of regulation of glycogen metabolism in microorganisms is apparent (Fig. 10). Conditions which favour the accumulation of glycogen, i.e. high energy charge and the carbon source in excess of the requirements for growth and maintenance, are those which Iead t o the activation of the enzymes of glycogen synthesis and t o the inhibition or inactivation of glycogen phosphorylase. All the microbial glycogen phosphorylases so far examined are inhibited by the substrate for glycogen synthetase in the particular organism, UDP-glucose in eukaryotes and ADP-glucose in prokaryotes. Likewise, conditions favouring glycogen degradation, namely low energy charge and low concentrations of ADP(UDP)-glucose, tend to inhibit or inactivate the glycogen-synthesizing systems. Thus a fine balance of control is achieved, closely linked to the prevailing conditions in the external environment, and so designed as to avoid the metabolic short circuits which would otherwise occur in a cyclic pathway and which would lead to a rapid loss of energy and subsequently, viability. I n Section IV G it will be seen that an analogous system of controls operates for the regulation of poly-/3-hydroxybutyrate metabolism. Certain microbial systems, such as those found in Tetrahymena pyriformis and Neurospora crassa, are apparently more complex than others, resembling the highly complicated mammalian glycogen

ENERGY RESERVE POLYMERS I N MICRO-ORGANISMS

A

D -t Mg2+ P

Y

177

lGLYCOGENl

ADP-Glucose 1

l k

( l u c o s e 1-phosphate

/TP (+) ATP

(+)

i

Pructose 1,G-diphosphate NADPH2 Pyridoxal phosphate

ADP ATP Glucose 6-phosphate NADPH2(-)

Fructose 6-phosphatc G-Phosphogluconate ADP

cp15’-AMP(-)

Fructose 1,G-diphosphate

3

Glyceraldehyde 3-phosphate

Dihydroxyacetone phosphate

I

Phosphoenolpyruvate

Pyruvat,e

FIG.10. Regulation of glycogen metabolism in prokaryotes. Positive and negative effectors are represented as (+) and (-) respectively, and broad arrows indicate their sites of action.

metabolizing systems found in muscle tissue. Cyclic-AMP was shown t o affect these systems in a similar manner t o the muscle system and it is thus possible that a crude form of adrenergenic/seretoninergic control may exist in lower eukaryotes. I n assessing the role of glycogen in microbial survival it would be

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interesting, for example, to know whether the glycogen-deficient mutants of E . coli are less able to withstand conditions of starvation than the wild-type organism. Such experiments might enable the true value of glycogen as a reserve of carbon and energy in the prokaryotes to be evaluated. However, it must be borne in mind that Tempest and Strange ( 1966) demonstrated that glycogen-rich nitrogen-limited A . uerogenes organisms have a significantly higher magnesium content than carbonlimited glycogen-poor cells, and Strange (1968) reported that if magnesium ions are added to the environment then there is no appreciable difference between the survival of nitrogen-limited and carbon-limited E . coli. I n eukaryotes it is possible that glycogen serves a dual function, first as a source of carbon and energy during conditions of starvation, and second, by its synthesis and degradation comprising an integral part of the life cycle of both yeasts and D. discoideum. The co-ordination of these activities will clearly require a more complex regulatory system than that found in prokaryotes.

111. Polyphosphate

A. STATUS OF POLYPHOSPHATE AS A RESERVE MATERIAL The status of polyphosphate as a,n energy reserve compound is by no means clearly established, and indeed the present balance of evidence seems to be against this function and to favour its role as a reserve of phosphorus. However, there is no question that polyphosphate can in some micro-organisms fulfil the function of ATP and Lipmann (1965) has advanced the suggestion that the earliest organisms used polyphosphate or pyrophosphate as their prime energy carrier, the role of ATP as the universal energy carrier in contemporary organisms having arisen during the course of evolution. Miller and Parris (1964) have discussed the ways in which abiogenic processes, such as the condensation of inorganic orthophosphate a t high temperatures, and the reaction of cyanate with calcium phosphate, could give rise to polyphosphates as consitituents of the “primordial soup”. I n this connection Schramm (1965) has shown that ethyl metaphosphate promotes the formation of polynucleotides, polypeptides and polyglucoses from the appropriate monomers under very mild conditions. The formation of the peptide bond of diglycine by reaction of aqueous solutions of glyciiie with cyclic or linear polyphosphates a t room temperature, and within the pH range of 7-8, was investigated by Rabinowitz et ul. (1969), who obtained a maximum yield of 36% with trimetaphosphate. They suggest that polyphosphates may thus have

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played an important role in the prebiotic synthesis of peptides and other substances of biological importance. Such observations led Harold (1966) to conjecture that polyphosphates might comprise a metabolic fossil which throughout the ages has lost its original role in polymer synthesis and has assumed new functions which still elude us. Certainly, during the intervening six years since Harold’s speculation, little has been achieved to give greater clarity and precision to the biological role of polyphosphate. We propose to discuss the chemical structure of polyphosphates, their occurrence in micro-organisms, the enzymes concerned with polyphosphate metabolism and to examine the role of polyphosphates as reserve materials.

B. OCCURRENCE OF POLYPHOSPHATES IN MICRO-ORGANISMS Marly bacteria and yeasts possess intracellular granules that stain metachromatically with basic dyes but which can also be seen in living cells by phase-contrast microscopy. These so-called “volutin” granules are generally conceded to contain polyphosphate in association with other components and Widra (1959),surveying the available information, has indicated that volutiri granules contain RNA, lipid, protein and Mg2+ in addition to polyphosphate. Subsequent work with other organisms has supported this conclusion. Martinez (1963), however, claimed that the metachromatic granules of Spirillum vclutuns are composed not of polyphosphate but of poly-P-hydroxybutyrate ; i t is possible, therefore, that bacterial metachromstic granules do not necessarily contain polyphosphate, although i t is beyond question that polyphosphate deposits exhibit metachromasy. I n Myxococcus xanthus polyphosphate granules are associated with glycogen inclusions, dispersed in the cytoplasm or embedded within the nucleoid (Voelz et aE., 1966). Electron micrographs of polyphosphate from the myxomycete PhysarunL polycephalum revealed groups of spherical particles nieasuriiig from 40 to 1 1 0 nm in diameter, the larger particles possibly being aggregates of the smaller (Goodman et al., 1969).Some idea of the distribution of polyphospliate in micro-organisms may be gained from Table 5 which, however, is not an exhaustive survey and the reader is referred t o the extensive compjlation by Kuhl (1960) for further information.

c. CI-IEMICAL STltIJCTURB O F POLYPHOSPHA4TES The detailed chemistry of the polyphosphates has been reviewed by ‘l’hilo (1962a, b) and van Wazer (1958). Here we shall confine our atten9

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tion to the essential features of these condensed inorganic phosphates necessary for an understanding of their biological role.

1 . Cyclic Condensed Phosphates :Metaphosphates Only tri- and tetrametaphosphates are known and they occur in certain melts. They are hydrolysed to orthophosphate (Pi)when heated TABLE5. Occurrence of Polyphosphate in Micro-organisms Organism

Aerobacter aerogenes Anabaena variabilis Azotobacter agilis Azotobacter vinelandii Chlorella sp. Corynebacterium xerosis Corynebacterium diphtheriae Chlorobium thiosulfatophilum Euglena gracilis Escherichia coli Hydrogenomonas eutropha Micrococcus lysodeikticus Mycobacterium chelonei Mycobacterium phlei Mycobacterium smegmatis Mycobacterium thamnopheos Myxococcus xanthus Nitrosomonas europeae Physarum polycephalum Rhodopseudomonas spheroides Saccharomyces cerevisiae Salmonella minnesota Streptococcus SL-1

Reference Duguid et al. (1954); Harold (1963) Carr and Sandhu (1966) Zaitseva and Li (1961) Zaitseva et al. (1960) Ebel et al. (1958) Muhammed et al. (1959) Sall et al. (1956) Cole and Hughes (1965); Hughes, Conti and Fuller (1963) Albaum et al. (1950). Kornberg et al. (1956). Kaltwasser and Sclilegel (1959) Friedberg and Avigad (1968). Mudd et al. (1958) Drews (1960) Winder and Denneny (1957) Mudd et al. (1958) Voelz et al. (1966) Terry and Hooper (1970) Goodman et al. (1969) Carr and Sandliu (1966) Wiame (1947a, b ) Miihlradt (1971) Tamer and Krichevsky (1970)

[See Kuhl (1960) for a more extensive survey].

in acid, but alkali converts them to the corresponding linear polyphosphate (Fig. 11).

2. Linear Condensed Polyphosphates These unbranched structures may vary in chain length from two units to about lo4 units and generally consist of mixtures of different molecular size. The average chain length is usually determined by titration of the second acid function (with pK of approximately 7) associated with the terminal groups.

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I n the absence of divalent cations, linear polyphosphates are stable to alkali. They are completely hydrolysed by N-acid to Pi within 15 min a t 100°C, and this technique forms the basis of their chemical determination. Since, however, acid hydrolysis also releases some Pi from other biological phosphate compounds which are usually regarded as being acid stable (e.g. nucleic acids), it is generally necessary to separate polyphosphates from other compounds prior to hydrolysis. Suitable techniques for doing this have been discussed by Harold (1966).

3. Irnidophosphate Polymers The work of Correll and his associates has shown that the “polyphosphate” isolated from Chlorella is not a simple phosphate anhydride

FIG.1 1 . Structures of trimetaphosphate and linear polyphosphate.

polymer. The entire polymer was hydrolysed by alkali under conditions which do not hydrolyse true polyphosphates. Complete hydrolysis released 1-2 moles of phosphate per mole of ammonia and the infra-red spectrum of the purified material showed an absorption a t 1400 cm-’ characteristic of imidophosphate linkages. On these grounds Correll (1966) deduced that the Chlorella polyphosphate contained both phosphate anhydride and imidophosphate bonding. Some possible repeating units in the structure are shown in Fig. 12. Correll (personal communication) has recently isolated from Chlorella a large amount of a diphosphate compound which contained one nitrogen atom per two atoms of phosphorus and has been characterized as imidodiphosphate. This material labels rapidly and is possibly an intermediate in the synthesis of the polymer. The physiological significance of the imidophosphate polymers is not yet known.

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4 . Trichloroacetic Acid Xoltd~ility The cellular fractionation schemes of Schmidt and Thaniihauser (1945) and Schneider (1945) yield polyphosphates in two fractions, those extractable with cold 5% trichloroacetic or perchloric acids and those that are not and are recovered in the nucleic acid fraction. These fractions are generally referred to as “acid-soluble’’ and “acid-insoluble” polyphosphates. The acid-soluble polyphosphates are of low niolecular

n

FIG.12. Some possible monomer units for the “polyphosphatc” of ChZoreZZa (Correll, 1966). (Copyright 1966 by the American Association for the Advancement of Science.)

weight whereas the acid-insoluble entities are high molecular weight species and are primarily concerned with the reserve material functions associated with polyphosphates.

5. Free Energy of Hydrolysis Polyphosphates thermodynamically are high energy phosphate compounds. Thus the free energy of hydrolysis of the anhydride linkage yields some 9 kcal per phosphate bond a t p H 5. Their effective energy storage function depends therefore on the ability of the bond cleavage

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reaction to effect phosphorylation a i d thus conservation of the energy associated with the reaction.

[PJn+ [Pi],-,

+ Pi

AGO' e --9 kcal/moIe

D. ACCUMULATION AKD UTILIZATION OF POLYPHOSPI-IATE The conditions which favour polyphosphate accumulation and degradation have now been charted in several micro-organisms and a characteristic feature is the extreme variation in polyphosphate content which may be encountered. I n general, the polyphosphate content is low during rapid growth but increases markedly when a nutrient imbalance causes the growth rate to decline; there are, however, some exceptions to this pattern. The concept of an antagonistic relationship between polyphosphate and nacleic acid metabolism has been propounded by Mudd et al. (1958) for mycobacteria and by Smith et al. (1954) and Wilkinson and Duguid (1960) for A . aerogenes. The extensive work of Harold and his associates with A . aerogenes, described subsequently, also supports this postulate.

1. Aerobacter aerogenes Some of the earliest work on the variation of polyphosphate content in response to the phase of growth and nutritional conditions was carried out with this organism by Duguid, Wilkinson and their colleagues (Duguid et nE., 1054; Smith et al., 1954). They showed that exponentially growing bacteria are devoid of polyphosphate, as are stationary phase cells when growth is limited by a deficiency in either the carbon and energy source, the phosphorus source or the potassium source. If, however, growth is limited by exhaustion of the nitrogen or the sulphur source, or by the development of a low pH value in the medium, substantial polyphosphate deposition occurs ; Mg2+ is required for this process. These experiments demonstrated the net synthesis of polyphosphate in the stationary growth phase provided there is an excess of the carbon and energy source, together with phosphate, potassium and magnesium ions. When polyphosphate-rich bacteria were transferred t o fresh medium the accumulated polymer was utilized. If bacteria which had been subjected to phosphate starvation were then provided with inorganic phosphate in an otherwise complete medium, a very rapid formation of polyphosphate ensued, followed by its more gradual utilization. Harold and his associates extended this work with A . aerogenes arid demonstrated that the accumulation of polyphosphate under conditions of nutrient imbalance displayed two separate patterns (Fig. 13 ; Harold, 1963; 1964). One pattern involved the cessation of nucleic acid synthesis

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due to exhaustion of an essential metabolite, as for example in sulphate starvation, and under these conditions a slow deposition of polyphosphate occurred as Pi was assimilated from the medium. Similar results were observed when auxotrophic mutants requiring amino acids or uracil were deprived of their essential nntrilite. When growth was allowed to resume polyphosphate was degraded rapidly and it was shown isotopically that the phosphate appeared in the nucleic acid (RNA) fraction. By dissociating protein synthesis with chloramphenicol a dual competitive relationship between polyphosphate accumulation I

1

Time

FIG.13. Patterns of polyphosphate accumulation in Aerobacter aerogenes. (a) Nutrient deprivation : cells were placed in a medium devoid of sulphur a t zero time. (b)Phosphate “overplus” :cells were placed in a medium devoid of phosphate a t zero time ; inorganic phosphate was restored a t 4 h. The initial bacterial density was lo9 cells/ml in both experiments. Polyphosphate, 0 ; polyphosphate kinase,

o ; polyphosphatase, A. From Harold (1963, 1964).

and nucleic acid synthesis became apparent such that resumption of nucleic acid synthesis decreased the rate of concurrent polyphosphate synthesis and simultaneously stimulated its degradation (Harold, 1965 ; Harold and Harold, 1965). The specificity of environmental factors was investigated by Harold and Sylvan (1963) who found that in media containing low concentrations of inorganic phosphate, polyphosphate accumulated whenever nucleic acid synthesis ceased due to a nutritional deficiency, irrespective of its nature, whereas in high-phosphate media only sulphur starvation induced polyphosphate accumulation. They attributed this observation to the presence of an intracellular inhibitor of polyphosphate deposition which was depleted in sulphur starvation and was identified as oxidized glutathione or a closely related compound. The second pattern was observed when phosphate was added to

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bacteria previously starved of phosphate; as already noted, a rapid and massive polyphosphate accumulation ensues. Harold has coined the term “polyphosphate overplus” for this phenomenon, a not very happy translation of the “Polyphosphat Uberkompensation” of Liss and Langen (1962).Polyphosphate is slowly degraded when growth and nucleic acid synthesis are resumed and the phosphate is transferred to nucleic acids. 2. Yeast Historically, yeast was the first micro-organism from which polyphosphate was isolated (Liebermann, 1888) and subsequently much of the pioneer work on the polymer was carried out with yeast by Schmidt et al. (1946) and Wiame (1947a, b ; 1948). The identity of the metachroniatic or volutin granules with polyphosphate (Wiame, 1947b) was a crucial advance linking chemical and cytological investigations. The polyphosphate content of Sacch. cerevisiae is minimal during the exponential phase of growth but increases as the growth rate declines (Katchman and Fetty, 1955). Irradiation has also been shown to induce polyphosphate accumulation (Spoerl and Looney, 1958; Katchman et al., 1959). Langen and his colleagues have elucidated the biosynthesis of polyphosphate in yeast by their discovery of four distinct polymer fractions of average chain lengths 4,20, 55 and 250 units corresponding to molecular weights (as the K + salts) of 530, 2400, 6500 and 30,700 daltons (Langen et al., 1962). 32Piwas most rapidly incorporated into the highest molecular weight fraction when added to non-proliferating suspensions in which fermentation had been initiated by glucose ; incorporation into the other fractions progressively decreased with decrease in molecular size. When yeast cells were labelled by brief exposure t o 32Piand fermentation was inhibited by iodoacetate, polyphosphate degradation was induced yielding an equivalent quantity of Pi. However, the Pi released was not labelled and although the highest molecular weight fraction was the first to be degraded it was evident that 32Piderived from it passed into the smaller polyphosphate fractions. These findings led Langen et al. (1962)to the concept of a “polyphosphate cycle” in yeast, in which biosynthesis results in the formation of long chains (260 units) which are subsequently cleaved to shorter ones, thus : ATP

t Pi

+ Poly P 260

-

__f

Poly P 55

I Poly P 4

t--

PolyP 20

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E . A. DAWES AND P. J . SENIOR

The discovery of Polyphospliate Uberkcompensation, the “overplus” phenomenon discussed in Section 111, D . l (p. 184), was made with yeast by Liss and Langen (1962).When the organism was starved of phosphate and then subsequently exposed to adequate amounts of Pi, a marked increase in the rate and extent of polyphospliate synthesis occurred and, again, the largest molecules were synthesized first and served as precursors for the smaller chain length compounds. The polyphosphate kinase (Section 111, E . l ) of yeast is presumably derepressed under these conditions. I n Saccharomyces mellis phosphate starvation also results in derepression of the acid phosphatase but Weirnberg and Orton (1965) could demonstrate no direct relationship between this enzyme and polyphosphate metabolism. The distribution of phosphorus compounds in fractions isolated from metabolically lysed yeast protoplasts has been examined by Indge (1968). About 40% of the acid-soluble phosphorus compounds was associated with a particulate fraction which sedimented a t 2000 g and was rich in vacuoles ; these compounds were comprised essentially of polyphosphates of relatively high molecular weight and were apparently contained in the vacuole sap, although the evidence was not conclusive. The phosphorus of the crude vacuole fraction was labelled only slowly when protoplasts were allowed to take up 32P-orthophosphate,and there appeared to be low molecular weight precursor(s) present in the 2000 g supernatant fluid from which the radioactivity was derived. Although these findings would appear to be contrary to the operation of the polyphosphate cycle proposed by Langen et al. (1962), direct comparison is not possible since Indge used unstarved yeast while Langen and Liss used starved cells. Stahl (1969) carried out investigations to discover the precise intracellular location of polyphosphate synthesis in Xacch. cerevisiae harvested from the exponential and stationary phases of growth in complete and phosphate-deficient media. Fractionation of extracts was carried out by differential centrifugation and the highest synthetic activity was found in the fraction of highest density associated with cell walls and membranes. The mitochondrial fraction also possessed activity but intact mitochondrial structure was not essential. I n all cases the biosynthetic activity was greatest with cells grown in a phosphate-deficient medium. When the growth of yeast cells is resumed after phosphate starvation, polyphosphate is broken down via the polyphosphate cycle and phosphorus is ultimately transferred to nucleic acids and phospholipids. The available evidence indicates that polyphosphate provides a flow of phosphorus into the precursor pools for RNA synthesis and the pools of P, and ATP of yeast (Stahl et al. 1964a, b). The enzymology of polyphosphate utilization in yeast is still not

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clarified for although polyphosphate kinase is reversible (Section 111, E.2(a), p. 194); Hoffman-Ostenhof, 1962 ; Hoffman-Ostenhof and Slechta, 1957), Langen (1965) could not demonstrate the direct conversion of polyphosphate to ATP in vivo. It is possible, therefore, that in yeast sequential hydrolysis of polyphosphate to the smaller chain length moieties, and ultimately Pi, occurs via the mediation of polyphosphatases as described by Mattenheimer (1956a, b, c).

3. Corynebacteria The accumulation and degradation of polyphosphate in Corynebacterium xerosis follows the conventional pattern. Hughes and Muhammed (1962) found that, on transfer to fresh medium, polyphosphate accumulated during the lag phase, was consumed during the phase of exponential growth, and accumulated again during the stationary phase. These authors and Muhammed (1961) have shown that polyphosphate accumulation is mediated by polyphosphate kinase and is an energydependent process. Evidence for the route of breakdown of polyphosphate in C . xerosis is less satisfactory although Hughes and Muhammed (1962) suggest that the polyphosphatase (Muhammed et al., 1959) of the organism represents the principal pathway. However, Dirheimer and Ebel (1962) have shown that C. xerosis possesses a polyphosphate glucokinase (see Section 111, E.2(c), p. 195) with an affinity for polyphosphate high enough in extracts effectively to compete for the substrate in the presence of the polyphosphatase. Thus a physiological role for polyphosphate in Corynebacteria may well be the phosphorylation of glucose, which would be in accord with the observation of Sall et al. (1956) that polyphosphate accumulation in C . diphtlzerine is stimulated by substrates such as malatc but suppressed by glucose. Sall et al. (1956) also showed that nitrogen deficiency induced polyphosphate deposition. This same group obtained a synchronous culture ofC. diphtheriae by temperature shock and observed marked fluctuations in the polyphosphatc content with maxima just prior to cell division; in contrast, the DNA content increased steadily throughout the cell cycle to the time of division (Sall et al., 1958). Corynebacterium xerosis also contains a polyphosphate-AMP-phosphotransferase (Dirheimer and Ebel, 1965) which, as discussed in Section 111, E.2(b) (p. 195), has been suggested as a means whereby ADP may be formed independently of ATP. 4 . Mycobacteria I n many respects the polyphosphates of the Mycobacteria display similar behaviour to those of the Corynebacteria. The polyphosphate contents of Mycobacterium phlei (Drews, 1960), M . chelonei and M .

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thamnopheos (Mudd et al., 1958)and M . smegmatis (Winder and Denneny, 1957) were all lowest in rapidly growing cells and increased rapidly in the stationary phase. Mudd et al. (1958) demonstrated a reciprocal relationship between nucleic acid synthesis and polyphosphate accumulation ; azaserine by inhibiting purine synthesis promoted polyphosphate deposition. The use of ethionine (Drews, 1960) as a metabolite analogue and growth in a zinc-deficient medium (Winder and O’Hara, 1962) had similar effects. If growth was subsequently allowed to resume, the accumulated polyphosphate was broken down and used as a source of phosphorus for the synthesis of nucleic acids and phospholipids (Winder and Denneny, 1957; Mudd et al., 1958). A curious finding, which does not accord with the foregoing observations and which has not been adequately explained, was the stimulation of polyphosphate deposition by 2% tetrahydrofurfuryl alcohol without concomitant inhibition of nucleic acid synthesis (Winder and Denneny, 1957). The mechanism of polyphosphate synthesis is presumably via polyphosphate kinase found by Winder and Denneny (1955) in extracts of M . smegmatis. The degradative pathway may be via polyphosphatases or via the polyphosphate glucokinase which Szymona and associates have demonstrated in M . phlei (Szymona, 1962; Szymona et nl., 1962; Szymona and Ostrowski, 1964) and which, as previously mentioned in connection with the Corynebacteria, offers a physiological function for the polymer. Possible additional evidence for the role of hexose phosphorylation comes from the demonstration that M . phlei grown on fructose may contain an inducible polyphosphate-dependent fructokinase (Szymona and Szumilo, 1966; see Section 111, E.Z(d), p. 196). Finally, M . smegwatis also possesses the polyphosphate-AMP-phosphotransferase (Winder and Denneny, 1957) representing the fourth possible route for polyphosphate metabolism.

4 . Hydrogenomonas eutropha Kaltwasser (1962), by studying the uptake of 32P0,3-by washed suspensions of phosphorus-deficient cells of Hydrogenornonas, observed that in the absence of NH,Cl, and with either air or H2-0, gas mixtures, the principal storage products were acid-insoluble polyphosphates. Under these conditions the synthesis of nucleic acids and other acidinsoluble phosphates was very limited. It appeared that in the absence of hydrogen the necessary energy for phosphate uptake was furnished by the oxidation of storage material, presumably poly-P-hydroxybutyrate. Under anaerobic conditions no inorganic phosphate was taken up nor was previously accumulated polyphosphate degraded. If it nitrogen source (NH,Cl) was added to the suspensions, thus

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permitting growth to occur, the synthesis of stable acid-insoluble phosphate compounds took place, stored polyphosphates were consumed and a transfer of 32Pfrom unstable to stable acid-insoluble fractions was observed. I n a bacterial suspension containing NH,C1 and inorganic phosphate, polyphosphates were accumulated so long as external phosphate was present but, following its consumption, polyphosphates were used for the continuing synthesis of organic phosphate compounds and the polyphosphate fraction displayed a rapid turnover of 32P. Kaltwasser (1962) concluded from his investigations that the function of polyphosphate in Hydrogenomonas is that of a phosphorus storage material, being involved in the regulation of the inorganic phosphate level of the cell. The high energy content of the polyphosphate bonds he considered represents an accessory property which biologically is of minor importance.

5 . Rhodopseudomonas spheroides and Anabaena variabilis Carr and Sandhu (1966) studied the role of polyphosphates in the endogenous metabolism of two photosynthetic micro-organisms, R. spheroides and A. variabilis, which grow anaerobically, photosynthetically in the light, and aerobically in the dark to yield non-pigmented forms. The polyphosphate content of washed suspensions of aerobicallygrown cells incubated under anaerobic conditions declined, as did that of photosynthetically-grown cells incubated anaerobically in the dark. I n contrast, photosynthetically-grown cells incubated in the light showed a marked increase in polyphosphate. Thus when the organisms had a source of ATP formation but lacked carbon for growth, polyphosphate accumulated whereas deprivation of ATP-forming mechanisms resulted in degradation of the polymer which was unaffected by 8-azaguanine as an inhibitor of nucleic acid biosynthesis. However, since the changes in polyphosphate content observed with A . variabilis did not involve more than 20% of the total polyphosphate present, Carr and Sandhu (1966) reached the conclusion that although polyphosphate does appear to act as an energy reserve during endogenous metabolism, this cannot be its sole function. It seems probable, therefore, in the light of evidence secured with other micro-organisms, that its other function relates to the phosphate economy of the cell. 6 . Nitrosomonas europaea When this chemoantotroph, which oxidizes ammonia to nitrite, was transferred from a stationary phase culture to fresh medium it displayed a rapid increase in cellular inorganic orthophosphate content, reaching the unusually high concentration of 800 pmoles per gram wet weight, whereas the acid-insoluble polyphosphate content decreased rapidly to

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a low value (Terry and Hooper, 1970). As active growth proceeded, the inorganic orthophosphate content decreased whereas the polyphosphate content increased to 60 to 90 pmoles per gram. This increase in polyphosphate content during growth was related to a decreased availability of the carbon source caused by the effect of the decreasing p H value of the culture on the solubility of carbonate; thus, cultures titrated to pH 7.6 with sterile 50% (w/v)K,CO, did not accumulate the polymer. The decrease in p H during growth, as a result of nitrite formation, also appears to uncouple net cellular synthesis from ammonia oxidation and ATP production which continue a t an increased rate ; consequently the growth efficiency declines. These conditions favour the deposition of polyphosphate either because of the increased intracellular steady state concentration of ATP or because polyphosphate degradation no longer supports biosyrithetic reactions. Polyphosphate formation, however, accounts for onIy a proportion of the ATP generated. I n contrast to many other micro-organisms polyphosphate was not broken down in the stationary phase of growth. Since N . europaea possesses polyphospliatase activity, presumably substrate and enzyme are not accessible to one another in the stationary phase but become so on transfer of the bacteria to fresh medium ; utilization of polyphosphate under these conditions appears to be initiated primarily by the higher pH of the fresh medium.

7 . Streptococcus SL-1 Tanzer and Krichevsky (1970), with a caries-conducive Streptococcus designated SL-1, have shown that, contrary to earlier belief, streptococci do form polyphosphates. Deposition occurred only in washed cell suspensions furnished with glucose as an energy source and, by a doublelabelling technique involving the use of [U-I4C]glucose and NaH,32P0,, they demonstrated conclusively that [32P]polyphosphate devoid of I4C was synthesized, and that considerable turnover of phosphate took place in the nucleic acids of these non-proliferating cells. The polyphosphates formed were of relatively small molecular size, comprising between 10 and 20 units. There is no clear indication o f a physiological role for polyphosphates in such bacteria but the authors suggest that they might serve as a continuous sink for environmental phosphate provided that a source of energy, such as fermentable carbohydrate, is present.

8. Physarum polycephalum This slime mould offers an opportunity for investigating the possible role of polyphosphate in cell differentiation. Goodman et al. (1969) found acid-soluble and acid-insoluble polyphosphate present but during

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growth the combined level was never greater than 2% of the total cellular phosphorus. Despite these low concentrations, statistically significant changes in both types of polyphosphate were obtained during the intermitotic period. I n contrast, during starvation, which leads to the formation of spherules, only the acid-insoluble polyphosphate underwent appreciable fluctuation, decreasing from 12% to 2.5% in 3 h, increasing to 21% a t 9 h, decreasing to 7 % a t 24 h and increasing again up t o 30% of the total cellular phosphorus a t 30 hours, by which time spherule formation was almost complete. These observations suggest that polyphosphate is stored in the spherule, possibly for use when a favourable environment subsequently induces the emergence of the plasmodium. During growth and the early stages of spherule formation polyphosphate may be principally concerned with nucleic acid synthesis but later in the process of spherulation may help to maintain osmotic equilibrium in the plasmodium by sequestering inorganic phosphate in polymeric form. Again the competitive interaction between polyphosphate formation and nucleic acid synthesis is manifest since deposition of the acid-insoluble polymer does not occur during the known period of DNA and RNA replication that immediately follows mitosis (Mohberg and Rusch, 1969; Nygaard et al., 1960).

9. Atypical Polyphosphate Accumulation and Degradation: Micrococcus lysodeikticus Some micro-organisms which possess polyphosphate do not display the pattern of accumulation and degradation normally characteristic of reserve materials, thus raising the question of the significance of polyphosphate deposition in these cases. An example is Micrococcus lysodeikticus. Friedberg and Avigad (1968) have now shown the presence of polyphosphate in three strains of M . lysodeilcticus where it occurs in metachromatic granular structures (40 to 80 nm in diameter) which appear to be associated with more complex subcellular structures containing substantial amounts of protein and lipid and smaller amounts of RNA, carbohydrate and CaZf and Mg2+ ions. The polyphosphate accumulated during the exponential phase of growth and disappeared gradually during the stationary phase. During the starvation of washed cell suspensions the particles gradually disappeared and bacteria shaken aerobically in a carbohydrate-free salts solution lost most of their dense particles after 6 hours incubation. The authors do not give quantitative data, however, nor is it clear what parameter caused cessation of exponential growth in the Casamino acids-yeast extract-glucose medium they used.

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10. Bacillus m e g a t e r i u ~ I n a general study of the effect of nutrient limitation on the synthesis of possible carbon and energy storage polymers in the asporogenous KM strain of B. megaterium, Wilkinson and Munro (1967) found that polyphosphate was accumulated only under conditions of sulphur limitation. Carbon, nitrogen and potassium limitations were without effect. Even with sulphur limitation deposition of the polymer occurred only a t low growth rates; thus a t a dilution rate of 0.1 h-I polyphosphate accounted for 23% of the total cellular phosphate but for only 5.4% a t a dilution rate of 0.2 h-I.

E. POLYPHOSPHATE METABOLISM : ENZYMOLOGY Knowledge of various enzymes involved in the metabolism of polyphosphate has now been obtained and these are discussed under the headings of biosynthesis and degradation.

1. Biosynthesis of polyphosphate (a) A T P -Polyphosphate Phosphotransferase (Polyphosphate kinase). Polyphosphate is synthesized by a unique pathway involving the enzyme polyphosphate kinase, which catalyses the transfer of the terminal phosphoryl group of ATP to polyphosphate (Pi).

+ ATP

+ ADP

+(pi)"+i

This enzyme was first detected in yeast by Yoshida and Yamatalia (1953) and subsequently isolated and purified by Kornberg et al. (1956) from Escherichia coli. It is Mg2+-dependentand inhibited by fluoride, and the biosynthetic reaction is strongly inhibited by ADP, due to reversal of the reaction, i.e. the conversion of ADP to ATP by polyphosphate. Kornberg (1957) determined the K , for ADP to be 47 pM and that for polyphosphate 26 pM, while the K , for ATP was much higher a t 1.4 mM. These K , values suggest that a t low ATP:ADP ratios in viwo polyphosphate would phosphorylate ADP by reversal of the polyphosphate kinase reaction, although Kornberg (1957) observed that different preparations of polyphosphate varied in their suitability as substrates. Muhammed (1961) found the reaction catalysed by the enzyme from Corynebacterium xerosis was not readily reversible in contrast to that of Corynebacterium diphther~aestudied by Kornberg ( 1957). More recently Miihlradt (1971)has purifiedsome 80-fold the kinase from arough mutant of ~almonellaminnesota and shown it t o be very similar t o the enyzme from E . coli. The K," for ATP was 1-25mM. No evidence has been obtained for the existence of short chain intermediates and the phosphoryl group appears to be transferred to a primer molecule of polyphosphate.

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However, the detailed mechanism of action of the kinase remains to be investigated. Mutants of Aerobacter aerogenes which lack polyphosphate kinase are unable to synthesize the polymer and this pathway of synthesis must therefore be unique in the organism (Harold and Harold, 1963; Harold, 1964). The wide distribution of polyphosphate kinase in micro-organisms has now been established (Table 6 ) . TABLE6. Distribution of Polyphosphate Phosphotransferases in Micro-organisms ATP-Polyphospliate Phosphotransferase (Polyphosphate Kinase) Harold (1964) Aerobacter aerogenes Nishi (1960) Aspergillus niger Zaitseva et al. (1960) Azotobacter vinelandii Cole and Hughes (1965) Chlorobium thiosulfatophilum Szulmaster and Gardiner (1960) Clostridium Kornberg (1957) Corynebacterium diphtheriae Muhsmmed (1961) Corynebacterium xerosis Kornberg et al. (1956) Escherichia coli Winder and Denneny (1957) Mycobacterium smegmatis Yoshida and Yamataka (1953) Saccharornyces cerevisiae Muhlradt (1971) Salmonella minnesota 1,3-Diphosphog1ycerate -Polyphosphate Phosphotransferase

Escherichia coli Micrococcus lysodeikticus Neurospora crmsa Penicillium chrysogenum Propionibacterium shermanii

Kulaev et al. (1971) Kulaev et al. (1971) Kulaev and Bobyk (1971) Kulaev etal. (1971) Kulaev et al. (1971)

(b) 1,3-Diphosphoglycerate-PolyphosphatePhosphotransferase. Kulaev et al. (1968), working with an a,denine-deficient mutant of Neurospora crassa, obtained evidence for the existence of a polyphosphate-synthesizing system other than polyphosphate kinase. It was stimulated 100-fold by the addition of a system comprising fructose 1,g-diphosphate, aldolase, glyceraldehyde 3-phosphate dehydrogenase and NAD and was virtually completely inhibited by iodoacetate (2 mM) and by a mixture of arsenate (50 mM) and fluoride (20 mM). Azide and 2,4-dinitrophenol were without effect while ATP and ADP strongly inhibited. They proposed that the polyphosphate molecule was elongated a t the expense of the high energy phosphate group potential of 1,3-diphosphoglycerate (which is greater than that of ATP), thus COOPO,H,

I CHOH

I

CH,OPO,H,

+(PI),

-

COOH

I ‘HOE

I

CH2OPO3HZ

+ (Pi)ntl

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E. A . DAWES AND P J . SENIOR

Subsequently Kulaev and Bobyk (1971) reported the presence of a new phosphotransferase, in enzyme, 1,3-diphosphoglycerate-polyphosphate N . crassa which carries out the above reaction. They used 1,3-diphosphoglycerate labelled with 32Pin the 1-position and demonstrated the incorporation of radioactivity in the polyphosphate. They went on to seek the new enzyme in other species of micro-organisms (Table 6) and endeavoured to assess its significance in relation t o the ATP-polyphosphate phosphotransferase (Kulaev et al., 1971).PenicilliunL chrysogermm was found to possess only the 1,3-diphosphoglycerate enzyme and no activity with the ATP-requiring enzyme could be detected; somewhat surprisingly, in view of their original work with this organism (Iiulaev et al. 1968), they claimed that N . crassa also possessed only the 1,3diphosphoglycerate enzyme. However, both enzymes were demonstrated in E . coli, N . lysodeikticus and Propionihacterium sh,ermanii, although the activities of the ATP-polyphosphate phosphotransferase were generally some three- to ten-fold higher than those of the diphosphoglycerate enzyme. The results obtained with N . crassa and E . coli indicate that enzymic activity is a function both of the growth phase of the culture and the conditions of growth. With E . coli the 1,3-diphosphoglycerate enzyme activity increased as growth progressed. The significance of the new enzyme in those organisms which also possess the ATP-polyphosphate phosphotransferase remains to be elucidated but the generally lower activity suggests that it may not be of major importance. Since 1,3-diphosphoglycerate is an intermediate in the conversion of glyceraldehyde 3-phosphate to pyruvate, and this metabolic sequence is common to all the known pathways of glucose metaboIism, which differ essentially in the means whereby glyceraldehyde 3-phosphate is formed, there is no apparent metabolic guide t o the occurrence of the enzyme. Presumably in those micro-organisms that lack the ATP-requiring enzyme, the 1,3-diphosphoglycerate-polyphophate phosphotransferase could represent the principal pathway of polyphosphate biosynthesis, but investigations with mutants lacking the enzyme now seem desirable to assess this possibility.

2. Degradation of polyphosphate Several enzymes have been found in micro-organisms which cataljrse the degradation of polyphosphates and, as Harold (1966) has pointed out, problems are consequently posed as to the contribution of each to the total rate of polyphosphate breakdown under physiological conditions in the intact cell. We shall consider these enzymes in turn. (a) Polyphosphate kinase. This enzyme is generally reversible and may therefore participate in degradation as well as biosynthesis. Kornberg

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(1957) demonstrated the high affinity of the E . coli enzyme for ADP which suggests that the polyphosphate content of the organism could be controlled by the ATP:ADP ratio of the cell. Such a dual role for the enzyme would not, of course, be in keeping with the general concept of biosynthetic and degradative reactions occurring by different routes. (b) Polyphosph,ate-adenosine monophosphate phosphotransferase. The transfer of phosphate from polyphosphate to AMP was first observed with crude extracts of M . smegmatis (Winder and Denneny, 1957) and was subsequently confirmed by Szymona (1962)

P,)"+l +AMP

-

P,)"+ADP

The enzyme was also found in C. xesosis by Dirheimer and Ebel (1965) who clearly distinguished i t from the polyphosphate kinase, polyphosphate glucokinase (see below) and adenylate kinase of the organism. The enzyme was Mg2+-dependentand specific for AMP and polyphosphate of fairly high molecular weight. Since, however, the K,, for AMP was very high (20 mM), the physiological role of the enzyme seems open to doubt. (c) Polyphosphate glucokinase. This Mg2+-dependent enzyme, which catalyses the phosphorylation of glucose by polyphosphate according to the reaction Glucose

+

+

glucose 6-phosphate + (Pi),

was discovered in glucose-grown M . phlei, other mycobacteria and C. diphtheriae, but could not be detected in E . coli, A . aerogenes or Asp. niger (Szymona, 1962 ; Szymona et al. 1962).Glucosamine was also phosphorylated but not fructose or mannose. Subsequent work by Szymona axid Ostrowski (1964) with the enzyme from M . phlei enabled them to separate it partially, but not completely, from the ATP-specific glucolrinase also present in the bacterial extracts. However, their evidence indicates that distinct enzymes are involved and the reaction is not an indirect one via the intermediary formation of ATP resulting from polyphosphate kinase activity. Dirheimer and Ebel (1968) also adduced evidence that the enzyme they purified two hundred-fold from C. xerosis catalysed a direct reaction between poIyphosphate and glucose or glucosaniine. The optimum pH was 8.2 and the apparent K , values for glucose and polyphosphate were, respectively, 312 and 8.3 pM. The polyphosphate glucokinase of M . yhlei required a high concentration of neutral salts for optimum activity and displayed a K , for polyphosphate of 175 pM (Szymona and Ostrowski, 1964). Szymona et al. (1967) surveyed nineteen species of micro-organism for

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E. A. DAWES AND P. J. SENIOR

the presence of polyphosphate glucokinase and detected it in five species of Proactinomyces (Nocardia) and in five species of Actinomyces but not in Azotobacter vinelandii, Rhodospirillum rubruna, Chlorella vulgaris, Torula utilis, Neurospora crassa, Claviceps paspali, Phycomyces blakesleanus or Agaricus bisporus. All the organisms studied did possess an ATP-glucokinase and Szymona et al. (1967) suggest that the occurrence of the polyphosphate glucokinase might be of taxonomic value, although they did not detect the enzyme in Nocardia paragvensis. These same authors purified the polyphosphate glucokinase 10- to 20-fold and investigated some of the properties of the enzymes from Nocardia and Actinomyces. A partial separation of the polyphosphateand ATP-glucokinases was achieved by fractionation on Sephadex G-100. A survey of the occurrence of polyphosphate glucokinase has also been carried out by Uryson and Kulaev (1968) who found the enzyme in Micrococcus lysodeikticus, Sarcina lutea, Xtaphylococcus aureus, Xtreptococcus faecalis, Propionibacterium shermanii and Propionibacteriurn freidenreichii but not in various Lactobacteria and Acetobacteria. (d) Polyphosphate fructokinase. Extracts of Mycobacterium phlei, which had been grown on fructose instead of glucose, phosphorylated fructose using either ATP or polyphosphate as the phosphate donor. While these activities towards fructose could be separated from the polyphosphate glucokinase and ATP-glucokinase activities by filtration through Sephadex G-100, it proved difficult to demonstrate rigorously the existence of two separate fructokinases aIthough there is strong presumptive evidence for them (Szymona and Szumilo, 1966). (e) Polyphosphatases. The polyphosphatases catalyse the hydrolysis of long chain polyphosphates to inorganic phosphate

+ HzO

(pi)n+~

+

(Pi), + Pi

Quite distinct enzymes have been found to hydrolyse metaphosphates, tetrapolyphosphate, tripolyphosphate and pyrophosphate (Mattenheimer, 1956a, b, c). The polyphosphatase of Corynebacterium xerosis was purified some 100-fold by Muhammed et al. (1959) and shown to be specific for long chain polyphosphates ; no short-chain intermediates could be detected and it appears the polymer is attacked from the end groups. This enzyme was inhibited by Mg2+whereas a similar enzyme from Aerobacter aerogenes was shown by Harold and Harold (1965) to require both Mg2+ and a high salt concentration (about 150 mM) for optimum activity. A physiological role for the polyphosphatase of A . aerogenes in polyphosphate degradation was strongly suggested by the same workers’ observation that a mutant which accumulated poly-

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phosphate in the normal way but was inhibited in its degradation, was devoid of the enzyme. There was evidence to indicate that the phosphate acceptor in the reaction was other than water but Harold (1966) has pointed out that this possibility must remain, especially since no convincing mechanism for the control of polyphosphatase activity has yet been found. The polyphosphatase of baker’s yeast is insensitive to freezing and thawing of the cells, treatment which results in degradation of polyphosphate to inorganic phosphate. The enzyme is, however, affected by p H and NaF in a similar manner to overall polyphosphate breakdown and Souzu (1967a) suggested that in the intact cell, enzyme and substrate are spatially separated by some barrier which is broken down by freezing and thawing. Xouzu (1967b) went on to substantiate this view by showing that polyphosphatase was located inside the cell membrane while polyphosphate and acid phosphatase were in the cell wall. Freezing and thawing damaged the cytoplasmic membrane but not, apparently, the cell wall. The status of polyphosphate which occurs externally to the cytoplasmic membrane in the overall cellular economy remains obscure. Variation in the polyphosphatase activity of Sacch. cerevisiae was observed by Felter and Stahl (1970) as a function of the composition of the medium and growth cycle. The polyphosphatase activity increased a t the beginning of exponential growth, whether the cells had accumulated polyphosphates or not, and then declined. It also increased markedly in phosphorus-deficient cells when phosphate was added to the growth medium, coinciding with the rapid synthesis of polyphosphate. Since inorganic phosphate is a product of polyphosphatase activity, an increase in the phosphate concentration might have been expected to have the opposite result to that observed; the authors suggest that perhaps a stimulation of biosynthesis of the enzyme occurred. Felter et al. (1970) subsequently purified a fraction from Sacch. cerevisiae which displayed general polyphosphatase activity towards all linear polyphosphates tested but not towards pyrophosphate, ATP or cyclic metaphosphates. The enzyme was activated by Mg2+ and Co2+ ions, had a K , for triphosphate of 0-345 mM and displayed its optimum activity a t p H 7.5.

F. REGULATION OF POLYPHOSPHATE METABOLISM Although our understanding of the overall regulatory patterns of polyphosphate metabolism is incomplete, the principal contributions t o our present knowledge have been made by Harold and his associates

TABLE7 . Mutants of Aerobacter aerogenes deficient in polyphosphate metabolism (Harold, 1966) Enzymes

Mutant

Physiological characteristics

Polyphosphate kinase

Polyphosphatase

Alkaline phosphatase ~-

Wild type A3(0)

Pn-1 Pn-2

Pn-3

Pn-4

Polyphosphate not detectablo i n growing cells ; accumulates both by nutrient deprivation and “overplus” No “overplus” but accumulates polyphosphate upon nutrient deprivation Does not accumulate polyphosphate under any conditions Transient accumulation of polyphosphate in growing cells Blocked in polyphosphate degradation

I

Control

7-

All enzymes derepressed by phosphate starvation

F ?

uj,

4 -1-

T

-1-

-

t

Enzymes present but not derepressed by phosphate starvation Kinase absent, other enzymes dercprossod by phosphate starvation AU enzymes constitutively elevated Polyphosphatase absent, other enzymes derepressed by phosphate starvation

? +‘1 0

‘d 4

g 0

ESERGY RESERVE POLYMERS I N MICRO-ORGANISMS

199

with Aerobacter aerogenes. They first isolated a series of mutants which lacked the various enzymes of polyphosphate metabolism (Table 7 ) . The mutant Pn-2, which lacked polyphosphate kinase, was completely unable to accumulate polyphosphate under any conditions (Harold, 1964) while mutant Pn-4, which was devoid of polyphosphatase, could accumulate the polymer but not significantly degrade it (Harold and Harold, 1965). The conclusion drawn therefore was that the only pathway of biosynthesis was mediated by polyphosphate kinase, and the degradative route was via hydrolysis catalysed by polyphosphatase. The “polyphosphate cycle” envisaged is shown in Pig. 14. The polyphosphate content of the cell a t any time must clearly represent the balance between the rates of synthesis and degradation, and control in A . aerogenes appears to be exerted on both enzyme synthesis and activity. Organisms harvested from the exponential phase contained low levels of polyphosphate kinase, polyphosphatase and alkaline phosphatase but if subjected to phosphate starvation differential synthesis of these three enzymes occurred, the kinase level increasing markedly (Fig. 13). Defects in the regulation of enzyme synthesis were observed with two mutants: Pn-1 possessed all three enzymes but repression of their synthesis was not released when they were starved of phosphate, and only when nucleic acid synthesis was prevented by nutrient deficiency did polyphosphate accumulate. The polyphosphate overplus phenomenon (Section 111, D . l , p. 184) was not displayed by this mutant. I n the second mutant, Pn-3, all three enzymes were constitutively derepressed, giving elevated enzyme levels, which correlated with the observed transient accumulation of polyphosphate during the exponential phase of growth (Harold and Harold, 1965). On the basis of his findings with mutants, Harold has proposed that these three enzymes share a common regulator gene but do not occur within a single operon; the genetic control proposed is shown in Fig. 14. The rate of polyphosphate synthesis has been shown to be proportional to the specific activity of polyphosphate kinase and since this enzyme requires ATP for its action, concurrent nucleic acid synthesis, probably by competing for the available ATP, inhibits accumulation of the polymer. The rate of polyphosphate breakdown is similarly proportional t o the specific activity of polyphosphatase, and is stimulated by eoncurrent nucleic acid synthesis. Although the mechanism of coupling between synthesis and degradation is still obscure, these observations do permit a general understanding of the effects of nutritional conditions on polyphosphate accumulation in A. aerogenes. During exponential growth nucleic acid synthesis inhibits polyphosphate deposition and stimulates its degradation, so that no significant polyphosphate accumulation occurs. Cessation of growth and nucleic

E. A . DAWES AND P. J . SENIOR

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201

acid synthesis, as a result of the deprivation of an essential nutrient, leads simultaneously to inhibition of polyphosphate degradation and to promotion of polyphosphate synthesis a t a rate determined by the specific activity of the kinase, since the synthesis of nucleic acids now no longer effectively competes for ATP. As we have already seen, bacteria which have been subjected to phosphate starvation possess elevated levels of polyphosphate kinase as a consequence of differential enzyme synthesis (Harold, 1964) and thus when inorganic phosphate is restored, they enter a phase of rapid polyphosphate synthesis, the basis of the phenomenon of “phosphate overplus”. The activity of the polyphosphate kinase of baker’s yeast has been shown by Felter and Stahl(l970) to increase a t the onset of growth, but when phosphate was added to a phosphorus-deficient growth medium the enzymic activity decreased a t the time when a marked stimulation of polyphosphate synthesis occurred. It was not possible to demonstrate repression of the enzyme by inorganic phosphate and the authors suggest that Pi and polyphosphate ions might have a regulatory effect on polyphosphate kinase activity.

G. PHYSIOLOGICAL FUNCTIONS OF POLYPHOSPHATES 1 . Microbial Xurvival While two of the essential criteria for designating polyphosphate a,s a storage compound have been realized, namely its accumulation under conditions when the supply of energy from exogenous sources is in excess of essential cellular requirements, and its subsequent utilization when the exogenous energy supply no longer fulfils these demands, there is as yet no convincing evidence that possession of polyphosphate enables a cell to survive better than one not so endowed. Attempts by Harold (1965) to demonstrate such biological advantage by comparing wild-type A . aerogenes with a polyphosphate-less mutant proved inconclusive, although he suggests that possibly the capacity to synthesize polyphosphate does confer a small selective advantage, which is not readily detected but is sufficient to ensure the preservation of the enzymes of polyphosphate metabolism and to account for the fact that they have not been eliminated during the course of evolution. 8. Function as an Energy Reserve: the Phosphagen Hypothesis

Hoffman-Ostenhof (1962) has marshalled the arguments in favour of the role of polyphosphate as a microbial phosphagen. These may be

202

E. A . DAWBS AND P. J . SENIOR

summarized as follows : (i)enzymes exist which transfer phosphate from polyphosphate to ADP ; (ii) polyphosphates are linear phosphate anhydrides with an overall heat of hydrolysis of 9 kcal per phosphate bond a t pH 5 ; (iii) polyphosphates accumulate under conditions where ATP production exceeds cellular demands for work and biosynthesis and they are consumed when growth and energy demands are resumed; (iv) 2,4-dinitrophenol abolishes their deposition ; and (v) from the standpoint of comparative biochemistry, polyphosphates occur in microorganisms which lack the conventional phosphagens such as guanidinophosphates and phytin. As Harold (1966) has pointed out, if the term “phosphagen” is interpreted strictly to mean any naturally-occurring phosphorylated compound which can function as a reserve of high energy phosphate and which can be used to phosphorylate ADP to form ATP, then some evidence can be adduced against polyphosphates functioning generally as phosphagens. Thus Harold (1962) working with Neurospora crassa, and Kaltwasser ( 1962) with Hydrogenomonas, have shown that polyphosphate is not degraded in these organisms if the energy supply of the cell is limited or blocked, nor is the ATP pool maintained a t the expense of polyphosphate under such conditions. I n the case of Chlorobium, which Shaposhnikov and Fedorov (1960) did find degraded, to some extent, its polyphosphate when incubated with CO, in the dark, the amount of polymer mobilized was only about one-tenth of the total present, which does not offer powerful support for the phosphagen concept. Further, Harold and Harold (1965) obtained direct evidence that the breakdown of polyphosphate in A . aerogenes is hydrolytic and does not involve conservation of the phosphate group transfer potential. On the other hand, conservation of the phosphate group transfer potential of polyphosphate has been clearly demonstrated in Mycobacteria and Corynebacteria (see Section 111,E.2(c) p. 195),which carry out the phosphorylation of glucose by polyphosphate, although this reaction does not accord with the strict definition of a phosphagen. But perhaps the most telling argument against polyphosphate functioning generally as an energy reserve is that in A . aerogenes (Harold and Sylvan, 1963),Hydrogenomonas (Kaltwasser, 1962) and yeast (Liss and Langen, 1960) the polyphosphate accumulated accounts for but a small proportion of the total ATP generated by these organisms.

3. Function as a Phosphorus Reserve It is now well-established that polyphosphate can supply phosphorus for the biosynthesis of nucleic acids and phospholipids when various organisms are subjected to phosphorus starvation (for a review, see Harold, 1966), and Nishi (1961) has shown that a similar situation exists

E N E R G Y R E S E R V E POLYMERS IN MICRO-ORGANISMS

203

in the early stages of spore germination in Aspergillus niger. The experiments of Kaltwasser (1962) with Hydrogenomonas led him to conclude that polyphosphate in this organism functions as a phosphorus storage material and that its high energy function is biologically of minor importance. The properties of the polymer are clearly in keeping with this role since the osmotic equilibrium of the cell will undergo much less change than if inorganic phosphate were concentrated, which in turn might be expected to affect the equilibria involving the adenine nucleotides and thus the energy charge. As the phosphate content of many natural environments is low due to the insolubility of calcium phosphate (Gulick, 1965), the existence of a phosphorus-reserve material in free-living organisms, and the derepression of the enzyme responsible for its accumulation under conditions of phosphorus starvation, is eminently reasonable (Harold, 1966).

IV. Poly-P-Hydroxybutyrate A. HISTORY Lemoigne (1926,) isolated from Bacillus megaterium two compounds which he considered to be the hydrolysis products of a polymer of p hydroxybutyrate. Both compounds had the same empirical formula (C,H,O) but one was crystalline (m.p. 120°C) while the other was amorphous (m.p. 157°C). The latter compound was shown to be polyp-hydroxybutyrate (PHB).Lemoigne (lR26b ; 1927) found that during autolysis of the bacteria both 3-hydroxybutyrate and the crystalline compound were produced a t the expense of the polymer, and that the latter compound was a low molecular weight polymer of 3-hydroxybutyrate. The first suggestion of a physiological role for P H B was made by Lemoigne and Roukhelman (1940), who demonstrated the formation of 3-hydroxybutyrate during the sporulation of B. rnegaterium. Subsequently, a direct correlation was observed between 3-hydroxybutyrate production and the presence of lipid granules in members of the genus Bacillus (Lemoigne et aZ., 1943). The production of P H B and its degradation prior to encystment were also shown in Azotobacter chroococcurn (Lemoigne and Girard, 1943).Lemoigne et al. (1944) attempted t o classify the genus Bacillus on the basis of acetoin and P H B production and found four groups of organisms, namely PHB-positive and negative, and acetoin-positive and negative. The effect of growth conditions on PHB metabolism was first studied

204

E. A. DAWES AND P. J. SENIOR

by Macrae and Wilkinson (1958a) using an asporogenous strain of B. megaterium. They made the important observation that the quantity of P H B accumulated increased as the carbon to nitrogen ratio of the growth medium was increased. Their results suggested that, like polyphosphate and carbohydrate reserves, P H B accumulation occurred in response to an imbalance of growth brought about by a nutrient limitation. OF POLY-P-HYDROXYBUTYRATE B. OCCURRENCE

A wide variety of micro-organisms can accumulate reserves of polyP-hydroxybutyrate. It is found in Gram-negative and Gram-positive aerobic and photosynthetic species and in lithotrophs as well as many organotrophs. A list of PHB-containing genera is given in Table 8. The accumulation of P H B by an extensive range of Gram-negative bacteria was first demonstrated by Forsyth et al. (1958) and these workers subsequently suggested that the occurrence of P H B could be used as a taxonomic tool in the classification of aerobic Gram-negative organisms (Hayward et al., 1959; Hayward, 1959; Morris and Roberts, 1959). Although it now appears that the possession of P H B cannot be used for classification purposes among the major bacterial taxa, it has proved to be a very useful taxonomic character in classifying pseudomonads. The monumental taxonomic study of aerobic pseudomonads by Stanier et al. (1966) led them to the conclusion that P H B accumulation is probably the best single character for the primary subdivision of the genus Pseudomonas. The polymer producers are Ps. acidovorans, Ps. lemoignei, Ps.mallei, Ps. multivorans, Ps.pseudomnllei, and Ps. testosteroni while this character is completely absent from the fluorescent group, Ps. stutzeri and Ps. maltophila. The alcaligenes group is variable although predominantly positive : two out of six strains of Ps. pseudoalcaligenes failed to produce PHB. Delafield et al. (1965b) isolated a number of pseudomonads capable of accumulating P H B and degrading the polymer when presented exogenously, and two strains of Hydrogenomonas possessing this capacity were also isolated. The extracellular depolymerase system was shown to be distinct from the enzyme system responsible for the endogenous hydrolysis of the reserve polymer. Nodule bacterioids of the genus Rhizobium accumulate PHB (Hayward et al., 1959) although Schlegel (1962a) could not detect it in nodules of Alnus species. Kutty et al. (1969) have isolated P H B from Streptomyces antibioticus, and the formation of P H B by actinomycetes has been the subject of an article by Kannan and Rehacek (1970).

ENERGY RESERVE POLYMERS I N MICRO-ORGANISMS

205

The aerobic nitrogen-fixing organisms of the genera Azotobacter, Beijerinckia and Ilerxia all produce PHB within wide limits (Stockdale et al., 1968), and A . beijerinckii is particularly notable for its ability to TABLE 8. Occurrence of poly-B-hydroxybutyrate in micro-orga.nisms Genera Actinomycetes Azotobacter Bacillus

Beijerinckia Chlorogloea Chromatiurn Chromobocterium Derxia Ferrobacillus Hydrogenomonas Hyphomicro bium Lampropaedia Micrococcus Nocardia Pseudomonas

Rhizobium Rhodop8eztdomonos Rhodospirillum Sphaerotilus Spirillum Streptomyces Tetrahymena Zoogloea

Reference Kannan and Rehacek (1970) Lemoigne and Girard (1943) ; Stockdale et al. (1965, 1968) Wilkinson and Munro (1967) ; Lemoigne (1925, 1946) ; Kominek and Halvorson (1965) Stockdale et al. (1968) Carr (1966); Jensen and Sicko (1971) Schlegel (196213) Forsyth et al. (1958) Stockdale et al. (1968) Lundgren et al. (1965) ; Wang and Lundgren (1969) Schlegel et al. (1961) Hirsch and Conti (1964) Lundgren et al. (1965) Sierra and Gibbons (1962a. b ; 1963) Davis (1964) Forsyth et al. (1958); Doudoroff and Stanier (1959); Delafield et al. (1965 a, b ) ; Stanier et al. (1966) Forsyth et al. (1958) ; Hayward et al. (1959) ; Schlegel (1962a); Fottrell and O'Hara (1969);Wong and Evans (1971) Williamson et al. (1962); Carr and Lascelles (1961) Doudoroff and Stanier (1959) ; Moskowitz and Merrick (1969) Mulder et al. (1962) ;Rouf and Stokes (1962) ; Stokes and Parson (1968); Stokes and Powers (1967) Hayward et al. (1959) Kutty et al. (1969) D. B . Lloyd (personal communication) Crabtree et al. (1965)

accumulate up to 74% of its dry weight of the polymer under certain growth conditions (Ritchie, 1968). A strain of ~ ~ d r o g e n o m osurn~ passes this level; Schlegel et d . (1961) have reported the accumulation of 86% (w/w) of PHB in Hydrogenomonas eutropha.

206

E. A. DAWES AND P. J . SENIOR

c.

!kE

NATUREO F POLY-P-HYDROXYBUTYltATE

1 . Physical and chemical properties Poly-P-hydroxybutyrate is a straight chain homopolymer of u(-)-3hydroxybutyrate, the formula of which is r

1

L When it is extracted with neutral solvents n = 600 t o 2,500, representing a molecular weight of between 60,000 and 250,000 (Lundgren et al., 1965). Lower molecular weights are recorded if the polymer is extracted with sodium hypochlorite (Williamson and Wilkinson, 1958). The purified polymer displays a range of melting points but most determinations have fallen within the span of 157°C (Schlegel and Gottschalk, 1962) to 188°C (Schlegel, 1962b). Polymer purified from A . heijerinckii by alkaline hypochlorite digestion of whole cells, followed by washing with water, acetone and ether, had a melting point of 178"-182OC (E. A.Dawes and P.J. Senior, unpublished observation). The variations of melting point and molecular weight may be attributed to differences in polymer chain length, caused by either the extraction procedure or the stage of growth of the culture from which the polymer was isolated. Alkaline hypochlorite and acid hydrolysis treatment of bacteria gave consistently lower melting point samples, whereas direct neutral solvent extraction of lyophilized cells always gave higher melting points. Presumably the latter extraction procedure incurred less degradation of the polymer (KBpBs and Pdaucl-Lenoel, 1952 ; Alper et al., 1963; Ellar et al., 1968; Lundgren et al., 1965). The chemical and physical properties of PHB are summarized in Table 9. The poly-P-hydroxybutyrate isolated from all the genera studied by Rouf and Stokes (1962), Lundgren et al. (1965) and, in our own laboratory, by Stockdale et al. (1U68), displayed essentially identical infra-red spectra with the major absorption peak a t 17.30 to 17.35 m-' corresponding to the ester carbonyl group. Low molecular weight samples showed distinct -OH stretching absorption peaks a t 34 m-I indicative of free end groups. Investigations of solid PHB were made by Alper et al. (1963) and Lundgren et al. (1965). X-Ray diffractograms of all PHB samples from various micro-organisms had the same patterns and PHB granules in situ were found to be crystalline. The determination of the absolute conformation of the polymer from Rhixobium was reported by Okamura

TABLE9. Chcmical and P h p c a l Properties of Poly-P-hydroxybut3rata.After Ritchie ( 1968) Property -.

Empirical formula Elementary analysis

Melting point Specific gravity Intrinsic viscosity (7) Molecular weight -Idn(g mol-')

Solubility

Reference ~

_

~

_

~

~

.

(C4H60Z)n

% C "OH Theoretical polymer 55.81 7.03 46.15 7.69 p-Hydroxybutyric acid (Most published values approximate t o those of the theoretical polymer) R'ange 160"-1 72°C (Lowest recorded, 157°C;highest, 188°C) 1.23-1.25 g

Yo0 37.16 46.15

Lemoigne (1927); Schlegel and Gottschalk (1962); Schlegel (196213) Williamson and Wilkinson (1958); Okamura and Marchessault (1967) KQp& and P6aud Lenoel (1952)

In 0.2 to 1.5% (w/v) chloroform solutions, measured in an Ubbelhode viscometer. 0.04- 1.05 dl g-' Alper et al. (1963); Lundgren et al. (1965); Hypochlorite isolation Neutral solvent extraction 2.60-1 1.45 dl g-' Okamura and Marchessault (1967) Calculated from [ q ]or by sedimentation using the Archibald method. Hypochlorite isolation : ( I t o 22) x 103 Alper et a2. (1963) Neutral solront extraction: (59 to 256) x lo3 Lundgren et al. (1965) Thioglycollate-chloroform: (140 to 400) x l o 3 Nuti et al. (1972) Soluble in : Chloroform Triolein Ellar et al. (1968); KAppBs and PBaud Lenoel Trichloroethylene Camphor (1952); Williamson and Wilkinson (1958) Dichloroacetate Acetic acid, glacial Trifluoroethanol Phenol, 0.5 M aqueous Dimethylformamide NaOH, M Ethyl acetoacetate Hyamine hydroxide, M Partially soluble in.: Dioxane Toluene Pyridine Octanol Insoluble in : Methanol Water Ethanol Dilute mineral acids Acetone Alkaline hypochlorite Ether Ethyl acetate Hexane Carbon tetrachloride

M

2z

F2 B

a w

C

zEt3

su1 w

Z

F2

@

? 0 0

*k5

B

cn

K'

s

208

E. A . DAWES AND P. J . SENIOR

and Marchessault (1967) who proposed that the PHB molecuIe is a compact right-handed helix with a two-fold screw axis along the chain. Conformational analysis by Cornibert and Marchessault (1972) led to the molecular model shown in Fig. 15.

0000 H C n

0

CH,

U

Model 1

FIG.15. Molecular model of poly-8-hydroxybutrate with thc lowest energy, based on X-ray and conformational analysis. The fibre repeat, 5.96 d,involves two residues related by a two-fold screw axis along the chain. (From Cornibert and Marchessault, 1972.)

Alper et al. (1963) have used electron microscopy to examine recrystallized PHB. Crystals were lath shaped and invariably folded. Lamellar morphology was also noted in larger crystals with lemellae 5 nm thick. These structures are single crystals of PHB with the polymer molecules folded and running a t 90" to the lamellar surface (Okamura and Mar-

ENERGY RESERVE POLYMERS I N MICRO-ORGANISMS

209

chessault, 1967). Marchessault et al. (1970) carried out investigations of the conformational aspects of P H B in solution by means of intrinsic viscosity, sedimentation velocity and optical rotatory dispersion measurements. Coil-like behaviour in chloroform, ethylene dichloride and trifluoroethanol was indicated by the hydrodynamic properties observed in each solvent alone, but comparisons of the different solvents supported a sharp helix-coil transition and the authors proposed a model involving folded helical segments which allows rigid rod behaviour but is reconcilable with the hydrodynamic data. To obtain more direct evidence on molecular shape, light-scattering studies with solutions of P H B in 2,2,2-trifluoroethanol were undertaken (Cornibert et al., 1970). The results were interpreted in terms of a relatively rigid, rod-like particle with a linear density greater than that of the crystallographic helix. This implies that the helical segments are folded on themselves, an hypothesis which appears to account for the hydrodynamic and lightscattering data recorded with solutions of P H B in trifluoroethanol.

2. Chemical synthesis of D-poly-P-hydroxybutyrate The first chemical synthesis of D-PHB was announced in 1971 (Shelton et al., 1971a, b ; Agostini et al., 1971). These workers investigated organometallic catalyst systems for the polymerization of DL-P-butyrolactone and found that triethylaluminium plus water (1 : 1)as co-catalyst produced highly crystalline samples of the polyester. The resulting racemic polymer was shown by a variety of techniques, including infra-red, NMR and ORD spectra, t o be essentially identical with the naturally-occurring polymer in all respects except optical activity. They then applied the same method to polymerize D(+)-P-hydroxybutyrolactone of 73% optical purity and obtained D-PHBwhich was essentially identical with the natural polymer from B. cereus and Rhixobium. The melting point of the synthetic P H B was 168°C and its intrinsic viscosity in chloroform solution 0.60 dI g-l, well within the range of molecular weights reported by Lundgren et al. (1965) and recorded in Table 9. X-Ray diffractograms were virtually identical with those of the bacterial polymer, from which it was assumed that the synthetic polymer contained the same helical structure in the crystalline state as natura,llyoccurring PHB.

3. Poly-P-hydroxybutyrate granules Granules in bacteria which could be stained with Sudan Black were suggested to contain P H B (Williamson and Wilkinson, 1958), a proposal which was confirmed by the subsequent work of Merrick and Doudoroff (1961). When bacteria which have accumulated P H B are viewed under the microscope with phase contrast or dark field illumination, granules

210

E. A. DAWES AND P. J. SENIOR

FIG.16 (a) Photomicrograph of Azotobacter beijerinckii N.C.I.B. 9067 showing capsule and intracellular poly-8-hydroxybutyrate granules. Bacteria were taken from the stationary phase of a nitrogen-free-1'YO(w/v) glucose culture, suspended in India ink, and viewed under phase contrast. From Senior (1972). ( b ) and (c) Electron micrographs of ~iltratli in sections of Hydrogenomonas eutropha H 16 revealing poly-B-hydroxybutyrategranules as optically empty areas. Exponential-pl-iasc cells (b)were incubated for 20 h with acetate under aerobic conditions in tho absence of R nitrogen source (c). Elcctrori micrography by P. Hillmer and 1'. Amelunxen. By courtcsy of Professor H. G. Schlegel.

of PHB may be visualized quite clearly due t'o their high refractivity (Fig. 16). Although the granules are intensely stained by Sudan Black in fixed bacterial preparations, isolated granules or purified polymer do not take up the dye. Presumably during cell disruption a compound of lipoidal nature, which binds at or near the granule surface, is removed.

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211

(For legend see facing page)

Poly-P-hydroxybutyrate granules isolated from B. megaterium are spherical and range in diameter from 0.2 to 0.7 pm with a number average value of 0.49 pm (Ellar et at., 1968). By use of light-scattering techniques these workers reported a weight-average particle weight of 3.57 x lo9 g/mol of particles. This means that each granule contains a t 10

212

E. A . DAWES A X D P . J . SEXIOR

least several thousand PHB molecules. All these measurements by Ellar et al. (1968) were carried out on native granules, that is granules which are susceptible to enzymic degradation under certain conditions, as described by Merrick and Doudoroff (1964) (see Section IV, FZ(b)(ii), p. 238). Electron microscopy of serial sections of protoplasts of Rhodospirillum rubrum revealed that the P H B granules present were surrounded by a membrane 4.5 nm thick (Boatman and Douglas, 1963). A similar observation was made by Pfister and Lundgren (1964) during their studies of thin sections of samples of Bacillus cereus treated by freezing and thawing. The coat around the B. cereus granules did not have the typical dark-light appearance of osmium stained unit membranes. Further work by Pfister et al. (1964) and Lundgren et al. (1964) demonstrated the presence of a membrane 6 to 8 nm thick around the granule, and which appeared to be continuous with the cytoplasmic membrane. Thin sections of Caulobacter (Poindexter, 1964) and Lampropaedia hyaline (Doudoroff, 1966) clearly demonstrate the presence of a membrane around the PHB granules in these organisms. Poly-/3-hydroxybutyrate granules synthesized by the chemolithotrophic bacterium Perrobacillus ferro-oxidans are also surrounded by a dense membrane 2.5 to 3.5 nm wide (Wang and Lundgren, 1969). The membranes of granules which have been isolated by alkaline hypochlorite solubilization of other cell components are destroyed and the granules are no longer “native”, i.e. they are not susceptible to enzymic degradation. Granules prepared from B. cereus by this method contain approximately 11% of the dry weight of a mixture of trigly cerides, free fatty acids and traces of phospholipids (Williamson and Wilkinson, 1958).With Pseudomonas nzethanica, Kallio and Harrington (1960) have shown that 92% of the chloroform-soluble constituents of dried cells was P H B and the rest monopalmitin. However, in view of the subsequent report that native granules from B. megaterium contain only about 0.5% lipid (Griebel et al., 1968), the validity of this earlier work seems doubtful and it is possible that lipids from other components of the cell contaminated the granules. Native granules from B. megaterium also contain protein and phosphorus; 2.1 mg and 0.2 pmo1 respectively per 100 mg of PHB (Doudoroff, 1966). Although no neutral lipid was detected, phosphatidic acid accounted for most of the phosphorus content. Treatment of native PHB granules with aqueous acetone removes the membrane coat and thereby exposes the granules (Ellar et al., 1968). Granules were observed to be unfolded revealing a fibrillar structure and long, parallel flexible fibres coalesced to form ribbons which, in turn, yielded the characteristic lath-shaped crystals of PHB.

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213

The association of protein with the native granule has been linked with the observation that in some cases part or the whole of the enzyme system(s) concerned with polymerization and depolymerization of P H B are attached to the native granule (Merrick and Doudoroff, 1961, 1964; Merrick, 1965). Griebel et al. (1968) noted that 2% (w/w) of the native P H B granule from B. megaterium was protein, together with approximately 0.5% (w/w) of lipid. The major component of the lipid fraction was phosphatidic acid. They confirmed the observation of Merrick et al. (1965) that extensive trypsin treatment of native granules yielded a granule inactive to enzymic degradation. It is almost certain that P H B synthetase is tightly bound to the granule in B. m e g a t e r i u ~and as such constitutes a significant proportion of the granule protein content recorded by Griebel et al. (1968). Ritchie (1968) also observed the association of P H B synthetase with purified native granules from A. beijerinclii. Griebel et al. (1968, 1971) have further shown that a labile factor involved in polymer depolymerization in B. megaterium is associated with the granule proteins. The properties of the various enzymes associated with native P H B granules will be discussed in detail in Sections IV, E (p. 228) and IV, F (p. 236) dealing with P H B biosynthesis and degradation. At this juncture it is suffkient to note that a fundamental biochemical problem is posed by the P H B granule. How does the microbial cell synthesize a granule of hydrophobic material in an aqueous environment? Obviously the surface components of the granule play an important role and, assuming that these systems are in close contact with their substratelproduct at all times, the native P H B granule would be an interesting model for the study of enzyme activities a t interfaces and the regulation of these activities.

4 . Determination of poly-P-hydroxybutyrate Lemoigne (1926~) introduced a gravimetric method for determining P H B based upon the fact that, of the common organic solvents, the polymer is soluble only in boiling chloroform and can therefore be purified by extraction with other solvents. Restrictions are presented by the relatively large quantity of polymer required (milligram amounts) and by the necessity for quantitative re-precipitation. A method affording greater sensitivity was devised by Williamson and Wilkinson (1958), in which the bacterial cells were dissolved completely in sodium hypochlorite solution and the turbidity of the remaining lipid granules measured. The method is calibrated by comparison with the gravimetric method but suffers from the disadvantages that calibration curves must be prepared for each organism investigated and it is appli-

214

2 . A . DAWES AND P. J. SENIOR

cable only to P H B present in the lipid granules. With Bacillus cereus a range up to about 210 mg PHB/ml was readily determined. Law and Slepecky (1961) introduced a convenient spectrophotometric determination of PHB involving the quantitative conversion of P H B to trans-crotonic acid by heating in concentrated sulphuric acid, and the fact that the U.V. absorption maximum of crotonic acid is shifted to 235 nm when concentrated sulphuric acid is the solvent. The PHB was prepared for assay by digesting the bacteria with sodium hypochlorite, as in the Williamson and Wilkinson (1958) technique, and collecting the granules by centrifuging. The granules were washed successively in distilled water, acetone and ethanol and the PHB was then extracted from the residue with hot chloroform and filtered, the filtrate being assayed for PHB. The assay procedure permitted a range of 5-50 mg of polymer to be determined. The molar extinction coefficient of crotonic acid under the conditions of the assay is 1-55 x lo4. Certain materials, notably carbohydrates, cause some interference in the assay and with some cells which do not contain PHB, an insoluble material is obtained after hypochlorite treatment which contains an interfering substance. The latter, unidentified material gives a spectrum which is, however quite different from the typical crotonic acid spectrum. The Law and Slepecky method has proved extremely useful and has been widely used. It suffers from the disadvantage that the repeated centrifuging needed to prepare samples for assay is time consuming and limits the number of samples which may easily be handled. Also the quantitative recovery of small amounts of P H B is difficult making the assay of small samples or samples with a low P H B content (less than 1.5% of the dry weight) inaccurate. To overcome these difficulties Ward and Dawes (1973) devised a disk assay which eliminated the need for centrifuging, and improved the accuracy. The bacterial suspensions were applied directly to glass fibre disks previously cleaned with concentrated sulphuric acid. After drying the disks were treated with sodium hypochlorite to digest the bacteria and then dried, followed by treatment with chloroform to ensure the P H B adhered to the disks. Further sodium hypochlorite treatment and washing with solvents preceded transfer of the disks to tubes containing concentrated sulphuric acid which were then heated as in the Law and Slepecky method. By this technique bacteria containing as little as 0.1% PHB could be assayed reproducibly.

D. POLY-P-HYDROXYBUTYRATE METABOLISM

I. Introduction The central theme of our review is the role of P H B as an intracellnlar reserve material and it is pertinent to note that, on purely chemical

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215

grounds, P H B would provide an excellent storage compound in that it exists in a highly reduced state as a virtually insoluble crystalline polymer exerting negligible osmotic pressure. The metabolic role of P H B will therefore be discussed in terms of its ability to serve as a n endogenous reserve of carbon and energy and in relation to the maintenance of viability during starvation, sporulation and encystment.

2. The accumulation of PHB in batch culture Lemoigne et al. (1950) observed that the quantity of P H B synthesized by B. megaterium was dependent on the composition of the medium. With an asporogenous strain of the same organism Macrae and Wilkinson (1958b)noted that as the ratio of the carbon and energy source (glucose) to the nitrogen source (NH,Cl) of the medium was increased, the amount of P H B accumulated per cell also increased. Nitrogen-limitation of exponential growth in the presence of excess carbon and energy source produced cells with four-fold the quantity of P H B recorded for glucoselimited cultures. Pyruvate, 3-hydroxybutyrate and glucose increased the yield o f PHB synthesized by washed cells of an asporogenous strain of B. megaterium KM and when acetate was added together with any of these substrates, the rate o f P H B synthesis was greatly increased (Macrae and Wilkinson, 1958a). The importance of acetate in P H B metabolism was thus established for B. megaterium. Macrae and Wilkinsoii (1958a) also showed that P H B accumulation was not a mechanism for the removal of toxic acidic catabolites by a process of neutralization. Accumulation and degradation of the polymer had a well-defined p H optimum a t 7.5. Glycerol, pyruvate or acetate added to the basal complex medium increased the quantity of P H B accumulated by the halophile Micrococcus halodelzitrificans (Sierra and Gibbons, 1962a) although aeration was a factor in determining the quantity of polymer accumulated; as the aeration rate was increased the quantity of P H B accumulated decreased. Nitrogen-limitation of chemolithotrophic growth of several strains of Hydrogenomonas resuIted in the accumulation of massive quantities of PHB (Schlegel et al., 1961). Rhodospirillum rubrum is an interesting example of an organism which can accumulate both P H B and glycogen. Stanier et al. (1959)showed that the nature of the carbon source and the conditions of growth influenced the flow of carbon into the different reserves. They concluded that when substrates were metabolized via acetyl-CoA, without the intermediate formation of pyruvate, PHB aecumulation predominated. Growth with substrates which were metabolized via pyruvate led t,o the formation of glycogen. The first reports of the involvement o f P H B in the sporulation process

216

E. A. DAWES AND P. J. SENIOR

of Bacillus species were made by Tinelli (1955a, b). Slepecky and Law (1961) expanded these observations with B. megaterium and concluded that while P H B accumulation was not a pre-requisite for spore formation, the polymer, which accumulated on the cessation of exponential growth, could serve as a source of carbon and energy t o drive the sporulation process. Kominek and Halvorson (1965) reported similar findings with B. cereus. P H B was accumulated to 10% of the dry weight on limitation of exponential growth and disappeared when sporulation occurred (Fig. 17). Prior to the onset of sporulation the p H of the medium fell

FIG.17. Changes in the p H value, turbidity and poly-8-hydroxybutyratecontent of a culture of Bacillus cereus T during growth and sporulation. From Kominck and Halvorson (1965).

throughout exponential growth from 7.4 to 4.6-4.9. At this minimum value P H B accumulation commenced. Acetic acid could serve as the carbon source for P H B synthesis during the first stage of accumulation (Fig. 17) and acetoin, produced after the attainment of the low p H in these cultures, for the second stage of accumulation. The pathway of acetoin utilization in this organism is via an inducible 2,3-butanediol cycle, identical with that described by Juni and Heym (1956). For each turn of the cycle two molecules of acetoin regenerate one of acetoin and two molecules of acetate. This intracellular acetate is then, according to Kominek and Halvorson (1965), incorporated into PHB. The accumulation of P H B in batch cultures of the sporogenous B. cereus was, unlike that reported by Macrae and Wilkinson (1958a) for B. megaterium,very sensitive to changes of the medium pH. Maintenance of B. cereus cultures a t high p H values resulted in an inhibition of P H B accumulation, whereas buffering a t a low p H inhibited P H B utilization during the sporulation period. Kominek and Halvorson (1965), although

ENERGY RESERVE 'POLYMERS I N MICRO-ORGANISMS

217

carrying out a detailed study of the relationship of medium pH, intermediate formation and the activities of pertinent enzymes to the degree of sporulation, did not suggest a role for P H B in sporulation other than the usefulness of such a compound in its role as a reserve of carbon and energy to the endergonic reactions of sporulation. The role of acetate, which serves as a substrate for P H B biosynthesis, in sporogenesis of Bacillus cereus was studied by Nakata (1966). When cells committed to sporulation were placed in a chemically-defined sporulation medium containing [2-14C]acetate, 65 to 70% of the initial radioactivity was incorporated into P H B while the remainder entered other cellular constituents. Virtually no radioactivity was lost as I4CO, during the first 5 to 6 h after replacement. Then followed a gradual release of 14C02, coincident with P H B degradation, until 9 h when the polymer was essentially depleted and the first spore structures were observed in stained preparations. However, the total radioactivity eliminated as CO, was only 20 to 25% and the major portion of the products derived from PHB was incorporated into the spores, the majority entering protein although substantial amounts were found in dipicolinic acid and the hot trichloroacetic acid-soluble fraction. On the basis of these findings Nakata (1966) concluded that the principal role of acetate, and subsequently of PHB, in the sporulation process is to provide carbon precursors and energy for sporogenesis. Stevenson and Socolofsky (1966) have reported on the formation and significance of P H B in Axotobacter with reference to the formation of cysts, a process common to this genus. Axotobacter vinelandii formed cysts a t the end of vegetative growth on solid nitrogen-free medium with either glucose, sucrose or butanol as the carbon and energy source. P H B accumulation and degradation followed a similar pattern for all the carbon sources; butanol is shown as a typical example in Fig. 18. After two days growth of A . vinelandii on butanol-supplemented nitrogen-free medium, 35% of the dry weight could be accounted for as PHB. Subsequently cyst formation commenced and the accumulated P H B was degraded (Fig. 18). Butanol and glucose were particularly good carbon sources for the stimulation of P H B accumulation and cyst formation in nitrogen-fixing populations. However, during growth on these substrates with a fixed source of nitrogen, P H B was accumulated to only approximately 18% of the dry weight and cyst formation occurred in less than 10% of the population. Stevenson and Socolofsky (1966) concluded that P H B was utilized as a carbon and energy source during encystment and, further, that substrates which encouraged extensive cyst formation promoted the accumulation of large quantities of PHB. They proposed that the accumulation of P H B by A. vinelandii reflect,ed an imbalanced growth

218

A. DAWES AND P. J . SENIOR

E.

condition, which is essential for the encystment process ; the cells assimilate the exogenous carbon supply much faster than they can fix the molecular nitrogen necessary for conversion into nitrogenous cell components. The cells, therefore, accumulate a large amount of nonnitrogenous material as PHB, which provides a source of carbon and energy for encystment. Lin and Sadoff (1968) extended the work of Stevenson and Socolofsky (1966) with A . vinelandii and found that although growth in nitrogenfree liquid medium supplemented with butanol encouraged cyst for+

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mation, crotonate and 3-hydroxybutyrate gave increased encystment. Butyraldehyde and butyrate were without effect. The significance of PHB metabolism was not, however, discussed. I n an extensive survey of species of aerobic nitrogen-fixing genera Stockdale et al. (1968) noted that in most cases maximal levels of PHB accumulation were obtained by increasing the glucose content of the nitrogen-free batch culture medium from 1 to 2% (w/v) (Table 10).The infra-red spectra of the P H B from these genera were essentially similar to those recorded by Slepecky and Law (1961) for P H B isolated from B. megaterium. Stockdale et al. (1968) measured the accumulation of P H B in batch cultures of various Axotobacter and found the presence of a constant percentage of PHB in the organisms during exponential growth, indicating a departure from the normal pattern of reserve material production. To explain this observation it was suggested that P H B accuniulation was the result of one or more of: (i) a permanent restriction of

219

ENERGY RESERVE POLYMERS IN MICRO-ORGANISMS

respiratory rate a t the level of pyruvate oxidase or beyond; (ii) gaseous nitrogen-limitation of growth ; (iii) oxygen-limited growth. Of significance was the observation that the dissolved oxygen concentration in a batch culture of A . insigne V J 5 fell to zero during growth, lending supTABLE10. Poly-8-hydroxybutyrate content of the Azotobacteriaceae Maximum poly -P-hydroxybutyrate content

Organism

Strain

1%glucose in medium

2% glucose in medium

15.3 10.0 9.1 34.4 10.3 43.3

22.3 9.2 10.1 50.3 6.4 48.6 23.4 14.6 67.7 39.9 42.4 30.7 42.4

__

Azotobacter A. macrocytogenes A . mucrocytogenes A . vinelandii A . vinelundii A. agilis A. agilis A . insigne A . insigne A . beijerinckii A . beijerinckii A . chroococcum A. chroococcum A . chroococcurn Beijerinckii B. lacticogenes B. indicus B. indicus B. mobile B. Jlurninensis Derxia Derxia gumrnosa

NCIB 8700 NCIB 9128 NCIB 8660 NCIB 8789 NCIB 9473 NCIB 8637 NCIB 9127 VJ 5 NCIB 9067 NCIB 8948 NCIB 8002 NCIB 8003 NCIB 9125 NCIB 8846 NCIB 8597 NCIB 8712

21.5

10.5 70.4 28.0 40.3 26.4 13.4

-

16.2 6.5 15.9 29.4 2.5

14.1 16.9 21.5 37.5

NCIB 9604

19.7

25.8

-

6.5

From Stockdale et al. (1968).

port to the proposal that oxygen limitation may contribute t o PHB accumulation. Growth of Azotobacter on a fixed source of nitrogen, while increasing the growth rate, did not change the quantity of PHB accumulated. The discovery that A . beijerinckii could accumulate PHB in excess of 70% of its dry weight led to the choice of this organism for detailed studies of PHB metabolism in our laboratory. Ritchie (1968) and Senior

220

E. A. DAWES AND P. J. SENIOR

et al. (1972) demonstrated that PHB did not comprise a constant percentage of the bacterial dry weight during the batch growth of A . beijerinckii. I n nitrogen-free medium with 0.5% (w/v) glucose, polymer was accumulated towards the end of exponential growth and

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during the stationary phase (Fig. 19a). The maximum level was 35% (w/w). Following glucose exhaustion, synthesis of the polymer ceased and it was rapidly degraded without a concomitant decrease in total cell dry weight. It was concluded that carbon derived from the degraded polymer is incorporated into cell constituents, a process probably sustained by the energy derived from oxidation of a proportion of the PHB.

ENERGY RESERVE POLYMERS I N MICRO-ORGANISMS

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With 2% glucose in the medium (Fig. 19b) polymer was again accumulated towards the end of exponential growth, but, unlike the pattern with limiting glucose, polymer accumulation continued during the stationary phase and a t the final measurement comprised 74% (w/w) of the dry weight. Figure 20 illustrates the deposition of P H B during the batch growth of A . be6jerinckii on 1% glucose in nitrogen-free cultures. Batch culture work did not, however, permit identification of the nature of the nutrient limitation which potentiated accumulation of PHB. Glucose-limitation of growth could be eliminated but as the culture was simultaneously fixing atmospheric nitrogen and utilizing atmospheric oxygen, the true nature of the growth limitation could not be ascertained under these conditions. However, experiments with an oxygen electrode suspended in a shaken culture revealed that P H B accumulation was initiated about the time when the culture dissolved oxygen concentration attained zero, coinciding with the transition from exponential to a slower rate of growth (Senior et al., 1972). The problem was further pursued by experiments with continuous cultures [Section IV, D.3(c), p. 2241.

3. Continuous culture experiments (a) Bacillus megaterium. Wilkinson and Munro (1967) appear to have been the first to study the production of P H B under conditions which attempt t o simulate the natural physiological environment, by employing a chemostat. They found that B. megaterium KM, grown with either the nitrogen, sulphur, potassium or carbon and energy source as the limiting factor, accumulated P H B with a maximum level a t a dilution rate of approx. 0.4 h-’ . They noted, as did Holme (1957) for the accumulation of glycogen by E . coli in continuous culture, that the specificgrowth rate of the culture had an important effect on the amount of PKB accumulated, because the patterns of accumulation, during all the growth-limiting conditions studied, were rather similar (Fig. 2 1). Appreciable quantities of P H B (a maximum of 12% of the dry weight) were accumulated by carbon and energy-limited populations, suggesting that PHB biosynthesis in B. megaterium is not a metabolic “shunt” whereby carbon in excess of cellular requirements is metabolized to a waste product. The strain of B. megaterium used by Wilkinson and Munro (1967) had, however, undergone several significant changes since its original isolation, having lost the ability to form spores and having acquired the ability t o grow in simple synthetic medium and under conditions of continuous culture. I n relation to the findings of Slepecky and Law (1961), the interpretation of these chemostat results is thus not straightforward since an asporogenous mutant is now being discussed.

222

E. A. DAWES AND P. J. SENIOR

FIG.20. Photomicrographs of Azotobacter beijerinckii during batch growth on nitrogen-free-1 %, (w/v) glucose medium, showing deposition of poly-j-hydroxybutyrate. Unstained preparations taken at (a)3 h, (b) 13 h , (c)27 h after inoculation at which times the contents of poly-j-hydroxybutyrate were respectively 10, 25 and 65% of the dry weight. Exponential growth ceased after 13 h a n d the stationary phase was attained a t 27 h. Unpublished photographs of P. J. Senior and E. A. Dawes.

(b) ~ y ~ r o g e n o m o eutropha. n~s Schuster and Schlegel (1967) designed a chemostat for growth of the autotroph Hydrogenomonas eutropha HI6 on limiting concentrations of hydrogen or oxygen, the gaseous components being produced by electrolysis of the mineral medium. In contrast to static culture, in which the rate of gas uptake and hydrogenase

ENERGY RESERVE POLYMERS I N MICRO-ORGANISMS

223

(For legend, see facing page)

activity declined concomitantly, the organism in continuous culture with hydrogen as the limiting factor displayed high specific activities for hydrogenase and glyceraldehyde 3-phosphate dehydrogenase ; the PHB content was negligible under these conditions. However, with oxygen

224

E. A. DAWES AND P. J. SENIOR

as the limiting factor the bacteria accumulated up to 23% of their dry weight of P H B and the activities of hydrogenase and glyceraldehyde 3-phosphate were low. Thus H . eutropha was shown to accumulate P H B under conditions of oxygen limitation as well as under the previously demonstrated nitrogen limitation (Schlegel et al., 1961). (c) Azctobacter beijerinckii. The difficulties experienced by Senior et al. (1972) in their use of batch culture experiments to determine the nature of the iiutrient limitation(s) which stimulate P H B accumulation and degradation in A . beijerinckii were noted in Section IV, D2 (p. 221).

Dilution rate (h-1 )

FIG.21. Effect of nutrient limitation on the cellular content of poly-B-hydroxybutyrate and glycogen in Bacillus megaterium K M over a range of dilution rates. Limitations were (a) potassium and sulphur (b) carbon and energy (glucose) and nitrogen. Potassium-limited, 0; sulphur-limited, A ;glucose-limited, ; glucoselimited plus excess acetate, 0; nitrogen-limited, Q ; nitrogen-limited plus excess acetate, x. Open symbols represent poly-8-hydroxybutyrate content, and solid symbols glycogen content. Adapted from Wilkinson and Munro (1967).

They therefore transferred this problem to the more easily controlled environment of the chemostat (Senior et al., 1972). Nitrogen-fixing cultures of A. beijerilzckii (NCIB 9067) were grown under three different nutrient limitations and over a wide range of specific growth rates. The essential findings (Fig. 2 2 ) were : (i) nitrogen-limited cultures did not accumulate P H B ; (ii) PHB was detected (approx. 3.0% (w/w))in slowgrowing glucose-limited cultures but the quantity decreased with increasing specific growth rate ; and (iii) oxygen-limited cultures produced large quantities of PHB. Under these oxygen-1imit.edconditions PHB accumulation decreased with increasing specific growth rate. Cell yields per mole of glucose utilized were greater than for carbon- or nitrogen-limited populations a t all growth rates. Although P H B comprised a significant proportion of the increased yields, subtraction

ENERGY RESERVE POLYMERS I N MICRO-ORGANISMS

225

of the polymer content from the total dry weight revealed that oxygenlimited growth yields were still substantially greater than those for nitrogen- or carbon-limited cells. The effect of the sudden imposition of oxygen-limitation on a nitrogenlimited chemostat culture was investigated (Fig. 2 3 ) . Simultaneous recordings of pH, dissolved oxygen concentration, redox potential (E,), glucose concentration in culture supernatant, bacterial dry weight and PHB content were made during the transition period. PHB was accumulated when the culture dissolved oxygen concentration had fallen t,o < 1.0% (100% = air saturation under these conditions of temperature and ionic strength). The behaviour of the culture redox &

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potential during the transition period was particularly interesting. After an initial drop from +15 mV to -50 mV an oscillation was observed, followed by a dramatic rise from -50 mV to t30 mV. Thus the redox potential of the oxygen-limited culture was more positive than that of the nitrogen-limited culture where dissolved oxygen was in excess of requirement. The limitations and pitfalls involved in the interpretation of E , measurements on bacterial cultures are patent, but it was suggested by Senior et al. (1972)that these findings were in accord with their proposed function for PHB accumulation in A . beijerinckii, namely that of an “electron sink” for excess reducing power which accumulates when the cell becomes oxygen-limited and is no longer able to oxidize NAD(P)H produced by glucose catabolism a t the high rates achieved by nitrogenor carbon-limited cultures (Senior and Dawes, 1971a; 1971b). Evidence to support this hypothesis was provided by the discovery that oxygen-limited cultures, which displayed very low rates of O2

226

E. A. DAWES AND P. J. SENIOR

utilization and carbon dioxide formation in situ compared with the in situ rates for nitrogen- and carbon-limited cultures, had lowered potential rates of gas exchange. A glucose-limited population (D = 0-10 h-l) had a manometrically determined rate of oxygen uptake

H

w

Time ( h )

FIG.23. Effect of imposition of oxygen. limitation on a nitrogen-limited chernostat culture of Azotobacter beijerinckii. The nitrogen-limited culture growth rate was 0.233 h-' and, a t the point indicated by the arrow, the oxygen supply rate was decreased from 100 ml/min to 25 ml/min, imposing an oxygen limitation; 40 min

a,

later, the oxygen supply rate was decreased further to 15 ml/min. (a) Culture dissolved oxygen concentration; H, redox potential in situ; (b), A, poly-phydroxybutyrate content ; A , culture glucose concentration ; (c) 0 , culture (d) 0, bacterial dry weight. From Senior et al. (1972). turbidity (E500);

(equal to the rate of CO, evolution) of 675 pl h-'/mg dry weight. Similar determinations with bacteria from an oxygen-limited culture (D = 0.102 h-l) yielded a value of 100 pl h-'/mg dry weight, thus displaying an apparent seven-fold difference in the ability of the organisms to utilize oxygen. This difference is accentuated further when correction is made for the PHB content (45% w/w) of oxygen-limited cells at this dilution rate.

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Recently Senior and Dawes (1973) have shown that when an oxygenlimited culture of A. beijerinckii, possessing PHB, has the oxygen limitation relaxed and is allowed to grow unrestrictedly in the presence of excess glucose, molecular nitrogen and oxygen, PHB accumulation

Time (h) after relaxation of oxygen limitation

FIG.24. Effect of relaxation of oxygen-limitation on an oxygen-limited cheinostat culture of Azotobacter beijerinckii. The oxygen-limited culture specific growth rate was 0.101 h-l and, a t zero time, the oxygen supply rate was increased automatically by the dissolved oxygen concentration controller to maintain a dissolved oxygen concentration of 10% of air saturation. The arrows indicate the time when glucose in the culture became exhausted. (a) Redox potential i n situ, A ; (b) culture dissolved oxygen concentration, 0 ; (c) culture dry weight; 0; culture dry weight minus poly-P-hydroxybutyrate content, A ; (d) bacterial poly-/3hydroxybutyrate content, 0. (From Senior and Dawes, 1973.)

ceases and subsequently, when the glucose concentration in the culture has fallen t o a very low value, degradation of the polymer commences (Fig. 24). The relaxation of oxygen-limitation also caused an immediate increase in the rates of oxygen utilization and CO, evolution in situ, without a significant concomitant decrease in the culture glucose con centration.

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These experiments with chemostat cultures of nitrogen-fixing Azotobacter reveal that nitrogen-limitation does not stimulate P H B deposition, in contrast to many genera of micro-organisms in which the accumulation of P H B and glycogen is initiated by nitrogen-limited growth conditions. When the growth of A . beijerinckii is limited by a fixed nitrogen source (NH,+), P H B is not accumulated; only when ammonium salts cultures become oxygen-limited can P H B be detected in the cells. Dr. A. C. Ward, in our laboratory, has shown that, as with nitrogen-fixing cultures, the PHB content of ammonium-grown, oxygen-limited bacteria decreased with increasing dilution rate, although was greater for such cultures, the maximum specific growth rate (pmax) and a t dilution rates greater than 0.3 h-' these cells had no detectable PHB. A discussion of the significance of this work with chemostat cultures will be found in Section I V G (p. 244) which deals with the physiological aspects of the regulation of PAB metabolism.

E. THEENZYMOLOGY OF POLY-/!-HYDROXYBUTYRATE BIOSYNTHESIS 1 . General The earlier work of Stanier et al. (1959) with Rhodospirillum rubrum had suggested that acetyl-CoA was the precursor of P H B with their demonstration that acetate, butyrate and DL-3-hydroxybutyratecould be incorporated into PHB without the intermediate formation of pyruvate. Macrae and Wilkinson (1958a) had shown that production of P H B by B. megaterium was stimulated by the addition of acetate to cultures in the presence of glucose and ~ ~ + h y d r o x y b u t y r a t ealthough , the polymer could not be formed from acetate alone. By the use of ~(-)-3-hydroxy[U-'~C]butyryl-CoA Merrick and Doudoroff (1961) were able to demonstrate the incorporation of radioactivity into the P H B of native granule preparations from both R. rubrum and B. megaterium KM, but there was no incorporation from labelled ~(-)-3-hydroxybutyric acid. The synthesis of /!-hydroxybutyryl-CoA residues could occur via acetoacetyl-CoA produced by two different routes : namely (i) a simpIe reversal of the Knoop /!-oxidation pathway by a condensation of acetylCoA catalysed by /!-ketothiolase ; or (ii) the condensation of acetyl-CoA and malonyl-CoA involving the sequential operation of acetyl-CoA carboxylase and a /!-ketoacyl-CoA synthetase, thus : CH~COSCCIA + C 0 2 + A T P $ COOHCH~COSCOA+ A D P + Pi COOHCH,COSCoA + CH&OSCoA + CH~COCH~COSCOA + CO, + CoASH

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Both these possible pathways were investigated in extracts of Hydrogenomonas by Schindler (1964) and although he detected acetyl-CoA carboxylase and P-ketoacetyl-CoA synthetase, their activities were too low to account for the requirements of a principal pathway of P H B biosynthesis. However, it is worth noting that there are problems associated with the assay of acetyl-CoA carboxylase which led to its assignment as the rate-limiting step in mammalian fatty acid synthesis. Subsequent work has shown that activities are increased and regulatory properties changed if the enzyme is assayed in the presence of phospholipids or microsomes (Poster and McWhorter, 1969; Iliffe and Myant, 1970); it is possible therefore that the activity of the microbial enzyme may be significantly higher i n vivo. Two separate P-hydroxybutyryl-CoA dehydratases have been demonstrated in R. rubrum (Moskowitz and Merrick, 1969), one specific for ~(-)-3-hydroxybutyryl-CoA and the other for the L(+)-isomer. CrotonylCoA was incorporated into P H B in the presence of the enzyme specific for the D(-)-iSOmer and a particulate fraction but substitution of the L(+)-isomer specific dehydratase for the D(-)-isomer specific enzyme in this system did not lead to incorporation. The following reactions for P H B synthesis in R. rubrum were proposed : CH3COCH2COSCoA+ CoASH

2CH3COSCoA

iliBH

f H20

CH3CH=CHCOSCoA

D(-)-CH,CHOHCH,COSCOA

L(+)-CH~CHOHCH~COSCOA

T

PHB

CoASH

There was no evidence for the presence of a specific ~(-)-3-hydroxybutyryl-CoA dehydrogenase in this organism and the product of acetoacetyl-CoA reduction was identified as ~(+)-3-hydroxybutyrylCoA. The last enzyme of the sequence, P H B synthetase, was shown to be granule-bound. Nicotinamide nucleotide-linked acetoacetyl-CoA reductases have been reported in several PHB-producing organisms : Hydrogenomonas H16 (Schindler, 1964); R. rubrum (Stern et al., 1956b); Rhodopseudomonas spheroides (Can and Lascelles, 1961) and B. cereus (Kominek and Halvorson, 1965). The enzyme from Hydrogenomonas favoured NADH, as coenzyme but NADPH, would support 50% of the initial rate obtained

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with NADH,. The B. cereus enzyme was inducible, appearing prior to sporulation and utilizing NADH, as coenzyme. I n most of these cases kinetic studies were not carried out and the reaction product(s) were not identified.

2. Poly-p-hydroxybutyrate biosynthesis in Axotobacter beijerinckii I n our own studies of P H B metabolism in A . beijerinckii detailed investigations were made of all the enzymes which were believed might be involved in P H B biosynthesis in this organism. Ritchie (1968) detected 13-ketothiolase,acetoacetyl-CoA reductase and PHB synthetase in the organism. The PHB synthetase was specific for ~(-)-Q-hydroxybutyryl-CoA and was granule-bound. Acetoacetyl-CoA reductase produced ~(-)-3-hydroxybutyryl-CoAfrom acetoacetyl-CoA and NADPH, was the preferred coenzyme, NADH, giving one-fifth the rate. P-Ketothiolase catalysed the condensation of two molecules of acetyl-CoA to give acetoacetyl-CoA and constituted the first reaction of P H B biosynthesis. Although the K,, for this reaction does not favour acetoacetyl-CoA formation, Ritchie et al. (1971) argued that under conditions where the concentrations of NAD(P)H, and acetyl-CoA were high and that of CoASH low, the equilibrium could be displaced in favour of PHB biosynthesis. On the basis of these findings the following scheme was proposed: CoASH

CH3COCHzCOSCoA NAD(P)H, CoASH

1

PHB

NAD(P)

d-D(-)-CH,CHOHCH,COSCOA

Attempts to detect enoyl-CoA hydratases in A . beeerinckii failed (Senior and Dawes, unpublished) and the possibility of a pathway similar to that proposed for R. rubrum by Moskowitz and Merrick (1969) was discarded. The pathway of PHB biosynthesis was shown to be quite distinct from that of fatty acid biosynthesis in A . beijerinckii. An acyl carrier protein was isolated and thiol esters of [U-14C]acetoacetateand D(-)-3hydr~xy['~C]butyrate were prepared. These esters were not active with the enzymes which readily catalysed incorporation of I4C from the corresponding labelled CoA esters (Ritchie, 1968; Ritchie and Dawes, 1969). Further work on the acetoacetyl-CoA reductase of A . beijerinckii

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(Ritchie et al., 1971) confirmed and extended the findings of Ritchie (1968). The reaction product was identified as ~(-)-3-hydroxybutyryICoA and the K , for acetoacetyl-CoA was in the range 2.9-21.0 pM. Subsequent studies with this enzyme (Senior and Dawes, 1973) have shown that the K , for acetoacetyl-CoA is in the range 1-2 pM but a t high concentrations of acetoacetyl-CoA (>lo pM) significant inhibition of activity occurs. The properties of the A . beijerinckii enzyme are similar to those of the pigeon liver enzyme (Wakil and Bressler, 1962)in respect of p H value optimum, coenzyme specificity, reaction product and the equilibrium constant of the reaction.

3. ,B-Ketothiolases of Azotobacter beijerinckii and Hydrogenornonus eutropha The P-ketothiolase of 8.beijerinckii, first noted by Ritchie (1968), has now been examined in detail (Senior and Dawes, 1973) and possesses several features of interest. When assayed in the direction of thiolysis of acetoacetyl-CoA, high concentrations of acetoacetyl-CoA inhibit the enzyme. However, increasing the concentration of the other substrate (CoASH)relieves this inhibition and increases the V,,, (Fig. 25a). When these results are presented as a Lineweaver-Burk double reciprocal plot (Fig. 25b), a t the lowest concentration of acetoacetyl-CoA used the K , for CoASH is about 15 pM. As the acetoacetyl-CoA concentration is increased then the affinity of the enzyme for CoASH decreases until, a t high concentrations, the double reciprocal plots become non-linear. When the enzyme was assayed in the direction of condensation the enzyme obeyed normal Michaelis-Menten kinetics with respect to acetyl-CoA, displaying a K,,, value for this substrate of approximately 0.9 mM. CoASH inhibits this reaction and the interaction of acetyl-CoA with the enzyme becomes complex, giving non-linear Lineweaver-Burk plots. NAD and NADP also inhibit the reaction but high, non-physiological concentrations are required. The B-ketothiolase of Hydrogenomonas has been subjected to a more detailed study by Oeding and Schlegel (1973), who were the first to discover the importance of this enzyme in the regulation of P H B metabolism, and it is clear that there are many features of similarity with the A . beijerinckii enzyme. The patterns of inhibition with CoASH and acetoacetyl-CoA are almost identical, the kinetic constants are very similar and so is the behaviour of the enzyme towards NAD(P). Oeding and Schlegel (1973) have examined the physical characteristics of the Hydrogenomonas enzyme which they purified 49-fold. The molecular weights derived from sucrose-gradient centrifugation and gel filtration were 148,000 and 147,000 daltons respectively. No evidence for isoenzymes was obtained and the kinetics indicated that the two-substrate

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I/[CoASH]

pM-'

FIG.25 (a).Plot of velocity versus acetoacetyl-CoAconcentration for j3-ketothiolase of Azotobacter beijerinckii in the presence of various concentrations of coenzyme-A. (b) Data from (a)replotted as a Lineweaver-Burk double reciprocal plot t o show the effect of coenzyme-A concentration, in the presence of various concentrations of acetoacetyl-CoA, on the activity of j3-ketothiolase. Concentrations (pM) of acetoacetyl-CoA were: A, 5.53; 0,11.05; 0, 22.1; A , 44.2; 0, 66.3. Concentrations (pM) of coenzyme A were : V , 17.2 ; m, 24.08 ; 0 , 34.4 ; A , 51.6 ; A. 68.8; 0 , 103.2; 0, 172. From Senior and Dawes (1973).

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cleavage reaction follows a “ping-pong” rather than a sequential mechanism ; there are two acetoacetyl-CoA binding sites per enzyme molecule and more than two CoASH binding sites. These kinetics suggest that the enzyme is product-inhibited in both directions, although an allosteric type interaction cannot be completely ruled out as an interpretation of the results. The work of the Gottingen and Hull laboratories clearly established that similar pathways for P H B metabolism exist in Hydrogenomonas and that the regulatory mechanisms central to these pathways involve the control of reactions catalysed by P-ketothiolase. These features of regulation are discussed further in Section IV G (p. 244).

4 . PHB Xynthetase (~(-)-3-Hydroxybutyryl-CoA Polymerase) It is a general conclusion that the final reaction of bacterial P H B biosynthesis is catalysed by a P H B granule-bound hydroxybutyryl-CoA polymerase. The substrate for this reaction is ~(-)-3-hydroxybutyrylCoA, and CoASH is released (Ritchie, 1968; Ritchie et al., 1971 ; Schlegel et al., 1970; Oeding and Schlegel, 1973; Senior and Dawes, 1973). Ellar et al. (1968) proposed a mechanism of granule biosynthesis in B . megaterium which is depicted in Fig. 26. They suggested that the polymerizing unit, in the form of protein subunits, aggregates into particle form. Within this protein cover polymer would be formed in flexible fibrils which would wind and coil on each other to produce the spherical granule. As the granule grows, more enzyme is added to the granule surface. A system of differential centrifugation and density gradient centrifugation enabled Griebel et al. (19681 to purify two-fold the P H B synthetase associated with native P H B granules from B. megaterium. During purification a small amount of enzyme was stripped from the granules. Re-addition of supernatant fractions to purified native granules did not re-establish full activity. The synthetase had a pH optimum of 7.5 and the K , for ~(-)-3-hydroxybutyryl-CoA was 9.25 x M. Addition of MgCl,, albumin or 2-mercaptoethanol stimulated enzymic activity; a five-fold stimulation was obtained if all three activators were simultaneously present. Thiol inhibitors such as p-chloromercuribenzoate (PCMB) and N-ethylmaleimide (NEM) were potent inhibitors. The A. beijerinckii enzyme is similar in its response to 2-mercaptoethanol, serum albumin, PCMB and NEM, although it is was unaffected by metal ions. The K , for ~(-)-3-hydroxybutyryl-CoA approx. 0.5 mM (Ritchie, 1968). Thus the enzymes from both B. megaterium and A . beijerinckii appear to have functional thiol groups. Griebel and Merrick (1971) have succeeded in separating the P H B synthetase from the native PHB granules of B. megaterium. Mild

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alkaline (10 mM-NaOH) extraction of PHB granules resulted in a complete loss of synthetase activity in either supernatant or granule fractions. However, recombination of the two fractions reconstituted the PHB-synthesizing system, but only t o 5-6% of the original activity.

PIC.26. Proposed mechanism of biosynthesis of extended chain fibrils of poly-/jhydroxybutyrate inside the domain of a micelle made by poly-/?-hydroxybutyrate polymerase. The monomer is transported across the enzyme membrane and polymerized a t the surface. Hexagons (a, b) are sub-units of the polymerizing system. (b) and (c) show the aggregation of units into a inicellar form. From Ellar et al. (1968).

This substantial loss of activity was attributed t o the extreme lability of the extracted synthetase when it was diluted with buffer. On fractionation of the solubilized granule protein, two protein fractions were obtained of which only one was active in reconstituting synthetase activity; the other was capable of preventing the hydrolysis of PHB by the depolymerase. The K , for ~(-)-3-hydroxybutyryI-CoA of the reconstituted granule was 3.12 x M, which is somewhat M, Griebel et al., greater than the value previously reported (9.25 x 1968) for purified native granules, and may reflect an altered confor-

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mation of the synthetase in reconstituted systems with a resultant loss of affinity for the substrate. It was also shown that treatment of the fractionated granule preparation (synthetase-free) with sonic oscillation or repeated centrifugation, resulted in some 90% loss of activity on re-constitution of the PHB-synthesizing system. They concluded that these treatments produced a conformational change in the surface structure(s) of the PHB granule, resulting in the loss of ability to reconstitute the PHB-synthesizing system on addition of P H B synthetase fractions. Griebel and Merrick (1971) proposed that polymerization is a twostage reaction involving the formation of an acyl-enzyme intermediate :

+

(1) ~(-)-3-Hydroxybutyryl-CoASynthetase-SH --f

3-Hydroxybutyryl-S-Synthetase+ CoASH

+

(2) 3-Hydroxybutyryl-S-Synthetase PHB primer (nunits) -t P H B primer (n + 1 units) Synthetase-SH

+

Further investigation of the final reaction of P H B biosynthesis may prove extremely fruitful, not only in elucidating the mechanism of polymerization but also on account of the possibilities of using this system as a model for investigations of the interaction of proteins with lipoidal substances, and for the study of enzymic catalysis a t interfaces.

5. Mutants defective in poly-P-hydroxybutyrate accumulation Schlegel et al. (1970) and Schlegel and Oeding (1971) have isolated five mutant strains of Hydrogenomonas H16 which were defective in P H B accumulation. They used the N-methyl-N’-nitro-N-nitrosoguanidine, ultraviolet irradiation and 32P-suicidetechniques and separated mutants by sucrose density gradient centrifugation since PHB-less cells have a higher specific gravity ; the mutants were differentiated from the wild type by staining with Sudan Black B. Only two of the mutants did not produce any P H B when incubated in the absence of a nitrogen source with either fructose, gluconate, acetate or CO, + H,, but the others yielded very much decreased amounts, never exceeding about one-third of the PHB accumulated by the wild type and usually considerably less. One of the PHB-less mutants, isolated after enrichment with [32P] phosphate, was able to grow on glucose, which the wild type cannot do, and generally grew faster on the substrates tested. The enzymic defects in these mutants have not yet been reported but since four of them were unable to synthesize P H B from S-hydroxybutyrate, presumably in these cases synthesis is blocked subsequent t o the biosynthesis of 3-hydroxybutyryl-CoA.

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F. THE ENZYMOLOGY OF POLY-/~-HYDROXYBUTYRATE DEGRADATION

1. Degradation of extracellular P H B It is generally assumed that P H B is a common carbonaceous substance in soil, liberated by the death and lysis of organisms such as Azotobacter and Hydrogenomonas which accumulate substantial amounts of PHB. Delafield et al. (1965b) isolated and characterized a number of aerobic pseudomonads capable of utilizing extracellular purified P H B as their sole source of carbon and energy. They presented the formal description of a new species named Pseudomonas lemoignei which, together with the other isolated strains, Ps. mallei, Ps. pseudomallei, Ps. multivorans, Ps. testosteroni and Ps. acidovorans, accumulated P H B in addition to hydrolysing the exogenous polymer. Chowdhury ( 1963)had previously isolated two strains of Pseudomonas which were capable of degrading extracellular PHB. One, designated Pseudomonas PL, was investigated in detail. Of the various substrates tested only P H B granules induced the PHB-degrading enzymes. The extracellular P H B depolymerase secreted by this organism had a p H optimum of 9.5 and ~(-)-3-hydroxybutyricacid was the sole degradation product. However, the enzyme was not specific for poly-/3-hydroxybutyrate and different substrates could be hydrolysed in the following order of efficiency: hydroxybutyrate propyl ester > P H B > ethyl acetate > tributyrin > p-nitrophenyl acetate > p-nitrophenyl caprylate. Earlier, Merrick et al. (1962) had reported that, a t least with one pseudomonad, the product of extracellular P H B degradation was dimeric 3-hydroxybutyrate. The dimer is then taken into the cell and hydrolysed to monomer by the intracellular dimer hydrolase present in this organism (Delafield et al., 1965a). Further studies on the enrichment of organisms capable of P H B degradation revealed that, besides certain Pseudomonas species and Xtreptomyces, an unidentified Bacillus and a number of Hydrogenomonas strains were capable of degrading extracellular P H B (Delafield et al., 1965b). Few of these strains degraded the polymer to monomer units and most produced a mixture of dimer, trimer and monomer, with the latter compound comprising some 20% of the total degraded polymer. Pseudomonas lemoignei secreted a P H B depolymerase when grown on succinate with vigorous aeration. The enzyme preparation from culture supernatants contained two enzymes capable of catalysing P H B degradation, and EDTA inhibited while the re-addition of CaZ+ or Mg2+ions re-established activity. The ~(-)-3-hydroxybutyrateand the dimeric ester that are produced in the culture media by the action of the extracellular depolymerase are absorbed by the cell and oxidized in part to CO, while part is utilized for the manufacture of cell constituents.

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Monomer produced intracellularly by the action of the dimer hydrolase is then oxidized to acetoacetate, a reaction catalysed by an NADspecific ~(-)-3-hydroxybutyratedehydrogenase (Delafield et al., 1965a). The dimer hydrolase and hydroxybutyrate dehydrogenase are considered in detail subsequently in Section IV, F.2 (pp. 240-241). While the yields of extracellular depolymerase were similar during growth on the various carbon sources tested, when growth occurred on purified polymer, yields were decreased due to adsorption of the enzyme on to the P H B granules. Growth on 3-hydroxybutyrate resulted in negligible formation of depolymerase. Indeed in cultures metabolizing ~(-)-3-hydroxybutyratenot only was extracellular enzyme synthesis suppressed but the enzyme was actively degraded either via reabsorption into the cell or by destruction at the cell surface ; a secreted proteinase was not involved. Delafield et al. (1965b) noted that in every pseudomonad which could utilize P H B as the sole source of carbon and energy, PHB-degrading enzymes were secreted constitutively, regardless of the nature of carbon source for growth. On account of the insolubility of P H B it seems difficult t o justify the belief expressed by Chowdhury (1963) that extracellular polymer induced the synthesis of the extracellular depolymerase secreted by the pseudomonad that he examined. More likely is the explanation offered by Delafield et al. (1965b) that small quantities of constitutively secreted depolymerase in this organism degrade extracellular P H B to monomer and dimer, which being soluble, can then enter the cell and induce further synthesis of depolymerase. Lusty and Doudoroff (1966) fractionated the P H B depolymerases of Ps.lemoignei and obtained two fractions, A and B, each homogeneous in ultracentrifugal and electrophoretic analyses and with respective molecular weights of 387,000 and 375,000 daltons. They displayed many similarities in physical properties and substrate specificities, in their products of PHB metabolism and susceptibility to chelating agents. Neither possessed sensitive thiol groups but, unlike many extracellular hydrolases, they both contained cystine. The enzymes differed, however, with respect to the course of P H B degradation: with A, little trimer accumulated during hydrolysis and the final monomer/dimer ratio was less than 0.2, whereas trimer accumulated as the principal product of B until the polymer had disappeared, and then was hydrolysed giving a final monomer/dimer ratio of about 0-75. Fraction B was tightly bound t o the polymer while A was more soluble. The fractions also differed in their response to divalent (Ca” and Mg2+) and monovalent cations in the presence of chelating agents, susceptibility to di-isopropylphosphofluoridate and in their immunological properties.

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Thus, although the pathway of degradation of P H B by Pseudomonas species has been delineated to the stage of acetoacetate, details of both the incorporation of acetoacetate into cell constituents and its oxidation t o CO, and water await elucidation.

2. The intracellular degradation of PHB (a) Introduction. The catabolism of intracellular P H B reserves is probably not initiated until all available exogenous carbon and energy sources are virtually exhausted. There is no evidence to suggest that P H B undergoes turnover, indeed this seems highly unlikely in view of its physical properties (Alper et al., 1963), and it is probable that the last monomers to be polymerized during the synthesis of P H B by R. rubrum are the first to be hydrolysed (Merrick and Doudoroff, 1961). Oxygen is necessary for the degradation of P H B by whole cells of B. rnegaterium (Macrae and Wilkinson, 1958a), a requirement also demonstrated with A . insigne (Stockdale, 1967). The addition of a nitrogen source to aerobic, washed suspensions of PHB-rich Hydrogenomonas organisms stimulated P H B degradation, indicating that P H B catabolism, while an oxidative process, served as a source of carbon for incorporation into cell constituents under these conditions (Schlegel et al., 1961). Stanier et al. (1959) had established that CO, was essential for P H B degradation by anaerobic, light-incubated cells of R. rubrum, under which conditions a large quantity of the carbon of PHB was incorporated into a glycogen-like material. At the time the only feasible explanation for these observations was that P H B could only be used as a source of carbon, energy and reducing power during starvation, when CO, was also available as an electron acceptor. However, with the discovery of the reduced ferredoxin-dependent carboxylation of acetyl-CoA to pyruvate in R. rubrum (Buchanan et al., 1967): Fd(Red)

+ CO, + CH~COSCOA+Fd(ox)+ CH3COCOOH + COASH

a reaction occurring anaerobically in the light, and the finding that pyruvate is the sole activator for ADP-glucose pyrophosphorylase, the first enzyme of glycogen biosynthesis (Furlong and Preiss, 1969a), an explanation of the results of Stanier et al. (1959) is apparent. These observations also suggest that the final product of P H B degradation in R. rubrum is the same as the initial precursor for biosynthesis, namely acetyl-CoA. (b) Initial hydrolysis of PHB. (i)Product of hydrolysis : ~(-)-3-hydroxybutyric acid. The various groups who have studied the degradation of PHB agree that degradation occurs via the free acid of 3-hydroxybutyrate derived from hydrolysis of PHB. I n the earliest studies ~ ( - ) - 3 -

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hydroxybutyrate was found to be the main product formed during the autolysis of B. megaterium (Lemoigne, 1927). I n the course of aerobic P H B degradation by B. megaterium, Macrae and Wilkinson (1958a) detected 3-hydroxybutyrate, acetoacetate and acetate. The initial reaction of P H B degradation in M . halodenitri$cans was found to be hydrolysis yielding 3-hydroxybutyrate (Sierra and Gibbons, 1963). (ii) PHB depolymerase. The first degradative step is catalysed by a P H B depolymerase. Such an enzyme has been isolated and characterized from soluble cell extracts of B. megaterium, R. rubrum and Hydrogenomonas. Native granules from R. rubrum are self-hydrolysing (Merrick et al., 1962) whereas those from B. megaterium are quite stable. However, a soluble extract from R. rubrum was active in the degradation of B. meguterium native granules; purified polymer or denatured native granules did not serve as substrates (Merrick and Doudoroff, 1964). A soluble activator protein was isolated from extracts of R. rubrum which, in the presence of depolymerase, activated P H B hydrolysis. D(-)-3Hydroxybutyrate was the major hydrolysis product but a significant quantity of dimeric ester was formed. The depolymerase from Rhizobium bacteroides has been shown to be particulate, and the reaction product was ~(-)-3-hydroxybutyrate(Wong and Evans, 1971). Following the initial investigations of Merrick and Doudoroff (1964), Gavard et al. (1966) isolated and purified a soluble P H B depolymerase from B. megaterium. The hydrolysis products were identified as a mixture of dimer and D (-) - 3-hydroxybutyrate . However, D (-) - 3-hydroxy butyrate proved to be the sole product of P H B hydrolysis in Hydrogenomonas catalysed by soluble depolymerase (Hippe and Schlegel, 1967). Merrick and Yu (1966) and Merrick and Doudoroff (1964) isolated a dimer hydrolase from R. rubrum which converted dimeric to monomeric ~(-)-3-hydroxybutyrate,and a similar esterase was purified from B. megaterium (Gavard et al., 1966), as described in Section IV, F.Z(c) (p. 240). The depolymerase system of B. megaterium has received detailed investigation by Griebel et al. (1968). Depolymerization required either the addition of an activator or trypsin treatment of native granules before the depolymerase would attack the granule. Freezing and thawing of granule preparations, repeated centrifuging or extensive trypsin treatment rendered the polymer granule either partially or completely inactive as a substrate for hydrolysis. These results suggest that while the PHB depolymerase is not granule-bound (unlike the P H B synthetase in this organism), there is some protein material associated with the granule which inhibits depolymerase activity. Extraction of the native granules with 10 mM-NaOH removes P H B synthetase (see Section I V E.4, p. 233 together with this inhibitor, rendering the granule susceptible to hydrolysis by the depolymerase (Griebel and Merrick, 1971). These

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workers also found that the depolymerizing system was extremely sensitive to salt concentration, an increase of the buffer concentration leading to extensive inhibition. Depolymerization of alkali-extracted granules could, however, be inhibited by addition of the extracted protein fraction ; this inhibition was reversed by either trypsin treatment or by addition of activator. The nature of the soluble activator is unknown. These findings suggest the possibility that the mechanism for “switching on” PHB hydrolysis in B. megaterium might be analogous to that of enzyme induction in bacteria. A small protein bound t o the granule may prevent the binding or activity of the soluble P H B depolymerase. Under the correct physiological conditions an activator is produced which in some manner neutralizes the inhibitor protein and thereby permits depolymerization t o begin. (c) 3-Hydroxybutyric acid dimer hydrolase. The extracellular formation of a dimer of 3-hydroxybutyrate during degradation of exogenous P H B by Ps. lemoignei and its subsequent entry to the cell and hydrolysis (Delafield et al., 1965a, b) has been noted (Section I V F.l, p. 236). These workers isolated and purified about 55-fold the dimer hydrolase of Ps. lemoignei and studied its properties. The enzyme was unusually specific in comparison with other bacterial lipases, displaying principal specificity for the CH,CHORCH,COOR’ portion of the substrate, which must be of the D(-) configuration. The terminal carbon may be either a free carboxyl group (as in the dimer) or esterified (as in the p-bromophenylacyl ester of the dimer) but the D-oxybutanoate moiety cannot be substituted by L-oxybutanoate, DL-oxypropionate, or n- or sec-oxybutane, Specificity was less rigid for the D-3-hydroxybutanoyl (R)moiety of the dimer which could be substituted by the L-isomer or by a butanoyl group. The latter substitution, however, significantly affected both the affinity of the enzyme for the substrate and the rate of hydrolysis. The trimer was hydrolysed a t only about 10% of the rate of the dimer. The K,, for the D-dimer was about 200 p M and V,,, was attained a t 500 pM. The enzyme did not require divalent metals, was not sensitive to EDTA and was rather unstable when stored a t 5°C or in the frozen state. The products of PHB depolymerase activity in B. megaterium represent some 80 to 85% of ~(-)-3-hydroxybutyricacid with the balance as soluble esters and Merrick and Doudoroff (1964)demonstrated the presence of an esterase which hydrolysed these short chain esters to the monomer unit. The activity of the enzyme was never great but they showed that it hydrolysed the dimer. Subsequently Gavard et al. (1966, 1967) isolated a hydrolase from B. megaterium which acted upon

ENERGY RESERVE POLYMERS IN MICRO-ORGANISMS

24 1

the di-, tri- and pentamers of 3-hydroxybutyric acid to yield the monomer. The enzyme, of the highest purification achieved, apparently also hydrolysed products of P.HB depolymerization (which displayed melting points in the range 88"-13OoC), to give the monomer. However, they showed that the PHB depolymerase of this organism forms principally the dimer and monomer so that the normal physiological function of the enzyme is presumably to hydrolyse the dimer. Merrick and Yu (1966) isolated and purified 70-fold a dimer hydrolase from R. rubrum. The enzyme exhibited a high degree of specificity for dimer possessing the D(-) configuration but attacked the trimer more rapidly and they suggested that the dimer may not be the physiological substrate for the enzyme. The hydrolase displayed no activity with other esters, nor with native PHB granules or purified PHB, and was inhibited by di-isopropyl-phosphofluoridate. (d) Further metabolism of ~(-)-3-hydroxybutyrate. Every bacterium which is capable of either accumulating P H B or degrading exogenous PHB as the sole carbon and energy source, has been shown to possess a constitutive NAD-specific ~(-)-3-hydroxybutyratedehydrogenase. I n all cases the products are NADH, and acetoacetate. The properties of the enzyme from several bacterial sources are summarized in Table 11, and a detailed account of the kinetics and properties of bacterial D(-)-3hydroxybutyrate dehydrogenases has been given by Bergmeyer et al. (1967).All the enzymes cited in Table 11 display similar pH optima for oxidation and K , values for substrates. In view of the close similarities of the ,l-ketothiolases of Hydrogenomonas eutropha H16 and A . beijerinckii (Section IV, E.3, p. 231), the similarity also of their ~(-)-3-hydroxybutyrate dehydrogenases is of particular interest. The recent work of Oeding and Schlegel(l973) has established that the H . eutropha enzyme is inhibited competitively by NADH,, pyruvate and oxaloacetate while 2-oxoglutarate is not effective. The A. beijerinckii enzyme is also inhibited competitively by pyruvate and NADH,, but differs in that oxaloacetate does not inhibit whereas 2-oxoglutarate does. Further, the A . beijerinckii enzyme is unaffected by NADPH, (Senior and Dawes, 1973). The enzymological and physiological significance of these regulatory phenomena are discussed in Section IV, G (p. 244). Poly-,l-hydroxybutyrate is degraded via ~(-)-3-hydroxybutyrate by Rhixobium bacteroids and iso-enzymes of ~(-)-3-hydroxybutyrate dehydrogenase have been found. In R. leguminosarum five iso-enzymes were demonstrated and it has been proposed that these iso-enzymes could be used as a taxonomic device for distinguishing the species of the genus Rhixobium (O'Hara and Fottrell, 1968; Fottrell and O'Hara, 1969). Experiments conducted by Klucas and Evans (1968) on the identity

TABLE11. Properties of some bacterial D (-) -3-hydroxybutyratedehydrogenases based on data principally assembled by Ritchie (1968) Sourcc

Arotobacter

bei]eriackii"

A%otobacter vineiandiib

Khodopseudomonas spheroidesc

Rhodospirillum mbrtinid

Pseudomonns lemoigneie

Hydrogenomonasf

Purification 6.0 320 170 120 29 180 factor pH Optimum 8.4-8.5 8.0 8.0-9?? 8.5 6.s8.5 8.0 for oxidation Xichaelis Constants 4.1 x 10-4 M 6 0 x 10-4 ST D(-)-38.77 x 10-4 N 8-4 x 10-4 31 Hydroxy-butyrste 2.8 x 10-4 1 1 2.0 x 10-4 M Acetoacetate 7.0 x 10-5 Y 8.0 Y 10-5 11 7.0 x 10-5 n i NAD 7.0 X I O - 5 ~II NADHz 5.4 x 10-5 11 Substrate ~(-)-3-hydroxybutrratc~!-)-3-hydrosybutyrate D(-)-3-hydroxybntyrste D(-)-3-hydroxybutyrate D(-)-8-hydroxybutyrate D(-)-3-hydroxybutyrate specificity dcetoacetate Acetoacetate Acetoacetate Acctoacetate Acetoacetate Acetoacetate Thiol reagents EDTA EDTA Inhibitors Pyruvzte D-lactate D-lactate 2-oxoglutarate pyruvste D~-2-hydroxybutyrate NADH2 DL-2-hydroxybutyrate Activators M@+, EDTA cysteine

-

c

-

Ahhreviatiow : EDTA. rthylenediamine tetra-acetate. Senior and D;Lwes (1973) Jurtshuk e l nl. (1968)

a

M

?

P P 2

U +d

F-'

Ki

3 5 w

243

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of an electron donor system for nitrogenase-dependent acetylene reduction by extracts of soybean nodules, suggested that the reducing power generated by the oxidation of ~(-)-3-hydroxybutyrateduring the degradation of PHB by R. japonicum could be utilized as a source of electrons for the reduction of nitrogen. They proposed the scheme shown in Fig. 27. The nature of the carrier “X” was not identified. Preliminary information concerning a similar scheme for nitrogen reduction in A . vinezandii was also mentioned (Klucas and Evans, 1968).

r(,,,,,,,/’

n (-) -3-Hydroxybutyrate

3CH,COCH>COOH

Diaphorase

Nitrogenase

2T\iH,

‘s’ (ox) ADP 4 pi

FIG.27. A scheme of electron transport from 3-hydroxybutyrato dehydrogenase to nitrogenase in Rhizobium japonicum. The nature of “X”is unknown but benzyl or methyl viologen effectively substitutes for it suggesting a redox potential in the region of -0.3 V. From Klucas and Evans (1968).

However, more recent work by Wong et al. (1971) has suggested that NADPH, is the most likely electron donor to the nitrogenase electron transport pathway in nodule bacteroids. If this is so it would preclude PHB serving as a source of electrons for nitrogen fixation. Wong and Evans (1971) have therefore concluded that P H B does not directly aid nitrogen fixation by glucose- and carbohydrate-starved cells. At the time of writing we believe the complete pathway of P H B degradation to have been elucidated in only one organism, namely A . beijerinclcii. The demonstration of the presence of acetoacetate : succinyl-CoA CoA-transferase (thiophorase) in this bacterium led to the suggestion that it is involved in the degradation of acetoacetate, as shown in Fig. 28 (Senior and Dawes, 1973).The enzyme obeys Michaelis-Menten kinetics with respect t o both acetoacetyl-CoA (IT,,,2.8 x M) and succinate (K,,,, 4.0 mM). When assayed in the direction of acetoacetylCoA hydrolysis, acetoacetate was a potent competitive inhibitor. Thiophorase was first demonstrated by Stadtman (1953) in extracts of Clostridiu,m kluyveri and was shown to be highly active in acetoacetate 11

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E . A. DAWES AND P. J. SENIOR

degradation in pig heart tissues (Stern et al., 1956a). Kinetic constants for the A. beijerinckii thiophorase are similar to those of the pig heart enzyme. Although the equilibrium constant for this reaction is not favourable for the formation of acetoacetyl-CoA from acetoacetate and succinyl-CoA, conditions of high CoASH, acetoacetate and low acetylCoA concentrations would be expected t o displace this equilibrium in favour of PHB degradation. The presence of thiophorase in A. beijerinckii suggests that PHB metabolism in this organism occurs via a cyclic process with acetyl-CoA fulfilling the role of both precursor and product and acetoacetyl-CoA functioning as an intermediate common to biosynthesis and degradation. Poly - P - hydroxybutyrate

------+ D

-3- Hydroxybutyrote

LNAD

( 6 )

NADHz

Succinyl-CoA

Succinate

Acetoocetote

:i I-C~ASH Acetoacetyl - C o A

2 Acetyl- CoA

FIG.28. The pathway of poly-8-hydroxybutyrate degradation in Azotobacter beijerinckii. From Senior and Dawes (1973).

P-Ketothiolase, the enzyme catalysing the first reaction of biosynthesis and the last reaction of degradation, occupies a key position and, as with H . eutropha (Oeding and Schlegel, 1973))there is no evidence to suggest that more than one P-ketothiolase is responsible for these activities in A . beijerinckii. Oeding and Schlegel (1973), while not reporting the presence of a thiophorase or an acetoacetyl-CoA thiokinase, have concluded that P H B degradation in Hydrogenomonas eutropha H16 proceeds via a pathway identical with that shown in Fig. 28. Consequently, they also have been led to the conclusion that P H B metabolism is a cyclic process in this organism, much the same as for A. beijerinckii, and with almost identical regulatory controls. Their detailed studies of P-ketothiolase clearly indicate its key role in the cyclic process and a full comparison of the two schemes is considered in Section IV, G (p. 245).

G. THEREGULATION OF POLY-P-HYDROXYBUTYRATE METABOLISM AND ITS PHYSIOLOGICAL SIGNIFICANCE Any regulatory scheme proposed to explain the control of P H B metabolism must take into account the accepted criteria for the definition of a reserve material, and must also recognize the observed physio-

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logical functions of P H B in activities such as sporulation in Bacillus species, encystment in the Azotobacter and survival during starvation, as observed with various organisms. At present two bacteria have been studied in detail with regard to the regulation of P H B metabolism, Hydrogenomonas eutropha by Professor H. G. Schlegel and his colleagues and A. beijerinckii in our owii laboratory, With A . beijerinckii we have shown that oxygen limitation is the initiating factor for P H B accumulation by cells fixing atmospheric nitrogen in the presence of excess glucose (Senior et al., 1972). Senior and Dawes (1971a, b ) found that among several oxido-reductases involved in glucose oxidation NAD(P)H, inhibition of activity was common. Of particular interest was the observation that, as with Axotobacter vinelandii (Weitzman and Jones, 1968), citrate synthase, the enzyme catalysing the first reaction of the tricarboxylic acid cycle, was inhibited by NADH,, while isocitrate dehydrogenase was inhibited by both reduced nicotinamide nucleotides. It was proposed that when A . beijerinckii became oxygen-limited the rate of NAD(P)H, oxidation decreased resulting in the accumulation of NAD(P)H,. Citrate synthase and isocitrate dehydrogenase would thus be inhibited and acetyl-CoA, no longer able to be oxidized a t a rapid rate via the tricarboxylic acid cycle, would accumulate and then be channelled into PHB biosynthesis. As revealed by the chemostat experiments involving transition from nitrogen-limited to oxygen-limited steady states, the initiation of P H B synthesis was accompanied by a pronounced rise in the redox potential of the culture with a concomitant fall in the potential rates of oxygen utilization and carbon dioxide evolution (Senior et al., 1972). The interpretation placed on these observations was that during oxygenlimitation the cells adjusted to their new environment by re-oxidizing excess reducing power in the process of synthesizing PHB. I n this case P H B would be acting as an “electron sink” in a quasi-fermentation process. Relaxation of oxygen-limited growth conditions led to inhibition of P H B biosynthesis and the rates of oxygen utilization and carbon dioxide evolution in sitzc increased rapidly without a concomitant increase in the rate of glucose uptake, indicating that substantial intracellular pools of glucose catabolites were available for immediate oxidation as soon as an adequate oxygen supply became available (Senior and Dawes, 1973). The scheme shown in Fig. 29(a) was proposed by Senior and Dawes (1973) to explain the regulation of P H B metabolism in A . beijerinckii, while that in Pig. 29(b) is the corresponding scheme for H . eutropha postulated by Oeding and Schlegel (1973); the two are almost identical in the suggested mechanism of regulation of biosynthesis and degrada-

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PPRUY-LTTE

(4

--

Acrloacetyl-CoA

/j-Hydro\\ hiit>ratti

\k

P o l y - p - h y d r o x yb u t y r a t e FIG.29. Cyclic scheme for the metabolism of poly-P-hydroxybutyrate and its control in (a)Azotobcccter be.ijerinckii (Senior and Dawes, 1973) and (b) Hydropnornonas eufropha (Oeding and Schlegel, 1973). The dotted linesin (a),and the solid lines from effectors in (b),indicate inhibition.

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tion of the polymer. The existence of a cyclic process raises the question of how the cell regulates metabolism to prevent the occurrence of energetically wasteful cycling whereby acetoacetyl-CoA formed by degradation is reconverted to PHB. I n the absence of any information concerning regulation of P H B depolymerase it seems quite feasible that polymer degradation could be controlled by the inhibition of ~(-)-3-hydroxybutyratedehydrogenase by NADH, (competing with NAD), pyruvate and 2-oxoglutarate ( A . beijerinckii) or oxaloacetate ( H . eutropha) (competing with ~ ( - ) - 3 hydroxybutyrate). ThisNAD-specificenzymeisnot affected by NADPH,. The regulation of the A . beijerinckii enzyme by pyruvate and 2-0x0glutarate is probably exerted when glucose catabolism and the tricarboxylic acid cycle are operating maximally and the need t o oxidize accumulated PHB, in its role as a reserve of carbon and energy, is minimal. I n H . eutropha pyruvate inhibits the enzyme, as does oxaloacetate, instead of 2-oxoglutarate. This difference between the two organisms may reflect the importance of 2-oxoglutarate as a key metabolite in the incorporation of reduced dinitrogen into amino acids by the nitrogen-fixing organism. Thus it has recently been shown that the major pathway of NH,+ incorporation into amino acids in A . vinelandii is via a reductive transamidation whereby one molecule each of glutamine and 2-oxoglutarate are converted t o two glutamate molecules with concomitant oxidation of NAD(P)H, (Nagatani et al., 1971), a reaction sequence first discovered in Escherichia coli by Tempest et al. (1970). A finer control of the A . beijerinckii enzyme is exerted by the competitive inhibition of NADH,, rendering the enzyme very sensitive t o changes in the NADH, :NAD ratio brought about, for example, by the imposition or relaxation of oxygen limitation of growth. The polymer would not be degraded on relaxation of an oxygen limitation until both the NAD concentration had increased and the pyruvate concentration decreased, the latter condition being achieved on exhaustion of exogenous glucose. During conditions of unrestricted growth in the presence of excess oxygen, i.e. with no P H B accumulation, the steady state concentration of coenzyme A would be expected to be high, and that of acetyl-CoA low, mediated by the activity of citrate synthase with citrate formation serving as a sink for acetyl groups and simultaneously releasing free coenzyme A. Consequently the rate of acetoncetyl-CoA synthesis would be low due to the sub-optimal concentration of acetyl-CoA and to the inhibitory effect of free coenzyme A on p-ketothiolase; P H B would therefore not be synthesized under such conditions. The relief b y coenzyme A of acetoacetyl CoA inhibition of the thiolysis of acetoacetylCoA catalysed by P-ketothiolase would, in turn, ensure that only when 11*

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coenzyme A was present a t high concentrations would degradation of P H B proceed. Restriction of citrate synthase activity as a result of NADH, accumulation during oxygen limitation (Senior and Dawes, 1971b) would lead to a decrease in the high steady state concentration of free coenzyme A, with a concomitant increase in the concentration of acetyl-CoA. A substantial increase in acetyl-CoA concentration would be required to saturate the 8-ketothiolase of A . beijerinckii ( K , for acetyl-CoA, 0.9 mM) and thereby initiate the synthesis of PHB. The ,&ketothiolase thus occupies a key position in metabolism of the polymer, governing both biosynthesis and degradation. The observation that acetoacetyl-CoA inhibited acetoacetyl-CoA reductase in A . beijerinckii (Ritchie et al., 1971)is probably explained on the basis that only during conditions when NAD(P)H, concentrations are high, for example during oxygen-limited growth, would reduction of acetoacetyl-CoA, and consequently synthesis of PHB, proceed. This inhibitory effect would counter the tendency for cycling, leading to energy wastage, The overall scheme of regulation may now be summarized. Conditions for PHB biosynthesis, namely high NAD(P)H,, high acetyl-CoA and low free coenzyme A concentrations, are produced during oxygen-limitation of growth, which thus stimulate biosynthesis of the polymer. Simultaneously these conditions inhibit the degradation of PHB and prevent unrestricted cycling of polymer metabolism. Situations which favour a high intracellular concentration of NAD(P),such as the relaxation of an oxygen limitation of growth, would restrict biosynthesis but would not stimulate degradation of PHB until the steady state concentration of acetyl-CoA decreased, and that of coenzyme A increased, as a result of the supply of glucose catabolites (e.g. pyruvate) becoming restricted. These latter effects would then enable the thioclastic cleavage of acetoacetyl CoA catalysed by 8-ketothiolase to proceed. Similar considerations can be applied to the regulation of P H B metabolism in organisms which accumulate the polymer under growth limitations other than oxygen, e.g. B. megaterium with nitrogen-, sulphur- and potassium-limitation (Wilkinson and Munro, 1967) and Hydrogenomonas with nitrogen-limitation (Schlegel et al., 1961). The cessation of protein synthesis, an endergonic process tightly coupled to ATP production via the electron transport chain, would presumably lead to an increase in the NAD(P)H, concentration of the cell, since then oxygen could serve as a terminal electron acceptor only via transhydrogenase and NADH, oxidase activities. Inhibition of citrate synthase by NADH, would result in an increase in the acetyl-CoA concentration and B decrease in coenzyme A concentration which, in association with the

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excess of reducing power, would favour PHB synthesis and so relieve the restriction on carbon flow imposed by the limitation of protein synthesis. It is possible that the reason why PHB synthesis does not occur when A . beijerinclcii is subjected to nitrogen-limitation is the necessity for the organism to preserve its respiratory protection o f the nitrogenase system (see Section IV, H.3, p. 252); under such conditions the NAD(P)H,/ NAD(P) ratio would be expected to be low even with a high ATP concentration.

H. FUNCTION OF POLY-/I-HYUROXYBUTYRATE 1. Role as a carbon andlor energy source during starvation and in survival Macrae and Wilkinson (1958a) were the first to claim that a high content of PHB delayed the autolysis and death of nitrogen-deficient B. megaterium. Their method o f estimating autolysis was not entirely satisfactory, however, (see Dawes and Ribbons, 1964)and it was evident that a substrate other than PHB was being utilized concurrently with the polymer since oxygen consumption was in excess of that required for complete combustion of the PHB; this substrate was not carbohydrate (Macrae and Wilkinson, 1958b). Since growth did not occur when PHB-rich cells were held in a medium lacking a carbon and energy source, it is clear that PHB may serve as a source of energy but not as a source of carbon skeletons for biosynthesis in B. megaterium. The role of PHB in the endogenous respiration and survival of the obligate halophile Micrococcus halodenitriJicans was studied by Sierra and Gibbons (1962a). The R.Q. of endogenous respiration was 0.87 rt 0.05 ; that required for complete combustion of PHB is 0.88. Aeration of washed suspensions at 25°C in phosphate buffer containing NaCl decreased the PHB content slowly from 55 to 29% in 127 hours. Under these conditions some lysis of cells occurred but the endogenous Q0, remained constant at 40 throughout this period. A comparison of the behaviour of polymer-rich cells containing 50% PHB and polymer-poor cells containing 10% PHB, revealed that the latter died rapidly on starvation and there were less than 10% survivors after 30 hours. I n contrast, the PHB-rich cells displayed 100% viability for 100 hours, by whichtime the PHB content had fallen to less than 30% of the dry weight, although too few PHB analyses were carried out to define the content accurately. Viability then fell rapidly to a negligible value a t 120 hours. These experiments demonstrate a clear relationship between survival and the PHB content of M . halodenitrijkans. In the chemolithotrophic Hydrogenomonas, PHB, which can be accumulated to very high levels, serves as both a carbon and energy

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E. A. DAWES AND P. J . SENIOR

source and can support protein synthesis in the presence of a suitable source of nitrogen (Schlegel et al., 1961). The endogenous metabolism and survival of Hydrogenomonas eutropha H16 have been studied by Hippe (1967); PHB was shown to be the preferentially utilized endogenous substrate and its presence inhibited the net degradation of nitrogenous cellular constituents and the release of ammonia. The P H B content of polymer-rich cells fell a t a steady rate to about half its initial value over an incubation period of 84 hours, and although the Qo, value decreased from 18 to 3 during this time, complete oxidation of the degraded polymer occurred. PHB-poor cells rapidly exhausted their polymer and the Qo, fell to a minimum value within 10 hours although they remained 80% viable after some 95 hours. The addition of uncoupling agents, such as methylene blue, carbonylcyanide-m-chlorophenylhydrazone and 2,4-dinitrophenol, increased both the Qo, and the rate of PHB degradation some five-fold, suggesting a high degree of coupling between oxidative phosphorylation and endogenous respiration in Hydrogenomonas. Provided the polymer content was high viability remained a t maximum levels over long periods of starvation, although it was evident that cells with low polymer content, and after their polymer was exhausted, could maintain viability a t the expense of cell protein. The filamentous, sheathed bacterium Sphaerotilus discophorus accumulates P H B during growth in glucose-ammonium salts medium and Stokes and Parson (1968) have demonstrated that polymer-rich cells survive better than cells with little or no P H B when they are starved in phosphate buffer suspensions. It was observed that exponential phase cells, which contained 23.8% of their dry weight as PHB, survived better than stationary phase cells containing 35% P H B ; after starvation for 8 hours, when the PHB contents had fallen to 16.2 and 18.6% respectively, the corresponding viabilities were 79 and 44%. A similar experiment with bacteria grown in the absence of glucose and, in consequence, devoid of PHB, reveded 37% survival of cells from the exponential phase and only 19% survival of those from the stationary phase. This finding is unusual insofar that stationary phase bacteria have generally been regarded as being more resistant to stresses such as cold shock, heat shock and desiccation (Postgate and Hunter, 1962). Increased oxygenation or addition of Mg2+,both of which stiniulate P H B oxidation (Stokes and Powers, 1967))led to more rapid death of PHBrich cells but the reason is obscure, especially since considerable amounts of the polymer were still present in the cells a t the end of the starvation period. Howevcr, the results do indicate that possession of P H B aids the survival of 8. discophorus. Although PHB is a substrate for endogenous respiration in Pseudo-

ENERGY RESERVE POLYMERS I N MICRO-ORGANISMS

251

monas saccharophila i t is not, apparently, converted to other cellular constituents when the organism is held in the absence of an exogenous carbon source (Doudoroff and Stanier, 1959) and thus serves solely as an energy source. I n contrast, the photosynthetic Rhodospirillum rubrum has been shown to utilize P H B as an endogenous source of carbon for biosynthesis, although the presence of CO, was essential (Stanier et al., 1959); the experiments were not designed to test the possibility that PHB also serves as an energy source in this organism. It was considered that the polymer functions as a store of carbon and of reducing power for further assimilation of carbon dioxide. With neither organism have viability studies been carried out. Axotobacter organisms possess the highest known respiratory rates in the presence of oxidizable exogenous substrates, but they display a low efficiency of oxidative assiinilation and a low endogenous Q,,,. The high respiratory rates have been associated with a scavenging role for removing oxygen which inhibits the nitrogenase system of these organisms (Bulen et al., 1963; Dalton and Postgate, 1969). Thus Oppenheim and Marcus (1970) and Oppenheim et al. (1970) have shown that A . vinelandii grown on atmospheric nitrogen possesses a highly complex internal membrane system which is not present in cells grown on a fixed source of nitrogen (NH,+) ; the development of the membranes, which bear respiratory enzymes, in association with the induction of nitrogenase, supports the role of respiration as a protective mechanism for the oxygen-sensitive nitrogenase. It might be considered that low endogenous respiratory activity would be a factor conducive to cell survival. Sobek et al. (1966) studied the respiratory quotient during endogenous metabolism of A . agilis but the range of values observed was of little value in predicting the nature of the primary endogenous substrate. They found that [U-I 4C]-glucosegrown, PHB-rich, or PHB-poor cells degraded their polymer a t a rate fa,ster than the loss of radioactivity from other cell constituents during starvation, and a similar conclusion was reached with both IJJ-l4C]acetate and [U- ''C]succinate-grown bacteria on starvation. However, they discovered that cells grown on these radioactive substrates died more rapidly than unlabelled cells and degraded their cellular constituents at a faster rate. High PHB contents exerted a sparing action on the degradation of cellular protein and probably delayed the utilization of the degradation products. Bacteria with low polymer contents were observed to degrade protein extensively and concomitantly with their PHB, although little ammonia production occurred. The viability of starving A. agilis appeared to be related to the initial P H B contents of the cells. Sobek et aZ. (1966) found that the viability of glucose-grown cells containing 18% P H B did not decrease

252

E. A . DAWES AND P. J. SENIOR

until over half of the polymer had been degraded whereas PHB-poor cells (4% of the dry weight) displayed a rapid and immediate decline in viability on starvation. However, in similar experiments with succinate-grown bacteria, in which the P H B contents were consistently lower (maximum of 12% of the dry weight) than for glucose-grown cells, the organisms degraded the polymer a t a slower rate and survived longer. It was suggested that these cells may use their polymer in a more efficient manner. Similar studies have been made by Stockdale (1967) with A . insigne. During carbon starvation in a basal salts medium PHB served as the immediate major endogenous substrate although other cell constituents were oxidized simultaneously and, after P H B exhaustion, a high Qo, was still manifest. Again with this organism, the initial polymer content alone did not determine the survival pattern of the cells. Stationary phase cells grown on limiting glucose and containing little P H B survived better initially than cells grown in the presence of excess glucose, although subsequently they died a t a faster rate. I n passing, it is worth noting that viability determinations with Axotobacter often present difficulties and the work of Smith and Wyss (1969) is helpful in this context. It seems clear, therefore, that under certain circumstances, cells with a lower content of P H B can survive longer than those with a higher one, either because they are not subjected to some additional adverse factor (since they may be better able to utilize alternative components, e.g. cellular protein, as endogenous substrates) or because they can utilize their reserve material more efficiently.

2. Role in sporulation and encystment The formation of spores in Bacillus species and of cysts in the Azotobacter represent specialized mechanisms for assisting the survival of these organisms under adverse conditions, usually when nutrient deficiencies are encountered. The possible relationship of P H B to both these processes has already been discussed in Section IV, D.2 (p. 215) and it may be concluded that the polymer probably serves as a reserve of carbon and of energy for the endergonic reactions involved in sporulation and encystment. 3. Role of Poly-P-hydroxybutyrate in the Axotobacteriaceae The natural habitat of the Azotobacter is soil, the oxygen concentration of which in both gaseous and liquid phases is variable (Greenwood, 1968). Oxygen concentrations in excess of about 20% of air saturation have been shown to be inhibitory to Axotobacter (Bulen et al., 1963; Dalton and Postgate, 1969) by inhibiting the nitrogenase system. These

ENERGY RESERVE POLYMERS I N MICRO-ORGANISMS

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growth-inhibitory effects may be countered by the organism increasing its oxidative activity, thus lowering the environmental partial pressure of oxygen to a more acceptable value. This process was called “respiratory protection” by Dalton and Postgate (1969) who proposed that the mechanism operated in growing Axotobacter organisms to protect the nitrogenase, whereas in non-growing bacteria the appropriate components of the system assumed a conformation that prevented access of oxygen to the sensitive sites. The scavenging role of augmented respiration in A . chroococcum was confirmed by Drozd and Postgate (1970) who showed that respiratory activities and cytochrome a, contents of nitrogen-fixing continuous cultures increased with increasing partial pressures of oxygen. Derxia gummosa also prefers a Iow partial pressure of oxygen for growth when fixing nitrogen (Hill and Postgate, 1969) and Hill (1971) has suggested that the inhibition by oxygen of nitrogen fixation in this organism can be overcome when small amounts of fixed nitrogen are present in agar medium, because the respiratory activity accompanying the initial phase of growth lowers the concentration of the local oxygen supply sufficiently to release the nitrogenase system from inhibition. The most suitable environment for these strict aerobes is therefore, paradoxically, one in which the oxygen concentration is low and probably limiting. Senior et al. (1972) found that growth yields of A . beijerinckii were greater and the oxygen requirement lower under oxygen-limited conditions in continuous culture. These conditions also resulted in the accumulation of PHB which confers advantages for maintenance of viability during periods of starvation. Further, the possession of PHB bestows an additional advantage in that it will permit a cell to increase its oxidative activity in the absence of an exogenous substrate and thereby secure respiratory protection. I n the absence of both exogenous substrate and intracellular reserve materials this process of respiratory protection would be impossible and viability would be threatened. All the Azotoba,cteriaceaeexamined by Stockdale et al. (1968) accumulated PHB and it may be speculated that the polymer serves a similar role in all these organisms, functioning both as a storage material and as a means of regulating the oxygen environment of their natural habitat for maintenance of viability. The pathways of P H B biosynthesis and degradation in A. beijerinckii seem particularly suited for the role of a redox-regulator. The two pathways are distinct, biosynthesis involving the coenzyme A esters and degradation occurring via the free acids ; reductive synthesis consumes NAD(P)H, while oxidative degradation produces NADH,. If the ratio of NAD(P)H, to NAD(P) was controlled by the activities of

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NAD(P)H, oxidase and transhydrogenase alone, then re-adjustment of a high ratio would require moleculm oxygen which, under conditions of oxygen limitation, would be difficult to achieve. However, a biosyntheticdegradative cycle for PHB would not consume electrons but would act as an insoluble store of reducing power.

V. Conclusions An important development in an evolving life form is the acquisition of characteristics which bestow upon the organism the ability to survive the adverse conditions imposed by nutrient starvation. Once these characteristics have been acquired the life form then has a more secure basis for further development and diversification. I n general, protection against starvation has been achieved by the organism accumulating reserve compounds during periods of nutritional plenty and then degrading these storage materials during times of famine, thereby maintaining viability. The two most commonly encountered reserve materials in the microbial world are poly-P-hydroxybutyrate and glycogen (or glycogen-like materials) and in some, but not all, cases it has been convincingly shown that glycogen and PHB function as carbon and/or energy sources, and their possession enables a cell to maintain its viability better than a corresponding cell not so endowed. However, the accumulation of reserve materials is not necessarily a pre-requisite for growth. I n the laboratory fermenter cells devoid of storage compounds grow quite readily under favourable conditions. When these cells are starved they do not usually die immediately, but death is preceded by a general degradation of other cell constituents, such as RNA and protein, to furnish small metabolites which may then be metabolized to provide energy of maintenance and presumably to facilitate the induction of enzymes required for the metabolism of new carbon sources in the environment. The value of an intracellular store of carbon and energy can be readily appreciated when the plight of a nitrogen-fixing organism is considered, for the reduction of dinitrogeii is energetically expensive. When new enzymes have to be synthesized after a period of starvation, some degree of nitrogen-fixation will probably be necessary, a process which requires both carbon skeletons and energy. I n the absence of a readily metabolizable exogenous source of carbon and energy, enzymes must be induced to deal with the novel substrate(s) and the necessary reactions would be virtually impossible were it not for the possession of substantial intracellular reserves.

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The study of mutants defective in the synthesis or mobilization of reserve polymers might be anticipated t o elucidate the physiological role of these storage compounds. Although a number of such mutants has now been isolated in various laboratories it is still not possible to give a precise unequivocal answer to the problem. Nonetheless, this approach appears fruitful and work currently in progress should extend our understanding. While our knowledge of the nature of the regulatory mechanisms which operate in the biosynthesis and degradation of reserve polymers is still incomplete in certain respects, an encouragingly large body of information has been obtained, especially in relation t o the modulation of enzyme activity and the role and function of allosteric effectors. The importance of “feed-forward’’ controls is apparent in glycogen synthesis. Recent investigations of the regulation of glycogen metabolism in microbes have revealed the evolution of a logical sequence of regulatory mechanisms. I n the lower evolutionary forms of the microbial world, typified by the Clostridia, it appears that glycogen synthesis is catalysed by non-allosteric enzymes with the signal for glycogen biosynthesis being a high energy charge, while a low energy charge inhibits synthesis. Likewise it is apparent that glycogen degradation may be regulated by the availability of carbon compounds. Further along the evolutionary path may be discerned the enteric organisms, which display more complex regulatory interactions involving “feed-forward” signals to allosteric enzymes. I n certain cases, such as R.rubrum, this type of system has been further developed t o support the requirements of a photosynthetic organism. The regulatory mechanisms controlling glycogen metabolism in the eukaryote Tetrahymena display features of even greater complexity. Besides allosteric interactions the system is probably augmented by the presence of a crude adrenergenic/seretonergenic system, mediated by cyclic-AMP. Ultimately the mammalian system presents, in adapted form, all the types of regulatory system previously encountered lower down the evolutionary scale, together with a complex system of hormonal control. However, there appears to be one common denominator in all these systems, namely the energy charge. The problems associated with our understanding of all these regulatory mechanisms may be resolved by a detailed examination of the role of energy charge in the individual cases. Since ATP is involved directly in the synthesis of microbial glycogen and polyphosphate the relevance of an energy charge study is obvious. The synthesis of PHB is unique amongst energy storage compounds in not requiring the direct participation of ATP. However, reducing

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power in the form of NAD(P)H, is essential and PHB formation may be regarded as a quasi-fermentation process, permitting the re-oxidation of reduced nicotinamide nucleotides under conditions where this would otherwise be restricted, either because of an oxygen limitation or, indirectly, on account of the cessation of protein synthesis which is tightly coupled via ATP formation and utilization to the electron transport chain. Poly-,!-hydroxybutyrate is thus a highly reduced carbon and energy storage compound which, according to organism, may additionally play a role in spore or cyst formation, themselves mechanisms for prolonging survival under adverse conditions. I n the case of nitrogen-fixing organisms in the soil we suggest that the possession of P H B could afford respiratory protection when readily oxidizable exogenous substrates are not available. Polyphosphate undoubtedly occupies the most controversial position as an energy reserve material although its function as a phosphate storage compound is unchallenged. However, in the Mycobacteria and Corynebacteria conservation of the phosphate group transfer potential of polyphosphate in the phosphorylation of glucose has been clearly demonstrated, while in other micro-organisms it seems that energystorage is incidental to the principal role of phosphate accumulation. The intracellular location of the enzymes concerned with polymer synthesis and degradation is an intriguing problem. The most detailed work has been carried out with PHB granules which have P H B synthetase and a factor involved in depolymerase activity associated with the membrane that bounds the granule. The depolymerase system is a, complex one which also involves a soluble fraction, underlining the importance of reactions a t liquid-solid interfaces in the process and emphasizing that relatively little work has been done to isolate polyphosphate or glycogen granules in their native state in order t o examine the possibility of regulation by a membranous system. There are, of course, difficulties inherent in the isolation of such entities and the precautions for polyphosphate isolation, for example, have been discussed by Sykes (1971). I n the case of P H B it is precisely such problems which have so far prevented the satisfactory investigation of the possible control of PHB depolymerase. However, the discovery of the association of glycogen synthetase with glycogen particles [FOX, quoted by Preiss (1969) ; Morris and Robson, unpublished observations] is indicative of the interesting relationships which may be revealed by pursuing these lines of investigation.

VI. Acknowledgements

It is a pleasure to record our gratitude to all those former and present colleagues who have contributed to the progress of our own research

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and understanding of microbial reserve polymers, and in particular to Douglas Ribbons, Grant Burleigh, Howard Stockdale, Graham Ritchie, Gordon Beech, Alan Ward and Michael Stephenson. We are greatly indebted t o the Agricultural, Science and Medical Research Councils and The Royal Society for their support of our researches at various times. We also thank Professor Anita D. Panek who read part of the manuscript, and Professor Hans G. Schlegel, Professor J. Gareth Morris and Dr. David L. Correll for kindly providing information prior to publication. The review of the literature for this article was concluded on 30th June, 1972. Note added in proof: A review of “ADP-glucose Pyrophosphorylase” by Dr. Jack Preiss will appear in Volume 8 of The Enzymes (Paul Boyer, ed.), Academic Press, New York. We are indebted to Dr. Preiss for an advance copy of his article. We are also grateful to Dr. R. H. Marchessault for a photograph of the most recent molecular model of poly-P-hydroxybutyrate in advance of publication; this now appears as Fig. 15. REFERENCES Agostini, D. E., Lando, J. B. and Shelton, J. R . (1971). J . Polymer Sci. A - 1 9, 2771-2787. Albaum, H. G., Schatz, A., Hutnor, S. H. and Hirschfeld, A. (1950). Archs Biochem. 29, 210-218. Alper, R., Lundgren, D. G., Marchessault, R. H. and Cote, W. A. (1963). Biopolymers 1, 545-556. Antoine, A. D. and Tepper, B. S. (1969a). A r c h s Biochem. B i o p h y s . 134,207-215. Antoine, A. D. and Tepper, B. S. (1969b).J . Bact. 100, 538-539. Antoine, A. D. and Tepper, B. S. ( 1 9 6 9 ~ )J. . gen. Microbiol. 55, 217-226. Ashworth, J. M. and Watts, D. J. (1970). Biocliem. J . 119, 175-182. Atkinson, D. E. (1968). I n “The Metabolic Roles of Citrate”, (T. W. Goodwin, ed.), pp. 23-40. Academic Press, London and New York. Atkinson, D. E. (1971). I n “Metabolic Pathways”, (H. J. Vogel, ed.), Vol. 5 , pp. 1-21. Academic Press, New York. Atkinson, D. E. and Walton, G. M. (1965). J . biol. Chem. 240, 757-766. Barry, C., Gavard, R., Milhaud, G. and Aubert, J. P. (1952).C. r . hebd. Se‘alzc.A c a d . Sci., Paris 235, 1062-1064. Baumann, P. (1969). Biochemistry, N.Y. 8, 5011-5015. Bergmeyer, H. U., Gawehn, K., Klotzsch, H., Krebs, H. A. and Williamson, D. H. (1967). Biochem. J . 102,423-431. Blum, J. J. (1967). Proc. natn. Acad. Sci. U.S.A. 58, 81-88. Blum, J. J. (1970). A r c h s Biochem. B i o p h y s . 137, 65-74. Boatman, E. S. and Douglas, H. C. (1963). J . a p p l . P h y s . 34, 2528. Buchanan, B.B., Evans, M. C. W. and Arnon, D. I. ( 1967).A r c h . Mikrobiol. 5 9 , 32-40. Builder, J. E. and Walker, 0. J. (1970). Carbohyd. Res. 14, 35-51. Bulen, W. A., Le Comte, J. R. and Bales, H. E. (1963). J . Bact. S5, 666-670.

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

A

Bandurski, R . S., 109,126 Barash,V., 57,77 Barile,M. F., 2,4,8,13,71,72,72,73,74 Barker, D., 37, 77 Barker,H.A., 91,127 Barlow, G. H., 101,103,127 Baron,C., 35,72 Barrera, C. R., 242,260 Barrnett, R. J., 9,23,76 Barry, C., 143,257 Barta, J., 122,126 Barton, L. L., 88,100,126 Bartsch,R. G., 101,129 Basu, D., 93,99,130 Baumana, P., 168,257 Beck, G., 124, 127 Beckman, B. L., 41, 73 Beech, G. A., 219, 220, 221, 224, 225, 226, 245,253,264 Bell, G. R., 100,126 Bell, F. P., 47, 73 Belly, R . T., 2,73 Belozerskii, 180,193,266 Benoit,H., 209,258 Berezina, F. S., 118,130 Bergmeyer, H. U., 241,242,257 Berlin, C. M., 72, 79 Bernal, J. D., 117, 126 Beriisteiri-Ziv, R., 7, 8, 9, 11, 23, 73 Berti, M., 119, 127 Bhattacharjee, S. B., 17, 18, 19, 74 Biberfeld, G., 8, 9, 12, 14, 73 Biberfeld, P., 8, 9, 14, 73 Billy, C., 118, 126 Birch Andersen, A., 8, 9, 14, 23, 74 Bjorklund, R. F., 36, 55, 73 Black, F. T., 14, 15, 73 Blum, J. J., 143, 145, 170, 175,257, 260 Boatman,E. S., 6,8,73,212,257 Bobyk, M. A., 193,194,261 Bode,H.R., 7,14,73,76 Bonissol, C., 3,6,79 Bonsen, P. P. M., 38,80 Booth,G.H.,85,119,120,121,126,132 Boughton, J. E., 67,79 Boroner, R . H., 144,261 267

Abbot, A., 37, 78 Abrams, A., 35, 72 Adams, M. E., 97,125 Agostini, D. E., 209, 257, 264 Akagi, J.M., 88,89,93,95,98,99, 107,112, 113,125,126,127,130,132 Albaum, H. G., 180,257 Alexander,M., 119,122,125 Alico, R. K., 84,125 Aheva,N. S., 121,122,131 Allan,D., 34,35,72 Allen,T. C., 3,9,15,23,72,75 Alper,R., 205,206,207,238,257,261 Ambler,R.P., 90,101,102,106,125,126 Ambron,R. T., 39,72 Anderson,D.L., 6,72 Anderson, D. R., 4,8,13,72 Anderson, K. E., 94,127 Anderson,M. L., 145,266 Ando,M., 125,132 Andrewes, C. H., 13,72 Antoine,A.D., 136,143,144,257 Aomine, G., 124,126 Argaman,M., 28,37,46,57,72,73,78 Armstrong, D., 2,73 Amon,D.I., 107,132,157,238,257 ARada,K., 109,125 Asahi, T., 109,126 Asato,R. N., 95,96,128 Ashworth, J. M., 143, 145, 168, 169, 257, 259,265 Atkinson, D. E., 109, 128, 138, 142, 153, 161,163,257,258,260,263,264 Aubert, J. P., 143,257 Auborn, J. J.,64,73 Avigad,G., 180,191,259 Azerad, R., 109,129

B Bak, A. L., 14,15,73 Bakes, J., 186,264 Bales, H. E., 251,252,257 Ballinger,D. G., 125,128

12

268

AUTHOR INDEX

Bov6, J. M., 3,6,79 Bowen,C. C., 143,258 Boyer,R.F.,99,126 Boyle, W. C., 205,258 Branton,D., 52,54,60,61,69,79 Bredt, W., 7,13,73,76 Bressler, R., 231,265 Brisou, J., 119,126 Brock,T. D., 2,4,73,74 Broda,E., 117,126 Brower, L. F., 6,72 Brown, C. N., 247,265 Brown,M.S.,89,126 Bruce, J., 19,21,73,75 Bruckdorfer, K. R., 48,74 Brunner, H., 7,73 Bruschi,M., 90,98,101,102,106,125,126 Bruschi-Heriaud, M., 96, 103, 105, 106, 107,126,127 Buchanan,B.B., 157,238,257 Builder, J. E., 143,145,158,160,257 Bulen, W.A.,251,252,257 Burgess, S. G., 122,126 Burleigh, I. G., 141,258 Burris, R. H., 85,130 Burton, C. P., 113,126 Busch,K. A,, 185,260 Butler, K. W., 48,73 Butler, T. F., 58, 73 Byrne, M. J., 140, 143, 145, 167, 258

C Cabasso,V., 124,126 Cabib,E., 145,163,164,165,166,174,263 Cahet,G., 118,126 Cairns, J., 14,73 CaIdes, G., 37,41,63,77, 78 Calinescu,I., 125,131 Calvin,M., 117,128 Camargo,E.P., 143,145,167,168,258 Camejo, G., 36,73 Campbell, J. J. R., 136,258 Campbell, L.L., 84,85,87,96,99,101,103, 108, 113, 114, 115, 126, 127, 128, 130, 131 Cantino, E. C., 143,258 Cantor, C. R., 107,129 Cardini, C. F., 150, 261 Carminatti, H., 163, 261 Carr, N. G., 180, 189,205,229,258 Carroll, W. R., 72, 79 Carski, T. R., 2, 72, 73, 74 Carstensen, E. L., 8, 49, 73 Carter, R., 52, 61, 79 Castrejon-Diez, J., 70, 73

CattanBo, J., 139, 145, 149, 258, 264 Chambers, L. A., 144,261 Chambost, J. P., 145,264 Chanock, R. M., 6, 7, 17, 37, 41, 54, 55, 60, 63, 73,74,77, 78,79 Chao, L., 143,258 Chapman,A.G., 138,258 Chapman, D., 48,50,73,77 Charba, J. F., 251,264 Chargaff, E., 143,258 Chen,G. S., 171,172,258 Chen,R.F., 163,258 Chen,T. A., 3,8,73,74 Chernick, S. S., 37,77 Chester,V. E., 140,143,145,167,258 Chiu, R., 3,76 Cho, H. W., 6,35,36,66,73 Choulas, G. L., 30, 32, 36, 55, 64, 68, 73, 80 Chowdhury,A. A., 91,127,236,237,258 Christiansen,C., 14,15,73 Chu,H.P.,31,73 Cinquina,C.L., 109,127 Citri, N., 63,64,78 Clark,D. J., 136,262 Clark, J., 18,79 Cleland,S., 168,258 Cleverdon, R. C., 29,30,31,32,33,37,77 Clyde, W. A., Jr., 9,74 Coe, E., 168,258 Cohen,A., 65,67,68,69,71,77 Colaeicco, G., 36,73 Colas, J., 180,259 Cole, B. C., 70,73 Cole, J. A., 180,193,258 Cole, R. M., 60,63,73 Collier,A.M.,9,11,12,73, 74 Combremont, R., 124,127 Conrad,H.E., 143,145,150,259,260 Connell, W. E., 118,120,127 Constantinesco, S., 87,130 Conti, S. F., 2,4, 74, 180,205,260 Conto Poulos, R., 205,236,237,240,258 Contopoulou, R., 136, 140, 228, 238, 251, 264 Cook, R. A., 163,263 Cooksey,K. E., 205,236,237,240,242,258 Coon,M. J., 93,99,126,129,130,244,264 Cooper,A. W.,85,120,121,126 Cooper,P.M., 120,121,126 Cornibert,J.,208,209,258 Correll,D.L., 181,182,258 Cosenza, B. J., 7,8,9,13,44,45,77 Costello, J. A., 84,126 Cote, W. A., 206,207,238,257 Crabtree, K., 205,268 Cronquist,A., 116,128 Croson,M., 203,215,261

269

AUTHOR INDEX

Crumpton,M.J., 34,35,72,74 Czarnecky,D.,3,75

D Dagley, S., 140,258 Dahinger, A., 239,240,259 Dalton, H., 251,252,253,258 Daly,D. J.,94,127 Damoglov, A. P., 140,258 Damotte,M., 139,258 Daniels,M. J., 15,56,79 Dark,F.A., 141,143,265 Darland, G., 2,4,74 Das, J., 17,18,19,74 Davidson, J.T., 110,111,129,130 Davidson,M., 9,80 Davis, J. B., 92,127,205,258 Davis, J. C., 180,187,263 Davis,R.E.,3,8,74 Dawes, E. A., 136, 138, 140, 141, 142, 172, 205, 206, 214, 218, 219, 220, 221, 224, 225, 226, 227, 230, 231, 232, 233, 241, 242, 243, 244, 245, 246, 248, 249, 253, 258,263,265 Dayhoff,M. O., 107,127 Deacon,T.E., 110,130 DeBertoldi,M., 207,262 De Gier, J., 45,48,74,76 De Kruiff, B., 43,74 Delafield, F. P., 205, 236, 237, 239, 240, 242,258,262 Delafuente, G., 138,264 Delaporte, B., 203,261 Del Campillo, A., 229,244,265 Del Giudice, R. A., 2,72,73,74 Demel, R. A., 48,74 Denneny, J.M., 180,188, 193,195,266 DerVartanian,D.V.,96, 101,103,105,106, 107,127,129 De Rautlin De La Roy, Y., 119,126 D’Eustachio,A. J., 88,127 Dicks, J. W., 140,141,258 Dienema, M. H., 143,205,262 Dirheimer, G., 187, 195,197,258,259 Dittbrenner,R., 91,127 Domermuth, C.H., 8,9,14,23,74 Dommergues,V., 124,127,128 Doudoroff,M., 136,138,140,157,205,209, 212, 213, 228, 236, 237, 238, 239, 240, 242,251,258,259,261,262,264 Douglas,H.C.,212,257 Dragoni,N., 98,99,105,107,108,129 Drews,G., 180,187,188,259 Dreyfus, J., 109,127 Drozd, J.,253,259 Drucker,H., 101,103,127

12*

Dubourdieu,M., 98,f06,108,109,126,127, 132 Dugan,P.R., 125,132 Dugle,D.L., 19,74 Dugle, J. R., 19,74 Duguid, J. P., 136, 180, 183, 259, 264, 266 Duncan,M. G., 136,258 Durner,G., 119,127 Dus, K., 106,126

E Eaton,M.D., 70,76 Eaton, W. A., 103,127 Ebel, J. P., 180, 186, 187, 195, 197, 258, 259,264 Eck, R. V., 107,127 Edelmann,P.L., 143,145,157,158,259 Edmundson, T. D., 145,259 Edward,D.G.,6,14,46,67,74 Edwards, G. A., 18,74 Edwards,V. H., 84,129 Ehret,A., 109,128 Ehrlich,H.L.,82,131 Ehrman, L., 3,80 Eidels,L., 143,145,157,259 Einlof, C. W., Jr., 8,49,73 Eisenberg, R. C., 33,74 Elford, L., 120,126 Ellar,D., 207,211,212,233,234,259 Engelman, D. M., 31, 36, 51, 57, 58, 59, 60, 62,64,74,76,79 Entner, N., 157,259 Evans,H. J . , 205,239,241,243,260,266 Evans,M. C. W., 157,238,257 Eylar, E. H., 64,74 Eyring, E. M., 64,73

F Fabricant, J.,6,74 Fall, L., 138,258 Falter, H., 24,25,26,74 Fanger,M. W., 103,127 Favard,A., 145,149,258,264 Fedorov, V. D., 202,264 Feldberg,R., 110,129 Feldman,H., 24,25,26,74 Felter, S., 197,201,259 FencheI, T. M., 119,127 Fernandez,L. J.,119,127 Fetty, W. O., 185,260 Fiechter, A., 140, 165, 166, 167, 169,261 Findley,J.E., 112,127 Fischer, E. H., 174,259 Fisher, R. J., 251,262

270

AUTHOR INDEX

Fischman,D.A.,31,80 Fisher,E., Jr, 70 Fisher,H., 24,25,26,75 Fisher, T. N., 70,73 Fitch, W. M., 101,127 Fitzgerald, W. A., 46,74 Florance, E. R., 9,23,72 Flores, J., 178,263 Fogh, J., 18,74 Folsome,C. E., 17, 18,74,79 Folsome, J.,17,74 Ford, D. K., 7 0 , 7 4 Ford, H. W., 227 Forget,N.,98,99,128 Forsyth, W. G. C., 205,259,260 Fosset,M., 174,259 Foster, J. W., 137,229,259 Fottrell,P.F., 205,241,259,262 Foust,G.P., 110,129 Foust, W. N., 251,264 Freeman, R . , 38,79 Freke, A. M., 123,127 F r e u n d t , E . A., 2, 6, 8 , 9, 12, 13, 14, 15, 23, 46,73,74, 75 Friedberg,I., 180,191,259 Fujimoto,D., 110,128 Fuller, R. C., 180,260 Furlong,C. E., 143,157,238,259 Furness,G., 12,13,17,75

G Gahan,L. C., 143,145,150,259 Gancedo, C., 138,264 Gardiner, 11.C., 193,265 Garwes,D. J., 19,21,73,75 Gascoyns, T., 163,260 Gattellier, M. C., 84,122,130 Gavaro, R., 143, 144,239,240, 257,259 Gawehn,K., 241,242,257 Genovess, S., 118,127 Gentner, M., 143, 145, 146, 147,259,263 Gentry, G. A., 69,79 German,R. J., 143,259 Germano, G. J.,94,127 Gest,H., 88,94,95,128,129 Ghosh, H. P., 149,259 Gianotti, J., 3,75 Gibbons,N.E., 205,215,249,264 Gibbons, R. J., 143, 158, 159, 160, 161, 239,259 Gibbs, C. J., 2,72,73 Gibson, Q. H., 109,131 Giraro,H., 203,205,261 Gittinger, L. B., 122,127 Goldenberg, S. H., 163,261 Goldstein, A., 143,258

Gonzalez,H., 119,127 Goodman,E.M., 179,180,190,259 Goodman, J., 174,259 Gordon,A.,36,80 Gorlenko,V.M., 118,121,128,129 Gotay, I., 174,263 Gottfried, L., 6,36,49,65,66,72,77,78 Gottschalk, G., 91, 93, 117, 127, 205, 206, 207,215,224,238,248,250,263 Gourlay, R. N., 8, 19, 20,21,22, 73, 75 Govons,S., 139,145,259 Grace, J. T., Jr., 25,75 Graf, L., 63, 77 Granados, R. R., 3, 8, 73, 74, 76 Gray, C. T., 94,95,128 Gray, W. R., 30, 32, 73 Grebner, P., 103, 132 Greenberg, E., 143, 145, 146, 147, 148, 149, 150,259,263 Greenwood,D. J.,252,259 Grelet, N., 215,261 Griebel, R . J., 212, 213, 233, 234, 235, 239, 259 Gronlund, A. F., 70,74,136,258 Grula, E. A., 58,73 Guarraia,L., 100,128 Guarraia, L. J.,98,99,128 Gulick, A,, 203,259 Gustafson, G., 168,263 Guttes, S., 191,262

H Hall,R.H., 25, 75 Hallberg, R. O., 84,123,128 Haller, 0.J., 19,22,75 Halvorson,H. O., 205,216,229,260 Hames, B. D., 143,168,259 Hammerberg, K., 70,80 Hampton, R. O., 3, 9, 23, 72, 75 Han, J., 117, 128 Hanawalt, P. C., 17, 18, 28, 69, 79 Harbury, H. A., 103,127 Harden, A., 143,259 Hardy, R. W. F., 88,127 Harold,F. M., 137, 138, 139, 180, 181, 183, 184, 193, 196, 197, 199, 200, 201, 202, 203,259,260 Harold, R. L., 184, 193, 194, 196, 199, 200, 202,259,260 Harrington,A.A.,212,260 Harris,E. J., 148,260 Hartzell, R. W., Jr., 46,78 Haschke,R.,96,108,113, 114,115,128 Hata,Y., 117,128 Hatchikian, E. C., 89,98,99, 100, 106, 108, 226,128,129

27 1

AUTHOR INDEX

Hathaway, J. A., 163,260,263 Hattingh,M., 84,118,132 Hauttecoeur, B., 239,240,259 Hayashi,H., 24,25,26,75 Hayflick, L., 6, 31, 46, 52,53,55, 62,67, 70, 74,75,78 Hayward, A. C., 205,259,260 Hecht,L., 185,264 Hempfling, W.P., 148,260 Henrikson, C. V., 32,38, 39,43, 49, 75, 77, 79 Herbert, 136,137,140,260 Herzberg,K., 31,78 Hespell, R. B., 87,128 Hettinger, T. P., 103,127 Heym, G. A., 2 16,260 Hill, S., 253,260 Hippe, H., 239,250,260 Hirsch, P., 205,260 Hirschfield, A., 180,257 Hirth, R. S., 8,75 Hirumi, H., 3,76 Hochstrasser,R.M., 103,127 Hodson,R.C.,113,131 Hofer,M., 148,260 Hoffman-Ostenhof,O., 157,201,260 Hollingdale, M. R., 28,29, 75 Holm,H. W., 118,128 Holme, T., 140,141, 143, 144,221,260 Honya,M., 95,132 Hooper,A. B., 180,190,265 Horne,R. W., 8,22,31,73,75 Horoszewicz, J . , 25,75 Horowitz, J.,32,24,25,26,75 Horswood, R.L., 17,79 Howell, L. G., 110,129 Hubbard, J. C., 7,75 Hubbell, W. L., 46,48.52,53,62, 75,78 Hughes, D. E., 180, 157,193, 196,258,260, 262 Hughes,J.E.,85,130 Hull, R., 19, 73,75 Hummeler, K., 31,75 Hunter, J. R., 142,250,263,265 Hiisler,D.,31, 78 Hutner,S. H., 180,257

I Iizuka, H., 85,128 Iliffe, J.,229,260 Indge, K . J., 186,260 Ingraham, J., 139,145,259 Inokuchi,H., 95,133 Ishida, M., 35,75 Ishimoto,M.,97,110,112,113,128,129

Ivanov,M. V., 118,122,128 Iverson, W.P., 83,116,120,128

1 Jackson, G., 93,125 Jacobs,R. E., 70,79 Jacq,V., 124,127,128 James, W.D., 37,41,63,77, 78 Jannasch,H. W., 85,132 Jansen,H.M., 141,143,160,265 Jenson,L.H., 109,132 Jensen, T. E., 205,260 Jin-Po-Lee, 113,114,115,128,129 Johnson, A. R., 140,258 Johnson, J.D.,22,24,25,26,75 Johnson,L., 25,75 Jones,A. S., 143,259 Jones,D., 245,265 Jones,H.E., 85,105,107,128 Jones-Mortimer,M. C., 125,128 Joseph, R., 87,128 Jukes, T. H., 107,129 Juni,E.,216,260 Jurtshuk, P., 242,260

K Kadota, H., 117,128 Kaganoff, M. F., 2,73 Kahane, I., 29,30,31,32,33,34,35,36,37, 40,42,56,57,62,63,64, 75, 76, 77 Kahn,V., 175,260 Kalica, A., 7,73 Kalkstein,A., 63,64,78 Kallio, R. E., 212,260 Kaltwasser, A., 180, 188, 189, 202, 203, 260 Kamen,M. D., 101,109,129 Kammer, G. M., 8,75 Kamin,H., 109,131 Kamura, T., 124,132 Kannan, L. V., 205,260,261 Kaplan,I. R., 122,130 Kapsimalis, B., 143, 158, 159, 160, 161, 259 Karavaiko,G.I., 118,128 Kasimer,P., 68,79 Kasprzycki,M. A., 126 Katchman, B. J., 185,260 Kato, G., 123,128 Keith, A., 52,54,61,69,79 Kelleher, J. J .,85,132 Kelly,D.P.,93,128 Kemp, J.D., 109,128 Kenny, G.E., 6,8,41,73

272

AUTHOR INDEX

KBpBs,A.,206,207,260 Kernaghan,R. P., 3,80 Keup,L.E., 125,128 Kidman,A.D.,95,96,128 Kimata,M., 117,128 Kindt, T. J., 150,260 King,R. A,, 84,120,126,128 Kingsbury,D. T., 14,75 Kinsky, S. C., 47, SO Kirk, G. R., 7,76 Kirk, R. G., 22,24,26, 75 Kitamara,H., 118,132 Kite, J. H., Jr., 7,75 Klainer, A. S., 8,75 Klein,R.M., 116,128 Kleineberger-Nobel, E., 6,8,12, 75 Kieinig, H., 46,75 Kleinschmidt,A., 14,19,75 Klemme, J. H., 88,129 Klotzsch, H., 241,242,257 Klucas,R.V., 241,243,260,266 Knight, E., 88,127 Knudson, D. L., 7,76 Knyszynski, A., 69,77 Kobayashi,K., 113,129 Koike,M., 180,183,188,262 Kominek,L.A., 205,216,229,260 Koostra, W. L., 32,37,39,40,58, 75, 79 Kornberg,A., 180,192,193,260 Kornberg, R. D., 57,76 Kornberg, S. R., 180, 192, 193, 194, 195, 260 Kovartovsky, J.,29,31,34,36,57,76,77 Kowalik,R., 84,125,129 Koyama, J.,97,112,128 Krassowski, B., 84,125,129 Krauss, I., 139,235,263 Krebs, H. A., 163, 205, 241, 242, 257, 260, 266 Krebsbach, R., 178,263 Krichevsky,M.I., 180,190,265 Kritchevsky, D., 46,78 Krivacic, J.R., 55,80 Kiienzi,M. T., 140, 165, 166, 167, 169,261 Kuhl,A., 179,180,261 Kulaev, I. S., 193, 194, 195, 196, 261, 265 Kulesza, S., 188,195,265 Kunisawa, R., 136, 140, 228, 238, 251, 264 Kuster,E., 118,129 Kutty,M. R., 205,261 Kuznetsova,V.A., 121,129

L Lafferty, R., 139,235,263 LaflBche,D., 3,6,79

Laishley, E. J., 98, 99, 107, 109, 128, 129, 130 Lammel, C., 145, 147, 150, 151, 152, 153, 154,155,263 Lando, J. B., 209,257,264 Langen,P., 185,186,187,202,261 LaRiviBre, J. W. M., 82,129 Larner, J., 164,165,261 Lascelles, J.,205,229,258 Laurent, T., 140,144,260 Law, J.H.,214,216,218,221,261, 263 Lazzarini, R. A., 109,128 Leban,M., 84,129 Lee, C., 3,75 LeComte, J. R., 251,252,257 Le Gall, J., 89, 90, 93, 94, 96, 98, 99, 100, 101, 102, 103, 105, 106, 107, lOS, 109, 114,115,125,126,127,128,129,132 Leinweber,F. J., 109,131 Leloir, L. F., 150,163,261 Lemcke, R., 6,7,28,39,76 Lemcke, R. M., 6, 28, 29, 37, 41, 74, 75, 77 Lemoigne, M., 203, 205, 207, 213, 215, 239, 261 Lepidi, A. A., 207,262 Le Roux, N. W., 123,132 Leterrier,F., 108,127 Levine, S., 144,261 Levine,Y.K.,48,76 Lewis, K., 125,129 Li, C. Y., 180,266 Liebermann, L., 185,261 Liebermeister, K., 12, 76 Liegey, F. W., 84,125 Lifshitz, Y., 69,77 Lin,C.-C., 83,125,129 Lin, K . C., 83,125,129 Lin,L.P.,218,261 Lin,P.M., 107,109,129 Lin, S., 3,76 Lipmann, F., 110, 131, 143, 174, 178, 261, 265 Liss,A.,21,22,76 Liss, E., 185,186,202,261 Lode,E.T.,99,126 Lohmann,K., 185,261 Looney,D., 185,264 Low, I. E., 70,76 Lowry, 0. H., 163,262 Lund, P., 163,260 Lund, P. G., 68,76 Lundblad,G.T., 143,145,173,174,264 Lundgren, D. G., 205, 206, 207, 209, 211, 212, 213, 233, 234, 238, 257, 259, 261, 262,263,265 Lusty, C. J., 205,236,237,240,258,261 Luzzati,V., 48,77 Lyalikova, N. N., 123,229

AUTHOR INDEX

Lynn, R . J., 19,22,75 Lyons,M. J., 13,76

M McCandlish, K. L., 70,74 McCandIish, L., 74 McConnell, H. M., 46,52,53, 57, 62, 76, 78 McCoy, E., 205,258 McDonald,C.C., 103,105,129 MacDonald, J., 70,74 McElhaney, R. N., 30, 32, 40, 41, 42, 43, 44,45,48,50,51,76,78,79,80 McIntosh, J. R., 57,78 McKenna, E. J.,99,129 Mackey,B.M., 141,161,261 McLain, G., 105,131 MacLennan,D.H., 37,79 MacLeod, R., 7,76 Macmillan, C. B., 125,132 McMurtrey,M. J., 12,13,75 Macrae, R. M., 215, 228, 238, 239, 249,261 Maori, G., 118,127 McWhorter, W. P., 229,259 Maddy,A.H., 33,76 Madsen,N. B., 143,146,261,262 Mager, J., 109,129 Makman, R. S., 170,262 Mallette, M. F., 136,262 Mandersloot, J. G., 48,74 Maniloff, J., 7,8,9,12,15,16,17. 18,19,21, 22,23,49,73,74,76 Manners, D. J., 138,144,262 Manning, S., 242,260 Mansour, T. F., 163,262 Mara,D. D., 83,129 Maramorosch,K., 3,7,76,80 Marchesi,V. T., 61,79 Marchessault, R. H., 205, 206, 207, 208, 209, 211, 212, 233, 234, 238, 257, 258, 259,261,262 Marcus,L., 251,262 Margoliash,E., 101,103,127 MaritzaGonzalez, M., 119,127 Marmion,B. P.,37,39,41,77 Maroc, J., 109,129 Marr,A. G., 136,262 Martin, C. H., 70,73 Martinez,R. J., 179,262 Maruyama, K., 101,103,132 Masotti, L., 55,80 Massey,V., 110,129 Matschiner, J.T., 109,132 Matsubara, H., 107,129 Mattenheimer,H., 187,196,262 Matthews, R. G., 110,129

273

Mayberry, W. R., 37, 38, 39, 42, 46,49, 58, 77,78,79 Mayer,T., 101,129 Mayhew, S. G., 110,129 Mama, G., 105,129 Meers, J. L., 247,265 Mekhtieva,N.A., 121,122,131 Melchior,D. L., 50, 76 Mellanby, J., 205,266 Merrick, J. M., 205,209,212,213,228,229, 233, 234, 235, 236, 238, 239, 240, 241, 259, 261, 262,263 Meselson,M., 14,76 Metcalfe, J. C., 62, 75, 76 Metcalfe, S. M., 62,75 Metz, T., 7,76 Meuser,R., 143,145,167,168,258 Mialhe, H., 46,78 Michaels, G. B., 110,111,129 Milhaud, G., 143,144,257,259 Miller, J. D. A., 84, 85, 89, 114, 119, 120, 126,128,129,130 Miller, S. L., 178,262 Millet, J.,130 Mindich,L., 27,76 Mitsui, S., 124,130 Mittelman, A., 25,75 Miyoshi,H., 117,128 Mizushima, S., 35,75 Model, P., 153,156,262 Mohberg, J., 191,262 Monty, K. J., 109,127,131 Moore, D. H., 143,258 Morey,A.V., 122,131 Mori,Y., 95,133 Morowitz, H. J., 6, 7, 8, 9, 12, 14, 15, 16, 18, 19,22,23, 24,31,32,33,35,36,50, 54,57,58,59, 60, 64, 66,68, 70, 73, 74, 75,76,77,79 Morris, J. A., 2,72,73 Morris, J. G., 141,145, 161,261,263 Morris, M. B., 262 Mortenson, L. E., 96,97,130 Mortlock, R . P., 87,128 Moses,V., 148,262 Moskowitz, G. J.,205,229,262 Mudo, S., 180, 183, 187, 188, 262,263 Muhammed, A., 180, 187, 192, 193, 260, 262 Mtihlradt,P.F., 180,192,193,262 Muir, L. W., 174,259 Mulder,E. G., 143,205,262 Muller,F., 110,129 Muller, S., 180,259 Muller-Felter, S., 186,264 Munoz, E., 35,76 Munro, A. L. S., 136, 137, 140, 142, 192, 205,221,224,248,266

274

AUTHOR INDEX

Murphy, W. H., 18,79 Murzaev,P. M.,122,130 Myant, N. B., 229,260

N Nadarajah,M., 143,259 Nagai,Y.,97,128 Nagatani,H.,247,262 Naide,Y.,31,76 Nakai,N., 109,130 Nakamura, T., 109,153

Ollat, C., 124,127 Operti,M. S., 174,262 Oppenheim, J.,251,262 Oppenheimer, C. H., 93,130 Ortigoza, R. O., 179,265 Orton, W. L., 186,265 Ostrowski, W., 188,195,265 Oulette, C. A., 85,130 Overath, P., 66,79 Owens, N. F., 48,73

P

Nakata,H.M.,217,262 Nakatsukasa, W., 112,113,130,132 Pace, B., 85,130 Nakos, G., 96,97,130 Paisley, H.M., 126 Nalewaik, R. P., 26,79 Palleroni, N. J.,205, 236, 237, 240, 258, Nauman, R. K., 24,76 264 Ne’eman, Z., 30, 31,32,33, 34,35, 36, 57, Palmstierna, H., 140,141,143,144,260 Panek,A.D., 174,262 58,59,60,64,65,70,76,77 Neimark,H. C., 15,77 Pankhurst,E. S., 83,84,121,130 Nelson, J.B., 13,77 Panos, C., 40,42,43,68, 75,77,78 Neumann, P.M., 89,129 Parris,M., 178,262 Ness,A. G., 141,143,265 Parson, W. L., 205,250,265 Newman,D. J.,99,107,108,130,132 Passoneau, J. V., 163,262 Newsholme,E.A., 163,260 Pastan, I.,170,262 Ng, M. H., 35,76 Patrick,W.H., 118,120,127 Nicholas,D. J.D., 109,131 Paule,M.R., 143,145,262 Nickless, G., 82,130 PBaud-Lendel, C., 206,207,260 Nielsen,L.D., 174,259 Peck, H. D.Jr., 82,88,90,98,99, 100,107, Nielsen,M. H., 8,9,14,23,74 109,110,111, 113,114,115,116,126, Nielson, S. W., 8,75 128,129,130,131,132 Nikolaev,N.N., 193,194,261 Perlman,D., 17,79 Nilson,E.H., 136,262 Perlman,R., 170,262 Nishi, A,, 193,202,262 Peterson, J. A,, 93,99,130 Nissenbaum, A., 122,130 Peterson, J.E., 1 1 , 1 2 , 1 5 , 2 6 , 2 7 , 5 6 , 6 7 , 6 8 , Novozhilova, L. P., 180,193,266 78 Novozhilova, M. I.,118,130 Petrovici, A., 87,130 Nuti, M.P., 207,262 Pfendt,E. A., 67,70,78 Nygaard,O.F., 191,262 Pfister,R.M., 209,212,213,261,262, 263 Philippot, J., 34,77 Phillips,M. C., 48,73 Phillips, W. D., 103,105,129 0 Pichinoty, F., 94,105,129,131 O’Brien,R. W., 91,130 Pieringer, A., 39,72 Ochynski, F. W., 85,130 Pipes, F. J.,12,13,75 Oedin,V.,262 Pirt, S. J.,136,263 Oeding,V., 139,231,233,235,241,244,245,Plackett, P., 36,37,38, 39,40,41,42,49, 246, 264 65,77,80 Ohad, I.,30,58,59,60,64,65,77 Plaut, G. W. G., 163,258 O’Hara,A.,205,241,259,262 Plescher,C., 31,78 O’Hara, C., 188,266 Plessis, A , , 84,122,130 Okamura, K., 206,207,208, 209,211,212, Pogell,B.M., 163,265 233,234,259,262 Poindexter, J. S., 212,263 Okazaki, H., 85,128 Pollack, J. D., 8, 29,30, 31,32,33, 38, 42, Okuda, S., 31,80 43,44,75,77,78 Olavarria, J.M., 133,261 Pollack, M.E., 26,29,30,79 Oldfield,E., 48,77 Pollock,M. E., 6,72,76,77

275

AUTHOR INDEX

Popkin, T.J., 60,63,73 Postgate, J. R., 82, 83, 84, 85, 87, 89, 92, 93, 94, 97, 99, 101, 105, 108, 110, 112, 115, 116, 117, 119, 123, 124, 125, 125, 126, 130, 131, 132, 142, 250, 251, 252, 253,259,260,263 Powers, M. T., 205,250,265 Pozsgi, N., 85,131 Prabhakararad, K., 109,131 Preiss, J., 138, 139, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 238, 256, 259, 262, 263, 264 Prescott, B., 37, 54,55,60,63,73,77,78 Pressman, B. C., 148,260 Presswood, R. P., 109,131 Proctor, P., 70,76

R Rabinowitz, J., 178,263 Racker, E., 65,77 Rader,R.L.,50,51,79 Ramaiah, A., 163,263 Rand, R. P., 48,77 Randles, C. I., 125,132 Rapport,M. M., 63,73,77 Raw, I., 229,265 Razin. S., 2, 6, 7, 8,9, 13,28,29, 30, 31, 32, 33, 34,35,36,37,39,40,41,42,43,44, 45,46,47,49,52,54,55,56,57,58,59, 60, 61, 62, 63,64, 65,66,67,68,69,70, 7 ~ 7 272, , 73, 74, 75, 76, rr, 7 8 , 79 Rehacek, Z., 205,260,261 Reich, P. R., 22,78 Reinert, J. C., 50,51,78,79 Reuss,K.,31, 78 Reynaud, C., 239,240,259 Reynolds, J.A., 35,62,64,78 Ribbons, D. W., 136, 138, 141, 142, 172, 205, 206, 218, 219, 249, 253, 258, 263, 265 Rib6reau-Gayon, G., 145, 147, 150, 151, 152,153,154,155,263 Rickard, D. T., 123,131 Riederer, M. A., 131 Riederer-Henderson, M. A., 90,94, 131 Ried1,R. J., 119,127 Rigano,C., 118,127 Riggs,D. B., 71,73 Ritchie, G. A. F., 205, 213, 219, 220, 221, 224, 225, 226, 230, 231, 233, 245, 248, 253,263,264 Rittenberg,D., 96,131,153,156,262 Rittenberg, S. C., 82,131 Robbins,P. W., 110,131 Roberts, J.B.,205,259,260,262

Robson,R.L., 143,145,161,263 Robson,R.M., 143,145,161,263 Rodgers,A., 180,187,196,262 Rodriguez,M.,174,263 Rodwell, A. W., 11, 12, 15, 26, 27, 31, 37, 38, 39,44,46,56,57,67, 68, 69, 70, 71, 77, 78 Rodwell, E. S., 11, 12, 15, 18,26,27,42,45, 56,68,78 Roemer,R., 85,119,123,127,131 Rogers,G., 178,263 Rohlich, G. A., 205,258 Romano,N., 37,38,39,42,46,78 Rosenberg, S. A., 57,78 Rosenthal, S., 143, 145, 173, 174, 264 Rosness,P.A., 168,263 Rothblat, G. H., 46,78,79 Rothman, L.B., 145,163,164,165,174,263 Rothman-Denes, L. B., 164, 165, 166, 263 Rothstein, A., 174,259 Rottem, S., 6, 7, 29, 30, 31, 32, 35, 36, 37, 40,42,43,45,46,47,49, 52,53, 54,55, 57, 58, 59, 62, 63, 64, 65,66, 67, 68, 69, 70,72,77,78 Rouf, M. A., 205,206,263 Roukhelman,N., 203,261 Roussel, H., 124,126 Rowland, S., 143,259 Roy, A. B., 82,131 Rozanova,E.P., 121,122,131 Rueger, 0.C., 109,131 Rusch, H. P., 179, 180, 190, 191, 259, 262 Russell, S., 243,266 Ruys, A. C., 14,80 Ryan, J. L., 16,24,79

5 Sabrow, A., 145, 147, 148, 150, 151, 152, 153,154,155,263 Sadana, J. C., 96,122,131 Sadoffe, H. L., 218,261 Sadurski, I., 84,125,129 Sagardia,F., 174,263 Saglio, M. P., 3,6,79 Salas, J., 163,263 Salas,M., 163,263 Salas,M. L., 165,264 Saleh, A. &I., 114,126,129 Sall,T., 180,187,263 Salton, M. R. J.,35,76 Samsonoff, W., 2,34,74 Samuni, A., 52,54,78 Sandhu, G. R., 180,189,258 Sanwal, B. D., 163,263 Sassine, J.,3,75

276

AUTROR INDEX

Sato,R., 109,133 Sauer,H.W., 179,180,190,259 Sauer,L., 179,180,190,259 Saunders, G. F., 85,130,131 Schaechter, M., 15,56,79 Schairer,H., 66,79 Schatz, A., 180,257 Schiff,J.A.,113,131 Schimke,R.T., 71,72,73,79 Schindler, J.,229,242,263 Schlege1,H. G., 139,180,205,206,207,215, 222, 224, 231, 233, 235, 238, 239, 241, 242, 244, 245, 246, 248, 250, 260, 262, 263,264 Schlender,K.K., 145,165,264 Schmidt,G., 182,185,264 Schnaitman, C., 205,206,261 Schneider, H., 48,73 Schneider,M. C., 244,265 Schneider, W. C., 182,264 Scholes,P.B., 105,131 Schor, M. T., 35,76 Schramm, G., 178,264 Schuman,M., 110,129 Schuster,E.,222,264 Schwarz,A., 119,131 Schwartz, C. J . , 47,73 Schwartz, J.L., 17,79 Schwartz,W.,85,119,123,127,131 Scott, R. E., 61,79 Sefer,M., 85,87,125,130,131 Segel, I. H., 143, 145, 146, 149, 171, 172, 173,174,175,258,264 Senez, J.C.,94,105,129,131 Senior,A.E., 37,79 Senior, P. J., 210, 219, 220, 221, 224, 225, 226, 227, 231, 232, 233, 241, 242, 243, 244,245,246,248,253,263,264 Senterfit,L. B., 29,30,31,38,42,77 Sergeev,N. S., 193,194,261 Seto, S., 85,128 Shafer,Z.,46,78 Shaposhnikov, 202,264 Sharp, P. B., 148,262 Shaw,E. J., 37,39,40,41,77 Shaw,N., 37,38,40,79 Shelton, J. R., 209,257,264 Shen,L., 143,145,146,147,156,263,264 Shen,L.C., 161,264 Shepherd, D., 143, 145, 173, 174, 175, 264 Shimizu, M., 247,262 Shinn,P.M., 120,126 Shioiri, M., 124,131 Shorb,M. S., 68,76 Shuster, D. W., 242,264 Sicko, L. M., 205,260 Siegel,L.M., 109,131 Sieker,L. C., 109,132

Sierra, G., 205,215,239,249,264 Sigal,N., 139,145,149,258,264 Silver, W. S., 94,131 Silverman, M. P., 82,131 Silverman, D. J.,24,76 Simms,E. S., 180,193,260 Sisler,F.D.,93,131 Skerman,V. B. D., 85,131 Slater, M. L., 18,79 Slechta,L., 187,260 Slepecky, R. A.,214,216,218,221,261,264 Sloan,H.R., 37,77 Smith, D. D., 252,264 Smith,D. W., 17,18,28,69,79 Smith, G. L., 58,73 Smith, I. C. P., 48,73 Smith,I. W., 136,180,183,259,264 Smith, K. O., 14,79 Smith, L., 105,131 Smith, P. F., 2, 14,32,35,37,38,39,40,41, 42,46,47,48,49,58,66,67, 70, 71, 75, 76, 77,78,79,80 Smith, Z., 212,213,233,234,239,259 Sneath, P. H. A., 85,131 Sobek, J.M., 251,264 Socolofsky,M.D.,217,218,265 Sokolova, G. A., 123,129 So11, D., 24,25,26,75 Sols,A., 138,163,165,263,264 Somerson,N. L., 6,29,30,31,42,74,71 Sonneborn,D., 143,145,167,168,258 SorokinYu,I.,91,92,131 Souzu, H., 197,264 Speake, R., 163,260 Spilker, E., 96,129 Spoerl,E., 185,264 Stachow, C. S., 163,263 Stadtman,E. C., 243,264 Staehelin,L. A., 60,79 Stah1,A. J. C., 186,197,201,259,264 Stah1,F. W., 14,76 Stahl, J. C., 186,264 Stanier, R. Y., 136, 140, 143,205,228,238, 251,259,264 Stead,A., 38,79 Steim, J.M., 50,51,78,79 Stein, O., 57,58,59,65,78 Steinberg,P., 17,79 Stenderup, A., 14,73 Stern, J.R., 91,130,229,244,265 Stevens, J. O., 3,9,23,72,75 Stevenson,H. J. R., 144,261 Stevenson,L.H.,217,218,265 Stock, D.A., 69,79 Stockdale, H., 205, 206,218, 219, 238,252, 253,265 Stokes, J. L., 205,206,250,263,265 Strange,R. E., 141,142,143,178,265

277

AUTHOR INDEX

Strasdine, G. A., 141,162,265 Sturtevant, J. M., 50,76 Su,C. J., 209,262 Suh, B. J.,88,98,99,112,132 Sullivan, P. A., 110,129 Sutherland,E. W., 170,262 Swartzendruber, D. C., 18,79 Sweeney,E. W.,72,79 Sykes, J., 256,265 Sylvan, S., 184,202,260 Szulmaster, J., 193,265 Szumilo,T., 188,196,265 Szymona,M., 188,195,196,265 Szymona, O., 188, 193, 194, 195, 196,261, 265

Tremblay, G. Y., 15,56,79 Trousil,E.B., 101,103,127 Trudinger, P. A., 82,114,115,131,132 Trueper,H. G., 85,116,132 Tsong, T. Y., 50,76 Tsuda,M.,95,133 Tully, J. G., 2,6,39,49,67,74,78,79 Tuttle, J. H., 125,132

U Ueda, T., 99,132 Urbina, J.,50,73 Urry, D. W., 55,80,103,132 Uryson,S. O., 193,194,196,261,265

T Tabor, E. C., 144,261 Tachibana, S., 113,129 Tagawa,K., 107,132 Takagi,A., 187,263 Takai,Y., 124,132 Taketa, K., 163,265 Takii,S., 118,132 Tamiya, N., 95,132 Tamura, G., 109,126,132 Tanford,C., 35,62,64,78 Tanzer, J. M., 180,190,265 Tasnadi, R., 124,132 Tate,D., 123,127 Taylor-Robinson,D., 28,80 Tempest, D. W., 140, 141, 178, 247, 258 265 Temple, K. L., 123,132 Tepper,B.S., 136,143,144,257 Terry,K.R., 180,190,265 Terry, T. M., 8, 31, 32, 33, 35, 36, 54, 57, 58,59,60,70,74,76,77,79 Tezuka,Y., 118,132 Thannhapser,S. J.,182,185,264 Thiel, P. G., 84,118,132 Thilo,E., 179,265 Thomas, L., 9,80 Thrower,K. J . , 8 , 7 5 Tillaok, T. W., 52,61,79 Tiller,A. K., 85,120,121,126,132 Tillez-Inon,M.T., 145,173,265 Tinelli, R., 216,265 Toerien,D. F., 84,118,132 Tomassini,N.,31,75 Torres,H. N., 145,173,265 Tourtellotte, M. E., 6, 7, 8, 26, 40, 41, 42, 43,44,45,50, 51,52,54,61,67,68,69, 70, 75, 76, 77,78, 79 Traut, R. R., 143,174,265 Travis, J., 107,129,130,132

V Vago, C., 3,75 Valdesuso, J., 63,78 Valentine, R. C., 247,262 Vamos, R., 124,125,132 Van Deenen, L. L. M., 30,32,38,45,48,74,

76,80 Van Demark, P. J., 32,36,65,70,71,80 Van Golde, L. M. G., 30,32,80 VanHoute, J., 141,143,160,265 Van Iterson, W., 14,80 VanVeen, W. L., 143,205,262 Van Wazer, J.R., 179,265 Verheij,H. M., 38,79,80 Vennes, J.W., 118,128 Verna,A.,89,125 Verna, J. E., 2,74 Villar-Palasi, C., 145, 164, 165,261,264 Vinogradov, S. N., 103,105,129,132 Vinopal, R., 139,145,259 Vinuela, E., 163,263 Voelz,H., 24, 76,179,180,265 Voelz,U., 179,265 Von Bartha, R., 205, 215, 224, 238, 248, 250,263 Vosjau, J. H., 100,132

w Wainio, W. W., 103,132 Wainwright, T., 109,132 Wakerley,D.S.,89,120,126,129 Wakil, S. J.,231,265 Walker,D.A.,48,73 Walker,G. J., 143,145,158,160,267 Walker, R. T., 25,48,80 Wallach, D. F. H., 36,55,80 Walton,G.M., 163,257

27 8

AUTHOR INDEX

Wang, W. S., 212,265 Ward,A.C.,214,265 Ward, J. R., 70,73 Ware,D.A.,87,110,132 Watenpaugh, K. D., 109,132 Watts, D. J., 143,257 Weber,M. M., 47, SO, 109,132 Weeks,G., 143,168,169,269,265 Wei, S. H., 145,165,264 Weihull, C., 70: 80 Weil, J.H., 186,264 Weill, G., 209,258 Weimberg, R., 186,265 Woinbaum, G., 31,80 Weitzman,P. J.D., 245,265 Welch, F. V., 13,72 Whitcomb, R. F., 3,8,74 Wiame, J.M., 180,185,265 Widra,A., 179,265 Wilke, C. R., 84,129 Wilkins, M. H. F., 48,76 Wilkinson, J. F., 136, 137, 140, 142, 161, 180, 183, 192, 205, 206, 207, 209, 212, 213, 214, 215, 221, 224, 228, 238, 239, 248,249,259,261,264,266 Williams,D. J.A., 83,129 Williams, M. H., 28,80 Williamson, D. H., 205, 206, 207, 209, 212, 213,214,241,242,257,266 Williamson,D.L.,3,4,80 Wilson,L. G., 109,126 Wilson,P. W., 85,94,130,131,251,262 Winder, F. G., 180, 188,193,195,266 Whittler, R. G., 6, 74 Wolanski, B., 7,80 Wolfe,R.S.,87,117,132 Wolin,M. J.,33,74,95,133 Wong,P., 243,266

Wong,P.P., 205,239,243,266 Wood,L.B., 122,126 Woody,R. W., 103,127 Woolfolk,C.A., 112,132 Wright,B. E., 145,168,263,266 Wyld,S.G.,21,75 Wyss, O., 252,264

Y Yagi,T., 90,95,101,103,106,132,133 Yagoda, A., 2,73 Yamashiroya, H. M., 2,74 Yamataka,A., 192,193,266 Yanagihara, R., 95,96,128 Yarborough, H. F., 92,127 Yarrison, G., 68,SO Yates, M. G., 59,93,94,132 Yoshida, A., 180, 183, 188, 192, 262,266' Yoshimoto,A., 109,133 Young, D. W., 68,SO Yu, B. H., 2,73 Yu, C. I.,239,241,262 Yu, L., 33,74,95,133

Z Zahler, P. H., 36,55,80 Zahn,R., 14,19,75 Zaitseva,G. N., 180,193,266 Zevenhuizen, L. P. T. M., 143, 144, 145, 205,262,266 Zink,M. W., 163,263 Zobell, C. E., 93,131 Zucker-Franklin,D., 9,SO

SUBJECT INDEX A Accumulation, of poly-/3-hydroxybutyrate in batch cultures, 215 of polyphosphates by micro-organisms, 183 Acetate production by sulphate-reducing bacteria, 88 Acetate as a substrate for synthesis of poly/3-hydroxybutyrate, 217 Acetoacetate :succinyl-CoA CoA-transferase in bacteria, 243 Acetoacetyl-CoA reductases in bacteria, 229 Acetokinase of sulphate-reducing bacteria, 89 Acetone, effect of on granules of poly-/3hydroxybutyrate, 212 effect of on mycoplasma membranes, 33 Acetyl phosphate production by sulphatereducing bacteria, 89 Acholeplasma sp.. osmotic lysis of, 28 spp., size of genome in, 14 A . aranthum, base composition of DNA of, 15 A . granularum, base composition of DNA of, 15 A . laidlawii, base composition of DNA of, 15 ecology of, 2 fatty-acid composition of, 44 fractionation of solubilized proteins from membranes of, 34 freeze-etched replicas of membranes of, 61 lipid of composition of membranes of, 38 membrane-bound enzymes of, 32 membrane composition of, 30 membrane synthesis in, 56 potassium accumulation by, 66 transfer RNA in, 24 transformylase of, 24 uptake of cholesterol by, 47 viruses of, 19 Acidophilic mycoplasmas, 2 Actinomycetes, poly-P-hydroxybutyratc accumulation in, 205 polyphosphate glucokinase in, 196 2 79

Activation of ADP glucose pyrophosphorylase from Salmonella typhimuriurn, 154 Activators of glycogen synthesis, nature of effect, 153 Active transport in mycoplasmas, 66 Activities, enzymic, localization of in mycoplasmas, 31 Acyl carrier protein in Azotobacter beijerinckii, 230 Acylated sugar residues in lipids in mycoplasma membranes, 40 Adenine nucleotide deaminase of sulphatereducing bacteria, 94 Adenosine diphosphate-glucose, pyrophosphorylase in Arthrobacter viscosus 156 pyrophosphorylase in bacteria, 144 pyrophosphorylases of bacteria, kinetic parameters of, 155 Adenosine triphosphatase, location of in mycoplasmas, 31 in mycoplasma membranes, 66 Adenosine triphosphate, effect of on synthesis of ADP-glucose by extracts of Salmonella typhimurium, 152 Adenosine triphosphate sulphurylase in sulphate-reducing bacteria, 110 Adenylate energy charge and energy storage in microbes, 138 Adenylyl sulphate reductase in sulphatereducing bacteria, 110 Adsorption, of mycoplasmas t o cell surfaces 12 of mycoplasma viruses, 21 Aerated lakes, occurrence of sulphatereducing bacteria in, 118 Aerobacter aerogenes, ability of to accumulate more than one energy reserve, 136 accumulation of glycogen by, 143 degradation of accumulated polysaccharides by, 141 effect of ammonia limitation on, 142 effect of potassium ions on accumulation of glycogen by, 141 glycogen synthesis in, 150 mutants defective in biosynthesis of polyphosphate, 139 mutants of deficient in polyphosphate metabolism, 198

280

SUBJECT INDEX

Aerobacter aerogenes--continued polyphosphatases in, 196 polyphosphate in, 180 polyphosphate accumulation by, 183 polyphosphate cycle of, 200 polyphosphate kinase in, 193 pyrophosphorylases of, 145 A . cloacae, pyrophosphorylases of, 145 A. wuriubilis, polyphosphate in, 180 Agnricus bisporus, polyphosphate glucokinase in, 196 Agglutination of Mycoplasma pneumoniae, 54 Agrobacterium tumefaciens, accumulation of glycogen by, 143 glycogen synthcsis in, 157 pyrophosphorylases of, 145 Alanine dehydrogenase of sulphate-reducing bacteria, 94 Albumin, incorporation of into mycoplasma membranes, 63 Algal blooms, and sulphate-reducing bacteria, 124 Alkaline phosphatase of Aerobacter aerogenes, 198 Amino-acid, analyses of mycoplasma membrane proteins, 36 composition of cytochrome c553 of sulphate-reducing bacteria, 106 composition of ferredoxins of sulphatereducing bacteria, 107 requirements of mycoplasmas, 68 sequences of proteins in sulphatereducing bacteria, 100 Aminoacylphosphatidylglycerol synthetase, location of in mycoplasmas, 32 Ammonia limitation, effect of on glycogen synthesis by yeast, 163 Anabacna wariabilis, polyphosphate metabolism in, 189 Anaerobic nature of sulphate-reducing bacteria, 82 Andrenergic control of glycogen synthesis in Tetruhymena pyriformis, 170 Animal mycoplasmas, 3 Animals, effect of sulphate-reducing bacteria on, 124 mycoplasmas in, 2 Anodic attack by bacteria, and corrosion, 120 Antibiotics against mycoplasma diseases, 3 Antisera, and mycoplasma membranes, 63 Apoflavodoxin of sulphate-reducing hacteria, 109 n-Arabinitol 1,5.diphosphate as an activator of ADP-glucose pyrophosphorylase in bacteria, 154 Arginine deiminase in mycoplasmas, 71

Arginine dihydrolase pathway in mycoplasmas, 72 Arginine as an energy source in mycoplasmas, 71 Arthrobacter viscosus, accumulation of glycogen by, 143 glycogen synthesis in, 156 pyrophosphorylases of, 145 Aspergillus niger, polyphosphatc kinase in, 193 polyphosphate in spore germination in, 203 Assimilatory sulphate reduction by bacteria, 109 Authentication of nitrogen-fixing ability of sulphate-reducing bacteria, 9 4 Azotobucter spp., accumulation of poly-phydroxybutyrate in, 205 A. agilis, polyphosphatc in, 180 A . beijerinckia, accumulation of poly-phydroxybutyrate in continuous cultures of, 224 biosynthesis of poly-P-hydroxybutyrate in, 230 cyclic scheme for metabolism of poly-flhydroxybutyrate in, 246 degradation of poly-p-hydroxybutyrate by, 244 p-ketothiolase of, 231 A . vinelandii, polyphosphate in, 180 polyphosphate glucokinase in, 196 polyphosphate kinase in, 193 Azotobacteriaceae, role of poly-p-hydroxybutyrate in, 252

B Bacillus spp., accumulation o f poly-phydroxybutyrate in, 205 B. cereus, accumulation of glycogen by, 143 B. megaterium, ability of to accumulate more than one energy reserve, 136 accumulation of glycogen by, 143 accumulation of poly-p-hydroxybutyrate in continuous cultures of, 221 effect of substrate limitation in on accumulation of energy reserves, 140 poly-j-hydroxybutyrate in, 203 poly-p-hydroxybutyrate depolymerase for, 239 poly-8-hydroxybutyrate granules in, 210 polyphosphate metabolism in, 192 Bacteria, sulphate-reducing, physiology of, 81 Bacterial glucans, synthesis of, 144 Bacterial 3-hydroxybutyrate dchydrogen ases, properties of, 242 ~

SUBJECT INDEX

Base composition of DNA in sulphatereducing bacteria, 85 Base composition of mycoplasma DNA, 15, 16 Beijerinckia spp., accumulation of poly-13hydroxybutyrate in, 205 Bilayer, lipid, evidence for in mycoplasmas, 49 Binary fission in mycoplasmas, 12 Biochemical activities of mycoplasmas, 2 Biosynthesis, of glycogen-like reserves by microbes, 144 of poly-13-hydroxybutyrate, 228 Binding of magnesium to mycoplasma membranes, 59 Bisulphite reduction by extracts of sulphate-reducing bacteria, 112 Blackening of leather, and sulphatereducing bacteria, 125 Blustocladiella ernersonnii, glycogen accumulation by, 143 Blastocladiella ernersonii, glycogen synthesis in, 167 pyrophosphorylases of, 145 Blebs in mycoplasmas, 9 Branching in microbial glycogens, 144 Brij, effect of on mycoplasma membranes, 33

C Calcium, effect of on reconstitution of mycoplasma membranes, 58 Calorimeter scans of lipids in mycoplasma membranes, 51 Capacitance of mycoplasmamembranes, 49 Capacity for thermophily among sulphatereducing bacteria, 86 Carbamoyl phosphokinase in mycoplasmas, 71 Characters used to classify sulphatereducing bacteria, 86 Carbohydrate in mycoplasma membranes, 30 Carbon dioxide fixation in snlphatereducing bacteria, 91 Carbon metabolism of sulphate-rcducing bacteria, 88 Carbon monoxide-binding pigment from Desulfotornuculurn nigr'ijkans, 114 Carotenoids in mycoplasma membranes, 46 Catabolism of polysaccharide in Streptococcus mitis, 160 Catalase, activity of sulphate-reducing bacteria, 100 in mycoplasmas, 71 Cell membrane of mycoplasmas, 28

28 1

Cell surfaces, adsorption of mycoplasmas to 12 Cell yield of mycoplasmas a3 affected by fatty-acid supplement, 44 Centrifugation, effect of on ribosomal helices in mycoplasmas, 24 Cetyltrimethylammonium bromide, effect of on mycoplasma membranes, 33 Character, primitive, of sulphate-reducing bacteria, 116 Characterization of membrane enzymes from mycoplasmas, 35 Chemical composition of mycoplasma membranes, 30 Chemical properties of poly-,5-hydroxybutyrate, 206 Chemical structure of polyphosphates in micro-organisms, 179 Chemical synthesis of poly-,5-hydroxybutyrate, 209 Chemistry of sulphate-reducing bacteria, 100 Chemostat cultures of yeast, glycogen synthesis in, 166 Chemostats, use of in studies on accumulation of poly-,5-hydroxybutyrate by bacteria, 221 Chitin-decomposing bacteria, 119 Chloramphenicol, effect of on mycoplasmas, 28 Chloride, effect of on glycogen synthesis by yeast, 163 Cholesterol, as a mycoplasma growth factor, 67 in mycoplasma membranes, 46 role of in mycoplasma membranes, 47 requirement as a diagnostic factor in mycoplasma taxonomy, 46 requirement for in mycoplasmas, 37 as a sugar carrier in mycoplasmas, 66 Cholesteryl esters, absence of from mycoplasma membranes, 46 Cholesterylglucoside in mycoplasmas, 41 Chromate-resistant sulphate-reducing bacteria, 84 Chromatiurn spp., accumulation of poly-8hydroxybutyrate in, 205 Chrornobacteriurn spp., accumulation of poly-13-hydroxybutyrate in, 205 Circular dichroism spectra of mycoplasma membranes, 55 Circularity of mycoplasma genomes, 14 Cistrons, number of in mycoplasma genome, 15 Citrate synthase, role of in regulation of metabolism of poly-,5-hydroxybutyrate, 248 of sulphate-reducing bacteria 91

283

SUBJECT INDEX

Citrobacter freundii, pyrophosphorylases of, 145 Citrus crops, loss of caused by activities of sulphate-reducing bacteria, 124 Claviceps paspali, polyphosphate glucokinase in, 196 Chlorella sp., diphosphate in, 181 Chlorella sp., polyphosphate in, 180 C. vulgaris, polyphosphate glucokinase in, 196 Chlorobium thiosulfatophilum, polyphosphate in, 180 polyphosphate kinase in, 193 Chlorogloea spp., accumulation of poly-flhydroxybutyrate in, 205 Classification of sulphate-reducing bacteria, 84 Clostridia, primitive nature of, 116 Clostridium kluyveri, thiophorase in, 243 C. pasteurianum, accumulation of glycogen by, 143 glycogen degradation in, 172 glycogen synthesis in, 161 granulose synthesis in, 141 pyrophorylases, 145 Cooling towers, and sulphate-reducing bacteria, 121 Co-ordination, of macromolecular synthesis in mycoplasmas, 26 of membrane proteins and lipids in mycoplasma, 56 Composition, of mycoplasma membranes, 30 of mycoplasma ribosomes, 22 Computer analysis of circular dichroism spectra of mycoplasma membranes, 55 Conformation, of membrane proteins in mycoplasmas, 53 of proteins in mycoplasma membranes, 65 Conformational analysis of poly-flhydroxybutyrate, 208 Conformational changes in cytochromes c3 of sulphatc-reducing bacteria, 105 Content of glycogen in Dictyostelium discoideurn, 169 Control, of glycogen metabolism in microorganisms, 177 of lipid composition of mycoplasma membranes, 37 Control processes in sulphate-reducing bacteria, 87 Copper, toxicity of to Desulfovibrio, 123 Corynebacteria, polyphosphate metabolism in, 187 Corynebacterium diphtheriae, polyphosphate in, 180 polyphosphate kinase in, 193

C. freundii, kinetic parameters of ADPglucose pyrophosphorylase from, 155 C. xerosis, polyphosphate kinase in, 193 polyphosphate metabolism in, 187 polyphosphate in, 180 polyphosphate synthesis in, 195 Corrosion of metals, and sulphate-reducing bactcria, 119 Criteria for energy-storage function in microbes, 137 Cultivation of sulphate-reducing bacteria, 83 Cyclic-AMP, activation of glycogen phosphorylase in A'eurospora cra,wa by, 173 and glycogen synthesis in Dictyostelium discoideurn, 168 a role for in glycogen biosynthesis, 148 Cyclic condensed phosphates in microorganisms, 180 Cyclic scheme for metabolism of poly-phydroxybutyrate in bacteria, 246 Cyclic sulphite reduction by bacteria, 113 Cytochrome c, binding of to mycoplasma membranes, 52 cc3 of Desulfovibrio gigas, 106 c553 of sulphate-reducingbacteria, 105 Cytochromcs, location of in mycoplasmas, 32 b-type, in sulphate-reducing bacteria, 107 of sulphate-reducing bacteria, importance of in classification, 85 in sulphate-reducing bacteria, 97 in mycoplasmas, 70 c3 of sulphate-reducingbacteria, 101 Cytoplasmic division in mycoplasmas, 12 Cysteine content of membrane proteins in mycoplasmas, 36 Cysteine residues, high content of in cytochromes c3 of sulphate-reducing bacteria. 103

D Dark repair of DNA damage in mycoplasmas, 17 Death, of humans, and snlphate-reducing bacteria, 124 thymine-less, in mycoplasmas, 28 Decarboxylation of malate by sulphatereducing bacteria, 89 Defatted serum as a growth medium for mycoplasmas, 67 Defined media for growth of mycoplasmas, 67 Degradation, of accumulated polysaccharides by microbes, 141

283

SUBJECT INDEX

Degradation-continued of glycogen in Dictyostelium discoideum 169 of glycogen by micro-organisms, 170 of poly-p-hydroxybutyrate, enzymology of, 236 of polyphosphate, 194 Dental caries, and sulphate-reducing bacteria, 125 3-Deoxy-2-oxogluconate 6-phosphate as an activator of ADP-glucose pyrophosphorylase in bacteria, 154 Deoxyribonuclease, location of in mycoplasmas, 32 production of extracellular by, 69 Deoxyribonucleic acid, base composition of in mycoplasmas, 16 of mycoplasmas, 19 Depolarization by bacteria, 119 Depolymerase for poly-/?-hydroxybutyrate, 239 Deposits, oil, importance of sulphatereducing bacteria in, 121 Dcrxiu spp., accumulation of poly-phydroxybutyrate in, 205 Desulfotomaculum, problem of estimating, 83 D. nigrijcans, ferredoxin of, 107 properties of sulphite rcductase from, 115 pyruvate metabolism by, 88 Desulfovibriospecies, amino-acid sequences of cytochromes c3 of, 101 sp., estimation of, 84 toxicity of copper to, 123 D . nfricnnus, description of, 85 D.desulfuricans, pathways of mixotrophy in, 92 pyrophosphatase of, 87 U . gtgas, cytochrome cc3 of, 106 properties of sulphite reductase froin, 115 rubredoxin from, 108 D. vulgaris, cytochrome c553 of, 106 menaquinone of, 109 properties of sulphite reductase from, 115 sulphitc reductases of, 114 Desulfoviridin, ability of sulphate-rcducing bacteria to form, 86 in Desulfovibrio gigm, 1 13 Desulphurization of petroleum hydrocarbons, role of sulphate-reducing bacteria in, 122 Detergents, effect of on mycoplasma membranes, 33 Determination of poly-/3-hydroxybutyrate, 213

Dextrin phosphorylase in Escherichia coli, 172 Diameter of mycoplasmas, 6 Dichloroisoprenaline, effect of on synthesis of glycogen synthetase in Tetruhymenu pyrqormis, 170 Dictyostelium discoideum, glycogen accumulation by, 143 glycogen synthesis in, 168 pyrophosphorylases of, 145 Differential scanning calorimetry, and mycoplasma membranes, 50 Differentiation in Dictyosteliumdiscoideum, role of glycogen synthesis in, 169 Digitonin, lysis of mycoplasmas by, 29, 47 Dilution rate, in a chemostat, effect of on glycogen synthesis by yeast, 166 effect of on accumulation of poly-phydroxybutyrate in continuous cultures of Azotobacter beijerinckia, 224 Dimer hydrolase for poly-/?-hydroxybutyrate, 239 Dirncric 3-hydroxybutyrate, production of by pseudomonads, 236 Diphosphatidylglycerol in mycoplasma membranes, 37 Diphosphoglycerate-polyphosphate phosphotransferase in micro-organisms, 193 Disaggregation of the mycoplasma membrane, 57 Diseases in plants, and mycoplasmas, 3 Dissimilatory sulphate reduction by bacteria, 109 Distribution of sulphate-rcducingbacteria, 82 Disulphide bridges, absence of from membrane proteins in mycoplasmas, 36 Disulphur dioxide, production of by bacteria, 116 Dithionite, ability of sulphate-reducing bacteria to metabolize, 95 reduction by bacteria, 112 Divalent cations, requirement of for reconstitution of mycoplasma membranes, 58 Divalent ions, effect of in reconstitution of mycoplasma membranes, 58 Drosophila, and mycoplasmas, 3

E Ecology of mycoplasmas, 2 of sulphate-reducing bacteria, 82, 117 Economic activities of sulphate-reducing bacteria, 119

284

SUBJECT INDEX

Electron carriers, non-haem iron, in sulphate-reducing bacteria, 107 in sulphate-reducing bacteria, 97 Electron micrographs of thin sections through mycoplasmas, 4 Electron microscopy of poly-,!I-hydroxybutyrate granules, 212 Electron paramagnetic resonance spectra, of cytochromes c3 of sulphate-reducing bacteria, 103 of derived membranes of Acholeplasrna laidlawii, 53 Electron paramagnetic resonance of spinlabelled fatty-acid residucs in mycoplasma lipids, 52 Electron transport, from 3-hydroxybutyrate dehydrogenase from Rhizobium japonicum, 243 in mycoplasmas, 70 of sulphate-reducing bacteria, 97 Electrophoresis, of membrane proteins from mycoplasma membranes, 36, 57 Electrophoretic properties of cytochromes c3 of sulphate-reducing bacteria, 103 Elemental sulphur, production of by bacteria, 116 Elongation of fatty acids by mycoplasmas, 43 Encystment, of Azotobacter vinelandii, and accumulation of poly-/3-hydroxybutyrate, 218 of bacteria, role of poly-/3-hydroxybutyrate in, 252 Endogenous respiration, and poly-/3-hydroxybutyrate, 249 Energetic state of the microbial cell, assessment of, 138 Energy, carrier, polyphosphate as in microorganisms, 178 charge, role of in regulating glycogen synthesis in microbes, 153 and synthesis of glycogen phosphorylase in NeuTospora crassa, 174 Energy, of maintenance in microbes, 136 reserve function of polyphosphatcs, 201 reserve polymers, role of in microbes, 135 sources in mycoplasmas, 71 storage and adenylate energy charge in microbes, 138 storage compounds in microbes, 137 function, criteria for in microbes, 137 yielding pathways in mycoplasmas, 70 Enteric organisms, glycogen synthesis in, 146 Enterobacteriaceae, glycogen synthesis in, 146 Entner-Doudoroff pathway in glycogensynthesizing bacteria, 157

Enzymology of poly-P-hydrohydroxybutyrate biosynthesis, 228 Enzymes associated with granules of polyp-hydroxybutyrate, 2 13 Enzymic activities in mycoplasmas, localization of, 31 Enzymology, of polyphosphate metabolism in micro-organisms, 192 of polyphosphate utilization in yeast, 186 Episomes inmycoplasmas, 19 Escherichia aurescens, pyrophosphorylasos of, 145 E. coli, accumulation of glycogen by, 143 degradation of accumulated polysaccharides by, 141 degradation of glycogen in, 17 1 diphosphoglycerate-polyphosphatephosphotransferase in, 193 energy charge of, 138 glycogen-deficient mutants of, 146 glycogen synthesis in, 146 glycogen synthetase in, 149 mutants of defective in glycogen metabolism, 139 polyphosphate in, I80 polyphosphate kinase in, 193 pyrophosphorylases of, 145 Esterase activity of poly-/3-hydroxybutyrate, 239 Estimation of sulphate-reducing bacteria, 83 Ethane oxidation by sulphate-reducing bacteria, 92 Ether, sensitivity of mycoplasnia viruses to, 21 Eugbna gracilis, polyphosphate in, 180 Eukaryotes, degradation of glycogen in, 173 glycogen synthesis in, 163 Evolutionary significance of microbial sulphate reduction, 116 Exacting anaerobic nature of sulphatereducing bacteria, 82 Extracellular depolymerases produced by pseudomonads for poly-@-hydroxybutyrate, 237 Extracellular poly-/3-hydroxybutyrate, degradation of, 236 Exacting nature of mycoplasmas, 2

F Facilitated diffusion of sugars in mycoplasmas, 66 Fatty-acid synthases of mycoplasmas, 42 Fatty acids, of lipids in mycoplasmas, 42 requirement €or in mycoplasmas, 37

285

SUBJECT INDEX

Fermentation of sugars by mycoplasmas, 70 Ferredoxin of sulphate-reducing bacteria, 89,107 Ferrobacillus spp., accumulation of poly-13hydroxybutyrate in, 205 Fibrils, poly-P-hydroxybutyrate, mechanism of extension of, 234 Filaments produced by mycoplasmas, 7 Filipin, lysis of mycoplasmas by, 47 Filtration of mycoplasmas, 6 Fish, effect of sulphate-reducing bacteria on, 124 Fixation, of carbon dioxide by sulphatereducing bacteria, 91 of nitrogen by sulphate-reducing bacteria, 93 Flagella, ability of sulphate-reducing bacteria to form, 86 Flavodoxin, amino-acid composition of, from sulphate-reducing bacteria, 108 from Desulfovibrio gigas, 108 of sulphate-reducing bacteria, 98 Flexibility of mycoplasmas, 6 Fluidity, in mycoplasma membranes, 46,48 Fluorescence measurements on mycoplasma membranes, 62 Formats, dehydrogenase of sulphatereducing bacteria, 90 ferricytochrome cSs3 of sulphate-reducing bacteria, 90 oxidation by sulphate-reducing bacteria, 90 Formation, of hydrocarbons by sulphatereducing bacteria, 92 of minerals, importance of sulphate reducing bacteriain, 122 of vesicles in mycoplasma membrane preparations, 60 Fraction&tion, of membrane proteins in mycoplasmas, 33 of poly-8-hydroxybutyrategranules, 234 Fracture faces of membranes in mycoplasmas, 60 Fragility of mycoplasmas, 6 Free energy of hydrolysis of microbial polyphosphates, 182 Freedom of motion of fatty-acyl chains in mycoplasma lipids, 52 Freeze etched replicase of mycoplasma membranes, 61 Fructose l,f%diphosphate, as an activator of ADP-glucose phosphorylase in bacteria, 154 as an activator of ADP-glucose pyrophosphorylase in Escherichia coli, 146 Fuel cells, and sulphate-reducing bacteria, 125

Fumarate, dismutation by sulphate-reducing bacteria, 89 hydratase of sulphate-reducing bacteria, 89 Function, of poly-8-hydroxybutyrate in micro-organisms, 249 of polyphosphates as a phosphorus reserve, 202

G Galactosamine in mycoplasma membranes, 31 Genetics ofmycoplasmas, 14,17 Genome, ofmycoplasmas, 14 mycoplesma, base composition of, 15 nature of, 14 Geological strata, sulphate reduction in, 116 Glucan synthesis in prokaryotes and eukaryotes, 142 Glucans, biosynthesis of in microbes, 138 Glucose, 1,6-diphosphate as an activator of ADP-glucose pyrophosphorylase in bacteria, 154 effect of on accumulation of poly-fihydroxybutyrate by Bacillus megaterium, 2 15 metabolism in Desulfotomculurn nigrijL c a m , 93 Glucosamine in mycoplasma membranes, 31 15-Glucosidase,location of in mycoplasmas, 32 Glutamate dehydrogenase in mycoplasmas, 68 Glutaraldehyde, effect of on mycoplasmas, 6 Glyceraldehyde 3-phosphate, as an activator of ADP-glucose pyrophosphorylase in bacteria, 154 Glycerol deprivation, effect of on mycoplasmas, 26 Glycerol 1,3-diphosphate as an activator of ADP-glucose pyrophosphorylase in bacteria, 154 Glycerol labelling of mycoplasma membranes, 56 Glycogen accumulation, in Arthrobacter viscosus, 156 by Streptococcus rnitis, 158 Glycogen-deficient mutant of Escherichia coli, 148 of Saccharomyces cerevisiae, glucose transport in, 167 Glycogen degradation by micro-organisms, 170

286

SUBJECT INDEX

Glycogen-likercserves in microbcs, 140 Glycogen, as a microbial energy reserve, 140 phosphorylase of Saccharomyces cerevisiae, 174 in Tetrahymenapyrijormis,175 ofyeast, 166 phosphorylases in micro-organisms, 170 synthesis ofinmiorobes, 146 synthetase in Blastocludiella emersonii, 168 in Dictyostelium discoideum, 168 in Escherichia coli, 149 Glycolipids in mycoplasma membranes, 40 Glycoproteins, absence of from mycoplasmamembranes, 31 Glycosylated sterols in mycoplasmas, 41 Glycosyldiglycerides in mycoplasma membranes, 41 Glycosylation of cholesterol by Mycoplasma gallinarum, 46 Glycolysisin mycoplasmas, 70 Granule, poly-P-hydroxybutyrate, biosynthesisof, 233 Granules, in mycoplasmas, 8 occurrence of in micro-organisms, 179 of poly-p-hydroxybutyrate, nature of, 209 Granulose, biosynthesis in Clostridium pmteuriunum, 141,161 degradation in Clostridium pasteurianum, 172 Greening agent of citrus, and mycoplasmas, 3 Growth media for mycoplasma-like organisms, 3 Growth rate, and glycogen content, relationship between, 140 of mycoplasmas, as affected by fattyacid supplement, 44 Guanosine plus cytosine content of mycoplasma tRKA, 25

H Haems in cytochromes c3 of sulphatereducing bacteria, 101 Halophilic sulphate-reducing bacteria, 82 Heat, sensitivity of mycoplasma viruses to, 21 stability of glycogen phosphorylasc in Tetrahymena pyriformis, 175 Helices of ribosomes in mycoplasmas, 23 Hexosamines in mycoplasma membranes, 31

Hibitane resistancc of sulphate-reducing bacteria, 85 Homologies in ferredoxins froni sulphate reducing bacteria, 107 Host specificity of mycoplasmas, 2 Hydrocarbon oxidation by sulphate-reducing bacteria, 92 Hydrogen-ion concentration, effect of on mycoplasma ribosomal helices, 24 cffect of on synthesis of mycoplasma membranes, 56 Hydrogen, metabolism by sulphate-reducing bacteria, 94 oxidation by sulphate-reducing bacteria, 91 Hydrogenase, of Desulfovibrio desulfuricans, 95 role of enzyme in sulphate-reducing bacteria in metal corrosion, 119 of sulphate-reducing bacteria, 89 Hydrogenases of sulphate-reducing bacteria, properties of, 96 Hydrogenomonas H 16, mutants of defective in synthesis of storage polymers, 139 Hydrogenomonas spp., accumulation of poly-P-hydroxybutyratein, 205,229 polyphosphate as a reserve in, 203 H . eutropha, accumulation of poly-Phydroxybutyrate in continuous cultures of, 222 cyclic scheme for metabolism of poly-Phydroxybutyrate in, 246 P-ketothiolase of, 231 polyphosphate accumulation in, 180, 188 Hydrolysis of poly-/I-hydroxybutyrate, 238 Hydrophobicity of mycoplasma membrane proteins, 36 3-Hydroxybutyrate dehydrogenase in micro-organisms, 241 3-Hydroxybutyrate dehydrogenasc, bacterial, properties of, 242 3-Hydroxybutyrate, metabolism of, 241 3-Hydroxybutyric acid dimer hydrolase, 240 P-Hydroxybutyryl-CoA, biosynthesis of, 228 dehydratases in Rhodospirillum rubrum, 229 polymerase in bacteria, 233 3-Hydroxybutyratc, formation of from poly-P-hydroxy butyrate by Bacillus megaterium, 203 Hypochlorite, solubilization of poly-/Ihydroxybutyrate by, 212 Hyphomicrobium spp., accumulation of poly-/3-hydroxybutyrate in, 205

287

SUBJECT INDEX

I

L

Imidophosphate polymers in micro-organisms, 181 Immunogenicity of reconstituted mycoplasma membranes, 63 Immunological properties of cytochromes c3 of sulphate-reducing bacteria, 103 Incorporation of fatty acids into mycoplasmas, 43 Induction of enzyme synthesis in mycoplasmas, 18 Infrared spectra of sulphate-reducing bacteria, 85 Inhibition, of glycogen synthesis by ammonium ions in yeast, 165 of RNA synthesis in mycoplasmas, 24 of sulphate-reducing bacteria, 84 Inhibitors of protein synthesis in mycoplasmas, 27 Inorganic ions, transport of in mycoplasmas, 66 Insulation barrier in mycoplasma mernbranes, 50 Intracellular degradation of poly-p-hydroxybutyrate by pseudomonads, 238 Intracellular granules of poly-p-hydroxybutyrate in Azotobacter beijerinckia, 210 Intrinsic viscosity of poly-p-hydroxybutyrate, 207 Iron, requirement of sulphate-reducing bacteria for, 96 Iron phosphide, and metallic corrosion by bacteria, 120 Iron-sulphite agar medium for snlphatereducing bacteria, 84 Irradiation, effect of on polyphosphate accumulation by yeast, 185 Isoenzymes of 3-hydroxybutyrate dehydrogenase in micro-organisms, 241 Isolation of mycoplasma membranes, 28 Isoelectric points of cytochromes c3 of sulphate-reducing bacteria, 105

Lactate dehydrogenase of sulphate-reducing bacteria, 88 Lactate oxidation by sulphate-reducing bacteria, 88 Lakes, occurrence of sulphate-reducing bacteria in, 118 Lamellae in mycoplasma membranes, 59 Lampropaedia spp., accumulation of polyp-hydroxybutyrate in, 205 Latent period of mycoplasma viruses, 21 Leakiness of mycoplasmas, 29 Leather, blackening of, and sulphatereducing bacteria, 125 Lecithin in mycoplasma membranes, 39 Light scattering and mycoplasma membranes, 55 Linear condensed polyphosphates in microorganisms, 180 Lipase in mycoplasma membranes, 68 Lipid bilayer, evidence for in mycoplasmas, 49 Lipid content, of Escherichia coli, role of substrate limitation on, 140 of mycoplasma membranes, 30 Lipid granules in bacteria, nature of, 203 Lipid in granules of poly-p-hydroxybutyrate, 213 Lipid requirements of mycoplasmas, 67 Lipid synthesis, in mycoplasmas, 26 in Mycoplasmasp., 27 Lipids, as energy-storage compounds in microbes, 137 membrane, in mycoplasmas, 37 Lipolytic enzymes, effect of on mycoplasmamembranes, 62 Lipoprotein particles in mycoplasma mcmbranes, 64 Lipoprotein subunits of the mycoplasma membrane, 57 Liposomes of mycoplasma lipids, 45 Localization, of enzymic activities in mycoplasmas, 31 of membrane proteins in mycoplasmas, 53 Location of polyphosphate in Saccharomyces cereuisiae, 186 Locomotion of mycoplasmas, 13 Low G + C content of mycoplasma genome, 16 Lubrol, effect of on mycoplasma membranes, 33 Lysis, of mycoplasmas, 47 of mycoplasmas by digitonin, 29 osmotic, of mycoplasmas, 28 Lysophospholipase, location of in mycoplasmas, 32

K p-Ketoacetyl-CoA synthetase in Hydrogenomonas sp., 229 ,8-Ketothiolases of bacteria, 231 Key t o classification of sulphate-reducing bacteria, 86 Kinetic parameters of ADP-glucose pyrophosphorylases in bacteria, 155 Kinetics of cholesterol uptake by mycoplasmas, 47

288

SUBJECT INDEX

Lysozyme, binding of to the mycoplasma membrane, 52

M Macromolecular synthesis in mycoplasmas, co-ordination of, 26 Magnesium ions, binding of to mycoplasma membranes, 59 effect of in reconstitution of mycoplasma membranes, 58 Malate dismutation by sulphate-reducing bacteria, 89 Maltose, stimulatory effect of on dextrin phosphorylase in Escherichia coli, 172 Mapping of the mycoplasma genome, 17 Marine lagoons, occurrence of sulphatereducing bacteria in, 118 Marine sulphate-reducing bacteria, 119 Markers, genetic, in mycoplasmas, 18 Mechanical stress, and morphology of mycoplasmas, 7 Mechanism, of biosynthesis of chain fibrils of poly-/3-hydroxybutyrate, 234 of motility of mycoplasmas, 13 of transport in mycoplasmas, 65 Mechanism of membrane reconstitution inmycoplasmas, 64 Media for sulphate-reducing bacteria, 83 Melting point of poly-/3-hydroxybutyrate, 207 Membrane, attachment of mycoplasma genome to, 14 cell, of mycoplasmas, 28 lipids in mycoplasmas, 37 as location of enzyme activities in mycoplasmas, 3 1 proteins, characterization of in mycoplasmas, 31,35,36 fractionation of in mycoplasmas, 33 release offrom mycoplasmas, 31 solubilization of in mycoplasmas, 32 round poly-/3-hydroxybutyrate granules, 212 Membranes, cellular, of mycoplasmas, 28 Menaquinone of sulphate-reducing bacteria, 109 Metabisulphite reduction by extracts of sulphate-reducing bacteria, 112 Metabolism, of 3-hydroxybutyrate,241 of mycoplasmas, 67 of nitrogenous compounds by sulphatcreducing bacteria, 93 of organic compounds by sulphatereducing bacteria, 88 of poly-j3-hydroxybntyrate,214

Metabolism-continued of sulphur-containing compounds by sulphate-reducing bacteria, 109 Metabolites as activators of ADP-glucose phosphorylase in bacteria, 154 Metachromatic granules in micro-organisms, 179 Metal sulphide ores, and sulphate-reducing bacteria, 123 Metal tolerance ofbacteria, 119 Metals, corrosion of, 119 Metaphosphates in micro-organisrns, 180 Methane formation by sulphate-reducing bacteria, 92 Microbial phosphagen, 201 Microbial polyglucans, structure of, 142 Microbial survival, role of polyphosphates in, 201 Microbiological processes for producing elemental sulphur, 122 Micrococcus spp., accumulation of poly-phydroxybutyrate in, 205 M . ZactyZiticus, metabolism of metabisulphite by, 112 M . lysodeikticus, polyphosphate in, 180 polyphosphate glucokinase in, 196 polyphosphate metabolism in, 191 Mineral carbonates, formation of, and sulphate-reducing bacteria, 123 Minerals, formation of, importance of sulphate-reducing bacteria in, 122 Mine waters, and sulphate-reducing bacteria, 125 Minimal reproductive unit of mycoplasmas, 6 Mini-methane system of Desulfovibrio sp., 117 Mixotrophy in sulphate-reducing bacteria, 91 Mobility of hydrocarbon chains of phospholipids in mycoplasma membranes, 48 Mode of reproduction of mycoplasmas, 6, 12 Modified nucleosides in mycoplasma RNA, 25 Molecular hydrogen, ability of sulphatereducing bacteria t o metabolize, 94 Molecular organization in the mycoplasma membrane, 57 Molecular properties of mycoplasma membrane proteins, 36 Molecular weight of mycoplasma membrane proteins, 36 of poly-/3-hydroxybutyrate,206 Molybdenum, presence of in hydrogenase of sulphate-reducing bacteria, 96 Morphology, of mycoplasmas, 6,7 of mycoplasmas deprived of glycerol, 27

289

SUBJECT INDEX

Motility of mycoplasmas, 13 Muds, occurrence of sulphate-reducing bacteriain, 118 Multiplicity of energy reserves accumulated by microbes, 136 Multi-step assembly of mycoplasma membranes, 65 Mutagens, effects of on mycoplasmas, 17 Mutants, of Aerobacter aerogenes deficient in polyphosphate metabolism, 198 defective in poly-/3-hydroxybutyrate accumulation, 235 in storage polymer synthesis in microbes, 139 of mycoplasmas, 17 Mycobacteria, metabolism of polyphosphate in, 187 Mycobacterium chelonei, polyphosphate in, 180 M . phlei, ability of to accumulate two different energyreserves, 136 accumulation of glycogen by, 143 polyphosphate in, 180 polyphosphate glucokinase in, 195 M . smegmuiis, accumulation ofglycogen by, 143 polyphosphate in, 180 polyphosphate kinase in, 193 polyphosphate synthesis in, 195 M . thamnopheos, polyphosphate in, 180 M . tuberculosis, accumulation of glycogen by, 143 Mycoplama arginini, ecology of, 2 M . arthritidis, base composition of DNA of, 15 viruses in, 19 M . bowigenitalium, membrane composition of, 30 M . canis, ecology of, 2 Mycoplasma DNA, base composition of, 16 Mycoplasma gallinarum, glycolipids in, 41 M . gallisepticum, base composition of DNA of, 15 blebs in, 9 morphology of, 7 ribosomal helices in, 23 ribosomes of, 22 Mycoplasma genetics, 17 Mycoplasma hominis, mode of reproduction of, 13 ribosomes of, 22 Mycoplasma-like organisms, 3 Mycoplama meleagridis, base composition of DNA of, 15 Mycoplasma membranes, capacitance of, 49 composition of, 30

Mycoplasma membranes-continued freeze-etched replicas of, 61 nitroxide probes in, 53 reconstitutionof, 5 7 , 5 8 red shift distortion and, 56 Mycoplama rnycoides, effect of magnesium ions of reconstitution of membranes of, 58 membrane composition of, 30 ultrastructure of, 8 M . neurolyticum, base composition of DNA of, 15 M . pneumoniae, agglutination of, 5 4 base composition of DNA of, 15 terminal structure of, 9 M . pulmonis, modified bases in RNA of, 25 motility of, 13 Mycoplasma, transport mechanisms, 66 viruses, 18 Mycoplasmas, biochemical activities of, 2 ecology of, 2 genetics of, 14 genome of, 14 leakiness of, 29 nietabolism of, 67 motility of, 13 nutrition of, 67 physiology of, 1 resistance of to virus infection, 22 striated rods of, 12 ultrastructure of, 8 Myxococcus xanthus, polyphosphate in, 179,180

N Nature of mycoplasma genome, 14 Neurospora crasm, degradation of glycogenin, 173 diphosphoglycerate-polyphosphate phosphotransferase in, 193 glycogen accumulation by, 143 glycogen phosphorylase from, 173 polyphosphate glucokinase in, 196 pyrophosphorylases of, 145 N-Formylmcthionyl-RNA in mycoplasmas, 25 Nicotinic acid, adenine dinucleotide, reduced, as a n activator of ADPglucose phosphorylase, 153 as a mycoplasmagrowth factor, 69 Nitrogen content, eifect of in medium on glycogen accumulation by Streptococcus mitis, 159 Nitrogen fixation by sulphate-reducing bacteria, 87,93

2 90

SUBJECT INDEX

Nitrogen limitation, effect of on accumulation of poly-/3-hydroxybutyrate by Azotobacter beijerinckia, 228 Nitrogen metabolism of sulphate-reducing bacteria, 93 Nitrogen supply, effect of on accumulation of glycogen by microbes, 140 Nilrosomonas europeae, polyphosphate in, 180 polyphosphate metabolism in, 189 Nitroxide probes in mycoplasma membranes, 53 Nocardia spp., accumulation of poly-/3hydroxybutyrato in, 205 Nomenclature of sulphate-reducing bacteria, 85 Non-haem iron electron carriers insulphatereducing bacteria, 107 Non-magmatic sulphidc ores, and sulphate-reducing bacteria, 123 Non-specific reconstitution of mycoplasma membranes, 62 Nostoc muscarurn, glycogen accumulation by, 143 Nuclear magnetic relaxation measurements on mycoplasma membranes, 62 Nucleic acid, precursor requirements of mycoplasmas, 69 synthesis and polyphosphate synthesis inmycobacteria, 188 Nucleoside composition of mycoplasma transfer RXA 24 Nucleoside requirements of mycoplasmas, 69 Nucleosides, modified, in mycopIasma RNA, 25 Nucleotidase, location of in mycoplasmas, 32 Nucleotidc diphosphate pyrophorylases in microbes, 145 Nucleotide triphosphates in glycogen synthesis, 146 Numerical taxonomy of sulphate-reducing bacteria, 87 Nutrition of mycoplasmas, 67 Nutritional requirements of mycoplasmas, 67

0 0-Amino acyl esters of phosphatidylglycerol iu mycoplasma membranes, 39 Occurrence, of glycogen in microbes, 142 of poly-P-hydroxybutyrate in microorganisms, 204 of polyphosphates in micro-organisms, 179

Oil technology, importance of sulphatereducing bacteria in, 121 Optical techniques, use of in studies of mycoplasma membranes, 55 Ores, metal sulphide, and sulphate-reducing bacteria, 123 Organelles of mycoplasmas, 8 Organic matter, effect of on growth of sulphate-reducing bacteria, 84 Organization, of components of the mycoplasma membrane, 55 of lipid in mycoplasma membranes, 49 of protein in the mycoplasma membrane, 49 Orientation of lipids in mycoplasnia membranes, 48 Ornithine carbamoyltransferase in mycoplasmas, 71 Osmotic lysis of mycoplasmas, 28 Oxidation, of formate by sulphate-reducing bacteria, 90 of hydrocarbons by sulphatc reducing bacteria. 92 Oxidative pentoso cycle, role of NADP in governing, 153 Oxidative phosphorylation in mycoplasmas, 71 Oxidative phosphorylation in sulphatereducing bacteria, 99 Oxygen limitation, effect of on accumulation of poly-8-hydroxybutyrate, 246 effect of on accumulation of poIy-/3hydroxybutyrate in Azotohacter beijerinckiw, 225

P Packing of lipids in mycoplasma membranes, 45 Palmitate labelling of mycoplasma niembranes, 56 Pankhurst tube for cultivation of sulphatereducing bacteria, 83 Para-Nitrophenyl phosphatase, location of in mycoplasmas, 32 Pathway of microbial glycogen synthesis, 146 Penetration of mycoplasma viruses, 21 Penicillinase, incorporation of into mycoplasma membranes, 63 Penicillium chrysogenum, diphosphoglycerate-polyphosphate phosphotransferase in, 193 Peptidase, location of in mycoplasmas, 32 Peptidases of mycoplasma membranes, 30 Peptides, role of polyphosphate in probiotic synthes is of,179

SUBJECT INDEX

Permeability of mycoplasmas to glycerol, 45 Petroleum deposits, occurrence of sulphatereducing bacteria in, 119 Phosphagen hypothesis, 201 Phosphate starvation, effect of on accumulation of polyphosphate by Aerobacter acrogen.es, 183 effect of on Aerobucter aerogenes, 199 Phosphatidic acid in mycoplasma membranes, 37 Phosphatidylglucose in mycoplasma membranes, 38 Phosphatidylglycerol in mycoplasma membranes, 37 3’-Phosphoadenylyl sulphate in sulphatereducing bacteria, I09 Phosphoenolpyruvate, as an activator of ADP-glucose pyrophosphorylase in bacteria, 154 phosphotransferase mechanisms in mycoplasmas, 65 Phosphofructokinase, activation of by cyclic AMP, 149 Phosphoglucolipid synthetase, location of in mycoplasmas, 32 6-Phosphogluconate as an activator of ADP-glucose pyrophosphorylase in bacteria, 154 2-Phosphoglycerate as an activator of ADP-glucose phosphorylase in bacteria, 154 3-Phosphoglycerato as a n activator of ADP-glucose phosphorylase in bacteria, 154 Phosphoglycolipids in mycoplasma membranes, 38 Phospholipases of mycoplasma membranes 30 Phospholipids of mycoplasma membranes, 37 Phosphorus reserve, polyphosphates as, 202 Phosphorus storage, role of polyphosphates as, 189 Phosphorylation by sulphate-reducingbacteria, 97 Phosphosphingolipid in mycoplasma membranes, 39 Photoreactivation of ultraviolet damage in mycoplasmas, 17 Phycomyces blakesleunus, polyphosphate glucokinase in, 196 Phylogenotic tree, and sulphate-reducing bacteria, 100 P h y s a r u m polycephalum, polyphosphate in, 180 polyphosphate metabolism, 190

291

Physical properties of poly-/3-hydroxybutyrate, 206 Physiological functions of polyphosphates, 201 Physiology, of mycoplasmas, 1 of sulphate-reducingbacteria, 81 Plants, damage to caused by sulphatereducing bacteria, 123 mycoplasmas in, 3 Plasticity of mycoplasmas, 7 Polluted waters, and corrosion by sulphatereducing bacteria, 121 Polyglucan phosphorylases in Escherichia coli, 171 Polyglucan synthesis by microbes, 142 Poly-/3-hydroxybutyrate, biosynthesis in Azotobucter beijerinckiu, 230 chemical synthesis of, 209 content of Azotobacter spp., 219 depolymerase for, 239 as an energy reserve in micro-organisms, 203 enzymology of degradation of, 236 function of in micro-organisms, 249 metabolism of, 214 Poly-/3-hydroxybutyrate metabolism regulation of, 244 mutants deficient in accumulation of, 235 role of as a carbon and energy source, 249 synthetase in bacteria, 233 Polymerization of /3-hydroxybutyrate, 235 Polymers, energy-reserve, role of in microbes, 135 Polyphosphatases in micro-organisms, 196 Polyphosphate accumulation by microorganisms, conditions which favour, 183 Polyphosphate-adenosine monophosphate phosphotransferase in micro-organisms, 195 in Corynebacteriurn xerosis, 187 Polyphosphate, biosynthesis of, 192 cycle of Aerobaeter aerogenes, 200 cyclein yeast, 185 degradation of, 194 fructokinase in micro-organisms, 196 glucokinase activity in micro-organisms, 195 glucokinase in mycobacteria, 188 kinase from micro-organisms, 192,194 metabolism in micro-organisms, 192 metabolism, regulation of, 197 overplus, nature of, 185 in prebiotic evolution, 178 as a reserve material in micro-organisms, 178

292

SUBJECT INDEX

Polyphosphates, chemical structure of in micro-organisms, 179 as energy-storage compounds in microbes, 137 microbial, solubility of in trichloroacetic acid, 182 occurrence of in micro-organisms, 179 physiological function of, 201 Polysaccharides as energy-storage compounds inmicrobes, 137 Potassium ions, effect of on glycogen accumulation in Aerobacter aerogenes, 141 Precursor activation of glycogen synthesis, 157 Primates, mycoplasmas in, 2 Primitive character of sulphate-reducing bacteria, 97,116 Pristine nature of sulphate-reducing bacteria, 117 Products of the solubilization of the mycoplasmamembrane, 57 Prokaryotes, degradation of glycogen in, 171 glycogen synthesis by, 146 Prokaryotic nature of mycoplasma ribosomes, 22 Pronase digestion of mycoplasma membranes, 62 Properties, of bacterial 3 -hydroxybutyrate dehydrogenases, 242 of mycoplasma ribosomes, 22 of viruses of Acholeplasma laidlawii, 21 Propionibacterium shermanii, diphosphoglycerate-polyphosphate phosphotransferasein, 193 polyphosphate glucokinase in, 196 Protein composition of mycoplasma membranes, 30 Protein, membrane, digestion of in mycoplasmas, 52 synthesis inmycoplasmas, 22,26 Proteins, in granules of poly-/3-hydroxybutyrate, 213 membrane, co-ordination of with lipid synthesis, 56 membrane, properties of in mycoplasmas, 36 membrane, solubilization of in mycoplasmas, 32 in mycoplasma membranes, 31 transport, in mycoplasmas, 65 Proteolytic attack on mycoplasma membranes, 5 4 Proteolytic enzymes, effect of on mycoplasma membranes, 62 Protoplasts, yeast, distribution of phosphorus compounds in, 186

Pseudomonads, importance of poly-8hydroxybutyrate accumulation in taxonomy of, 204 utilization of extracellular poly-p-hydroxybutyrate by, 236 Pseudomonas spp., accumulation of poly-phydroxybutyrate in, 205 aeruginosa, inability of to accumulate energyreserves, 136 P8. Zemignei, poly-/3-hydroxybutyrate depolymerase of, 236 Ps. saccharophila, endogenous respiration of poly-/3-hydroxybutyrate in, 251 Psychrophilic sulphate-reducing bacteria, 82 Purification of membrane enzymes from mycoplasmas, 35 Pyridoxal, as a n activator of ADP-glucose pyrophosphorylase in bacteria, 154 5'-phosphate, as an activator of ADPglucose phosphorylase in bacteria, 154 phosphate in glycogen phosphorylase of Escherichia coli, 172 Pyrophosphatase, in sulphate-reducing bacteria, 87,110 Pyruvate, ability of sulphate-reducing bacteria t o use, 86 as a n activator of ADP-glucose pyro. phosphorylase in Arthrobacter viscosus, 157 phosphoroclastic reaction of sulphatereducing bacteria, 88

Q Quaternary ammonium compounds, effect of on sulphate-reducing bacteria, 84 Quinones in mycoplasmas, 71

R Rate of growth, and glycogen synthesis in yeast, 166 Re-aggregation of solubilized membrane components from mycoplasmas, 57 Reconstituted membranes of mycoplasmas, ultrastructure of, 59 Reconstitution, of mycoplasma membranes, 57,58 possible mechanism of in mycoplasmas, 65 Recycling sulphite pool in sulphatereducing bacteria, 112 Red-shift distortion and mycoplasmamembranes, 56

293

SUBJECT INDEX

Redox potential of culture, effect of on accumulation of poly-,&hydroxybutyrate by Azotobacter bcijerinckia, 225 Redox potentials of cytochromes cj of sulphate-reducingbacteria, 103 Regulation, of energy-reserve polymers in microbes, 135 of glycogen metabolism in microorganisms, 176 of glycogen synthesis in yeast, 164 of poly-/3-hydroxybutyrate metabolism, 244 of polyphosphate metabolism in microorganisms, 197 Removal of proteins from the mycoplasma membrane, 5 4 Repair of ultraviolet damage in the niycoplasma genome, 16 Replication of the mycoplasma genome, 14 Reproduction of mycoplasmas, 6 Reproductive unit, minimal, of mycoplasmas, 6 Reserpine, effect of on synthesis of glycogen synthetase in Tetrahymena pyriformis, 170 Respiratory chains in mycoplasmas, 71 Respiratory pathways in mycoplasmas, 70 Respiratory system, location of in mycoplasmas, 32 Rhodospirillum rubrum, pyrophosphorylases of, 145 Rhodopseudomonas spheroides, polyphosphate in, 180 Rhizobium spp., accumulation of poly-/3hydroxybutyrate in, 205 R. japonicum, electron transport from 3hydroxybutyrate dehydrogenase of, 243 Rhizosphere, activities of sulphate-reducing bacteriain, 124 Rhodanese activity in Desulfotomaculum nigri$cans, 1 13 Rhodopseudomonas spp., accumulation of poly-/3-hydroxybutyrate in, 205 R. capsulatus, accumulation of glycogen by, 143 glycogen synthesis in, 157 pyrophosphorylases of, 145 spheroides, polyphosphate metabolism in, 189 Rhodospirillum spp., accumulation of poly-/3-hydroxybutyrate in, 205 R. rubrum ability of to accumulate more than one energy reserve, 136 accumulation of glycogen by, 143 biosynthesis of poly-P-hydroxybutyrate in. 229 glycogen synthesis in, 157

R. rubrum-contimed poly-/3-hydroxybutyrate dimer hydrolasefrom. 240 polyphosphate glucokinase in, 196 Riboflavin as a mycoplasma growth factor, . 69 Ribonuclease, location of in mycoplasmas, 32 Ribonucleic acid, transfer, in mycoplasmas, 24 Ribose 5-phosphate as an activator of ADP-glucose pyrophosphorylase in bacteria, 154 Ribosomal helices in mycoplasmas, 23 Ribosomes of mycoplasmas, 22 Rice, damage to caused by sulphate-reducing bacteria, 123 Rivers, occurrence of sulphate-reducing bacteria in, 118 Role of cholesterol in mycoplasma membranes, 47 Role of energy-reserve polymers in microbes, 135 Rubredoxin, amino-acid composition of, from Desulfovibrio gigas, 108 in sulphate-reducingbacteria, 99 Ruminants, and sulphate-reducing bacteria, 125

s Saccharomyces cerevisiae, glycogen accumulation bv. 143 glycogendegraiition in, 174 glycogensynthetase from, 164 - . mutants of defective in glycogen metabolism, 140 polyphosphatase of, 197 pyrophosphorylases of, 145 polyphosphate kinase in, 193 polyphosphate in, 180 S. mellis, polyphosphate metabolism in, 186 Salmonella minnesota, polyphosphate in, 180 polyphosphate kinase in, 192 S. typhimurium, effect of ATP on synthesis of ADP-glucose by, 152 glycogen biosynthesis in, 150 kinetic parameters of ADP-glucose pyrophosphorylase from, 155 pyrophosphorylases of, 145 Salt-resistant sulphate-reducing bacteria, 119 Sarcina lutea, polyphosphate glucokinase in, 196

294

SUBJECT INDEX

Sckizoplasma, as a geiius of mycoplasmas, 13 Sedimentation cocfhieuts, of mycoplasma ribosomes, 22 Sensitivity of mycoplasmas to ultraviolet radiation, 16 Sera against mycoplasma membranes, 63 Serine, biosynthesis of by sulphate-reduciug bacteria, 94 Scrrcitia mnrcesccns, pyrophosphorylascs of, 145 Serum albumin, incorporation of into mycoplasma membranes, 63 Sewage, occurrence of sulphate-reducing bacteriain, 118 Shape of mycoplasma viruses, 21 Shock, osmotic, in mycoplasmas, 28 Size of mycoplasma genome, 14 Size of mycoplasma viruses, 2 1 Size of mycoplasmas, 6 Size of poly-P-hydroxybutyrate granules in bacteria, 210 Slime moulds, polyphosphate metabolism in, 190 Sodium chloride, effect of on growth of sulohate-reducing bacteria. 84 Sodium deoxycholate, effect of on mycoplasmamembranes, 33 Sodium dodecyl sulphatc, effect of on mycoplasma membranes, 33 Soils, occurrence of sulphate-reducing bacteriain, 119 Solubility of cytochromes c3 of sulphatrreducing bacteria, 105 of poly-/3-hydroxybutyrate,207 Solubilization of membrane proteins in mycoplasmas, 32 of mycoplasma membranes, 57 Solubilized membrane comaonents from mycoplasmas, re-aggregation of, 57 Sonic oscillation, effect of on granules of poly-P-hydroxybutyrate,235 Special structures in mycoplasmas, 9 Specialized reserves in micro-organisms, 136 Speciation in the genus Desulfovibrio, 85 Species of mycoplasmas, numbers of, 2 Specific gravity of poly-/3-hydroxybutyrate, 207 Specificity, of mycoplasma viruses, 22 of resolution phenomenon and mycoplasma membranes, 62 of transfer RNA from mycoplasmas, 24 Spermosphere, activities of sulphatereducing bacteriain, 124 Sphaerotilus spp., accumulation of poly-phydroxybutyrate in, 205

8. discophorus, role of poly-P-hydroxybutyrate in, 250 Sphingomylelin in mycoplasma membranes, 39 Spin-labelled fatty acids in mycoplasma membranes, 52 Spirillurn spp., accumulation of poly-Phydroxybutyrate in, 205 S p . volutans, volutin granules in, 179 Spore-forming sulphate-reducing bacteria, classification of, 84 Spores, ability of sulphatc-reducing bacteria to form, 86 Sporulation of bacilli, and accumulation of poly-P-hydroxybutyrate, 215 of bacteria, role of poly-p-hydroxybutyrate in, 252 Staphylococcus aureus, polyphosphate glucokinase in, 196 Starvation, glycogen degradation during in Escherichiu coli, 172 Starvation of micro-organisms, role of poly -P-hydroxybutyratein, 249 Sterol-requiring mycoplasmas, osmotic lysis of, 29 Sterols. function of in membranes. 48 Storage, function of polyphosphates, 201 polymer synthesis in microbes, mutants defectivc in, 139 of town gas, importance of sulphatereducing bacteria, 121 Strand breakage in mycoplasma DNA, 17 Streptococcus SL-1, polyphosphate in, 1SO polyphosphate metabolism in, 190 S. faecalis, polyphosphate glucokinase in, 196 S . rnitis, accumulation of glycogen by, 143 degradation of accumulated polysaccharides bv. 141 glycogen synthesis in, 158 pyrophosphorylases of, 145 . . ~ Streptomyces spp., accumulation of poly-Phydroxybutyrate in, 205 Streptomycin, resistance in mycoplasmas, 18 Striated rods of mycoplasmas, 12 Structural proteins, possible presence of in mycoplasma membranes, 36 Structure of microbial polyglucans, 142 Substrate limitation, effect of on accumulation of poly-/3-hydroxybutyrate by Azotobucter beijerinckia, 225 Succinate formation by sulphate-reducing bacteria, 89 Sudan Black, use of t o stain poly-P-hydroxybutyrate granules in bacteria, 210 Sugar transport in mycoplasmas, 66 " ,

295

SUBJECT INDEX

Sulphate-reducing bacteria, ecology of, 117 physiology of, 81 Sulphate-reducing metabolism of bacteria, 88 Sulphate reduction, in geological strata, 11 mechanism of in bacteria, 110 microbial, evolutionary significance of, 116 Sulphate, reduction of t o sulphite by bacteria, 110 Sulphide, accumulation by sulphate-reducing bacteria, 110 mechanism of formation of from sulphite in bacteria, 11 1 oxidizing bacteria, primitive nature of, 117 Sulphides of iron, occurrence of in sulphatcreducing bacterial cultures, 123 Sulphite, formation of from sulphate by bacteria, 110 Sulphite pool, recycling, in sulphatereducing bacteria, 112 Sulphite reductase of sulphate-reducing bacteria, 112 Sulphite reductases, properties of from sulphate-reducing bacteria, 115 Sulphite reduction, mechanism of in bacteria, 11 I Sulphur deposits, importance of sulphatereducing bacteria in relation to, 122 Sulphur metabolism of sulphate-reducing bacteria, 109 Sulphur oxides, formation of in town gas by sulphate-reducing bacteria, 121 Sulphur, production of by bacteria on economic scale, 122 Sulphur starvation, effect of, on accumulation of polyphosphate by Aerobactcr aerogenes, 184 Sulphuretum, as a n ecosystem, 117 Surface location of proteins in mycoplasma membranes, 54 Survival of micro-organisms, and glycogen content, 178 Survival of Streptococcus mi& in relation to glycogen content, 160 Swellingrates of mycoplasmas, 45 Synchronous cultures of yeast, and glycogen synthesis, 166 Synthesis of membrane proteins and lipids inmycoplasmas, 56 Synthesis of phospholipids in mycoplasmas, 38 Synthesis of proteins in mycoplasmas, 22, 26 Synthetic capabilities of mycoplasmas, 67 Syntrophy of sulphate-reducing bacteria, 118

T Taxonomy of bacteria, importance of accumulation of poly-/3-hydroxybutyrate in, 204 Terminal oxidases of sulphate-reducing bacteria, 98 Terminal structure of MycopZasma pneumoniae, 9 Tetrahydrofurfuryl alcohol, stimulatory effect of on polyphosphate deposition in mycobacteria, 188 Tetmhymena spp., accumulation of poly-,8hydroxybutyrate in, 205 T.pyrqorrnis, glycogen degradation by, 174 glycogen accumulation by, 143 glycogen synthesis in, 170 pyrophosphorylases of, 145 Theophylline, effect of on glycogen synthesis in Tetmhymena pyrijormis, 170 Thermophilic mycoplasmas, 2 Thermophilic sulphate-reducing bacteria, 82 I’herrnoplasma ucidophilum, ecology of, 2 fine structure of, 4 Thiamine as a mycoplasma growth factor, 69 Thickness of mycoplasma membranes, 5 4 Thioglycollate medium for sulphate-reducing bacteria, 83 Thiophorase, presence of, in bacteria, 243 Thiosulphate : cyanide sulphur transferase activity of bacteria, 113 as a n intermediate in sulphite reduction in bacteria, 11 1 in Desulfovibrio sp., 112 Thiosulphate reductase of Desulfovibrio vulgaris, properties of, 113 Thiosulphate reduction by Desulfovibrio desulfuricans, 99 Threonine, biosynthesis of by sulphatereducing bacteria, 94 Thymine deprivation, effect of on mycoplasmas, 26 Thymine-less death in mycoplasmas, 28 Tonicity of medium, effect of, on mycoplasma morphology, 7 Torula utilis, polyphosphate glucokinase in, 106 Torulopsis utilis, effect of environment on glycogen content of, 140 Town gas, storage of in relation to activities ofsulphate-reducingbacteria, 121 Trans-crotonic acid formation of in determination of poly-/3-hydroxybutyrate, 214 Transfer RNA in mycoplasmas, 22,24 Transformation in mycoplasmas, 18

2 96

SUBJECT INDEX

Transformylase of Acholeplasma laidlawii, 24 Transport mechanisms in mycoplasmas, 65 Trehalose, as an energy store in microbes, 137 Tricarboxylic acid cycle in mycoplasmas, 71 Triiodothyronine, effect of on synthesis of glycogen synthetase in Tetrahyrnena pyrqorrnis, 170 Trimetaphosphate in micro-organisms, 181 Trithionate reductase of sulphate-reducing bacteria, 113 Triton X-100, effect of on mycoplasma membranes, 33 Turnover of lipids in mycoplasma membranes, 40 Tween 80, as a mycoplasma growth factor, 68 Typos of citrate synthase in sulphatereducing bacteria, 91

Uridine diphosphate glucose, as a ratelimiting factor in glycogen synthesis in Dictyostelium discoideurn, 169 Utilization of polyphosphates by microorganisms, 183

V Vacuoles, yeast, polyphosphate in, 186 Vesicle formation in mycoplasma membrane preparations, 60 Viral nucleic acid, and mycoplasmas, 21 Viruses of Acholeplasma laidlawii, 20 ofmycoplasmas, 18 Volcanic lakes, occurrence of sulphatereducing bacteriain, 118 Volutin granules, nature of, 179 Vitamin requirements of mycoplasmas, 69

W Wilting of rice crops, and sulphate-reducing bacteria, 124

U Ultrasound, effect of on mycoplasmas, 28 Ultrastructure, of mycoplasmas, 8 of reconstituted membranes of mycoplasmas, 59 Ultraviolet inactivation of mycoplasma viruses, 22 Ultraviolet irradiation of mycoplasma genome, 16 Unit membranes in mycoplasmas, 8 Unsaturated fatty-acid requirement of mycoplasmas, 68 Unsaturated fatty acids, synthesis of by mycoplasmas, 42 Uptake of cholesterol by mycoplasmas, 47 Urea requirement of mycoplasmas, 69 Urease activity of mycoplasmas, 69 Uridine diphosphate glucose pyrophosphorylase in eukaryotes, 144

X X-Ray diffraction as applied to mycoplasmamembranes, 61 X -Ray diff r actogmms of poly - P-hydroxy bntyrate, 206

Y Yeast, glycogen synthetase, diffcrcnt forms of, 165 polyphosphatase of, 197 polyphosphate accumulation by, 185 Yeasts, glycogen synthesis in, 163

Z Zoogloea spp., accumulation of poly-,3hydroxybutyrate in, 205

CUMULATIVE INDEX OF TITLES Adaptive responses of Escherichia coli to a feast and famine existence, 6, 147 Aflatoxins, 1, 25 Aliphatic amidases of Pseudomonas aeruginosa, 4, 179 Aliphatic hydrocarbons, utilization of, by micro-organisms, 5, 1 Amoebae encystment in, 4,106 Antimicrobial agents and membrane function, 4,46 Aromatic compounds, catabolism of, by micro-organisms, 6, 1 Assimilatory and dissimilatory metabolism of inorganic sulphur compounds by micro-organisms, 3, 11 1 Bacteria, high-energy electrons in, 5, 135 Bacterial endospore, biochemistry of, 1, 133 Bacterial exopolysaccharides, 8, 143 Bacterial flagella, 6, 219 Bacterial lipids, comparative aspects of, 8, 1 Bacterial photosynthetic apparatus, 2 , l Bacterial ribosomes, “life cycle” of, 2, 89 Bacterial wall content and composition, effects of environment on, 7 , 8 3 Bdellovibrios, physiology of, 8, 215 Biochemical and physiological aspects of differentiation in the fungi, 5,45 Biochemical aspects of extreme halophilism, 1, 97 Biochemistry of the bacterial endospore, 1, 133 Biosynthesis of secondary metabolites : roles of trace metals, 4, 1 Branched electron-transport systems in bacteria, 5, 173 Budding of yeast cells, their scars and ageing, 2, 143 Carbohydrate utilization, catabolite repression and other control mechanisms in, 4, 25 Catabolism of aromatic compounds by micro-organisms, 6, 1 Catabolite repression and other control mechanisms in carbohydrate utilization, 4, 252 Chemolithotrophic bacteria, roles of exogenous organic matter in the physiology of, 3, 159 Comparative aspects of bacterial lipids, 8, 1 Comparative biology of prokaryotic resting cells, 9, 153 Differentiation in the fungi, biochemical and physiological aspects of, 5,45 Ectrophic mycorrhizas, physiology of, 3,53 Effects of environment on bacterial wall content and composition, 7 , 8 3 Electrophoretic mobility of micro-organisms, 9, 1 297

298

CUMULATIVE INDEX OF TITLES

Encystment in amoebae, 4,106 Energy conversion and generation of reducing power in bacterial photosynthesis, 7, 243 Energy reserve polymers in micro-organisms, role and regulation of, 10, 135 Enzymes during the cell cycle, synthesis of, 6,47 Escherichia coli, adaptive responses of, to a feast and famine existence, 6, 147 Escherichia coli, F-pilus of, 3, 2 Extreme halophilism, biochemical aspects of, 1, 97 F-pilus of Escherichia coli, 3, 2 Fungi, biochemical and physiological aspects of differentiation in, 5 , 4 5 Generation and utilization of energy during growth, 5, 213 Halophilism, extreme, biochemical aspects of, 1, 97 High-energy electrons in bacteria, 5, 135 Irradiated bacteria, repair of damaged DNA in, 2, 173 /3-Ketoadipate Pathway, 9, 89 “Life cycle” of bacterial ribosomes, 2, 89 Membrane function and antimicrobial agents, 4, 46 Metabolism of knallgasbacteria, regulatory phenomena in, 7, 205 Metabolism of one-carbon compounds by micro-organisms, 7, 119 Methane, microbial formation of, 6, 107 Micro-organisms,oxygen metabolism by, 3, 197 Micro-organisms,electrophoretic mobility of, 9, 1 Micro-organisms,utilization of aliphatic hydrocarbons by, 5, 1 Microbes under minimum stress, viability measurements and the survival of, 1, 1 Microbial formation of methane, 6, 107 Microbiological research, place of continuous culture in, 4,223 Mycoplasmas, physiology of, 10, 1 Nitrogen fixation, pathways of, 8, 59 Nucleic acid and protein formation in bacteria, regulation of, 1, 39 One-carbon compounds, metabolism of, by micro-organisms, 7, 119 Oxygen metabolism by micro-organisms, 3, 197 Pathways of nitrogen fixation, 8 , 5 9 Physiology of ectotrophic mycorrhizas, 3, 53 Physiology of mycoplasmas, 10, 1 Physiology of sulphate-reducing bacteria, 10, 81 Physiology of the bdellovibrios, 8, 215 Place of continuous culture in microbiological research, 4,223

CUMULATIVE INDEX OF TITLES

299

Plasmids of Staphylococcus aureus and their relation to other extrachromosomal elements in bacteria, 2,43 Prokaryotic resting cells, comparative biology of, 9, 153 Pseudomonas aeruginosa, aliphatic amidases of, 4, 179 Rapid detection and assessment of sparse microbial populations, 8, 105 Reducing power in bacterial photosynthesis, energy conversion and generation of, 7, 243 Regulation of nucleic acid and protein formation in bacteria, 1, 39 Regulatory phenomena in the metabolism of knallgasbacteria, 7,205 Repair of damaged DNA in irradiated bacteria, 2, 173 Role and regulation of energy reserve polymers in micro-organisms, 10, 135 Roles of exogenous organic matter in the physiology of chemolithotrophic bacteria, 3, 159 Secondary metabolites, biosynthesis of, roles of trace metals in, 4, 1 Serotype expression in Paramecium, 4, 132 Staphylococcus aureus, the plasmids of, and their relation to other extrachromosomal elements in bacteria, 2,43 Sulphate-reducing bacteria, physiology of, 10,81 Survival of microbes under minimum stress, and viability measurements, 1, 1 Synthesis of enzymes during the cell cycle, 6,47 Thermophilic bacteria and bacteriophages, 3, 83 Trace metals, roles of, in biosynthesis of secondary metabolites, 4, 1 Utilization of aliphatic hydrocarbons by micro-organisms, 5, 1 Viability measurements and the survival of microbes under minimum stress, 1, 1 Walls and membranes in bacteria, 7 , 2 Yeast cells, budding of, their scars and ageing, 2, 143

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