Advances in Microbial Physiology Volume 22

Advances in

MICROBIAL PHYSIOLOGY

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Advances in MICROBIAL PHYSIOLOGY edited by

A. H. ROSE School of Biological Sciences University of Bath England

J. GARETH MORRIS Department of Botany and Microbiology University College of Wales Aberyst wyth

Volume 22

1981

ACADEMIC PRESS London . New York . Toronto . Sydney. San Francisco A Subsidiary oj' Harcourt Brace Jovanovich, Publishers

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Copyright 0 1981 by ACADEMIC PRESS INC. (LONDON)

AN Rights Reserved

N o part of this book may be reproduced in any form by photostat, microfilm, or any other means, without written permission from the publishers

British Library Cataloguing in Publication Data

Advances in microbial physiology VOl. 22 I . Micro-organisms - Physiology 576.11 QR84 ISBN 0-12-027722-0 LCCCN 67-19850

Filmset by Northumberland Press Ltd. Gateshead. Tyne & Wear Printed in Great Britain by Fletcher and Son Ltd. Nornich

Contributors HOWARD BUSSEY Department of’ Biology, McGill University, Montreal, Quebec, Canada H3A IBI GERHART DREWS Institut fur Biologie 11 (Mikrobiologie), Universitat Freiburg, 0 - 7 8 Freiburg, Federal Republic of Germany A, FIECHTER Swiss Federal Insiitute of Technology, ETH-Honggerberg, CH-8093 Zurich, Switzerland G. F. FUHRMANN Institute of Pharmacology and Toxicology, Phillips University, Lahnberge, 0-3550 MarburglLahn, Germany 0. KAPPELI Swiss Federal Institute of Technology, ETH-Honggerberg, CH-8093 Zurich, Switzerland JURGEN OELZE Institut f u r Biologie IZ (Mikrobiologie), Universital Freiburg, 0-78 Freiburg, Federal Republic of Germany P. D. J. WEITZMAN Department of Biochemistry, University of Bath, Bath, England

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Contents Organization and Differentiation of Membranes of Phototropic Bacteria by GERHART DREWS and JURGEN OELZE I. Introduction . 11. Electron-transport systems . 111. Supramolecular Organization of the Membrane System . A. Fine structure of membranes . B. Functional subunits . . C. Topography of intracytoplasmic membranes IV. Differentiation of the cellular membrane system . A. Biogenesis of the photosynthetic apparatus . B. Influence of external factors on membrane differentiation C. Regulation of differentiation . V. Conclusions . VI. Acknowledgements . References . .

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1 4 9 9 16 30 36 37 53 71 78 80 81

Physiology of Killer Factor in Yeast by HOWARD BUSSEY I. Introduction . 11. Encapsidated double-stranded ribonucleic acid plasmids in killer yeast . A. Double-stranded ribonucleic acid B. Capsid . C. Other fungal double-stranded ribonucleic acid . . D. Physiology of plasmid replication E. Mutants in nuclear genes essential for plasmid maintenance and control . . F. Replication of double-stranded ribonucleic acids . . 111. Killer Toxin . A. Structure and properties . . B. Toxin synthesis and secretion . C. Physiology of toxin action . D. Cell-wall receptor for toxin . E. The membrane-damaging event . F. Toxin immunity . G . Other killer toxins . . H. Ustilago killer system . I. Action of killer toxins on pathogenic yeasts . References . .

93 95 95 98 99 100 101 103 104 104 106 108 109 111 114 1 I4

116 117 118

CONTENTS

viii

Regulation of Glucose Metabolism in Growing Yeast Cells by A. FIECHTER, G. F. FUHRMANN and 0. KAPPELI I. Introduction . . A. Control of growth . . B. Physiology of growth . 111. Molecular background of regulation A. Crabtree effect . B. Pasteur effect: Sols mod . . C. Energetical considerations . IV. Sugar transport . A. Introduction . . B. Transport systems , C. Concluding remarks . V. Conclusions . VI. Acknowledgements , References . . 11. Growth

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. . . . . .

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. . . . . . . .

123 125 125 132 142 142 153 157 159 159 162 175

176 171 117

Unity slid Diversity i n some Bacterial Critic Acid-Cycle Enzymes by P. D. J. Weitzman I. A view of the critic acid cycle . 11. Citrate synthase . . A. Energy controls . B. Biosynthetic controls . C. Molecular-size patterns . D. Allosterism: kinetic and molecular features. . E. Enzyme characteristics as an aid to bacterial taxonomy F. Exceptions t o the enzyme patterns . . G. Mutants: dysfunction as a clue to function. 111. Succinate thiokinase . A. Molecular-size patterns . B. Nucleotide-specificity patterns . IV. Isocitrate dehydrogenase . V. Pyruvate and a-oxoglutarate dehydrogenases . VI. Malate dehydrogenase . . VII. Multipoint control of the cycle VIII. Evolutionary aspects . IX. Concluding remarks . X. Acknowledgements . References . . Author Index Subject Index

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185 191 192 198 202 204 . 207 . 209 . 211 . 218 . 219 . 220 . 223 . 227 . 230 . 231 . 234 . 237 . 238 . 238

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,

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245 258

Organization and Differentiation of Membranes of Phototrophic Bacteria GERHART DREWS and JURGEN OELZE lnstitut fur Biologie I1 (Mikrobiologie), Universitat Freiburg, D- 78 Freiburg Federal Republic of Germany

I. Introduction . . . . 11. Electron-Transport Systems . 111. Supramolecular Organization of the Membrane System . A. Fine structure of membranes. . . B. Functional subunits . . . . C. Topography of intracytoplasmic membranes . . . IV. Differentiation of the Cellular Membrane System . A. Biogenesis of the photosynthetic apparatus . B. The influence of external factors o n membrane differenitiation C. Regulation of differentiation. . V. Conclusions . . . . . . . . VI. Acknowledgements . . . . . . . References . . . .

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9 9 16 30 36 37 53 71 78 80 81

I. Introduction A relatively small number of prokaryotic micro-organisms can satisfy their cellular energy requirements by light-driven processes. Among those are the cyanobacteria or blue-green algae, the prochlorophyta, the halobacteria, and members of the Rhodospirillales. The last group comprises, in a classical sense, the phototrophic bacteria. Cyanobacteria perform an oxygenic type of photosynthesis, as do eukaryotic plants. Most members of this group exhibit an obligatory photo-autotrophic metabolism (Carr, 1973; Stanier, 1

2

G. DREWS AND J. OELZE

1973, 1974). The photosynthetic apparatus (containing chlorophyll a) is localized in thylakoids (intracytoplasmic sac-like double membranes). Accessory antenna pigments, i.e. the phycobiliproteins, are organized in phycobilisomes attached to the thylakoids. Biological and systematic aspects of the cyanobacteria have been reviewed recently (Stanier et al., 1971; Stanier and Cohen-Bazire, 1977; Waterbury and Stanier, 1978). Recently the Prochlorophyta have been established (Lewin, 1976). The cells of Prochloron spp., which is an unicellular, prokaryotic marine member of this group, contain chlorophylls a and b in partially stacked thylakoids which are free of phycobilisomes (Withers et a/., 1978). Halobacterium sp., an “archaebacterium” (Woese et at., 1978) grows chemotropically. It also can produce ATP by a light-driven process under anaerobic conditions. The acceptor for light quanta is a bacteriorhodopsin crystal which is embedded in the cytoplasmic membrane and works as a proton pump (Oesterhelt and Stoeckenius, 1973; Dundas, 1977). However, the contribution of the light-dependent processes to the overall energy metabolism of those organisms is not known. Therefore, including halobacteria in the phototrophic micro-organisms seems to be premature. The above-mentioned organisms will not be treated in this article. We will restrict ourselves to the fourth group of phototrophic prokaryotes, i.e. the purple and green bacteria which are characterized by an anoxygenic type of photosynthesis and the presence of bacteriochlorophyll (Bchl) a or b as photochemically active pigments. Accessory pigments are one or more bacteriochlorophylls and carotenoids but never phycobiliproteins. Comparative, systemic surveys of the four major groups of phototrophic bacteria have recently been published (Pfennig and Triiper, 1974; Pfennig, 1977; Triiper and Pfennig, 1978). Members of the Rhodospirillaceae family prefer a photoheterotrophic mode of growth. But many species are also able to grow either chemoheterotrophically (aerobically in the dark) or photo-autotrophically. This family comprises a large spectrum of cell types, a wide range of DNA base ratios (Table I), and of chemotypes with respect to their cell wall macromolecules (Drews et al., 1978). The photosynthetic apparatus of most members is localized on tubular, vesicular, or lamellar intracytoplasmic membranes (Table 1; Fig. 1 in Oelze and Drews, 1972; Remsen, 1978). Exceptions are Rhodospirillum tenue and Rhodopseudomonas gelatinosa which have the photosynthetic apparatus in the cytoplasmic membrane. The principal mode of growth in members of the Chromatiaceae family is photo-autotrophy in which sulphide is oxidized to sulphate via elemental sulphur. A wide variety of morphological and chemical types have been described (Table 1; Pfennig and Truper, 1974; Triiper and Pfennig, 1978). The photosynthetic apparatus is localized on vesicular, tubular, or lamellar

TABLE 1. Survey of phototrophic bacteria (Rhodospirillales)”

Bacteria Rhodospirillaceae

Chromatiaceae Giant forms Chlorobiaceae

Chloroflexaceae

Cell shape

DNA base ratio (mole yo Principal mode of C ) photometabolism

+

Screw, rod sphere and stalk

62-72

Rod, sphere, screw, ovoid

62-72

Rod, ovoid, vibrio, sphere, prosthecae Flexible filaments

Aerobic or microaerophilic growth in the dark

Heterotroph

+

Motility

+

Intermediary Location of occurrence the photoBacterio- ofelemental synthetic chlorophyll sulphur apparatus aorb

Polar or peritrichousd flagella 45-50 49-58

Heterotroph or autotroph Autotroph Autotroph

53-55

Heterotroph

“ D a t a from Pfennig and Triiper (1974) and Triiper and Pfennig (1978). ICM indicates intracytoplasmic membrane; CM. cytoplasmic membrane c CM types include Rhodospirillum tenue, Rhodospeudomonas gelatinosa. Peritrichous: Rhodomicrobium. ‘Most of the antenna pigments are in chlorosomes (chlorobium vesicles).

-

+

(+I

(-1

-

+

uorh

Polar flagella -

ajc: aid: ale

+

alc

Gliding

-

+ + +

ICMb (CM)b ICM ICM CMb

CMb.‘

4

G . DREWS AND J. OELZE

intracytoplasmic membranes and contains only one Bchl species but some carotenoids. The green or brown sulphur bacteria (Chlorobiaceae) are non-motile, obligatory anaerobic and phototrophic organisms. They depend for their growth on hydrogen sulphide, which is oxidized to sulphate via elemental sulphur. The photosynthetic apparatus is localized in the cytoplasmic membrane and always contains two Bchl species. Most of the antenna pigments are localized in vesicles attached to the cytoplasmic membrane (chlorosomes). Members of the Chloroflexaceae family are more variable in their metabolism. Most of them are thermophilic. The photosynthetic apparatus is organized in the same way as in the Chlorobiaceae. Knowledge about the photosynthetic bacteria, especially concerning the primary events of bacterial photosynthesis, has been reviewed by Clayton and Sistrom (1978). The following chapter is confined to recent progress on structure, composition and differentiation of the membrane system in photosynthetic bacteria. It will be shown that, with phototrophic bacteria, components of electron-transport chains are differently compartmentalized and that different modes of adaptation to growth conditions have been developed.

11. Electron-Transport Systems

Many phototrophic bacteria are able to perform either a phototrophic or chemotrophic mode of energy metabolism. Although phototrophy depends on the presence of a photosynthetic apparatus, chemotrophy depends on the respiratory chain. In this context, a chemotrophic but fermentative metabolism is negligible in terms of energy production. In principle, both electron-transport systems and, as in the case of the photosynthetic apparatus, the accessory pigments are contained in the cellular membrane system. On the basis of the early investigations of Schachman etal. (1952), Frenkel(1954), Niklowitz and Drews (1955) and Vatter and Wolfe (1958), it is generally accepted that the photosynthetic apparatus is localized in the intracytoplasmic membrane system which on cell homogenization is broken down into pigmented membrane vesicles designated chromatophores. The respiratory system, on the other hand, has been postulated to be largely localized in the cytoplasmic membrane (Throm et al., 1970; Niederman et al., 1972; Oelze and Drews, 1972; Parks and Niederman, 1978), which is the only type of membrane present in cells depleted by photopigments. After expanding the investigations to diverse species and different culture conditions, however, it has become evident that there are numerous exceptions to the abovementioned concept of an independent and intracellularly confined distribu-

ORGANIZATION AND DIFFERENTIATION OF MEMBRANES

5

tion of the different electron-transport chains within the cellular membrane system: examples will be presented in Section 1V.A (p. 37). Much work has been done to elucidate mechanisms and pathways permitting photochemical and respiratory electron-transport reactions. For detailed descriptions of the present state of knowledge, the reader is referred to recent reviews (Parson, 1974; Jones, 1976; Dutton and Prince, 1978; Keister, 1978; Smith and Pinder, 1978). However, for a better understanding of membrane differentiation in the light of differentiation of their functional patterns, we shall subsequently summarize basic principles of the two energygenerating systems (Fig. 1). The photosynthetic apparatus contains antenna pigments and the photochemical reaction centre which is coupled to a chain of different redox carriers. The energized state, which is formed in the course of electron- and proton-transport processes, drives production of ATP via the coupling factor adenosine triphosphatase (ATPase) (Wraight et af., 1978a; BaccariniMelandri and Melandri, 1978; Baltscheffsky, 1978). Light energy is absorbed by the antenna pigments and funnelled, as excitation, to the reaction centre (Zankel, 1978) where charge separation and electron transport are initiated. In several species of phototrophic bacteria, as will be detailed later (Section IV, p. 36), the ratio of antenna bacteriochlorophyll (Bchl) to the reaction-centre components is subject to variation, depending on culture conditions. This ratio, for convenience, has been termed “photosynthetic unit” (Aagaard and Sistrom, 1972). It should be noted, however, that this unit does not refer to a functionally closed entity. Light

-

a BChl

Succinate dehydrogenase

+

N A D H dehydrogenase

4

BChl’

f

/

-

+c340 +6410 +O2

%b

+b 2 6 0

0 2

FIG. 1. A working scheme for electron-transport pathways in membranes of Rhodopseudomonas capsulata. The arrows represent possible steps involving some of the identified 6- and c-type cytochromes in aerobic and photosynthetic membranes. The box enclosing ubiquinone (UQ) represents a pool of ubiquinone-I0 and b-type cytochrome which may be reduced by electrons from any of several sources, and which may in turn reduce subsequent redox components in either of the terminal oxidase pathways indicated. Abbreviations: Bchl, bacteriochlorophyll; c and b, cytochromes; e, electron. From Zannoni rf af. (1978).

6

G. DREWS AND J. OELZE

On the contrary, excitation energy can be transferred from the antenna to different reaction centres (Clayton, 1966; Monger and Parson, 1977). On excitation, the reaction-centre Bchl a, which acts as a specialized dimer, may become oxidized and donate an electron, via an intermediary bacteriophaeophytin, to the primary electron acceptor (Clayton, 1978). This acceptor has been described as a quinone-iron complex (Bolton, 1978). The electron transfer from reaction-centre Bchl to bacteriophaeophytin takes between 10 and 200 picoseconds to be transferred to the primary acceptor. Oxidation of reaction-centre Bchl a can be followed spectrophotometrically by bleaching of an absorption peak at about 865 nm. Because the exact wavelength of the breachable peak varies among different species, the reaction-centre Bchl a is referred to in its literature as either P 865 or P870. The abbreviation P 960 is used for reaction centres containing Bchl b with a photobleachable peak at 960 nm. Bleaching also provides a means of quantitatively determining the amounts of reaction centre in cells, isolated membranes, as well as subchromatophore fractions (Aagaard and Sistrom, 1972; Straley et al., 1973). Studies on isolated reaction-centre preparations (see Section III.B.1, p. 18) have revealed that the quinone moiety of the primary acceptor is ubiquinone I in Rhodospirillum ruhrurn and Rhodopseudomonas sphaeroides (Slooten, 1972;Jolchine and Reiss-Husson, 1974; Okamura et al., 1975) and menaquinone in Chromatium vinosum and Rhodopseudomonas viridis (Okamura et al., 1976; Pucheu et al., 1976). The iron moiety is believed to function as an "iron wire" channelling electrons from the primary to the secondary acceptor, both of which have been identified as quinones (Feher and Okamura, I978b). From the secondary acceptor, the electron migrates, via a quinone-cytochrome b-c oxidoreductase, to a c-type cytochrome which has meanwhile been oxidized by the oxidized reaction-centre Bchl. There is good evidence, at least in Rp. capsulata, Rp. sphaeroides and Rs. rubrurn, that a third quinone, designated as Z, is involved in transfer of electrons to cytochromes c2 (Baccarini-Melandri and Melandri, 1977; Dutton et al., 1978; Gromet-Elhanan and Gest, 1978; Prince and Dutton, 1978). In addition, recent results suggest that quinone Z is involved in the regulation of electron and proton transport (Prince and Dutton, 1978, and Fig. 8). Different c-type cytochromes participate in the photochemical electron transport of different species of phototrophic bacteria (Fig. 1). On the basis of this knowledge, Bartsch (1978) has proposed a division of the group into at least three categories. The first would include all members of the Rhodospirillaceae family that contain the readily solubilizable cytochromes c2 (Rs. rubrurn, Rp. capsulata, Rp. sphaeroides and Rp. palustris). The second category would include organisms with a high redox potential cytochrome c-556 and a low redox potential cytochrome c-552, both of which are firmly bound to the membrane (e.g. Chr. vinosum, Thiocapsa pfennigii, Rp. gelatinosa, Rp.

ORGANIZATION AND DIFFERENTIATION OF MEMBRANES

7

viridis). Organisms in the third category would exhibit membrane-bound cytochromes c-553 or c-554 (members of the Chlorobiaceae as well as Chloroflexus aurantiacus). A multiplicity of spectroscopically different cytochromes b could be identified among phototrophic bacteria, even in one single strain. For example, up to five different b-type cytochromes were reported for Rp. sphaeroides (Saunders and Jones, 1975). But, according to Dutton and Jackson (1972), only a cytochrome b with an E6 value of f 5 0 mV is included in the lightdependent cyclic electron-transport system of this organism. The presence of light-dependent, open-chain electron-transport reactions has been proposed to occur predominantly in cells and chromatophores of Rs. rubrum and Rp. capsulata. There are, in principle, two different types of reactions. One proceeds via a reaction centre which also initiates cyclic electron flow, and the second is postulated to involve an independent second reaction centre. The first of the two activities has been known since the early investigations of Vernon and Kamen (1953), who showed that artificial electron donors were photo-oxidized at the expense of oxygen. This reaction has been re-investigated by several groups (Feldman and Gromet-Elhanan, 1972; del Valle-Tascon et a/., 1975; Hochman et a/., 1977). Zannoni and his colleagues (1978) demonstrated that, in Rp. capsulata, oxygen reduction in the light depends on the presence of cytochrome b260-containingpathway of the respiratory electron transport (Fig. 1). Gimenez-Gallego et al. (1976) proposed a physiological role for this reaction by which overreduction of the photochemical reaction centre, occurring on transfer of semi-aerobically dark-grown cells to anaerobiosis in the light, might be prevented by drainage of electrons to oxygen or other external electron acceptors. Thereby, the redox potential of the photosynthetic system is kept at its optimum level. The second hypothesis concerning the presence of two functionally independent reaction centres was originally proposed by Sybesma and his colleagues (reviewed by Sybesma, 1970) and more recently by van Grondelle et a/. (1976). Data from the latter group indicate that the second reaction centre amounts to only 5% of the total reaction centres. In addition, this reaction centre was shown to be especially active under low light intensities. But, while Sybesma (1970) proposed that the second reaction centre catalysed photoreduction of nicotinamide nucleotides, van Grondelle and his colleagues stated that the centre had an unknown function (Duysens e t a / . , 1978). Nevertheless, Picorel et a / (1977) considered a second reaction centre to be responsible for phototrophic growth of a mutant of Rs. rubrum which possessed an altered reaction centre. The respiratory chain has been studied in several facultative phototrophic bacteria (Keister, 1978; Smith and Pinder, 1978). But the presence of respiratory electron-transport reactions has also been described (Cusanovich and

8

G DREWS AND J OELZE

Kamen, 1968; Takamiya ct al., 1976) in obligate anaerobes such as Chr. vinosum. Through studies using respiratory mutants of Rp. capsulata, it has become evident that NADH and succinate are oxidized via a branched electrontransport system with the branching point at the level of ubiquinonecytochrome b-47 (Eb = f 4 7 mV) (Baccarini-Melandri et al., 1973; Marrs and Gest, 1973a; La Monica and Marrs, 1976; Zannoni et a/., 1976a, b). The electrons are then transferred via cytochrome c2 (EL = + 342 mV) to either a high-potential h-type cytochrome (Eb = +413 mV), which might act as a cyanide-sensitive terminal oxidase, or to an oxidase that is relatively insensitive to cyanide inhibition. Only the cyanide-sensitive branch was shown to be capable of energy conservation (Baccarini-Melandri et al., 1973). It has been suggested that the cytochrome h-cz section of the respiratory chain was also active in photochemical electron-transport chain (Fig. 1 and Jones, 1976). In fact, participation in both of the electron-transport chains of cytochrome c2 has been demonstrated by immunological methods (Prince et al., 1975; Baccarini-Melandri rt al., 1978 and Fig. 8). Branching of the respiratory chain has also been reported in Rp. palustris (King and Drews, 1975). In addition to two terminal oxidases, however, it was reported that the respective sites for ubiquinone,o are also different in the NADH-dependent chains as compared with the succinate-dependent respiratory chains (King and Drews, 1973). Similarly, inhibitor studies with cells and membrane preparations of Rs. ruhrum suggest participation of largely separate ubiquinonel o moieties in either NADH- or succinate-dependent respiration (Oelze and Kamen, 1975). Furthermore, the inhibitory action of 2-hydroxibiphenyl on electrontransport in vitro on the one hand, and oxygen uptake and growth of whole cells on the other, suggest that cellular respiration is best represented by NADH-dependent respiration which in turn limits the growth rate on malate under chemotrophic conditions (Oelze e t a / . , 1978). Thus, as shown previously with mutants of Rp. capsulata (Marrs and Gest, 1973a) the succinatedependent respiratory chain is obviously not needed for growth in malatecontaining medium. Nevertheless, under such conditions, Rs. rubrum forms the pathway that is potentially active in succinate oxidation. Reduction of nicotinamide nucleotides is known to be one of the major functions of photochemical reaction sequences in plant-type photosynthesis. In phototrophic bacteria, photoreduction of NAD has been reported for Chlorobium limicola (Buchanan and Evans, 1969), Rp. capsulata (Klemme, 1969) and Rs. rubrum (Feldman and Gromet-Elhanan, 1972; Sybesma, 1970; Govindjee and Sybesma, 1972). But, opposing the concept of a non-cyclic, light-dependent NAD+ reduction, Bose and Gest (1962) suggested that NAD+ is reduced via an energy-linked reverse electron transport (Fig. 1). +

ORGANIZATION AND DIFFERENTIATION OF MEMBRANES

9

This hypothesis has been confirmed by several authors for various organisms (Knoblochetal., 1971; Gest, 1972; Knaff, 1978). Succinate has beenemployed most frequently (Gest, 1972; Govindjee and Sybesma, 1972; Irschik and Oelze, 1976) to investigate NAD reduction in the light by a physiologically significant electron donor. This reaction includes succinate as well as NADH dehydrogenases. In evaluating the significance of succinate-dependent NAD+ reduction for living cells of JLF. rubrum, Oelze et al. (1978) specifically inhibited this reaction in growing cells and found virtually no influence on growth rate. On the contrary cells grew well on malate or succinate in the light and in the presence of 2-hydroxibiphenyl, even when NAD+ reduction was no longer measurable with membrane preparations from such cells. This suggests that succinate-dependent NAD reduction is not essential for phototrophic growth of Rs.rubrum. A similar conclusion can be drawn from the findings that mutants of Rp. capsulata, which lack NADH or succinate dehydrogenases, or both are still able to grow phototrophically, at least on malate-containing medium (Marrs and Gest, 1973a). Under optimum conditions, phototrophic growth of phototrophic bacteria depends primarily on light-dependent cyclic electron flow, whereas chemotrophic growth of facultative phototrophic members requires NADHdependent respiratory chain. Besides these principal electron-transport reactions, several others, the physiological significance of which is uncertain, have been detected in vitro and in some instances also in vivo (an example of the latter is presented by van Grondelle et al., 1976). It is possible that some of the reactions have been created only through application of sophisticated methods by skilful investigators. However, it may also be that a proper combination of the various reaction sequences has enabled organisms to adapt to extremely different environmental conditions. Thus, from a biological point of view, we suggest that one of the future goals in research on phototrophic bacteria should focus on investigations of the physiological significance of the various reactions described so far. +

+

111. Supramolecular Organization of the Membrane System A . F I N E S T R U C T U R E OF M E M B R A N E S

The findings that some phototrophic bacteria form intracytopiasmic membranes as sites for the photosynthetic apparatus, while others contain the photosynthetic apparatus more or less exclusively in cytoplasmic membranes and adjacent structures, suggest some diversity in the ultrastructure of membranes. Since the introduction of freeze-etching as well as freeze-

10

G. DREWS AND J. OELZE

fracturing techniques, considerable information on the ultrastructure of membranes has been obtained through electron microscopy. At present, it is generally accepted that particles visible on membrane-fracture faces represent (functional) protein units embedded in the lipid bilayer of the membrane (Miihlethaler, 1971; Branton et al., 1975). Thus, differences in the density (i.e'. number of particles per unit of face) as well as in the sizes of particles can be interpreted in terms of differences in membrane structure and function.

1. Types of Membrane Differentiation One cytologically defined group includes all organisms that form only occasionally, if at all, intracytoplasmic membranes. This is the situation with Rhodospirillum tenue and Rhodopseudomonas gelatinosa. The latter organism, however, generally has fewer irregular membrane invaginations than the former (De Boer, 1969; Weckesser et al., 1969). In our studies, we have never observed membrane invaginations in Rs. tenue growing actively under either chemotrophic or phototrophic conditions. In addition, except for mesosomal elements, no typical intracytoplasmic membranes in members of the Chlorobiaceae have been described (Cohen-Bazire et al., 1964). Generally, the possibility exists that irregular membrane invaginations are formed by cells growing under unbalanced conditions (Schon and Jank-Ladwig, 1972; Maudinas et al., 1973; Oelze et al., 1977). It remains to be seen, of course, if this also applies to the examples mentioned above. Rhodospirillum tenue, although exhibiting no significant quantities of intracytoplasmic membrane, is able to increase its cellular content of cytoplasmic membrane when adapting from chemotrophic to phototrophic conditions (Wakim et al., 1978). This is achieved through alterations in the lengths and diameters of cells, i.e. through alterations in the relative amount of cell envelope per cell volume. Chemotrophic and phototrophic cells exhibit distinct differences in the supramolecular architecture of the cytoplasmic membrane (Fig. 2). On both the exoplasmic (EF) and plasmic (PF) faces of the cytoplasmic membrane, the density of particles is higher in phototrophic than in chemotrophic cells. The difference in particle number, however, is much more pronounced when observed on exoplasmic fracture faces (Fig. 2 ) . With these faces, there is also a considerable dissimilarity in the size of particles. The exoplasmic fracture faces of the cytoplasmic membranes of chemotrophic cells exhibit particles up to about 10 nm in diameter, whereas the corresponding faces of membranes of phototrophic cells exhibit, additionally, equal quantities of particles of about 13 nm or greater diameter. Thus, it becomes apparent that the exoplasmic fracture face represents predominantly the exoplasmic leaflet of the membrane in which differentiation

ORGANIZATION AND DIFFERENTIATION OF MEMBRANES

11

FIG. 2 . Exoplasmic fracture face (EF) of the cytoplasmic membrane (PM) of freezefractured cells of Rhodospirillum tenue grown chemotrophically (a) or phototrophically (b). The arrow in the upper left-hand corner indicates the direction of shadowing. The bar represents 100 nm. The micrograph was taken by J. R. Golecki. takes place during reversible adaptation from a chemotrophic to a phototrophic mode of energy metabolism. So far, nothing is known about the functional properties of the different particles, but there is every likelihood that they are somehow related to the photosynthetic apparatus. Last, but not least, it should be noted that, during adaptation of cells from chemotrophic to phototrophic conditions, all of the particles are homogeneously distributed on the membrane fracture faces. N o areas can be distinguished which, on the basis of particle distribution, might suggest a localized incorporation of particles into the membrane. This, however, is a necessary prerequisite for membrane synthesis de n o w . The organization of the membrane system in members of the Chlorobiineae differs considerably from those in the Rhodospirillineae. In ultrathin sections through Chlorobium limicola and Chlorc?fle.uus aurantiacus, flat,

12

G. DREWS AND J . OELZE

elongated sac-like structures. closely appressed to the cytoplasmic membrane, are visible (Cohen-Bazire et al., 1964; Holt et a/., 1966; Pierson and Castenholz, 1974). These structures have been designated as chlorobium vesicles (Cohen-Bazire et al., 1964). At the contact point of a vesicle and the cytoplasmic membrane, the dense cytoplasmic leaflet of the unit membrane appears to be fused with the envelope (Holt et a/., 1966; Staehelin et al., 1978, 1980). The term “chlorobium vesicles” was proposed to be substituted by the term “chlorosome” for the following reasons (Staehelin et a/., 1978, 1980). (i) The structures are not limited by a classical bilayer membrane, and are therefore not vesicles in a strict sense. (ii) These structures have been found in organisms other than the Chlorohium species. (iii) They seem to perform a light-harvesting function (see Section III.B.2, p. 19) comparable to the phycobilisomes of cyanobacteria and red algae. Chlorosomes of Chlorcflesus aurantiacus measure 106 x 32 x 12 nm. Those of Chlorobium limicola show a considerable variation in size from about 40 x 70 nm to 100 x 260 nm. The height of Chlorobium chlorosomes is 10 nm. The supramolecular structure of chlorosomes and their attachment to the cytoplasmic membrane have been studied by freeze-fracture electron microscopy (Staehelin ei al., 1978, 1980). Each chlorosome consists of a core and of an approximately 2-3 nm thick envelope layer that lacks substructure. The core is filled with rod-shaped elements (5 nm in diameter in Chloruflexus sp., or 10 nm in diameter in Chlorohium sp.) embedded in a smooth unetchable matrix. The rod elements are closely packed and extend the full length of the chlorosome. A crystalline baseplate (in Chlorobium 5-6 nm thick) connects the chlorosome to the cytoplasmic membrane. The main striations of the baseplate lattice make an angle of between 40” and 60” with the longitudinal axis of the chlorosome and have a repeating distance of 6 nm. The fracture faces of the cytoplasmic membrane are covered with numerous intramembrane particles. Regions of the P-faces adjacent to the baseplates of chlorosomes are enriched in large intramembrane particles, most of which belong to the 10 nm and 12.5 nrr: particle-size categories. Each chlorosome attachment site in Chlorobium sp. contains between 20 and 30 very large ( > 12.0 nm diameter) intramembrane particles. These results of freezefracture electron microscopy, summarized in Fig. 3 (Staehelin et a/., 1980), are discussed together with biochemical data on the possible functions and composition of the structures in Section III.C, p. 35.

2 . I n t racy t oplasmic Mend rane Type

Intracytoplasmic membranes in cells of members of the Rhosospirillineae appear as vesicles, thylakoid-like flat lamellae, usually in the form of stacks

ORGANIZATION A N D DIFFERENTIATION OF MEMBRANES

13

and tubular membranes (Oelze and Drews, 1972; Pfennig and Truper, 1974; Remsen, 1978; Truper and Pfennig, 1978). These structures are connected to each other and/or to the cytoplasmic membrane. This has been demonstrated electron microscopically with ultrathin sections of whole cells (Boatman and Douglas, 1961; Drews and Giesbrecht, 1963, 1965; Holt and Marr, 1965a; Tauschel and Drews, 1967), with osmotically shocked sphaeroplasts (Giesbrecht and Drews, 1962; Boatman, 1964) and with membrane preparations isolated after a mild disruptive treatment of the cells (Hurlbert et al., 1974). The continuity of the cytoplasmic-intracytoplasmic membrane system is a consequence of morphogenetic processes (Lascelles, 1968; Peters and Cellarius, 1972; see Section IV.A.2, p. 41). The dynamic state of the membrane system in a living cell, however, implies that single membrane structures can be reversibly detached or fused by membrane flow (Singer, 1974). There are two consequences of the origin of intracytoplasmic membranes. Firstly, the interior of the intracytoplasmic membrane is an extracytoplasmic space which might be connected to the periplasmic space; secondly, the orientation of intramembranous structures is inverse in intracytoplasmic Rod elements (- 10 nm in diameter

Cytoplasm-

mem brane (lipid bilayer -5 nm)

Y Large intramembrane particles (mostly 1014 nm in diameter, reaction centre-noncrystalline L H bacteriochlorophyll a complexes?)

Crystal line baseplate (major periodicity -6 nm, minor periodicity -3 nm, bacteriochlarophyll aprotein?)

FIG. 3. Model of a chlorosome (otherwise a chlorobium vesicle) and its associated cytoplasmic membrane of Chlorohium Iimicola based on the freeze-fracture observation by Staehehn et al ( 1 980) and from data of Cruden and Stanier ( 1 970); Fowler et al. (1971); Olson (1978) and Olson et al. (1976). Chlorosomes are functionally light-harvesting complexes. From Staehelin rt al. (1980).

14

G. DREWS AND J. OELZE

compared with cytoplasmic membranes (Michels and Konings, 1978; see Section III.C, p. 30). Although intracytoplasmic and cytoplasmic membranes are connected to each other, and parts of the intracytoplasmic membrane are formed in invagination of the cytoplasmic membrane, both membrane systems differ in composition, function and in the kinetics of biosynthesis (Oelze and Drews, 1970a; Lampe et al., 1972; Oelze et al., 1975a; Niederman and Gibson, 1978; Parks and Niederman, 1978; see Section IV, p. 36). In ultrathin sections, both cytoplasmic and intracytoplasmic membranes show the same diameter and the same double-track structure. The surfaces of both membrane structures appear to be smooth in negatively stained preparations (Oelze et al., 1969a; Takacs and Holt, 1971; Lampe et a[., 1972; Hurlbert et al., 1974). However, when membranes are isolated under conditions that preserve ATPase activity, knob-like structures appear on the cytoplasmic surface of both membranes (Low and Afzelius, 1964; Lampe et al., 1972; Reed and Raveed, 1972). Although ATPase has been isolated from these membranes, the knob-like structures have not yet been functionally identified (see Section III.C, p. 30). In some preparations, regular surface structures were described in membranes after freeze-etching, negative staining, or shadow casting (Holt and Marr, 1965b; Reed et al., 1975). These structures, observed in Rp. viridis (Giesbrecht and Drews, 1966) or in Rs. rubrum (Oelze and Golecki, 1975), showed a centre-to-centre spacing with a periodic pattern of approximately 10 nm. It was assumed, but not proven, that this pattern is expressed by intramembrane particles which are exposed on the surface after drying of the membrane preparations. A structural order was observed in chromatophores of RF.rubrum and Rp. sphaeroides by X-ray studies. It was assumed that the protein molecules form two-dimensional crystalline clusters (Ueki et al., 1976). As in other electron-transport membranes, the intramembrane-fracture faces, visualized by freeze-fracture electron microscopy of both cytoplasmic and intracytoplasmic membranes, are covered with particles. It is striking that the protoplasmic fracture face (PF) is more densely populated than the extracellular fracture face (EF) (Figs. 4 and 5). In cells of Rp. sphaeroides, grown under high light intensities, the EF of both cytoplasmic and intracytoplasmic membranes contains 500 particles (1 1 nm diameter) per pm2. The PF, on the other hand, contains particles of 11-12 nm diameter at a density of 3130 particles per pm2 on the intracytoplasmic membranes and 1460 particles per pm2 on the cytoplasmic membrane (Lommen and Takemoto, 1978). The higher density of particles on the PF, i.e. the convex fracture face of the cytoplasmic membrane and the concave fracture face of the intracytoplasmic vesicles, was, in contrast with Rs. tenue, also observed in cells of Rp. capsulata (Golecki et a/., 1979), Chloroflexus aurantiacus (Staehelin et al.,

ORGANIZATION AND DIFFERENTIATION OF MEMBRANES

15

FIG. 4. Rhodopseudomonas pulustris. The photosynthetic apparatus of cells grown phototrophically is localized particularly in lamellae arranged intracytoplasmic membranes (ICM). The protoplasmic fracture face (PF) of ICM, exposed after freezefracturing of cells (a), is densely covered with particles, whereas on the extraplasmic face (EF) of fractured membranes (b) only few particles are detectable. The bar represents 100 nm. The micrograph was prepared by J. R. Golecki. The arrow in the upper left corner indicates direction of shadowing.

FIG. 5 . Rhodopsrudomonuspalustris.Freeze-fracture electron microscopy of the cytoplasmic membrane, enriched in respiratory functions. The protoplasmic fracture face (PF) is densely populated with particles (a), whereas the exoplasmic fracture face is relatively smooth (b). The bar represents 100 n m . The micrograph was prepared by J. R. Golecki.

16

G. DREWS AND J. OELZE

1978), Chtorohium timicofu (Staehelin rt al., 19801, Rs. ruhrum (Golecki and Oelze, 1975) and ThiocupsaJloridunu (Takacs and Holt, 1971). The particle density and diameter can vary depending on culture conditions (Golecki et al., 1979). At present, the functional properties of these particles are unknown. However, it will be shown in Sections 1II.C and 1V.B (pp. 30 and 53) that the number of photosynthetic units and the number of particles per unit area of membrane are somehow correlated. Different size classes of particles seem to be present, which suggests a structural and functional heterogeneity among the particles. The asymmetrical localization of the particles in the membrane is important for vectorial distribution of functional subunits of electron-transport chains in the membrane. B . FUNCTIONAL SUBUNITS

Through the development of methods to disintegrate membranes into different protein constituents, and, moreover, to separate these proteins by means of sodium dodecyl sulphate-polyacrylamide-gel electrophoresis. it has become possible to characterize different types of membranes on the basis of their protein patterns (Fig. 6). Such different protein patterns, because of the fact that membrane proteins are involved in specific functions, are representative of differences in membrane-bound activities. Consequently, variations in the protein patterns of a given membrane indicate variations in its functional pattern. Thus, polyacrylamide-gel electrophoresis of membrane proteins has become one of the most informative and also popular means of studying membrane differentiation. A direct correlation between distinct proteins and specific functional units, however, is hampered by the fact that methods usually employed for membrane dissection also abolish these activities. In this context it is understood that the activities of functional units depend on the presence not only of proteins but also of cofactors. Nevertheless, the problem can be circumvented by isolation and purification of functional units followed by identification of their protein patterns within that of membranes after co-electrophoresis. Among the functional units that have been solubilized and purified from membranes of phototrophic bacteria, reaction-centre and light-harvesting bacteriochlorophyll (Bchl) preparations are the most adequate tools for following adaptation of the organisms from chemotrophic to phototrophic conditions and vice versa (see review by Drews, 1978). In addition, the isolation and purification of other membrane-bound functions have been described. All of these techniques are important for answering questions on membrane differentiation. Unfortunately only the proteins of Bchl complexes, ATPase and succinate dehydrogenase have been traced down so far in the protein patterns of the original membranes (Oelze, 1978; Fig. 6).

FIG. 6. Sodium dodecyl sulphate-polyacrylamide-gel electrophoresis of proteins from the membranes of various strains. Gel I , polypeptides of reaction centre (H = 28,000, M = 24,000, L = 20.500 molecular weights), isolated from Rhodopseudomonas capsulata, Ala . Gel 2, polypeptides of light-harvesting bacteriochlorophyll complex I1 (B 800-850) isolated from Rhodopseudomonas capsulata, strain Y5.Gel 3, proteins from membranes of Rhodopsrudomonas capsulata, strain St. Louis, wild-type. Bands I , 2,3,4indicatepolypeptidesoflight-harvestingcomplexes. Gel 4, polypeptides from membranes of Rhodopseudomonas capsulata Y 142 (reaction centre and light-harvesting I negative). Gel 5 , polypeptides from membranes of Rhodopseudomonas capsulata, Ala (carotenoid- and light-harvesting bacteriochlorophyll 11-negative). Gel 6, polypeptides from membranes of Rhodopseudomonas sphaeroides wild type. Gel 7, polypeptides from membranes of Rhodopseudomonas palustris le5, wild type. Gel 8, polypeptides from membranes of Rhodopseudomonas viridis F. Gel 9, polypeptides from membranes of Rhodospirillum rubrum. Molecular weights are as follows: reaction-centre polypeptides, H, 31,000; M, 24,500; L, 21,000; lightharvesting unit, 9,000 (A); ATPase, a, 55,000; /3, 51,000; succinate dehydrogenase (D), heavy subunit, 64,000. +

+

18

G. DREWS AND J. OELZE

1 . Photochemical Reaction-Centre Preparations

In 1963, Clayton defined a reaction centre “as the site where the quanta of energy bring about electron-transfer events that lead to the storage of stable chemical potential”. Aagaard and Sistrom (1972) extended this definition by including cytochrome as the primary electron donor. Consistent with the latter definition, all of the reaction-centre preparations initially isolated from different strains of phototrophic bacteria contained different amounts of cytochromes (summarized by Oelze and Drews, 1972). But refinement of methods for solubilization and purification of reaction centres from Rs. rubrum, Rp. sphaeroides and Chr. vinosum led to isolation of preparations with a minimum of constituents to satisfy the requirements of the primary photochemical process (Straley et al., 1973; Okamura et al., 1974, 1975; Feher and Okamura, 1978b; Mechler and Oelze, 1978b). With preparations from Rp. sphaeroides and Rs. rubrum, reaction centres contained four molecules of Bchl a, two molecules of bacteriophaeophytin, one to two molecules of ubiquinones, one ferrous iron ion, and, in the case of preparations of wild-type strains, one molecule of carotenoid per-molecule of reaction centre (Straley et al., 1973; van der Rest and Gingras, 1974; Cogdell et al., 1976). The function of carotenoid in reaction centres is probably protection of Bchl from destructive photo-oxidation (Cogdell et al., 1976; Boucher et al., 1977). Analyses by sodium dodecylsulphate-polyacrylamidegel electrophoresis of reaction centres from Rs. rubrum, Rp. capsulata, Rp. sphaeroides and Chr. vinosum revealed the presence of three protein subunits with molecular weights of between 20,000 and 3 1,000 (see reviews by Drews, 1978 and Feher and Okamura, 1978b). The three polypeptide subunits, designated as heavy (H), intermediate (M) and light (L), are always present in a stoicheiometry of 1 : 1 : 1 (Okamura et al., 1974). According to a note by Feher and Okamura (1978a), molecular weights of reaction-centre polypeptides might be underestimated because of preferential binding of sodium dodecylsulphate to hydrophobic proteins. Further purification of reaction centres from Rp. sphaeroides and Rp. capsulata resulted in separation of the H unit from the LM unit (Okamura et al., 1974; Nieth et al., 1975). The LM unit retained all of the constituents of the original HML complex required to perform the primary photochemical event. This is further supported by the findings that, after proteolytic digestion of the H subunit, isolated chromatophores still retain their full photochemical activity (Hall et al., 1978; Oelze, 1978). So far, no persuasive function has been found for the H subunit. Some reaction-centre preparations isolated from Rs. rubrum and Rp. sphaeroides contain two moles of ubiquinone, one of which is tightly bound

ORGANIZATION AND DIFFERENTIATION OF MEMBRANES

19

and the other loosely bound (Okamura et al., 1975; Slooten, 1972; Jolchine and Reiss-Husson, 1974). Only the tightly bound quinone is necessary for primary photochemistry. Recent investigations have indicated that this ubiquinone is bound to the M protein subunit of reaction centres. The loosely bound quinone, on the other hand, is thought to be in proximity, as a secondary acceptor, to the ferrous-iron moiety of the primary acceptor (Feher and Okamura, 1978a). Not all of the reaction centres isolated so far contain ubiquinone. As previously mentioned, preparations from Chr. vinosum and Rp. viridis contain menaquinone (Romijn and Amesz, 1977; Pucheu et al., 1976; Clayton and Clayton, 1978b). Reaction-centre preparations with properties different from those described above have been isolated from Rp. viridis, Rp. gelatinosa as well as from members of the Chlorobiceae. For a description of preparations from Chlorobium limicola, the reader is referred to recent reviews (Boyce et al., 1976; Olson et al., 1976; Prince and Olson, 1976; Olson, 1978). We will summarize major feactures of reaction-centre preparations derived from Rp. viridis and Rp. gelatinosa (Pucheu et al., 1976; Clayton and Clayton, 1978a, b). This is to demonstrate that, within the genus Rhodopseudomonas, significant diversity exists with respect to reaction-centre composition. Preparations from Rp. gelatinosa show spectral properties comparable to those known for Rp. sphaeroides, but differences can be observed in the polypeptide composition which contains only two subunits of 25,000 and 33,000 daltons. Reaction centres from Rp. viridis contain Bchl b instead of Bchl a which, on photo-oxidation, can be assayed by bleaching of an absorption peak at 960 nm. Consequently the pigment is designated P 960. Additional differences have been reported for the presence of menaquinone and of cytochromes 552 and 558 (Pucheu et al., 1976; Clayton and Clayton, 1978b). In addition, the molecular weights are different in that the three protein subunits amount to 31,000, 37,000 and 41,000.

2 . Light-Har vesting Antenna Pigment Complexes Absorption spectra in vivo of membrane3 from numerous photosynthetic bacteria show characteristic infrared absorption maxima (Biebl and Drews, 1969) which principally represent different spectral forms of light-harvesting Bchl (Thornber et a/., 1978). The absorption spectra show a remarkable red shift and an increase in the number of Bchl infrared absorption maxima compared with the respective absorption spectrum of Bchl in organic solvents (Fig. 7). It has been suggested that the different spectral forms of Bchl are due to either specific interactions of Bchl with proteins (Katz and Wassink, 1939; Wassink et al., 1939) or to aggregates of Bchl (Krdsnovsky et al., 1952;

20

G. DREWS AND J. OELZE

n

855

RC+~~~O+BBOO-B~S

-l

LOO

500

600

700

800

900 nm a55

RC + 8870

fd

(e 802

868

1

n

375

RC

L Loo

500

600

700

800

900 nm

L a

500

600

700

800

900nm

ORGANIZATION AND DIFFERENTIATION OF MEMBRANES

21

Katz et a/., 1966; Katz and Norris, 1973). The experimental evidence that Bchl in vivo is associated with polypeptides by ionic or hydrophobic interaction, but not by covalent linkages, has recently been summarized (Drews, 1978). Light-harvesting pigment-protein complexes have been isolated from numerous phototrophic bacteria, but few of them have been quantitatively analysed. Since the older literature has been reviewed (Oelze and Drews, 1972; Drews, 1978; Thornber et al., 1978; Cogdell and Thornber, 1979), the following description will be concentrated on four types of light-harvesting Bchl organization, which are represented by the following four species or genera: Rhodospirillum rubrum, Rhodopseudomonas, Chromatium and Chlorobium. a. Rhodospirifhm rubrum type. Wild-type strains of Rs. rubrum and carotenoid-less mutant strains of Rp. sphaeroides and Rp. capsulata contain one light-harvesting Bchl-protein complex which has one marked absorption maximum in the near infrared at 870-890 nm (B 875; Fig. 7; Sistrom et a/., 1956; Aagaard and Sistrom, 1972; Drews et a/., 1971; Oelze and Golecki, 1975; Cuendet and Zuber, 1977). The complex was solubilized from membranes of a carotenoid-less strain of Rp. capsulata by detergent treatment and purified by sucrose density-gradient centrifugation and column chromatography (Drews, 1976). In wild-type Rhodopseudomonas strains, the complex is difficult to separate from reaction centres. The Bchl is associated with one polypeptide in a molar ratio of 1 : I . It is not known whether the carotenoids of Rs. rubrum strains are bound to the same protein. Variable amounts of phospholipids were detected in the native complex (Table 2). The Bchl absorption spectrum in vivo of the B 875 complex, isolated from carotenoid-less strains, is not stable, in contrast with BchlkarotenoidFIG. 7. In vivo-absorption spectra of membranes and reaction-centre preparation isolated from Rhodopseudornonas capsulata. (a) membranes from the wild-type strain 37b4 showing the characteristic in vivo absorption bands of bacteriochlorophyll at 375. 590, 800, 855 and 870 nm. The maxima of the spectrum are attributable to reaction-centre, light-harvesting 1 (B 875) and I1 (B 80g850). The molar ratio of total bacteriochlorophyll per reaction centre is approximately 100 : 1 (compare with Fig. 6, gel 3). (b) Membranes from the carotenoid-less mutant Ala+. The dominating IR peak at 872 nm is attributable to light-harvesting bacteriochlorophyll complex I (B 875), the small peak at 800 nm to reaction centre. The molar ratio B 875 per reaction centre is approximately 25: 1. (For proteins, see Fig. 6, gel 5.) (c) Membranes from the photosynthetic-negative strain Y 5 which contains light-harvesting bacteriochlorophyll I1 complex (B 800-850) as the only bacteriochlorophyll-protein complex. (For proteins, see Fig. 6, gel 2.) (d) Reaction centre (RC) isolated from membranes of strain Ala+. ----, indicates the spectrum of the reversible bleached reaction centre when illuminated with actinic light. (For polypeptides, see Fig. 6, gel I ) . (e) Membranes from a photosynthetic-negative mutant of the strain Ala+. It shows the light-harvesting I spectrum (875).

TABLE 2. Light-harvesting-antenna-bacteriochlorophyll~arotenoid-protein complexes from members of Rhodospirillales

Strain Rhodopseudomonus cupsuluta Y5

Infrared maxima of Bchl in vivo (nm)

Carotenoids

802-855

+

Polypeptides” (M,x 8,10,14molar proportions 2:2: 1

Molar proportion of Bchl to carotenoid to protein

3 : I :2

Remarks

References

Thecomplex is an oliFeick and Drews (1978): gomeric form of subunits J . Shiozawa and containing three mol of G. Drews, unpublished Bchl and one mol of caro- observations tenoid bound to one mol of 10,000-mol. wt polypeptide, (no Cys + Arg), and one mol of 8000-mol. wt polypeptide (no Cys Trp). 1 pg lipid-P and 15 pg carbohydrates per mg protein are in the complex. The molecular weight of the complex is approx. 170,000.

+

Rhodopseudomonas cupsulatu A 1a

872

Rhodopseudomonas sphaeroides 2.4.1

80&850

-

I2,8 (?)

1:O:l

About 10

3 : I :2

Feick and Drews (1978)

+

Spheroidene spheroidenone

Subunit contains threemol of Bchl (two B 850), onemol ofcarotenoid. twomol of polypeptides.

Sauerand Austin, (1978); Cogdell and Crofts (1978):Cogdell and Thornber (1979); Cogdell et al. (1976)

Infrared maxima of Bchl in vivo (nm)

Carotenoids

Rhodopseudomonas sphaeroides R 26

855

-

8.5

Rhodopseudomonas sphaeroides

806850

+

about 10

+

12

-

14

+

8+ II

Strain

Rhodospirillum ruhrum

870

Rhodospirillum ruhrutn G 9

863

Chromatiurn ,itlosum

Polypeptides" (M,x

Molar proportion of Bchl to carotenoid to protein

Remarks

References

Subunit contains two polypeptides, 20"/, P-lipid, 2;b Bchl

Sauer and Austin (1978): Bolt and Sauer ( 1979)

No His and Cys

Fraker and Kaplan (1972); Huangand Kaplan (1973): Clayton and Clayton (1972)

2 : 1 : n.d.

N o Cys and Tyr. polarity 42%

Tonn er al. (1977); Cogdell and Thornber ( 1979)

I . I :0 : 1

N o Cys. 669, protein, 29:" P-lipid. 57, Bchl, polarity 40.4%

Cuendet and Zuber (1977)

2 :0 : I

1 : 5 : n.d. : 1

Spirilloxanthin

( a ) 80&82G850 (b) 8 0 6 8 5 0

+ 14

Separated by sodium dodecyl sulphateepolyacrylamide-gel electrophoresis. n.d. indicates that the value was not determined.

Mechler and Oelze (1978b)

24

G. DREWS AND J OELZE

protein complexes from wild-type strains. The shift of the 870 nm maximum to shorter wavelengths indicated a loosening of the Bchl-protein linkage and a phaeophytinization of Bchl (Cuendet and Zuber, 1977; Feick and Drews, 1978). The molecular weight of the polypeptide was determined by polyacrylamide-gel electrophoresis to be 10,000 to 14,000 (Table 2), but it might be higher when calculated on the basis of amino-acid composition (Tonn et a/., 1977). The polypeptide does not contain cysteine residues. The polarity of the amino acids is approximately 40% (Table 2). It was concluded, from molecular-weight determinations, that, in vivo, two molecules of Bchl and two polypeptides are associated to form a dimer (Sauer and Austin, 1978). b. Rhodopseudomonus type. Rhodopseudomonas sphaeroides, Rp. capsulata, Rp. palustris, and presumably all the other Rhodopseudomonas and Rhodospiriltum species (except for Rs. rubrum), contain two light-harvesting Bchlcarotenoid-protein complexes. The first one is light-harvesting Bchl I = B 875 (already described). The second one (light-harvesting Bchl 11; Lien eta/., 1973) is characterized by infra red absorption maxima at 800-805 nm (B 800) and 850-855 nm (B 850; Fig. 7). The native complex has been isolated from membranes of Rp. cupsulara after lauryl dimethylamine oxide solubilization, sucrose density-gradient separation, and chromatography on hydroxyapatite and DEAE cellulose (Feick and Drews, 1978). Different numbers of polypeptides have been shown to be constituents of the complex. The B 800-850 complexes of all strains of Rp. capsulata so far investigated contain three polypeptides with molecular weights of 14,000, 10,000 and 8000, respectively (determined by sodium dodecyl sulphate-polyacrylamide-gel electrophoresis, Fig. 6). Only the 10,000 and the 8000 molecular-weight polypeptides are associated with Bchl (Feick and Drews, 1978). The complex of Rp. palustris contains two polypeptides of apparent molecular weights 9000 and 1 1,000 (Fig. 6; Firsow and Drews, 1977). Data on the B 800-850 complex from Rp. spharroides are contradictory. Until recently, one polypeptide of apparent molecular weight 9000-10,000 (Fraker and Kaplan, 1972; Huang and Kaplan. 1973; Clayton and Clayton, 1972; Sauer and Austin, 1978), or two polypeptides of apparent molecular weight 9000 and 12,000 (Moskalenko and Erokhin, 1978). have been discovered (Fig. 6). The presence of two polypeptides in the B 800-850 complexes has been supported by comparison of results from spectroscopic data and isoelectric focusing (Cogdell and Thornber, 1979; Cogdell et a/., 1976). A molar ratio of Bchl to carotenoid close to 3 : 1 was found in the B 800-850 complex of wild-type strains of Rp. sphaeroides (Cogdell, 1978; Cogdell and Crofts, 1978; Cogdell and Thornber, 1979) and Rp. capsulata (Feick and Drews, 1978). Austin (1976) selectively removed the 800 nm-absorbing component from the light-harvesting Bchl I1 complex of Rp. sphueroidcs. The remaining 850 nm band still contained a pair of excitation-coupled Bchls.

ORGANIZATION AND DIFFERENTIATION OF MEMBRANES

25

Absorption and circular dichroism spectra were consistent with the presence of a strongly interacting pair of Bchl molecules (a dimer) in the B 850 component (Sauer and Austin, 1978). The fourth derivative of the infrared absorption spectrum of the light-harvesting Bchl I1 complex of Rp. sphaeroides showed two spectral forms in the 850 nm peak, whereas only one was present in the 800 band (Cogdell and Crofts, 1978). It was concluded that two molecules of Bchl gave rise to the 850 nm absorbance and one contributed the 800 nm peak. The fourth derivative of the infrared absorption spectrum of the B 800-850 Bchl complex of Rp. capsulata showed only one spectral form in both the 800 and 850 nm absorption bands (Talsky et al., 1980). This, however, does not contradict the idea that two molecules of Bchl are associated with the B 850 moiety, whereas the carotenoid and the third Bchl molecules are associated with the B 800 moiety, supposing that the two Bchl molecules of the B 850 moiety are close together and in the same surroundings. The Bchl molecules of the B 800-850 complex of Rp. capsulata are associated with two polypeptides of apparent molecular weights 8000 and 10,000, which are hydrophobic (the polarity of the amino acids is about 35%; J. Shiozawa and G. Drews, unpublished observations). Degradation of the 8000 molecular-weight polypeptide by trypsin treatment of whole membranes was accompanied by a proportional loss of absorbance at 800 nm. By contrast, the absorption peak at 855 nm and the content of the 10,000 molecular-weight polypeptide were, on trypsin treatment, stable for a longer time but were also lost simultaneously (Feick and Drews, 1979). Moreover, fluorescence emission spectra support the idea that two interacting Bchl molecules, bound to the heavy protein, are responsible for the flourescence emission. Separation of the B 800 and B 850 components with conservation of the absorption spectrum in vivo could not be obtained (Feick and Drews, 1978; Sauer and Austin, 1978). Although the pigment molecules seem to be bound separately to two polypeptides, protein-protein interaction seems to be important for stabilization of the absorption spectrum in vivo. The smallest subunit of the whole complex in Rp. capsulata in vivo seems to be an oligomeric form with a total molecular weight of approximately 170,000 (J. Shiozawa and G. Drews, unpublished observations). c. Chromatium type. Chromatium vinosum, which is the only known representative of this group, exhibits a complicated infrared absorption spectrum, which is due to the occurrence of shifts in the position of an absorption band. This organism forms a light-harvesting moiety, which is comparable to lightharvesting Bchl I of Rhodopseudomonas sp., with an absorption band at 880 nm. The second light-harvesting complex, on the other hand,, exhibits absorption bands of comparable magnitude at 800 and 850 nm when derived from cells grown at high-light intensity (auto- and mixotrophically) or high

26

G. DREWS AND J. OELZE

temperatures; when derived from cells grown at low light intensities or low temperatures, this second complex exhibits an absorption maximum at 800 nm with shoulders at 820 and 850 nm (Thornber, 1970; Ke and Chaney, 1971; Mechler and Oelze, 1978b, c). In any case, on treatment with Triton X-100, the absorption band at 850 nm is shifted towards 820 nm (Suzuki et al., 1969; Mechler and Oelze, 1978~).In spite of different absorption properties, the second light-harvesting Bchl complexes of Chr. vinosunz invariably show three polypeptide subunits of molecular weights 14,000, 1 1,000 and 8000, respectively, after electrophoresis on sodium dodecyl sulphate-polyacrylamide gels. The first spectrally invariable light-harvesting complex (B 880) of Chromatium sp. is associated with one polypeptide of apparent molecular weight 10,000 (Mechler and Oelze, I978b). d. Chlorobium type. This type seems to be present in all members of the Chlorobiaceae and Chloroflexaceae. In green bacteria, the ratio of total Bchl to reaction-centre Bchl is about ten-fold higher than in purple bacteria, i.e. Chlorobium sp. forms approximately 1000 molecules Bchl c per reaction centre (Fowler el al., 1971). This high ratio is one indication that lightharvesting Bchl complexes in green bacteria must be organized differently from in purple bacteria. Light-harvesting Bchl complexes of green bacteria fall into two categories. First, the water-soluble crystallizable Bchl a-protein complex (Olson, 1978), and secondly, the Bchl c, d o r e, and the bulk carotenoids which are localized in the chlorosomes. Analytical data are only available for Chlorobiurn limicola. The Bchl a-protein from Chlorobium limicola is a trimer. Each subunit contains seven Bchl molecules, and has a total molecular weight of approximately 46,000, corresponding to a trimer weight of approximately 140,000. All data on amino-acid composition, crystal properties and spectral properties have been recently compiled (Olson, 1978). The possible localization of the Bchl a-protein will be discussed later in this article. Chlorosomes have been isolated and analysed as relatively crude preparations. Data on composition vary considerably (Sykes et al., 1965; Schmitz, 1967; Cruden and Stanier, 1970): 17-63% protein, 9-18% lipids (much monogalactosyldiglyceride), 6.8-27.6% Bchl c, and 9-38% per dry weight of carbohydrate. It is unknown whether Bchl c is associated with protein as in the membrane-bound complexes (Bch1:protein ratios from 1 :9 to 1 : 1.2 were 0,bserved).The rod elements found in chlorosomes by freeze-fracture electron microscopy were interpreted to be Bchl c-protein-containing structures (Staehelin et al., 1978, 1980).

ORGANIZATION AND DIFFERENTIATION OF MEMBRANES

27

3. Coupling-Factor A TPases According to the chemi-osmotic hypothesis, the protonmotive force, which is formed across the membrane as a consequence of electron transport, drives ATP synthesis by membrane-bound coupling-factor ATPase. Coupling factors that exhibit ATPase (ATP-hydrolysing) activities can be operationally subdivided into two complexes, designated Fo and F1. The FOcomplex is a largely hydrophobic moiety embedded in the membrane, whereas FI is hydrophilic and is localized at the membrane surface. Mitochondria Fo and F 1 complexes are connected to each other by a protein that confers oligomycin sensitivity to the coupling factor. In phototrophic bacteria, except for Chr. vinosum (Gepshtein and Carmeli, 1974), membrane-bound coupling factors exhibit oligomycin sensitivities, although it is known that coupling factors of non-phototrophic prokaryotes are generally insensitive towards oligomycin (Haddock and Jones, 1977). A further characteristic of membrane-bound coupling factors is their activation by Mg2+ as well as by Ca2+ ions. Water-soluble ATPase (F1) preparations have been obtained from membranes of Rp. capsulata, Rs. rubrum, Rp. sphaeroides and Chr. vinosum. Adenosine triphosphatases (F,) from Rs. rubrum and Chr. vinosum were separated into five polypeptide subunits ( a , p, y , 6, E ) after treatment with sodium dodecyl sulphate followed by electrophoresis on polyacrylamide gels (Johansson and Baltscheffsky, 1975; Gepshtein and Carmeli, 1978). The presence of subunits a, p, 7, and 6 was also reported for ATPase preparations from Rp. sphaeroides (Jolchine, 1977). In the case of Rs. rubrum ATPase, the following molecular weights have been determined; ATPase complex (FI ), 350,000; subunits: a, 54,000; p, 50,000; y , 32,000; 6, 13,000; E , 7500 (Johansson and Baltscheffsky, 1975; Lucke and Klemme, 1976). Philosoph et al. (1977) reported selective solubilization of the p subunit by lithium chloride treatment of membranes from Rs. rubrum. Neither the isolated /3 subunit nor the residual ATPase depleted in this subunit exhibited ATPase activity. Re-incorporation of the p subunit, however, restored ATPase as well as photophosphorylation activities of lithium chloride-depleted membrane preparations. This indicated that the /3 subunit of ATPase ( F I )is essential for the function of coupling factors. All of the water-soluble ATPase ( F I ) fractions obtained so far do not exhibit sensitivities towards oligomycin (Baccarini-Melandri and Melandri, 1971; Johansson et al., 1973; Lucke and Klemme, 1976; Jolchine, 1977). Except for Chr. vinosum, oligomycin sensitivity could be restored, however, after re-incorporation of the solubilized ATPase into the ATPase-depleted membranes (Johansson et al., 1973; Jolchine, 1977; Baccarini-Melandri and

G. DREWS AND J. OELZE

28

Melandri, 1978). Interestingly enough, it was demonstrated that ATPase (F1) preparations from chemotrophically grown cells of Rp. capsulata can reconstitute photophosphorylation when incorporated into ATPase-depleted and, therefore, uncoupled chromatophores. Vice versa, ATPase from photosynthetically active chromatophores can lead to oxidative phosphorylation (Baccarini-Melandri and Melandri, 1971; Melandri et al., 1971). Differences in the properties of membrane-bound and solubilized ATPases could also be registered with respect to their Caz+ and Mg2+ activities. Adenosine triphosphatase preparations from Rs. rubrum possessed high Ca2+-dependent activities and, if at all, rather low Mg2+-dependent activities (Johansson et al., 1973; Liicke and Klemme, 1976; Melandri and BaccariniMelandri, 1976). In contrast to this, ATPases solubilized from Chr. vinosum and Rp. sphaeroides exhibited low Ca2+- and Mg2+-dependent activities (Gepshtein and Carmeli, 1977; Jolchine, 1977). Both activities could be stimulated if the ATPase from Chr. vinosum was treated with trypsin (Gepshtein and Carmeli, 1977). Coupling-factor ATPases, exhibiting properties comparable to those of complete membranes, have been isolated by treatment with Triton-X- 100 (Gromet-Elhanan and Oren, 1977; Oren and Gromet-Elhanan, 1977; Schneider et al., 1978). These preparations exhibited CaZ - as well as Mg2 dependent activities. In addition, oligomycin inhibited both activities. However, CaZ -dependent activity of the detergent-solubilized ATPase was more resistant towards oligomycin than that of membrane-bound ATPase (Oren and Gromet-Elhanan, 1977). Moreover, NN’-dicyclohexylcarbodiimide, which inhibits membrane-bound ATPases of mitochondria, Escherichia coli and Rs. rubrum, and which does not inhibit the F, complex, also exerts an inhibitory effect on the detergent-solubilized ATPase from Rs. rubrum (Gromet-Elhanan and Oren, 1977; Haddock and Jones, 1977). Sodium dodecyl sulphate-polyacrylamide-gel electrophoresis reveals the presence in detergent-solubilized ATPases of at least thirteen different protein subunits including those of the water-soluble ATPas (F1). The results reported above indicate that it is possible to solubilize selectively and isolate from membranes the subunit p, complex F1 as well as a complex with properties comparable to those of complete membrane-bound coupling factors (Baccarini-Melandri and Melandri, 1978). It should be noted, however, that only the two heaviest subunits (a and 8) of ATPase can be localized within the protein patterns of Rs. rubrum membranes obtained after solubilization with sodium dodecylsulphate followed by polyacrylamide-gel electrophoresis (Oelze, 1978). +

+

+

ORGANIZATION AND DIFFERENTIATION OF MEMBRANES

29

4. Constituents of Electron-Transport Chains and Other Isolated Functional Subunits Although many electron-transport carriers were identified and characterized, solubilization and isolation from membranes of functional subunits was seldom successful. Succinate dehydrogenase has been solubilized and purified from Rs.rubrum chromatophores (Hatefi et af., 1972). The enzyme contains eight g-atoms of iron and eight mol of acid-labile sulphide per mol of flavin. The flavin is covalently linked to the larger subunit (molecular weight 60,000). Both the large and the small (molecular weight 25,000) subunits contain ironsulphur proteins (Davis et al., 1977). As with other solubilized succinate dehydrogenases, two iron-sulphur centres are of the ferrodoxin type which are paramagnetic in the reduced state, and one iron-sulphur centre of the high-potential type that is paramagnetic in the oxidized state (Carithers et al., 1977). An additional iron -sulphur centre of the ferredoxin type was identified by electron paramagnetic resonance spectroscopy. Potentiometric titrations of the enzyme showed three components with midpoint potentials of about +50 mV, -160 mV, and -380 mV (centre 4) (Carithers et a f . , 1977). Ferredoxin I11 ( - 3 14 mV, n = 1; molecular weight 8500) and ferredoxin IV (two Fe4-S4 clusters, with oxidation-reduction potentials of 355 mV and - 380 mV, respectively, g = 2.01, molecular weight 14,000) were isolated and purified (Yoch et al., 1977). Ferredoxin-like centres (+50 mV, - 250 mV and + 80 mV) were removed together with succinate dehydrogenase activity by a single alkaline wash from chromatophores of Rp. sphaeroides (Ingledew and Prince, 1977). A light membrane fraction was isolated from Rp. sphaeroides which was enriched in NADH
+

G. DREWS AND J . OELZE

30

with apparent molecular weights of 30,500, 25,500, 12,200 and 9500, were identified (King and Drews, 1976). A NAD reductase, which was shown to be loosely bound to the membrane of Prosthecochloris aestuarii, was isolated and purified (Shioi et al., 1976). The enzyme was a FAD-containing flavoprotein with a molecular weight of 119,000. Artificial electron donors used were photoreduced spinach ferredoxin or reduced benzylviologen. The enzyme also catalysed reduction of cytochromes and dyes such as benzylviologen and 2,6-dichlorophenolindophenol with NADH or NADPH as the electron donor. Energy-linked transhydrogenase activity (NADH -+ NADPI) is bound to chromatophores of Rs. rubrum (Keister and Yike, 1967). The enzyme is comprised of a membrane-bound component and an easily dissociable soluble factor. The soluble transhydrogenase factor stimulates transfer of hydrogen catalysed by the insoluble membrane component (Fisher and Guillory, 1971). An active transhydrogenase complex has been solubilized by treatment of chromatophores with lysolecithin (Jacobs et al., 1977). Protection was afforded by NADP+ and NADPH to membrane-bound components. The pyruvate dehydrogenase complex of Rs. rubrum is associated with membranes from cells grown chemotrophically or phototrophically (Luderitz and Klemme, 1977). Membrane-bound hydrogenase, catalysing both oxidation and evolution of molecular hydrogen with suitable mediators, have been solubilized by detergents and purified from Rs. rubrum (Adams and Hall, 1977), Chromatium vinosum (Gitlitz and Krasna, 1975; Kakuno et al., 1977) and Thiocapsa roseopersicina (Gogotov et al., 1976). The enzymes from Rs. rubrum and Chr. vinosum are relatively insensitive to oxygen, and Rs. rubrum hydrogenase is a thermostable (120 min, 70 C ) iron-sulphur protein (4Fe-4s; 4g-atoms F e per 65,000 g of protein). This hydrogenase catalyses evolution of molecular hydrogen from reduced benzylviologen, methylene blue, Janus green, FMN and Rs. rubrum ferredoxin, but not from other ferredoxins or NADH. The literature on membrane-bound iron-sulphur centres in photosynthetic bacteria has recently been reviewed (Malkin and Bearden, 1978). The hydrogenase from Chr. vinosum failed to react with ferredoxin from any sources tested. Hydrogen evolution driven by sodium dithionite was mediated by methylviologen and stimulated by FMN. The molecular weight was approximately 70,000 (oligomer) (Kakuno et al., 1977). +

C . T O P O G R A P H Y OF I N T R A C Y T O P L A S M I C M E M B R A N E S

The function of membrane-bound electron-transport chains, i.e. formation of an electric field and a proton gradient across the membrane, which finally

ORGANIZATION A N D DIFFERENTIATION OF MEMBRANES

31

leads to formation of ATP or the enabling of energy-dependent transport processes across the membrane, presupposes an asymmetrical arrangement of the functional units within the membrane. The observed influx of protons into intracytoplasmic membrane vesicles and the efflux of protons out of whole cells and membrane-ghost preparations (Scholes et al., 1969; Hochman et al., 1975; Michels and Konings, 1978) confirm the expected asymmetry. Analyses of electron-transport processes and isolation of functional subunits from the membrane indicate that these units are multipolypeptide complexes in the membranes. In this section, we will summarize results of studies on localization of subunits within the membrane (Fig. 8). Measurement of proton movement was used to control the integrity of vesicle membranes in experiments to study the topography of chromatophore membranes (Oelze, 1978). It should be kept in mind that membranes are dynamic systems. Membrane constituents can move laterally and perpendicularly to the plane of the membrane (Staehelin et al., 1978). However, lipid-protein associations in chromatophores immobilize about 60% of the negatively charged lipids (Birrell et al., 1978). The knob-like particles (diameter 9 nm), visualized on the cytoplasmic surface of cytoplasmic and intracytoplasmic membranes (Low and Afzelius, 1964; Lampe et al., 1972; Reed and Raveed, 1972), can be washed from the chromatophore membrane of Rp. sphaeroides with EDTA or Triton X-100 (Reed and Raveed, 1972). These 9 nm-diameter particles were isolated and purified from the wash solution. They showed ATPase activity which was lost from the membrane during the washing process. It was concluded that the knob-like 9 nm-diameter particles on the surface of the cytoplasmic membrane were identical to the ATPase (F1; Reed and Raveed, 1972). These particles seemed to inhibit sterically attachment of ferritin-labelled antireaction-centre antibodies to reaction-centre sites exposed on the membrane surface (Reed et al., 1975). It was concluded that reaction centres are localized below ATPase in the membrane (Reed et al., 1975). As might be expected, ATPase of Chlorobium thiosuljatophilum has been localized in the cytoplasmic membrane but not on chlorosomes (Burns and Midgley, 1976). The a- and p- subunits of coupling-factor ATPase (molecular weights 55,000 and 51,000) in chromatophores of Rs. rubrum were accessible to digestion with trypsin and chymotrypsin as well as to labelling by enzymic iodination (Oelze, 1978). Consequently, at least these subunits were exposed at the cytoplasmic surface of the membranes. Photochemical reaction centres (Section III.B.1, p. 18) have a total molecular weight of approximately 150,000 (Drews, 1978; Clayton and Clayton, 1978a). The pigment, i.e. the Bchl dimer, bacteriophaeophytin and carotenoid molecules, are bound to the medium (M) and light (L) polypeptides, at least in Rp. sphaeroides and R p . capsulata (Okamura et al., 1974;

32

G. DREWS AND J. OELZE

FIG. 8. Model for the topography of photosynthetically active intracytoplasmic membranes. (a) Scheme for an electron micrograph of a thin-sectioned representative (e.g. Rhodospirillum rubrum, Rhodopseudomonas sphaeroides or Rhodopseudomonas capsdata) of the phototrophic bacteria grown phototrophically. The figure demonstrates intracytoplasmic membranes ( E M ) which can be seen in micrographs. It is not possible to derive information from electron micrographs on the degree of physical contact between cytoplasmic (CM) and intracytoplasmic membranes. CW indicates cell wall. (b) is a model showing the grosscomposition of intracytoplasmic membranes (Oelze and Drews, 1972), indicating components representative of the respiratory and those characteristic of the photosynthetic apparatus (0). Note that chain the respiratory chain is contained largely in the cytoplasmic membrane, whereas intracytoplasmic membranes contain predominantly the photosynthetic apparatus. (c) is a model of the spatial arrangement of functional units in intracytoplasmic membranes. The mobility of acyl chains of phospholipids and by this the fluidity of the lipid bilayer of the membrane is greatly lowered by proteins embedded in the membrane (Fraley et a/., 1978; Birrell e t a / . , 1978). Proteins and polypeptides exposed at the cytoplasmic face of intracytoplasmic membranes: coupling factor ATPase ( F I ) (Reed and Raveed, 1972; Oelze, 1978), invariable light-harvesting complex (B 875) (Cuendet et al.. 1978; Oelze, 1978), heavy (H) subunit of photochemical reaction centres (Ziirrer et a / . , 1977; Feher and Okamura, 1978a; Oelze, 1978) succinate (Succ. DH) and N A D H dehydrogenase (NADH-DH) (Oelze, 1978, and unpublished observations). Proteins and polypeptides exposed at the periplasmic (external) face

(a)

ORGANIZATION AND DIFFERENTIATION

OF MEMBRANES

33

Nieth et al., 1975; Feher and Okamura, 1978b). Binding of ferritin-labelled anti-LM-antibodies at both surfaces of cytoplasmic and intracytoplasmic membranes was interpreted as indicating that reaction centres of Rp. sphaeroides span the membrane, whereas the heavy (H) subunit was only accessible to antibodies on the cytoplasmic side of the membrane (Feher and Okamura, 1978b). In contrast to these results, only the H polypeptide of reaction centres was labelled by enzymic iodination with isolated chromatophores of Rs. rubrum (Zurrer et al., 1977; Oelze, 1978). The same polypeptide was cleaved by trypsin and a-chymotrypsin (Oelze, 1978). It is concluded that the H polypeptide, but not the L and M polypeptides, is largely exposed on the cytoplasmic surface of the membrane. The H but not the L and M subunits of reaction centres from Rp. sphaeroides were susceptible to digestion with pronase. Pronase-treated chromatophores were fully active photochemically. It is concluded that only the H subunit is significantly exposed on the outer chromatophore surface, and that the H subunit is apparently unnecessary for primary photochemistry in situ (Hall et al., 1978). Cytochrome c2 is located in vivo in the periplasmic space or in the extracytoplasmic space of intracytoplasmic vesicles of Rp. sphaeroides and Rp. capsulata (Hochman et al., 1975; Prince et al., 1975). Cytochrome cz binds to the L and M subunits of reaction centres of Rp. sphaeroides (Feher and Okamura, 1978b). These results support the conclusions, derived from labelling experiments with ferritin-bound anti-LM antibodies (Feher and Okamura, 1978b), that L and M subunits of reaction centres reach the external surfaces of cytoplasmic and intracytoplasmic membranes of Rp. capsulata and Rp. sphaeroides. Reed and Raveed (1972) proposed that 12 nm-diameter particles, visualized on the fracture faces of membranes of Rp. sphaeroides, are composed of reaction centres aggregated with other components of the photosynthetic apparatus. However, comparative studies have shown that the population of integral membrane particles must be heterogeneous. Particles of similar of intracytoplasmic membranes include: light and intermediate subunit of reaction centres (Feher and Okamura, 1978a), cytochrome cz (Baccarini-Melandri et al., 1978) light-harvesting complex (B 800-850) (J. J . Shiozawa, unpublished observations). This simplified version of the photochemical electron-transport chain is adapted from Prince and Dutton (1978). ----, represents a path of the respiratory electrontransport chain which includes cytochrome c2 (Baccarini-Melandri et al., 1978). It remains to be seen if this cytochrome c2 is independent of or identical with the respective cytochrome coupled to the reaction centre. The model does not take into account branching of the respiratory chain that has been reported (see Fig. I , p. 5). Note that sizes of units and constituents shown are not to scale. Abbreviations: reaction-centre-Bchl-dimer, P 865; bacteriophaeophytin, Bph; ubiquinone, Q; hypothetical carrier, Z; cytochrome cz, cz; cytochrome b. Cyt b; flavoprotein, FP; ironsulphur protein, FeS; terminal oxidase, Cyt 0.

34

G. DREWS AND J OELZE

diameter have been observed both in membranes from cells of Rp. capsulata grown chemotrophically (free of Bchl) and phototrophically (high Bchl content) (Golecki et al., 1979). Thus, functionally different subunits of the membrane can form integral membrane particles of similar-size classes. Nevertheless, variable percentages of the population of integral membrane particles in Rp. capsulata seem to consist of photosynthetic units, because the number of photosynthetic units and the number of integral membrane particles correlate, i.e. they increase or decrease co-ordinately after induction or repression of formation of the photosynthetic apparatus of Rp. capsdata (Golecki et a[., 1979). Supposing that, in Rp. capsdata, photosynthetic units form integral membrane particles of approximately 10 nm in diameter, there must be an explanation as to why the diameter of the particles is relatively constant under different culture conditions while the size of the photosynthetic unit changes. Light-harvesting Bchl (B 875) and reaction-centre Bchl, plus the polypeptides that belong to these complexes, have always been synthesized in a molar ratio of approximately 25 : 1 (Lien et af., 1973; Schumacher and Drews, 1978). the total molecular weight of which amounts to about 450,000. A particle of this molecular weight may very well exhibit a diameter of about 10 nm. The probability that such a particle, consisting of light-harvesting Bchl I and reaction-centre complexes, did exist is supported by (i) the observation of excitation energy transfer from light-harvesting Bchl I1 (B 800-850) to reaction centres via B 875 (Monger and Parson, 1977), as well as by the findings that (ii) the whole complex can be solubilized by detergent treatment and enriched by sucrose density-gradient centrifugation (Nieth et al., 1975; Firsow and Drews, 1977; Mechler and Oelze, 1978b), and (iii) that the constituents of the whole complex are cosynthesized. The light-harvesting antenna Bchl I1 complexes are supposed to be localized in between the membrane particles, and connect them when these cells are cultivated under phototrophic conditions (Monger and Parson, 1977; Drews, 1978; Pradel et a[., 1978).The heavy polypeptide (apparent molecular weight 14,000; polarity 44% of the light-harvesting Bchl complexes I1 of Rp. capsulata, which is not associated with Bchl, is readily digested by low concentrations of trypsin and chymotrypsin without impairing the integrity of the membrane, whereas the smaller hydrophobic polypeptides are digested slowly when membranes are incubated with high concentrations of trypsin or chymotrypsin (Feick and Drews, 1979). Presumably, the heavy polypeptide is exposed on the cytoplasmic membrane surface. The orientation of bulk pigments in the chrotnatophores was studied by measuring linear dichroism spectra of oriented membrane samples prepared from different photosynthetic bacteria. The far-red Qy transition moment of Bchl molecules in membranes of Rp. palustris and Rp. sphaeroides was

ORGANIZATION AND DIFFERENTIATION OF MEMBRANES

35

oriented nearly parallel to the plane of the membrane, and the Qx transition moment (590 nm) was tilted out of the membrane plane at an angle greater than 45'. The transition moment of the carotenoid molecules was shown to point out of the membrane plane (Breton, 1974). In membranes of Rp. viridis, the antenna Bchl molecular plane was nearly perpendicular to the membrane. The Qy and Qx transition moments of these molecules made respective angles of 20 and 70 with the membrane plane. The antenna carotenoid molecules made an angle of 45 with the membrane. The 970-nm transition moment of the primary electron donor was parallel to the membrane plane, and the 850-nm transition was tilted out of the plane (Paillotin et af., 1979). An orientation of reaction-centre and light-harvesting Bchl and carotenoid molecules relative to each other, and to the plane of the membrane, was observed in different organisms (Morita and Miyazaki, 1978; Vermeglio et al., 1978). Since the pigment molecules were associated with proteins, the pigment complexes must have been oriented within the membrane. The orientation of pigment-protein complexes seems to have been influenced by lipid-protein and protein-protein interactions (Sandermann, 1978). The enzymes of the respiratory chain, especially the dehydrogenases, are thought to be in the cytoplasmic leaflet of the membrane system (Drews, 1978 and unpublished results; Ingledew and Prince, 1977). Trypsin or achymotrypsin completely cleaves the heavy subunit of succinate dehydrogenase (apparent molecular weight 64,000) of Rs. rubrum. This protein was also labelled by enzymic iodination. It is concluded that succinate dehydrogenase is exposed on the cytoplasmic surface of intracytoplasmic membranes (Oelze, 1978). The localization of other participants of electron-transport chains in membranes of photosynthetic bacteria has not as yet been determined. The supramolecular structure of the cytoplasmic membrane and their attached chlorosomes (Staehelin et af., 1978, 1980), and the composition of the pigment complexes of green bacteria (Olson, 1978; Pierson and Castenholz, 1978), have been described in Sections 1II.A and B (pp. 9 and 16). An interpretative model of a chlorosome and the attached part of cytoplasmic membrane is discussed (Fig. 3, p. 13). It is proposed that the Bchl c is complexed with protein(s) and localized in the rod elements of chlorosomes. Whether the 10-nm rod elements are related to the 10-nm diameter ring-like structures, as described by Cruden and Stanier (1970) in the isolated chlorosomes, is unknown. However, the fact that the number and size of the two structures coincide, suggests that there may be more than a causal relationship between the two. Since the core is often less resistant to fracture cleavage than the adjacent cytoplasmic membrane, and negative stains are excluded from the interior of the chlorosomes (Holt et al., 1966), a hydrophobic nature of the chlorosome core is suggested. The boundary layer between the chloro-

36

G. DREWS AND J. OELZE

some and the cytoplasmic membrane is formed by a plate-like structure with a crystalline substructure (Staehelin ef al., 1980). Since Bchl a-protein complexes aggregate into crystals and function as an intermediary in transfer of excitation energy from the light-harvesting Bchl c to the photochemical reaction centre, it is most likely that Bchl a-protein complexes form a planar lattice configuration in the crystalline baseplate. The large particles in the attachment sites of chlorosomes to the cytoplasmic membrane may correspond to complexes containing reaction centres.

IV. Differentiation of the Cellular Membrane System As detailed above (Section 11, p. 4), many members of the phototrophic bacteria are able to adapt to different types of energy metabolism, i.e. to phototrophy or chemotrophy. In addition, as a response to changing conditions, a given energy metabolism may also be adapted in its efficiency which, within limits, means in the relative proportions of the various constituents involved. Both phototrophic and chemotrophic energy metabolisms are coupled to functionally separate electron-transport pathways which are contained either in identical or in cytologically distinct parts of the cellular membrane system (Sections I and 111, pp. 1 and 9). This means that any changes in the ratio of the photochemical and respiratory electrontransport chains or even in the composition of one or the other of them can be considered as differentiation of the entire cellular membrane system and, consequently, as differentiation of the cell. In this context, the term differentiation is used in the sense of developmental physiology to describe structural and functional alterations of a living system. Thus, from the standpoint of developmental biology, phototrophic bacteria provide excellent and also experimentally convenient systems to study several aspects of cellular differentiation on the basis of membrane differentiation. In particular, the process of adaptation of chemotrophic cells to phototrophic conditions has stimulated a good deal of research activities. This can be related mainly to the fact that the acquisition of photosynthetic activities by bacteria is considered an essential step in the development of life on earth but also, less pretentiously, that the inducible formation of bright pigments by the originally colourless chemotrophic cells is a rather spectacular process. Only recently have studies been initiated on differentiation of phototrophically growing cells including differentiation of the photosynthetic apparatus. Accordingly, far more information is available on the formation of the photosynthetic apparatus than on its differentiation. Nevertheless, it should be realized that there is every likelihood that differentiation of phototrophically grown cells is physiologically much more relevant to organisms living on their natural environment

ORGANIZATION AND DIFFERENTIATION OF MEMBRANES

37

than is formation of the photosynthetic apparatus de novo. Developmental processes always include steps of differentiation. However, for the sake of clarity, we will use the unorthodox term “formation”, as far as possible, for those processes leading to an acquisition of structures and functional systems, while the term “differentiation” will be used to describe alterations of structures and functional systems already present.

A. B I O G E N E S I S O F T H E P H O T O S Y N T H E T I C A P P A R A T U S

1. Formation of Constituents

a. Bacteriochlorophyll. In Section IIIB (p. 16) of this article. it was shown that different members of phototrophic bacteria may produce spectrally different Bchl derivatives and, moreover, that one single derivative associated with different polypeptides also exhibits different absorption properties. Individual pathways involved in the formation of spectrally distinct Bchl derivatives or polypeptide complexes are far from known. So far, research activities in this field have been concerned almost exclusively with clarifying the pathway leading to Bchl a. However, it is likely that the initial steps in formation of magnesium tetrapyrroles are identical. Information on intermediates in Bchl a production have been derived mainly from studies with either pigment mutants or specific inhibitors affecting Bchl synthesis (Lascelles, 1968, 1975, 1978; Pudek and Richards, 1975; Saunders, 1978). In the context of this review, we do not want to describe present knowledge on the pathway of Bchl synthesis; the interested reader is referred to relevant reviews (Lascelles, 1968, 1978; Sandy et al., 1975; Jones, 1978). Instead, we would like to focus on data obtained on regulation of Bchl synthesis. It can be concluded that Bchl synthesis is regulated on the basis of enzyme activities as well as on the basis of enzyme synthesis. This follows from findings that after transfer of cells to the appropriate conditions, (i) Bchl production starts or stops immediately, (ii) key enzymes involved in Bchl synthesis can be repressed or de-repressed and (iii) blockage of transcription with actinomycin inhibits Bchl synthesis (Biedermann et al., 1967). Studies on enzyme levels suffer from the instability of the enzymes concerned. Key enzymes in Bchl synthesis which are subject to regulatory processes have been described as 8-aminolaevulinate synthase (EC 2.3.1.37) and dehydratase (EC 4.2.1.24). Both enzymes initiate the tetrapyrrole biosynthetic pathway. In addition, enzymes that appear directly after the branching point separating the magnesium from the iron-tetrapyrrole pathway, i.e. magnesium chelatase and methyltransferase, are thought to be regulated (Lascelles, 1978).

G. DREWS AND J. OELZE

38

Numerous studies have shown that intracellular levels of Bchl depend mainly on the oxygen tension (Lascelles, 1959; Cohen-Bazire and Kunisawa, 1960; Higuchi and Kikuchi, 1963; Biedermann et al., 1967; Dierstein and Drews, 1974). Oxygen tension influences both synthesis and activity of key enzymes, and also effects synthesis of proteins that form complexes with Bchl (Sections I11 and IV.B, p. 9 and p. 53). It seems to be clear that oxygen tension regulates Bchl synthesis indirectly, perhaps by influencing the redox state of an effector molecule which triggers the signal chain (Fig. 9). Indeed, low-molecular-weight activators of 6-aminolaevulinate synthase have been isolated and identified as cystine trisulphide and glutathione trisulphides (Davies et al., 1973; Neuberger et al., 1973a, b; Sandy et al., 1975).

p

High activity enzvme

Reduced trisulphide

Oxidized trisulphide

Low activity enzyme

-scz>/14

i

--+ Oxygen

: i p

Light

FIG. 9. Model for regulation of 8-aminolaevulinatesynthase (enzyme) by trisulphides. From Davies et al. (1973).

Hayasaka and Tuboi (1974) described four forms of 6-aminolaevulinate synthase which differed in activity. For activation, an enzyme and L-cystine were required. Disulphide-bond formation within the S-aminolaevulinate synthase was proposed as the mechanism of activation (Hayasaka and Tuboi, 1974) (Fig. 9). Besides activation and inactivation triggered by oxygen tension, feed-back inhibition of 6-aminolaevulinate synthase by hemin and Mg-protoprophyrin (Yubisui and Yoneyama, 1972; Hayasaka and Tuboi, 1974), and induction and repression of enzyme synthesis (Lascelles, 1964; Drews, 1965; Drews and Oelze, 1966) have been shown to be responsible for rates of tetrapyrrole production. The multi-enzyme complex magnesium protoporphyrine chelatase and methyl transferase also exhibits sensitivity towards higher oxygen tensions (Gorchein, 1972). The Bchl molecule is immediately associated with one or more specific proteins of the photosynthetic apparatus. All precursor molecules are excreted when Bchl synthesis is blocked by mutation or because the specific proteins of the pigment complexes have not been synthesized (Lascelles, 1968;

ORGANIZATION AND DIFFERENTIATION OF MEMBRANES

39

Oelze el al., 1970; Drews et al., 1971, 1976). The formation of reaction-centre and light-harvesting Bchl complexes is strictly dependent on sufficient production of both Bchl and protein moieties. This interdependency and the regulation of these complex biosynthetic processes are far from clear and require further study. Five clusters of mutations affecting carotenoid biosynthesis and two affecting Bchl biosynthesis, are arranged in one linkage group of the chromosome of Rp. cupsulata (Yen and Marrs, 1976). Genes for proteins, forming complexes with Bchl, seem to be included in this linkage group (Drews er al., 1976). Genes necessary for the formation of coloured carotenoids and of light-harvesting Bchl I1 proteins seem to be linked somehow (Drews et al., 1976; Yen and Marrs, 1976; Marrs, 1978). b. Cytochromes. Cytochromes participate in different light-driven or respiratory electron-transport chains (Section 11, p. 4; Bartsch, 1978). Some cytochromes share two electron-transport paths. It is not easy to determine the concentrations of cytochromes in complex mixtures such as membrane fractions or cell-free extracts (Bartsch, 1978). Midpoint oxidation-reduction potentials and their pH-dependence, absorption spectra and extinction coefficients are needed. Furthermore, some cytochromes are water-soluble and are eluted during isolation and purification of membranes. Thus, quantitative data on cytochromes in membranes are restricted primarily to groups of cytochromes such as c- or b-type cytochromes. The concentration of these cytochromes in membranes don’t vary more than 2-3-fold (King and Drews, 1975). Although single cytochromes clearly vary in their cellular concentrations under different growth conditions, the change in concentration of iron tetrapyrroles is much smaller than changes in Bchl concentration (Irschik and Oelze, 1976). The concentrations of iron in the medium and of hemin in the cell influence the cellular cytochrome level (Lascelles, 1978). Other regulatory mechanisms must exist to adapt the rate of cytochrome synthesis to the requirement of specific electron-transport subunits. c. Quinones. Quinones have been found in all photosynthetic bacteria studied (Parson, 1978). Although quinones are active in different electronand proton-carrier systems of photosynthetic and respiratory electrontransport chains, most phototrophic bacteria contain only one benzoquinone, primarily ubiquinone, and some produce an additional naphthoquinone. Most of the ubiquinone is present in large amounts in a pool localized in the hydrophobic interior of the membrane. The other quinone molecules are bound to specific systems in stoicheiometric amounts, e.g. 1-2 molecules of ubiquinone are bound to the M subunit of the reaction centre in Rp. sphaeroides (Feher and Okamura, 1978b), or to the succinate dehydrogenase of Rp. palustris (King and Drews, 1973).

40

G DREWS AND J. OELZE

Cellular levels of quinone vary with different culture conditions. Under phototrophic conditions, ubiquinone 10 and Bchl are formed concomitantly. Transfer of Rs. rubrum cultures grown chemotrophically to photosynthetic conditions leads to an increase of both pigments on a cellular protein basis but with different kinetics (Oelze et al., 1975a). Since quinones are active in both respiratory and photosynthetic electron-transport chains, the cellular concentration roughly follows the amount of membranes present per cell. d. Carotenoids. The photosynthetic apparatus of all wild-type strains contain carotenoids (Pfennig and Truper, 1974; Schmidt, 1978). They act as accessory light-harvesting pigments and protect Bchl from sensitizing the photo-oxidative killing which occurs in the presence of light and oxygen (Griffith et al., 1955; Sistrom et al., 1956; Krinsky, 1968; Cogdell, 1978). Carotenoids are also associated with reaction centres and antenna pigmentprotein complexes. The light quanta absorbed by the carotenoids are transferred to Bchl by a singlet-singlet energy transfer (Goedheer, 1959). In the presence of oxygen, the metastable state of Bchl produced by strong light is delayed via a triplet state of antenna carotenoids (Monger et a/., 1976). A stoicheiometric relationship between Bchl and carotenoids of 3 : 1 to 2 : 1 has been determined for some purple bacteria (Niederman et a/., 1976; Cogdell and Crofts, 1978; Cogdell and Thornber, 1979; Feick and Drews, 1978; Schumacher and Drews, 1978). A change of the molar ratio of carotenoids to Bchl seems to be correlated to variations in the size of the photosynthetic unit (Sistrom, 1978). The constant ratio of carotenoids to Bchl and protein in the isolated Bchlkarotenoid complexes is thought to be due to a stoicheiometric association of Bchl and carotenoids with the polypeptides of the pigment complexes. The chemistry and biosynthesis of carotenoids have been reviewed (Liaaen-Jensen, 1978; Schmidt, 1978). The molecular basis of regulation of carotenoid synthesis is unknown. e. Proteins. The major proteins of the photosynthetic apparatus are components of the pigment complexes in the membranes. Minor proteins are apoproteins of redox components, enzymes and adenosine triphosphatase (ATPase). Primary and secondary structures, distribution of polar and nonpolar domains, and charges in the polypeptides determine protein-protein and protein-lipid interactions and localization of the polypeptide complexes in the membrane (see Section 11, p. 4). Many polypeptides of the membrane are synthesized and covalently bound or non-covalently associated in stoicheiometric ratios with non-protein components. On the basis of generation time, the turnover rates of membrane lipids (Oelze and Drews, 1970a; Gorchein et a/., 1968) and of membrane proteins (Takemoto, 1974) are low. Accordingly, differentiation and growth of the membrane are determined from the rates of co-ordinated incorporation of membrane components. In synchronously dividing cells of Rp. sphaeroides proteins and pigments were

ORGANIZATION AND DIFFERENTIATION OF MEMBRANES

41

continuously incorporated into the intracytoplasmic membrane, whereas phospholipids were discontinuously incorporated (Lueking et al., 1978; Fraley et al., 1978). It is unknown whether membrane proteins of photosynthetic bacteria are synthesized on membrane-bound or free polyribosomes.

2. Assembly of the Photosynthetic Apparatus

a. Reconstitution experiments. The minimum requirements for transformation in vitro of the photosynthetically inactive and Bchl-free membranes from cells grown chemotrophically into photosynthetically active membranes have been studied independently by Jones and Garcia in collaboration with their respective colleagues. Incorporation of isolated reaction-centre Bchl complexes and coupling factors into membranes of a chemotrophically grown mutant of Rp. capsulata which, even under phototrophic conditions, was unable to form Bchl, resulted in the restoration of low but significant lightdependent activities for forming ATP and for reducing NAD+ with succinate (Garcia et a/., 1974, 1975). In addition, these processes were-shown to involve photo-oxidation of reaction-centre Bchl and photo-oxidation and photoreduction of endogenous c-type and b-type cytochromes, respectively. On the basis of the findings that membranes from cells of Rp. sphaeroides, grown strictly chemotrophically or phototrophically, contained the same cand 6-type cytochromes, Jones and his colleagues presumed that the systems for respiratory electron and the photosynthetic electron transport might have the same composition (Conelly et al., 1973).As a matter of fact, incorporation of photochemical reaction centres into membranes of cells grown chemotropically was sufficient to perform light-induced oxidation of cytochrome c2 and reduction of cytochrome b (Jones and Plewis, 1974). Hunter and Jones (1979a, b) improved the methods of incorporation of reaction centres and investigated the kinetics of electron flow induced by flash illumination. The behaviour of the reconstituted system showed some similarities and some differences compared with the photosynthetic apparatus formed in vivo. The authors attributed these differences to incomplete reconstitution and an incorrect orientation of the reconstituted reaction centres. On the other hand, it was possible to increase the degree of similarities after addition of ubiquinone, (C. N. Hunter, personal communication). Furthermore, incorporation of antenna pigment complexes, in addition to reaction centres, resulted in an effective energy transfer from antenna to reaction centres and also in a more efficient photo-oxidation of cytochrome c2. From these impressive results, the authors concluded that incorporation of reaction centres into Bchl-free membranes satisfied the requirements to perform light-driven

42

G. DREWS AND J. OELZE

electron transport and photophosphorylation in vivo and also in vitro (Garcia et al., 1975; Hunter and Jones, 1979a, b). Being aware of the facts, however, that extrapolation of results obtained in vitro to those operative in vivo might be misleading, we tend to favour the principal idea that energy limiting conditions which forces the cell to produce the photosynthetic apparatus permit a rather limited biosynthetic activity only. Thus, teleologically speaking, during early stages of adaptation, it would be rather uneconomical for the cell not to take advantage of the various electron-transport constituents already present in cells grown under chemotrophic conditions. Later on, however, after the phototrophic energy metabolism is working sufficiently well, the cells can afford to synthesize and thereby extend the cellular contents of all of the constituents of photosynthetic electron-transport chains. b. Assembly in vivo. The reconstitution experiments in vitro, described above, support the idea that incorporation of reaction centres into a Bchl-free membrane is sufficient to obtain functional photosynthetic units. To test this hypothesis and to study early steps in the formation of the photosynthetic apparatus, dense cell suspensions precultivated under high aeration were incubated under low aeration in the dark. These conditions induced formation of the photosynthetic apparatus and concomitantly lowered the growth rate (Biedermann et al., 1967; Takemoto, 1974; Nieth and Drews, 1975; Oelze and Pahlke, 1976). The latter condition was necessary for exclusion, as far as possible, of synthesis of all constitutents formed during growth. Actually, it was interesting to note that a detailed analysis of the events leading to the formation de n o w of the photosynthetic apparatus have been performed only under conditions of low aeration in the dark and not under phototrophic conditions, i.e. anaerobiosis in the light. In cells of Rs. rubrum, the ratio between light-harvesting and reactioncentre Bchl did not change (Aagaard and Sistrom, 1972; Oelze and Pahlke, 1976). However, the proteins related to the photosynthetic apparatus were synthesized with different rates during the first few hours of incubation. This applied in particular to the heavy subunit of the reaction centre. Thus, initially, the photosynthetic apparatus of Rs. rubrum was assembled through a multistep mechanism (Oelze and Pahlke, 1976). Following this period, however, synthesis of polypeptides associated with reaction-centre and lightharvesting complexes eventually became co-ordinated. In Rp. sphaeroides and Rp. capsulata, the size of the photosynthetic unit, i.e. the molar ratio of total Bchl per reaction centre is variable (Aagaard and Sistrom, 1972; Lien et al., 1973). During the first period of incubation of these organisms at low aeration, membrane proteins associated with reaction-centre Bchl were incorporated at a higher rate into the membranes compared with the proteins associated with total light-harvesting Bchl. The size of the photosynthetic unit decreased during the first 30 minutes of

ORGANIZATION AND DIFFERENTIATION OF MEMBRANES

43

incubation and increased thereafter (Takemoto, 1974; Nieth and Drews, 1975). The size of the photosynthetic unit, as well as the activity of photophosphorylation, increased per membrane protein but decreased per total Bchl during the first hours of incubation (Drews et al., 1969; Takemoto, 1974; Nieth and Drews, 1975); photophosphorylation also decreased per reaction centre (Nieth and Drews, 1975). It has been shown that during the first hour(s) of incubation at low aeration, not only reaction centres but also light-harvesting Bchl I( B 875) complexes were incorporated into the membrane at a higher rate compared with lightharvesting Bchl 11(B 800-850) complexes. Light-harvesting Bchl I1 complex afterwards became the dominant component (Niederman et al., 1976; Schumacher and Drews, 1978). The level of h- and c-type cytochromes remained constant per membrane protein during initial stages (Niederman et al., 1976). The efficiency of energy transfer in the photosynthetic apparatus increased during the multistep process of assembly to an optimal level (Pradel et al., 1978). Thus, interspersing of reaction-centre and antenna Bchl complexes into the pigment-depleted aerobic membrane establishes a photochemically active membrane. The membrane-bound pigment complexes are thought to be formed in three main steps: ( I ) the synthesis of polypeptides and pigments; (2) the binding of pigments to proteins and assembly of the complex; and (3) the organization of functional units in the membrane. The sequence of the partial processes and the sites where the components are synthesized and assembled are unknown. It has been postulated that the pigment complexes of the photosynthetic apparatus arise from a precursor structure (Shaw and Richards, 1971; Gibson et al., 1972). An upper pigmented band was isolated which sedimented more slowly than chromatophores during sucrose-density centrifugation. The upper band contained polypeptides of the same mobility as those identified with reaction-centre and light-harvesting complexes. This suggested that the upper pigmented band contained material which has to be interspersed into the chromatophore membrane. Similar results were obtained by Richards (Shaw and Richards, 1971; W. R. Richards, personal communication). The upper band contained photochemically active reaction centres which were able to slowly photo-oxidize cytochrome c,. The energy-transfer from B 850 to B 875 was less efficient, the photoinduced carotenoid band shift was markedly reduced, and cytochrome h was not photoreduced (in this fraction). These interesting observations on early stages of development were interpreted as the formation of complexes active in producing excited states, but not fully coupled to the electron transport and unable to synthesize ATP (Hunter et al., 1979). The results, however, cannot exclude that this “light fraction” consists of a membrane fraction containing smaller particles of lower density than the “heavy” membrane fraction (Collins and Niederman, 1976a, b;

44

G DREW’S A N D J OELZE

Oelze et al., 1975b; Lueking et al., 1978). Different membrane fractions might be produced by cell fractionation and differential centrifugation of a continuous membrane system containing regions with different phospholipid and protein patterns. The idea that the formation of pigment-protein complexes is a membranebound process is supported by the following observations. First, the last steps of Bchl synthesis seem to be membrane bound (Gorchein, 1972); second, polypeptides of the pigment complexes were not detectable in the supernatant of cell-free extracts (300,000 g ) (Dierstein et al., 198I); third, low-temperature absorption spectra of membrane fractions, but not of the supernatant fraction, showed the characteristic absorption maxima of the membrane-bound pigment complexes (Niederman et al., 1976; Schumacher and Drews, 1978). Under special conditions and in mutant strains the supernatant fraction contained Mg- or Fe-tetrapyrrols or other pigments but never Bchl (Lascelles, 1966; Oelze et al., 1970; Oelze and Drews, 1970~;Drews et al., 1971; Richards et al., 1975). Fourth, polypeptides that passed through or were incorporated into membranes were found to be synthesized on membrane-associated polyribosomes (Smith et al., 1977, 1978). Fifth, studies on the topography of pigment complexes (Feher and Okamura, 1978a; Oelze, 1978; Zurrer et al., 1977), and on isolated complexes suggest that Bchl and protein need an amphiphilic environment for assembly. The membrane seems to be a more suitable environment for allowing a non-covalent binding of Bchl and polypeptides than the soluble cytoplasmic fraction. To unravel the process of the biogenesis of pigment complexes, the synthesis and incorporation of reaction-centre and light-harvesting polypeptides into a light (sediment at 300,000 g) and a heavy (sediment at 1000,000 g) membrane fraction under conditions of very low oxygen partial pressure and growth limitation (0.7 mmHg p 0 =~ 93 Pa, in the dark) was studied with Rp. capsulafa (Dierstein et af., 1981). The heavy polypeptide of reaction centre (molecular weight 28,000) was incorporated rapidly into the light membrane fraction at a constant rate, but was incorporated slowly into the heavy membrane fraction after a lag phase of 5 minutes. Similar results were obtained with the light-harvesting polypeptides (molecular weights 10,000 and SOOO), but the kinetics were slightly different. The data reflect incorporation of the newly synthesized polypeptides at different sites and with different kinetics during membrane biogenesis. Pulse-chase experiments indicate that at least one polypeptide of the lightharvesting complex I1 with a molecular weight of 10,000 was produced by proteolytic processing from a precursor polypeptide of molecular weight 11,000. An earlier labelled protein band with a molecular weight of 32,000, synthesized at growth limitation, was thought to be an intermediate of other unprocessed light-harvesting polypeptides (R. Dierstein and G. Drews, un-

45

ORGANIZATION AND DIFFERENTIATION OF MEMBRANES

- 10 I

0

Time (h)

0.151

0

I

2

I 4 Time (h)

2

4 Time (h)

6

I

6

FIG. 10. Time-course of accumulation of photosynthetic components in particulate fractions of a synchronous culture of Rhodopseudomonas sphaeroides. (b) 0.cytochrome c; 0, cytochrome b; A,reaction centres. (c) Light-harvesting Bchl forms and 850 (0)nm wavelengths. From Wraight et a / . (1978a). at 800 (0)

published observations). The modified polypeptides might be favoured for incorporation into the membrane and for assembly with Bchl and carotenoid molecules in a stoicheiometric ratio. Several exported or membrane-bound proteins which are proteolytically processed have been found in Escherichia coli. In one instance, bacteriophage f l pre-coat protein underwent cotransitional processing during which biosynthesis of the nascent chains and endoproteolytic cleavage with a signal peptidase took place while the polyribosomes were membrane bound (Chang et al., 1978). Another example is the post-transitional cleavage of membrane-bound precursor of arabinosebinding protein (Randall et al., 1978). On the basis of the present knowledge, the following hypothesis on the formation of photosynthetic units in Rp. capsulata is proposed. After induction (or depression) of the genes of the photosynthetic apparatus, mRNA

G. DREWS AND J. OELZE

46

2.01

C 0)

P

0

E

E 0 ._

E

0 -

-

a 0

) .

P

t

c .v

.L L

3

a

FIG. 11. Time-course of changes in the cell density (a), ratio of intracytoplasmic membrane protein to phospholipid (b) and specific density of intracytoplasmic membrane (c) during synchronous growth of Rhodopseudomonas sphaeroides. From Lueking e f al. (1978).

for the polypeptides of pigment complexes are translated on membranebound polyribosomes. The polypeptides (i.e. the heavy reaction-centre polypeptides) are incorporated into the membrane either immediately or after co- or post-translational proteolytic processing. During assembly, Bchl and carotenoid molecules bind to the polypeptides. The synthesis and assembly of pigments and polypeptides is highly co-ordinated. Thus a mutual influence is indicated. It has yet to be studied as to whether the tetrapyrrole-protein complexes, excreted by numerous mutants (Lascelles, 1966; Oelze and Drews, 1970~;Richards et al., 1975), are real precursors or nonsense products. The lipid patterns of membranes strongly influence the physical structure and dynamics of membrane processes and can be important for activity of

ORGANIZATION AND DIFFERENTIATION OF MEMBRANES

47

membrane enzymes (Sandermann, 1978). The phospholipid patterns of photosynthetic intracytoplasmic membranes is slightly different from membranes of cells grown aerobically (Gorchein, 1968). Synchronously, under phototrophic conditions, growing cell populations of Rp. sphaeroides exhibited continuous patterns of incorporation of photosynthetic pigmentprotein complexes and cytochromes c and b into the membrane fraction, whereas phospholipids increased discontinuously (Figs. 10 and 1 I; Fraley et al., 1978; Lueking et al., 1978; Wraight et al., 1978b). It has yet to be studied as to whether the changes of protein : phospholipid ratios and the qualitative changes in phospholipid patterns are of possible significance in the control of membrane synthesis and the process of assembly of intramembrane particles.

3. Fornzation qf Photosyntheticdy Active Membrunes As previously mentioned, most members of the photosynthetic bacteria form,

in addition to cytoplasmic membranes, cytologically distinct intracytoplasmic membranes when adapting from chemotrophic to phototrophic conditions. This provides an interesting system to study. Besides the formation of the photosynthetic apparatus, there are also various questions about the biogenesis of biological membranes. In the context of cellular differentiation, however, biogenesis of intracytoplasmic membranes is an essential step to cause differentiation of the entire cellular membrane system. The findings that, under balanced growth conditions, cells form only intracytoplasmic membranes concomitant with Bchl, led to the postulate that Bchl synthesis was a necessary prerequisite for intracytoplasmic membrane formation (Oelze et al., 1970; Brown et al., 1972). That this is apparently not inevitably true has been demonstrated with chemotrophically growing cells of Rs. rubrum which entered the stationary-growth phase (Oelze et al., 1977). Such cells, although completely free from detectable amounts of photosynthetic pigments, exhibited intracytoplasmic membranes. In addition, aerobically grown cells of Rp. capsulata, which are largely depleted of Bchl, contained significant amounts of intracytoplasmic membranes (Lampe et al., 1972). Furthermore, non-dividing sphaeroplasts of chemotrophically cultured Rs. rubrum produce vesicular intracytoplasmic membranes which contain no detectable Bchl (Golecki et al., 1980). This indicates that an increase in the rate of membrane formation over the rate of cell-wall formation results in intracytoplasmic membrane formation. This may occur in cells cultivated chemotrophically under special conditions that slow down cell-wall synthesis and also in phototrophically growing cells in which, through localized or homogeneous incorporations of constituents of the photosynthetic apparatus, the cellular

48

G. DREWS AND J. OELZE

membrane contents are increased. Interestingly enough, Rs. tenue, which, on adaptation from chemotrophic to phototrophic conditions, does not form intracytoplasmic membranes but alters the cellular membrane content to a comparable extent as an intracytoplasmic membrane-containing species (see Section III.A, p. 9), produces cell wall co-ordinated with membrane material (Wakim et al., 1978). In summary, the data suggest that (i) intracytoplasmic membrane formation depends on the relative rates of cell-wall and membrane formation and (ii) the vesicular shape of intracytoplasmic membranes in Rs. rubrum does not inevitably depend on the presence of the photosynthetic apparatus. The physical continuity between intracytoplasmic and cytoplasmic membranes demonstrated on electron micrographs has been interpreted to indicate that intracytoplasmic membranes arise by invagination of the cytoplasmic membrane (for an evaluation of the evidences derived from electron micrographs, see Lascelles, 1968). This hypothesis has received considerable experimental support from biochemical investigations in combination with electron microscopy (reviewed by Oelze and Drews, 1972).On the other hand, biochemical investigations have led to an alternative hypothesis, according to which intracytoplasmic membranes are formed independently and do not directly involve the cytoplasmic membrane (Gibson, 1965a, b; Huang and Kaplan, 1973; Kosakowski and Kaplan, 1974; Kaplan, 1978; Parks and Niederman, 1978). This includes the possibility either that constituents of intracytoplasmic membranes are condensed on (instead of incorporated into) thecytoplasmic membrane (Kosakowski and Kaplan, 1974),or that intracytoplasmic membrane vesicles become attached secondarily to the cytoplasmic membrane (Gibson, 1965a, b; Kaplan, 1978). Evidence for this hypothesis has come from the following observations. (i) Isolated chromatophores derived from intracytoplasmic membranes of Rp. sphaeroides contain only about 5% of polypeptides characteristic of the cytoplasmic membrane (Huang and Kaplan, 1973). The authors have concluded that these polypeptides were not constituents of the intracytoplasmic membrane but were derived from the juncture between intracytoplasmic and cytoplasmic membranes (for another explanation, see below). (ii) Cytoplasmic membrane preparations from phototrophically grown Rp. sphaeroides were depleted of Bchl, suggesting that the cytoplasmic membrane of chemotrophic cells is also synthesized by phototrophic cells; in other words, phototrophy has practically no influence on the composition of the cytoplasmic membrane (Parks and Niederman, 1978). (iii) Quantitative differences in the lipid composition of intracytoplasmic and cytoplasmic membranes (Gorchein, 1968; Steiner et al., 1970) indicate a lack of lateral lipid diffusion between both types of membranes which, in the light of the fluid mosaic model for biological membranes (Singer and Nicolson, 1972), might be interpreted as a lack of

ORGANIZATION AND DIFFERENTIATION OF MEMBRANES

49

continuity between the two membrane systems (Kaplan, 1978). However, the present state of knowledge on distribution of lipids within continuous membrane systems allows for the presence of lipid domains with different compositions (Sackmann et al., 1977). 1

5

!

P

1

5

15

10

I I I I I I II I 1

I I I I II

10

12 13

16

Relative migration

4 FIG. 12. Pulse-chasing of cytoplasmic-membrane proteins of Rhodospirillum rubrum into intracytoplasmic membranes (i.e. chromatophores). Membrane proteins were separated by electrophoresis in 10% polyacrylamide gels. Protein patterns (A, B) and autoradiograms (a, b) were scanned densitometrically. Enriched membrane (i.e. envelope) fractions (A, a) containing cytoplasmic membranes, were isolated from Bchl-free cells. Intracytoplasmic membranes (B, b) were isolated from cells containing 2.7 pg of Bchl/mg of protein by means of Ficoll and sucrose equilibrium densitygradient centrifugation. Bands 2-3 contain succinate dehydrogenase, bands 5 and 6 ATPase, bands 8-10 cell-wall contaminants, bands 11, 14 and 15 photochemical reaction centre. As a control for the purity of chromatophore preparations (B, b) note that the cell-wall bands (8-10) cannot be “chased” into this fraction. From Oelze (1976).

50

G . DREWS AND J . OELZE

Thus, differences in lipid composition do not argue against the original hypothesis according to which intracytoplasmic membranes are derived from cytoplasmic membranes by invagination. This hypothesis claims, in other words, that intracytoplasmic membranes, at least initially, arise by differentiation of the pre-existing cytoplasmic membrane (Oelze and Drews, 1972). In support of this hypothesis, the following results have been described: (i) radioactive label incorporated into the lipids of cytoplasmic membranes could be “chased” into the newly formed intracytoplasmic membranes of Rp. 0.04

0

Fraction no.

FIG. 13. Sucrose-density-gradient separation of membranes isolated from Rhoduspirillum rubrum containing various amounts of bacteriochlorophyll (a) Bchl not detectable, (b) 0.3 and (c) 2.5 pg of Bchl/mg of protein. The bacteriochlorophyll contents (b, c) were measured at 875 nm (O-O), membrane-bound cytochromes were determinable only in the absence of bacteriochlorophyll (a) at 41 2 nm Sucrose concentrations (. . . .) are shown in (c); buoyant densities (g/cm3) of cytoplasmic (1.146) and intracytoplasmic membranes (1.165) are indicated by the arrows in (a). Note that Bchl at very low levels (b) is contained largely in the cytoplasmic membrane fraction. From Oelze (1976).

(*a).

0 RGAN lZATl0 N AN D D IFF ER ENTlAT10 N 0 F M EMBRAN ES

51

sphaeroides, Rp. capsulata and Rs. rubrum (Gorchein, 1968; Oelze and Drews, 1969; Lampe et al., 1972); (ii) in addition, polypeptides of the cytoplasmic membrane could be “chased” into intracytoplasmic membranes (Fig. 12; Oelze, 1976); (iii) Bchl synthesized immediately after transfer of cells from chemotrophic to phototrophic conditions, was incorporated into the cytoplasmic membrane (Fig. 13; Biedermann et al., 1967; Oelze rt al., 1969b; Golecki and Oelze, 1975; Oelze, 1976); (iv) in fully developed chromatophores, about 5% of the total polypeptides of Rs. rubrum and Rp. sphaeroides have been identified as being representative of polypeptides of the cytoplasmic membrane (Oelze et al., 1969a, b; Huang and Kaplan, 1973). Recent improvements in methods for identifying membrane proteins suggest that this low percentage is an underestimation. Although Kaplan arrived at a different interpretation (see above), this may well prove to be the developmental relationship of cytoplasmic and intracytoplasmic membranes. As at least coupling factors, for example, succinate and NADH dehydrogenases, as well as electron-transport constituents, are contained in cytoplasmic as well as in intracytoplasmic membranes, both membranes must exhibit partially identical polypeptide patterns (Oelze and Drews, 1970a, b, 1972; Niederman, 1974; Oelze et al., 1975b; Collins and Niederman, 1976a, b; Irschik and Oelze, 1976; Niederman and Gibson, 1978; Oelze, 1978; Parks and Niederman, 1978). The major objection brought forward against the significance of the results outlined in (ikiv) was the possibility that the membrane fractions used might not have been completely purified from cross contaminations (Collins and Niederman, 1976a, b). In principle, this canndt be completely denied. But it should also be kept in mind that rigorous methods used to purify membranes can also be applied to detach essential constituents from fractions under purification. For example, coupling factors ATPase ( F l ) , NADH and succinate dehydrogenases are readily solubilized by mild detergent treatment, by lowering the ionic strength, or by the addition of EDTA (Reed and Raveed, 1972; Boll, 1968a, b, 1969). Even light-harvesting Bchl complexes can be washed off the cells of Rs. tenue after such treatments (B. Wakim and J. Oelze, unpublished observations). It is evident that effects like these might become rather misleading especially if, for example, the Bchl content is used as a criterion to categorize a membrane preparation. Although inevitable under certain conditions, we feel that extensive purification of membranes is a rather limited approach to the study of membrane structure and function. It is probably more advisable to d o investigations on lesspurified preparations of which the impurities and their respective influences on the results are known. This is largely true for the data mentioned above. Additional support for the hypothesis that intracytoplasmic membranes arise, at least initially, by differentiation and invagination of the cytoplasmic membrane has come from two other independent experimental approaches.

52

G.DREWS A N D J. OELZE

(v) Reconstitutional experiments (see Section 1V.A.2, p. 41) have demonstrated that insertion of the Bchl moieties into membranes of cells grown under strict chemotrophic conditions have already led to the formation of a photosynthetically active membrane; as discussed above, it seems unlikely that organisms, instead of making use of the appropriate constituents already available, should synthesize these during early stages of adaptation to phototrophic conditions. (vi) Rs. tenue, which does not form intracytoplasmic membranes but exlends its cellular membrane content by changing the ratio of cell surface to cqll volume, differentiates the entire cytoplasmic membrane homogeneously bj! incorporation of specific particles (see Section IILA, p. 9) (Wakim et dl., 1978). On the basis of particle distribution, electron micrographs do not reveal the presence of differently composed membrane areas in Rs.tenue at different stages of adaptation to phototrophic conditions (see Fig. 2). This, however, should be the case if, as demonstrated in Halobacteria (Stoeckenius and Kunau, 1968; Blaurock and Stoeckenius, 1971), structurally and functionally separate membrane areas were also formed in Rs. tenue. Thus, the homogeneous distribution within the cytoplasmic membrane of Rs. tenue of newly arising particles is the most obvious and convincing evidence to disprove the hypothesis of an independent formation of photosynthetic and respiratory membranes. Of course the findings that in Rs. tenue the entire cytoplasmic membrane becomes differentiated should not be taken to represent the degree of cytoplasmic membrane differentiation in other phototrophic bacteria as well. In any case, cytoplasmic membranes contain significantly lower amounts of photosynthetic pigments than do intracytoplasmic membranes. This has been reported for phototrophically grown Rs. rubrum and Rp. sphaeroides (Oelze et al., 1969a, b; Parks and Niederman, 1978; Niederman and Gibson, 1978). Taken together, the data available so far on the morphogenesis of intracytoplasmic membrane suggest the following events. Differentiation is initiated by intercalation of relatively small amounts of Bchl complexes (reaction centre plus antenna) into the preexisting cytoplasmic membrane. We do not know at present if this is homogeneous or confined to certain areas, in any case, respiratory-chain constituents become included in the developing photochemical electrontransport chain. Following this, Bchl units are incorporated predominantly into those areas from which intracytoplasmic membranes invaginate. Preferential incorporation into the newly arising intracytoplasmic membranes of units necessary to form a functional photosynthetic apparatus results in an increase in the Bchl contents of these membranes until a defined level has been reached. Increasing the Bchl contents of cells above this level means extension of the constantly composed intracytoplasmic membrane system (Oelze et al., 1969b; Oelze and Drews, 1972). Elongation of the intracytoplasmic membrane reticulum takes place by incorporation of constituents

ORGANIZATION AND DIFFER ENTIATION 0 F MEMBRANES

53

along its length (Kosakowski and Kaplan, 1974). This probably includes, besides photopigments, other additional constituents like coupling factors and various electron-transport catalysts. The findings that intracytoplasmic and cytoplasmic membranes exhibit different ratios in succinate and NADH dehydrogenase activities infer that both dehydrogenases are incorporated into intracytoplasmic membranes and cytoplasmic membranes in different proportions (Irschik and Oelze, 1976; Niederman and Gibson, 1978). The same applies to the quantitative lipid composition which, as mentioned above, is not identical in intracytoplasmic and cytoplasmic membranes (Gorchein, 1968; Steiner et af.,1970). This in turn infers that elongation of intracytoplasmic membranes is at least largely independent. Thus, for later stages of intracytoplasmic membrane elongation, the two hypotheses on intracytoplasmic membrane development come into practically an indistinguishable proximity. This, however, is not surprising because the final production of stages, differing structurally and functionally from the original, is the purpose of any developmental process. At this point the question arises as to why those organisms that form intracytoplasmic membranes, like Rs. rubrum, Rp. capsulata or Rp. sphaeroides, do not differentiate the entire cytoplasmic membrane like Rs. tenue, and in this way use up, rather economically, all of the constituents of the respiratory electron-transport chain. Instead, those organisms can obviously afford to conserve the respiratory capacity of the cytoplasmic membrane, even under conditions that require only the function of the photosynthetically active intracytoplasmic membranes. An answer may be derived from the findings that the ratio of some respiratory activities of phototrophic to chemotrophic cells is considerably lower in Rs. tenue than in Rs. rubrum (Keister and Minton, 1969; Oelze and Weaver, 1971; Wakim et al., 1980). Obviously, the high respiratory capacity of phototrophic cells of Rs.rubrum and related species provides the organisms with a greater versatility for adjusting to different types of metabolism as well as different environmental conditions. However, the mechanisms that enable the organisms to establish and maintain this kind of micro-compartmentation in the cellular membrane system are completely unknown.

B . I N F L U E N C E OF E X T E R N A L FACTORS ON MEMBRANE DIFFERENTIATION

Since the early investigations on the physiology of phototrophic bacteria, it is known that various environmental factors, like light intensity under anaerobiosis or oxygen partial pressure under low aeration in the dark, influence the rate of Bchl synthesis (Cohen-Bazire et al., 1957). More recently,

54

G.DREWS AND J. OELZE

effects of nutritional factors such as the availability of nitrogen sources or specific carbon sources have also been reported (Schon and Ladwig, 1970; Dierstein and Drews, 1975). It is self-evident that light intensity is the factor which, to date, has been most often and most intensively investigated. CohenBazire and colleagues (1957) were the first to show that cellular Bchl contents vary in reversed proportion to incident light intensity (for subsequent publications on this see the reviews by Oelze and Drews, 1972 and Kaplan, 1978). In addition, ultrastructural investigations revealed that the cellular contents of intracytoplasmic membranes are also inversely related to light intensity (Cohen-Bazire and Kunisawa, 1963; Drews and Giesbrecht, 1963; Holt and Marr, 1965c; Trentini and Starr, 1967). This might be interpreted as indicating that the amounts of Bchl per intracytoplasmic membrane protein were constant. However, direct determinations of specific Bchl contents of chromatophores derived from different organisms led to somewhat contradictory results. Chromatophores exhibited constant Bchl contents when derived from the intracytoplasmic membranes of Rs. rubrum with cellular Bchl contents greater than about 12 pg of Bchl per mg of cell protein (Oelze et al., 1969b). However, when isolated from cells of Rp. sphaeroides or Rp. capsulata of different Bchl contents, pigment contents of chromatophores also exhibited different values (Worden and Sistrom, 1964; Aagaard and Sistrom, 1972; Lampe and Drews, 1972). This inconsistency, however, was unravelled by Aagaard and Sistrom (1972) who demonstrated that Rp. sphaeroides exhibited a variable ratio of reaction-centre to light-harvesting Bchl, whereas in Rs. rubrum this ratio was constant (see Section III.B.2, p. 19). Thus, depending on the organism, it was possible to change the cellular amounts of the photosynthetic apparatus, its composition, or even both. These effects were also determined later with other bacteria and with environmental factors other than light intensity. The fact that different organisms respond differently to changes in culture conditions will be taken into account in the following review via a separate description of the results obtained with representative species.

1. Light Intensity Unfortunately, the effect of monochromatic light on the formation and differentiation of the photosynthetic apparatus in bacteria has not been investigated. Only the total amounts of Bchl formed at different wavelengths has been measured (Gobel, 1978). Therefore, in qualitative terms, light generally means wavelengths emitted by incandescent bulbs. a. Khodospirillum rubrum. Under all conditions, this organism exhibits the same absorption spectrum in the near infrared region (Fig. 14). Accordingly,

ORGANIZATION AND DIFFERENTIATION O F MEMBRANES

,

400

1

500

1

1

600

1

I

700

I

I

800

I

I

55

I

900

Wavelength (nm)

FIG. 14. Low-temperature (77 K) absorption spectrum of membranes of Rhodospirillum rubrum. This organism exhibits one spectral type in the region of Bchl absorbance, regardless of culture conditions.

Aagaard and Sistrom (1972) reported that cells grown under different light intensities exhibited a rather constant ratio of reaction-centres to lightharvesting Bchl of 1 : 25-35 (Table 4). Nevertheless, Cohen-Bazire and Kunisawa (1960) showed a direct relationship between the rate of photophosphorylation per unit of Bchl and the intensity of light applied for cultivating Rs. rubrum. In support of findings by Nishimura (1962a, b), this suggested to Aagaard and Sistrom (1972) that a step within the photochemical electron-transport system and not the primary light reaction, was ratelimiting for photophosphorylation (see also Section 11, p. 4). On that basis, Irschik and Oelze (1973, 1976) performed a more detailed study on electrontransport reactions with membranes from Rs. rubrum transferred from a low to a higher light intensity. As originally shown by Cohen-Bazire ez a/. (1957), this resulted in a temporary inhibition of Bchl synthesis while growth was unaffected. Provided the difference in light intensity was sufficiently high, the specific Bchl contents of growing cultures could be lowered to that range ( < 12 pg of Bchl per mg of cell protein) where a linear relationship exists between Bchl contents of whole cells and isolated chromatophores (Oelze et al., 1969b; Oelze and Drews, 1972). Within this range, differentiation of the

56

G. DREWS AND J. OELZE

TABLE 3. Variations in membrane-bound enzyme activities after transfer of Rhodospirillum ruhrum from low- (4 W m - z ) to high- (400 W m-z) light intensity under anaerobic conditions. Specific bacteriochlorophyll contents of whole cells and chromatophores were 29.5 and I17 (pg of Bchl per mg of protein) before (sample no. 1) and 8.8 and 70, respectively, after 6 h of growth (about two doublings of cell mass) under high-light intensity (sample no. 2). Activities are presented as nmol of N A D H (line l), succinate (line 2 ) and horse heart cytochrome c (line 3) oxidized per mg of bacteriochlorophyll per min or as nmol of ATP (line 4) and NADH (line 5 ) formed per mg of bacteriochlorophyll per min. From Irschik and Oelze ( I 976) Functional system

Sample no.

Activity

1

NADHXytochrome coxidoreductase

2

0.4 0.7

Succinate-cytochrome c oxidoreductase

I 2

0.34 0.62

1

0.3 0.5

Cytochrome c oxidase

2 Photophosphorylation

1.7 3.1

1

2 Succinate-dependent N A D + reduction in the light

0.08 0.15

1

2

intracytoplasmic membranes, as determined with purified chromatophores, isolated from cells after growth at high light intensity, could be followed by (i) incorporation of polypeptides not related to the well-known Bchl complexes, as well as by increases on, a Bchl basis, of (ii) NADH- and succinate-dependent respiratory reactions; (iii) activity of photophosphorylation; (iv) activity of light-dependent NAD reduction with succinate (Table 3). All of these increases were of almost identical magnitude. With whole cells, the same was true for the increase in light-induced absorbance changes at 422 nm, representing the oxidation ofcytodhrome c2 of the photochemical electron-transport system. Thus, of the energy-generating electrontransport systems, the only known parameter that remained quantitatively unchanged was Bchl in its different associations with light-harvesting and reaction-centre units. In principle, the results are comparable to intracytoplasmic membrane differentiation taking place during adaptation of phototrophic cultures to chemotrophic conditions (see Section IV.B.2, p. 63; Oelze and Drews, 1970a, b, 1972; Oelze and Weaver, 1971). The essential difference between both processes, however, is the fact that intracytoplasmic membranes apparently do not become integrated into the cytoplasmic membrane system under conditions of high light intensity (Irschik and Oelze, 1973). +

0 RGAN IZATIO N AN D D I FF ER ENTIAT ION 0 F M EMBRAN ES

57

In summary, increasing the light intensity results in a decrease of the specific cellular Bchl contents in phototrophically growing cells of Rs. rubrum, a decrease which, above a defined value, does not alter the composition and function of intracytoplasmic membranes. Below this value, however, intracytoplasmic membranes become differentiated to such an extent that any further decrease in the Bchl content becomes partially compensated by an increase in the efficiency of light-dependent reactions. b. Rhodopseudomonas ( R p . ) capsulata, Rp. sphaeroides and Rp. palustris. These form photosynthetic units of variable size (see Sections 1II.B and IV.A, pp. 16 and 37). Extremely strong shifts of irradiance influence the growth rate. At low-incident light energy, the growth rate is limited. Rp. capsulura is not able to adapt to light intensities less than 3nE*s-'.cm-* at 860 nm (Gobel, 1978) or 20 lux of white light (Biebl and Pfennig, 1978). It was observed that at growth-limiting light intensities, both the potential rate of photophosphorylation and the respiratory oxidation of NADH were decreased (Schumacher and Drews, 1979). In a continuous culture limited in energy of monochromatic light, the quantum yield of growth reached a maximum at a low-growth rate and decreased towards high- and very lowgrowth rates (Gobel, 1978). The quantum absorption rate per light-harvesting pigment molecule per time, i.e. turnover number of quanta, was increased by increasing the specific growth rate, by decreasing pigment content and by decreasing quantum yield. It was concluded that the turnover of excitation states in the reaction centre becomes inefficient at high growth rates (Gobel, 1978). In the following section, the influence of light intensity on the ratio of the various pigment complexes in cells that have reached steady-state levels of pigmentation, will be considered. The specific cellular Bchl content and the Bchl concentration of membranes of Rp. sphaeroides vary in reversed proportion to the incident light intensity (Table 4; Cohen-Bazire and Kunisawa. 1963; Worden and Sistrom, 1964; Gobel, 1978). An analysis of the spectral forms showed that the content of reaction centres per membrane protein was constant. In addition, the molar ratio of light-harvesting Bchl I (B 875) to reaction-centre Bchl was fixed at about 20-25 and furthermore, the size of the photosynthetic unit increased as the specific Bchl content of the cells increased (Table 4; Aagaard and Sistrom, 1972; Sistrom, 1978). Consequently, the two species of antenna Bchl, B 875 and B 800-850, were controlled independently. In conclusion, Rp. sphaeroides adapt to different incident light intensities by variation of the cellular content of intracytoplasmic membranes and by variation of the size of the photosynthetic unit (Table 4). At very low specific Bchl contents of cells, however, the number of photosynthetic units per membrane area seems to be variable (Sistrom, 1978).

58

G.DREWS AND J. OELZE

TABLE 4. Effect of incident light intensities on total bacteriochlorophyll content and size of photosynthetic unit in membranes of photosynthetic bacteria grown anaerobically ~

~~

~~

Organism Rhodopseudomonas sphaeroides G a Rp. sphaeroides R 22 (crt-)

~

~

~

a

~

Bacteriochlorophyll content (pg per mg of membrane protein)

Photosynthetic unit (total Bchl reaction centre)

646 lux

46.5

250

2690 lux 64590 lux

19.3 2.5

67 55

77.9 27.3 18.2 40

270 120 80 80 50

Light intensity

323 lux 6454 lux 2 1530 lux Rhodopseudomonas I Wm-2 capsulata 2000 W m - 2 37b4, leuRhodopseudomonas 86 12 lux capsuluta Z-1 40900 lux Rhodopseudomonas 100 W m palustris le5 IOOOWm-? Rhodosp irillum 4Wm-2 ruhrum 400 W m - 2 Rhodopseudornonas sphaeroides G a

~

5

51 7.8 63 12 85 31

0.6d 1.2" 203 60 26

25

Reference Cohen-Bazire and Sistrom (1 966)

Aagaard and Sistrom (1972) Schumacher and Drews (1 979) Lien et ul. (1973) Firsow and Drews (1977) Aagaard and Sistrom (1972) Irschik and Oelze (1 976)

A,noIAnm

The molar ratio of carotenoids to Bchl decreased sharply from 1.0 to 0.5 when the specific Bchl content of Rp. sphaeroides increased from very low concentrations to about 30 pg Bchl per mg of protein. Above this level, the ratio remained approximately constant (Sistrom, 1978). This variation of the molar ratio of carotenoid to Bchl seems to be a consequence of the variation in the size of the photosynthetic unit. Each reaction centre from wild-type strains of Rp. sphaeroides contains one mol of carotenoid, whereas a carotenoid to Bchl ratio of l :3 was found in the antenna Bchl complex B 800-850 (Cogdell, 1978).The ratio of carotenoids to Bchl in the antenna Bchl complex B 875 seems to be 1 : 1 (see Section III.B.2, p. 19). Rp. capsulata and Rp. palustris follow the same principles of adaptation to different light intensities. In addition to the size of the photosynthetic unit and the content of membrane per cell, the number of photosynthetic units per membrane protein, i.e. the content of reaction centres per membrane

TABLE 5. Differentiation of the membrane system of Rhodopsrudornonus capsulutu 37b4, induced by an increase of light intensity from 7 W m - z to 2000 W m - 2 in anaerobic cultures. From Schumacher and Drews (1979) Bchl content

Photophosphorylation (nmol ATPjmin) - -_

Incubation nmol Bchli time mg of cell (h) protein

nmoi Bchll per mg mg of of membrane protein protein

----

per p o i of Bchl

Dehydrogenase (nmol substrate)

Oxidation of NADH (mol NADHjmin) -_ _ _ _ - per nmol per mg of of protein Bchl

_-

~

~

- --

per pnol ~ 6 6 nm 0 of I Ern Bchl -____ Succinate NADH -- - - -- - - _ _ ---- -_ _ _ 0.04 226 36 34 06

per mg of protein -_____ Succinate NADH

~

~

5

12

22.7

25

1100

72

3.2

303

56

13

2.5

0.07

8

8

13.7

40

3030

61

4.5

295

51

22

3.1

0.08

11

4

12.0

40

3270

82

6.8

308

57

26

4.8

0.15

0 h indicates the beginning of irradiation with high intensities of white light

60

G . DREWS AND J. OELZE

400 500 600 700 800 900 400 500 600 700 600 900 Wavelength (nm)

FIG. 15. Low-temperature (77 K) absorbance spectra of membranes of Rhodopseudomonus cupsulutu 37b4, cultivated at low-light intensity (a) and after three

doublings of cell mass at high-light intensity (2000 W m-2) (b). The pigment content and enzyme activities of membranes are listed in Table 5. The spectra show that the portion of light-harvesting Bchl I1 complex relative to complex I is decreased in (b) compared with (a). The size of the photosynthetic unit is lowered from 80 (a) to 50 (b). From Schumacher and Drews (1979).

protein, is variable (Firsow and Drews, 1977; Schumacher and Drews, 1979). The effect of light intensity on the process of membrane differentiation was studied in Rp. cupsulutu (Schumacher and Drews, 1979). An increase of the incident light intensity initiated a multistep process similar to that triggered by a change of oxygen tension. A 500-fold increase of light intensity to 2000 W m - 2 effected an immediate stop of Bchl synthesis. After two doublings of cell mass, Bchl synthesis resumed and reaction-centre and lightharvesting Bchl I were incorporated, with their respective proteins, into the membrane, whereas light-harvesting Bchl I1 was not, or only in small amounts, synthesized. Consequently, the size of the photosynthetic unit decreased and light-harvesting Bchl I (B 875) became the dominant pigment complex (Table 5; Fig. 15). The amount of intracytoplasmic membranes per cell and the number of photosynthetic units per membrane area were also lowered. The potential rate of photophosphorylation, calculated on the basis of Bchl and membrane protein, increased (Table 5). New functional units, particularly enzymes of the respiratory chain, were incorporated into the membrane system (Schumacher and Drews, 1979). A change from a high to a low light intensity (7 W m - 2 ) resulted in immediate cessation of growth. After a lag phase, the bacteria grew exponentially with a mass-doubling time of I 1 hours. During the lag phase of

ORGANIZATION AND DIFFERENTIATION OF MEMBRANES

61

growth, new intracellular membranes were synthesized, and reaction centres and light-harvesting Bchl I complexes were preferentially incorporated into the membrane. Later, concentrations of reaction centres per membrane and the size of the photosynthetic unit increased significantly. Following this period, the size of the photosynthetic unit increased only slightly and cellular Bchl content reached a steady-state ,level. During incubation at low-light intensity, the potential rate of photophosphorylation decreased significantly, whereas the respiratory activity remained nearly constant (Table 5). The results show that during adaptation of cells to different light intensities, the cellular membrane system is differentiated by a multistep process. Reaction-centre and light-harvesting Bchl I complexes were synthesized concomitantly; light-harvesting Bchl I1 complex and the intracytoplasmic membrane, however, were synthesized with different rates. The components of the respiratory chain were also synthesized independently. These data support the idea that all three parameters, i.e. size, number of photosynthetic units per membrane and amount of intracytoplasmic membranes per cell, increase when light intensity is decreased, but, aside from the strict co-ordination of synthesis of reaction-centre and light-harvesting Bchl I (Aagaard and Sistrom, 1972; Lien et al., 1973; Takemoto and Huang Kao, 1977; Schumacher and Drews, 1979), all three parameters can, within certain limits, be regulated independently. In cells of Rp. capsulata 37b4, the size of the photosynthetic unit was less markedly changed than in cells of Rp. palustris le5. In contrast, the amount of intracytoplasmic membrane per cell varied more pronouncedly in cells of Rp. capsulata than in cells of Rp. palustris, under different light regimes. Under anaerobic conditions, the flux of light quanta influences both growth rate and cell differentiation. Whether the effect of radiation on energy metabolism (“energy state” of the cell, redox state, pool of low molecularweight substances, etc.; Cohen-Bazire and Sistrom, 1966; Lien et al., 1973) has a direct influence on the regulation of cell differentiation, has yet to be studied. It was suggested (Drews and Jaeger, 1963; Kaplan, 1978), but not strictly proven, that single Bchl-protein complexes of the photosynthetic apparatus are the primary acceptors of light quanta for photosynthesis and for the signal chain which leads to cell differentiation. An investigation of the possibility of an effector for radiation which triggers light-dependent morphogenesis, independent of the activity of the photosynthetic apparatus, would seem to be worthwhile. c. Chromatium vinosum. It has been known for a long time that light intensity influences the formation of absorption spectra in Chr. vinosum (Wassink et al., 1939). Under high-light intensities, cells express spectra with near-infrared absorption peaks at 800 and 850 nm, the latter with a shoulder at 880 nm. Spectra of cells grown at low-light intensity exhibit only one peak

62

G. DREWS AND J. OELZE

at 800 nm with shoulders at 820, 850 and 880 nm. Mechler and Oelze (1978a) reported that Chr. vinosurn showed only light intensity-dependent spectral changes when growing either photo-autotrophically or photo-mixotrotrophically, but not when growing strictly photoheterotrophically. In spite of spectral changes, however, light intensity under any set of conditions was effective in altering the specific Bchl contents of cells, i.e. the cellular contents of intracytoplasmic membranes and of photosynthetic apparatus. The first indications that spectrally different forms of Chr. vinosurn also contained differently composed photosynthetic apparatus were given by Garcia and colleagues (1966) who solubilized spectrally different subfractions from chromatophores of cells grown autotrophically under low- and high-light intensity, respectively. Further evidence for differences was presented by Takahashi et al. (1972) who reported that cells grown at low-light intensities exhibited higher photosynthetic activities than those grown at high-light intensities. On a Bchl basis, the activities were comparable. A more systematic characterization of functional and structural differences of the spectrally alternative modifications was done by Mechler and Oelze (1978b). But, because of the fact that in Chr. vinosum differentiation is most effective through defined variations in growth temperature rather than light intensity, temperature-induced modifications were employed for the investigation (Fig. 16); the results will be discussed in the appropriate section below. It should be noted, however, that the data are also representative of differences in the photosynthetic apparatus induced by changes in light intensity. d. Green sulphur bacteria. The information available on the effect of light intensity on these organisms centres mainly on the chlorobium “vesicles” which have been renamed chlorosomes (Staehelin et al., 1978, 1980). Holt et a/. (1966) studying Chloropseudomonas ethylica reported changes in the cellular amounts of chlorosomes as well as of the Bchl contents of those structures as a response to changes in light intensity. Similarly, in Chlurobium limicola, chromosomes with greater sizes and higher densities were found at low- rather than high-light intensities (Broch-Due et a/., 1978). The ratio of Bchl c (probably of chlorosomes) and Bchl a (probably of membranes) was determined in Chloroflexus aurantiacus after growth at different light intensities (Pierson and Castenholz, 1974). This ratio changed from over 10 to about 2 as the light intensity was increased from 300 to 5000 lux: at higher light intensities it approached a value of 0.8. The changes were predominantly due to changes in Bchl c contents. Bchl a contents changed by only a factor of three. The intracellular distribution and specific functions of the two Bchl derivatives in Chlorojlexus are not known at present, thus an interpretation of the data in the light of functional terms awaits further investigations.

ORGANIZATION AND DIFFERENTIATION OF MEMBRANES

63

2. Oxygen The findings that Bchl synthesis is influenced either positively or negatively by suitable oxygen tensions, and that different cellular Bchl contents under certain circumstances represent differently composed photosynthetic apparatus, suggests that oxygen is involved in regulating the composition of the photosynthetic apparatus (see Section IV.A, p. 37). Although different species of facultative phototrophic bacteria exhibit specific sensitivities towards oxygen, it can generally be concluded that at low oxygen tensions approaching anaerobiosis, cells form high amounts of Bchl. Aeration of phototrophically (i.e. anaerobically) light-grown cultures of photosynthetic apparatus (see Section IV.A, p. 37). Although different illumination of aerobically dark-grown cultures inhibits residual Bchl synthesis (Cellarius and Peters, 1969; K. Arnheim and J . Oelze, unpublished observations). This indicates that the sensitivity towards oxygen of Bchl synthesis is higher in the presence than in the absence of light. Apparently, light enhances the inhibitory effect of oxygen. In order to study these effects, however, care has to be taken to apply defined oxygen tensions and light intensities which, with batch-cultures, require rather thin cell suspensions (K. Arnheim and J. Oelze; B. T. Wakim and J . Oelze, unpublished observations). In this section, because of the antithetical effects of oxygen on the photosynthetic apparatus, we will discuss both aspects, pigmentation and depigmentation, as a response to defined oxygen tensions. a. Rhodospirillum rubrum. Lack of a quantitatively variable light-harvesting Bchl unit in this organism means that changes in the total Bchl contents represent changes in the content of the invariable light-harvesting reactioncentre unit. In the dark, formation by aerobically growing cells of the photosynthetic apparatus is at its maximum when oxygen tensions are up to 5 Torr (665 Pa). At higher tensions, the cellular Bchl contents approach, rather quickly, undetectably low values (Biedermann et al., 1967). As detailed in Section 1V.A (p. 37), formation of the photosynthetic apparatus can initially be followed by a largely independent incorporation of the polypeptides characteristic of reaction centres and light-harvesting Bchl complexes (Oelze and Pahlke, 1976). This initial period is followed by a co-ordinated incorporation of all of the polypeptides. It is noteworthy that during early stages of production of the photosynthetic apparatus rather minor amounts of radioactive amino acids, if any, are incorporated into the major polypeptides (a and /3 subunits) of coupling factor ATPase (Fl). This infers that coupling factors pre-existing in cytoplasmic membranes can sufficiently serve photochemical reactions. In Rs. rubrum, an enhanced incorporation into

64

G. DREWS AND J. OELZE

intracytoplasmic membranes of the respiratory system as well as a shift of radioactively labelled phospholipids from intracytoplasmic to cytoplasmic membranes, under pulseechase conditions, indicated that during adaptation to aerobiosis, intracytoplasmic membranes are differentiated in such a way that they become integrated into cytoplasmic membranes (Oelze and Drews, 1970a, b, 1972). This is in contrast to the structural conservation of intracytoplasmic membranes when, on transfer from low- to high-light intensities, the formation of Bchl is temporarily inhibited (see Section IV.B.1, p. 54; Irschik and Oelze, 1973, 1976). During adaptation to aerobiosis, increases in respiratory-chain specific activities measurable with isolated membranes are significantly higher after growth of the organisms in the dark than after growth in the presence of light (J. Oelze, unpublished observations). This applies, in particular, to the terminal cytochrome c oxidase which shows the most drastic increases in activities, compared to other reactions of the respiratory system. Lack of Bchl synthesis in the presence of oxygen suggests that the photosynthetic apparatus is not used any more. However, it does not mean that it loses its activity. On the contrary, as determined on the basis of its polypeptide composition and light-induced bleachability, the ratio of reaction-centre to light-harvesting units remains constant even after four generations of growth under complete aerobiosis (J. Oelze, unpublished observations). Moreover and unexpectedly, on adaptation to aerobiosis in the dark, photophosphorylation and light-dependent NAD reduction by succinate also exhibit the highest increases in activities (Keister and Minton, 1969; J. Oelze and B. Georg, unpublished observations). An explanation for this can be derived from the following considerations. Because of the inhibition by light of the respiration of whole cells, it may be assumed that the photochemical and the respiratory systems mutually influence their activities. Furthermore, the possibility that a section between cytochromes h and c2 rather than the lightreactive section (i.e. reaction centre or antenna) limits the photochemical electron-transport system (Nishimura, 1962a, b), infers that this limiting step is formed under aerobic conditions, which prevent formation of Bchlcontaining units but favour formation of respiratory reactions. These considerations point to an intimate interrelationship between at least parts of the photochemical and the respiratory chain. It is likely that this is based on constituents common to both electron-transport chains (see Section 11, p. 4). Thus, by placing the rate-limiting step of the photochemical electrontransport system into a section that is regulated by oxygen as a positive effector, growing organisms may counteract diminution of the photosynthetic capacity under conditions unfavourable for Bchl synthesis. Another example for this has already been given in Section 1V.B.I , p. 54. b. Rhodopseudomonas. Changes of oxygen partial pressure in the range +

ORGANIZATION AND DIFFERENTIATION OF MEMBRANES

65

of 3 to 100 mmHg (400 Pa to 14 kPa) in the culture do not influence the growth rate but can induce differentiation of the membrane system (Dierstein and Drews, 1974). In a continuous dark culture of Rp. capsulata, the following Bchl concentrations have been measured at various oxygen partial pressures: at 100 mmHg, the cells contained 0.2 pg Bchl; at 40 mmHg, 2.5 pg; and at 10 mmHg, 4.5 pg Bchl per mg dry weight. A further decrease of p 0 to ~ 5 and 3 mmHg effected a sharp increase of the steady-state level of Bchl to 9.8 and 15.8 pg per mg dry weight, respectively (Dierstein and Drews, 1974). A lowering of oxygen tension in dark-batch cultures of Rp. capsulata induced a multistep process of formation of the photosynthetic apparatus, as described in Section IV.A.2 and 3, pp. 41 and 47. The degree of differentiation of the intracytoplasmic membranes was found to be different from that of the cytoplasmic membrane. In dark cultures at an oxygen partial pressure of 5 mmHg, a Bchl content of 1 10 pg per mg of protein was estimated in the purified intracytoplasmic membrane, whereas the cytoplasmic membrane contained only 10 pg Bchl per mg of protein. The Bchl concentration of intracytoplasmic membrane increased linearly with the specific cellular Bchl content (Lampe and Drews, 1972). Lowering the oxygen partial pressure from 150 to 5 mmHg effected not only an increase of the amount of intracytoplasmic membrane per cell and an increase of the number and size of the photosynthetic units per membrane area, but it also influenced the rate of synthesis of respiratory activities. In the purified intracytoplasmic membrane, the activity of NADH oxidation (kmol of NADH-mg protein-I-min-') decreased from 0.8 to 0.17. The corresponding activity in the cytoplasmic membrane, however, was 1.7 at 5 mmHg. The differentiation of the membrane effected a change of the potential activities of photophosphorylation and oxidative phosphorylation in the intracytoplasmic membrane in opposite directions. The ratio of photophosphorylation to oxidative phosphorylation rose from 1.2 at 150 mmHg, to 5.4 at 5 mmHg ( ~ O Z(Lampe ) and Drews, 1972). Although the biosynthesis of the photosynthetic apparatus was dominant during differentiation, after transition from high to low oxygen tension, components of the respiratory apparatus were still incorporated into the membrane. The capacity of oxidative phosphorylation in purified intracytoplasmic membranes from cells grown at 5 mmHg, compared with cells grown at 150 mmHg, was decreased by approximately 50%. In comparison, the Bchl content of those membranes varied 20-fold and the activity, on a protein basis, of photophosphorylation 10-fold. Under a very low, growth-limiting oxygen tension (0.5 mmHg), the membrane-bound succinate and NADH dehydrogenase activity, as well as the capacity to oxidize succinate and NADH with oxygen as the acceptor,

66

G . DREWS AND J OELZE

did not change significantly for 4 hours after transfer of cultures from 400 mmHg to 0.5 mmHg, whereas the photosynthetic apparatus, preferentially reaction-centre and light-harvesting Bchl I complex, was synthesized (Schumacher and Drews, 1978). Rp. sphaeroides reacts like Rp. cupsulata to changes in oxygen partial pressure. In membranes of aerobically grown cells, B 875 (light-harvesting Bchl I) is the dominant Bchl complex, indicating a small photosynthetic unit (Saunders and Jones, 1974). After lowering of oxygen partial pressure, the components of the photosynthetic apparatus were synthesized and incorporated into the membrane at different rates. Reaction-centre and lightharvesting Bchl complex B 875 were preferentially inserted into the chromatophore membrane during early stages of induction (Takemoto and Lascelles, 1973; Takemoto, 1974; Niederman et al., 1976; Pradel et nl., 1978). Analysis of kinetics of fluorescent induction during formation of the photosynthetic apparatus under semi-aerobic culture conditions lead to the conclusion that integration and formation of photosynthetic units proceed from discrete membrane sites. After 1.5 hours of pigment formation, transfer and trapping efficiencies reached a maximum. The units (26 B 875 + 11 B 800-850 per reaction centres) were still functionally separated. The increase in the unit size proceeded through B 800-850 incorporation. Energy transfer between units occurred 5 hours after induction (Pradel et al., 1978). A similar process of membrane differentiation after transfer from high (130 mmHg) to low (1.5 mmHg) aeration was observed in cells of Rp. palustrzs. The membrane and cellular content of all Bchl-protein complexes increased, and photosynthetic units were larger at 1 mmHg than at 5 mmHg. The capacity to oxidize succinate and NADH decreased (Firsow and Drews, 1977). When photosynthetically grown cultures of Rp. sphaeroides and of Rp. cupsulata were transferred to aerobic conditions, Bchl synthesis was greatly decreased and the in vivo absorption spectrum in the dark became markedly affected. This was due to the fact that the content of light-harvesting Bchl I1 (B 800-850) was lowered more than light-harvesting Bchl I. In spite of a very low total Bchl content, aerobically grown cells were enriched with respect to light-harvesting Bchl 1 and its reaction centre (Aagaard and Sistrom, 1972; Lien et al., 1973). Similarly, in cells of Rp. pulustris, the Bchl synthesis was greatly decreased, and the size of the photosynthetic unit decreased when the cells were transferred from anaerobic light-growth to aerobic dark-growth conditions. The membrane-bound activity of respiratory oxidation of succinate and NADH increased two- to four-fold (Firsow and Drews, 1977). Presumably, new functional subunits which belong mainly to the respiratory apparatus, and small amounts of reaction centre, as well as light-harvesting B 875 units,

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67

were incorporated into the membrane fraction, but light-harvesting B 800850 synthesis was totally suppressed. The content of cytochromes, flavins and ubiquinone changed only slightly during differentiation of the membrane induced by changes in the level of oxygen tension. This indicates first, that parts of the electron-transport chain are common to both the photosynthetic and the respiratory electrontransport chain and second, that these segments of the electron-transport path are not strongly influenced by modifying external factors such as oxygen partial pressure and light intensity (King and Drews, 1975; Niederman et al., 1976). Taken together, the data indicate that in the Rhodopseudomonas species, oxygen tension regulates the synthesis of reaction centres and light-harvesting complexes differently in extent. At relatively high oxygen tensions ( > 50 mmHg for Rp. capsulata 37b4), the reaction-centre and light-harvesting Bchl I units were preferentially synthesized, whereas at low oxygen tensions (5 mmHg for Rp. capsulata) the light-harvesting Bchl I1 complexes were dominant.

3. Temperature Like other enzyme-catalysed reactions, temperature also influences the velocities of reactions coupled to photochemical processes. As might be expected, it has been demonstrated that the activity of coupling factor ATPase exhibits a clear-cut temperature dependency (Gepshtein and Carmeli, 1977; Oelze et al., 1980).This infers that the very activity of the photosynthetic apparatus, photophosphorylation, is also temperature dependent. If, as will be discussed below, a temperature-dependent control device like the state-of-energy metabolism influences formation and composition of the photosynthetic apparatus, then temperature should also be an environmental factor potentially active in inducing differentiation of the photosynthetic apparatus. That this is indeed the case has been reported for Rp. capsulata and Chr. vinosum. a. Rhodopseudomonas capsulata. Lien et al. (1 973) studied the composition of the photosynthetic apparatus in the wild-type and the arsenateresistant mutant strain Z-1 of Rp. capsulata grown at selected temperatures between 22°C and 38°C. In principle, the results revealed that after growth at a low temperature the photosynthetic apparatus contained higher amounts of the quantitatively variable light-harvesting Bchl I1 moiety than after growth at a high temperature. It is also noteworthy that on a total Bchl basis, photophosphorylation activity was higher with membranes de-

G. DREWS AND J. OELZE

68

pleted in light-harvesting Bchl I1 than with membranes exhibiting the full complement of this unit. This indicates that at saturating light intensity, addition of light-harvesting Bchl I1 units does not confer higher activities to the entire photosynthetic apparatus represented by total Bchl (i.e. reaction centres, light-harvesting I and light-harvesting I1 units) (see Sections I1 and 111). On the contrary, even on a protein basis membranes of lower lightharvesting Bchl I1 contents exhibited higher rates of photophosphorylation. These unexpected results should be understood in the light of overall energy metabolism; at low-light intensities or decreased temperatures, the rate of ATP regeneration is also decreased. This apparently provides a signal for enhanced synthesis of light-harvesting Bchl I1 (Lien et al., 1973). The physiological and ecological significance of this regulating system would probably be sufficiently understandable if the quantum requirements at different temperatures of differently composed photosynthetic apparatus were known. Unfortunately, however, information on this is lacking at present.

’

,

O

.

O

350

I

750 I

Wavelength (nrn)

I

I

’

950 ”

FIG. 16. Absorption spectra of membranes isolated from Chromatium vinosum, strain D, grown heterotrophically either at 3 3 T (-) or at 39’C (----). Spectra were taken at room temperature and at - 196’C (77 K) (insert). From Mechler and Oelze (l978b, c).

b. ~ h r o ~ avinosum. t i ~ ~Besides light intensity, temperature also induces the organism to express different spectral characteristics, especially within the near infrared absorption region (Kronenberg, 1969; Mechler and Oelze, 1978a). In contrast to light intensity, however, temperature also induces spectral changes in cultures of Chr. vinosum grown photoheterotrophically

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69

(Mechler and Oelze, 1978a), and, in this case, there is a threshold at 36.5”C. When cells are grown below this temperature, they exhibit a spectrum which is also characteristic of cells grown auto- or mixotrophically at low-light intensities; above 36.5‘C the spectrum formed is characteristic of cells grown auto- or mixotrophically at rather high-light intensities (Fig. 16). Solubilization, according to Thornber (1970), of chromatophores of the two extremely modified spectral types yielded in either instance a constantly composed reaction-centre light-harvesting preparation (ratio of reaction centre to light-harvesting Bchl 1 : 45). In addition, a second light-harvesting Bchl preparation could be obtained which varied not only quantitatively, relative to the reaction centre, but also showed different spectral properties. Preparations derived from cells grown below 36.5’’C had an absorption maximum at 800 nm with shoulders at 820 and 850 nm, whereas those from cells grown above 36.5”Cshowed an absorption maximum at 850 nm and a slightly smaller peak at 800 nm.

TABLE 6. Composition and activities of the photosynthetic apparatus of Chromatium vinosum strain D grown photoheterotrophically (3000 lux) at either 33 C or 39 C. Values were calculated on the basis of results obtained after measurements on membrane preparations or whole cells (sulphide oxidation). Functional tests were performed at 20 C. The values are presented on a cellular protein (mg) basis. From Mechler and Oelze (1978b). Contents (per mg of cell protein) Growth Reaction temperature Bchl centre Chromatophore ( C) (nmol) (nmol) protein (mg) 33 39

55 38

0.29 0.285

0.39 0.25

Photophosphorylation (nmol ATP/min)

Sulphate oxidation (nmol/min)

40.6 19.3

131 68

On the basis of these and additional functional analyses, temperaturedependent differentiation in Chr. vinosum grown under constant illumination could be defined on the basis of the following results (Table 6). When cells exceeded the temperature threshold at 36.5”C (i) intracytoplasmic membrane protein decreased from about 0.4 to 0.25 mg per mg of cell protein, (ii) cellular contents of reaction centres stayed constant although (iii) contents of light-harvesting Bchl decreased, due to the variable moiety, from 55 to 38 nmol per mg of cell protein. This was paralleled by decreases in (iv) photophosphorylation activity and (v) light-dependent sulphide oxidation. The full extent of the latter two differences in light-dependent activities can be estimated more adequately when presented on parameters of the photo-

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G. DREWS AND J. OELZE

synthetic apparatus. For example, on a total Bchl basis, both activities are just slightly higher at 33°C than at 39°C but on a reaction-centre basis, representing the photochemical system, they are twice as high. Overall, the data show that at a given light intensity Chr. vinosum responds to defined changes in temperature by changing the cellular amount of the variable light-harvesting moiety, but not of the invariably composed reactioncentre light-harvesting unit. In Chr. vinosum, when contrasting other photosynthetic bacteria with a variable second light-harvesting unit a relative increase in the proportion of this unit also increases the activities of physiologically significant reaction systems like photophosphorylation and sulphide oxidation. On the basis of quantitative cytochrome or coupling-factor ATPase determinations, no other alterations in the composition of photochemical electron-transport system and coupled reactions could be recorded. Therefore, it seems probable that Chr. vinosum, by a relatively slight variation in the amounts of the variable light-harvesting pigment unit, but a considerable alteration in its absorption properties, is capable of regulating, rather elegantly, the efficiency of the photosynthetic apparatus.

4. Nutrition The growth rate in a bacterial population is effected by substrate limitation. This section discusses whether the limitation of nutrition also influences differentiation. This was studied in a continuous culture regulated by the ammonium concentration in the inflowing medium. The formation of the photosynthetic apparatus was induced by a constant low-oxygen tension of 5 mmHg (667 Pa; Dierstein and Drews, 1974). The oxidative energy metabolism was not limited. When the growth rate was raised by increasing the ammonium concentration of the inflowing medium, the number of photosynthetic units per area membrane (which equals concentration of reaction centres) remained almost constant, but the size of the photosynthetic unit and the potential activity of photophosphorylation per reaction centre and per mg of membrane protein increased. The following steady-state concentrations and activities were observed at growth rates of 0.042 and 0.23 h-l, respectively: 4.8 and 24.2 pg total Bchl per mg of cell protein; 19.6 and 79.5 pg Bchl per mg of membrane protein; 0.18 nmol reaction centre per mg of membrane protein at both growth rates; 113 and 443 mol total Bchl per mol of reaction centre; and 30.5 and 178 nmol ATP per min per mg of membrane protein. Although the oxygen tension in all experiments was constant, and consequently the degree of induction was the same, the ammonium concentration influenced growth rate as well as differentiation. An increase in specific Bchl content was also observed in continuous

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anaerobic light-grown cultures of Rp. sphaeroides and Rs. rubrum when the growth rate was elevated (Cohen-Bazire and Sistrom, 1966). Rs. rubrum synthesized Bchl and intracytoplasmic membranes with pyruvate (0.2%) or fructose (2%) as substrates, when transferred from aerobic dark to anaerobic dark conditions. The formation of the photosynthetic apparatus was greatly enhanced when potassium ferricyanide was added in addition to fructose (Schon, 1972). Under nitrogen limitation and in the presence of acetate, the intracytoplasmic membranes in resting cells of Rs. rubrum were transformed into irregular membrane aggregates within 1-4 days, but the Bchl content of cells remained unchanged during this period. The rate of photophosphorylation and of respiration decreased (Schon and Jank-Ladwig, 1972). Influences on the differentiation by growth-limiting factors might be important during adaptation of photosynthetic bacteria to an ecological niche. Further studies on regulation of differentiation should take into consideration that besides the major factors, e.g. oxygen partial pressure and light intensity, the size of metabolic pools can also modify the process of differentiation. This could be due to direct limitation of a biosynthetic pathway influencing the regulation pattern involved in differentiation.

C . REGULATION OF DIFFERENTIATION

Membranes consist of numerous multi-component systems. Consequently, regulation of formation and differentiation of photosynthetically active membranes depends primarily on the regulation of individual biosynthetic pathways. This includes, for instance, biosyntheses of phospholipids, proteins, Bchl, carotenoids, and various electron-transport constituents like hemes, quinones and flavins. The state of affairs, however, becomes much more complicated when it is realized that the development of a functional membrane depends not only on the regulated synthesis of different classes of components but also on parameters like (i) synthesis of different derivatives of a compound, (ii) binding or association of derivatives with other constituents like proteins, as well as (iii) incorporation of the respective constituents into appropriate regions of the membrane. For example, different species of the phototrophic bacteria synthesize not only Bchl, they also form several Bchl derivates and/or Bchl-protein complexes in characteristic ratios, and they also incorporate the pigment complexes into the proper membrane (i.e. predominantly into intracytoplasmic membranes). Furthermore, an adequate ratio of different heme derivatives, including specific protein moieties, is required to make up a functional electron-transport chain; and finally, it is not sufficient only to synthesize quinones, the participation in

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G . DREWS AND J. OELZE

different sections of photochemical electron transport of a single derivative like ubiquinonel 0 also necessitates incorporation into distinct functional sections of even one single chain. However, lack of adequate information scarcely permits realistic speculations on most of the regulation problems detailed above. Much more is known about synthesis of various classes of components, particularly on the regulation of Bchl synthesis. This knowledge, because of the exclusiveness of Bchl in bacterial photosynthesis, is of outstanding importance in understanding the regulatory processes involved in membrane differentiation. This can be postulated on the basis of findings which indicate, first, that, concomitant with Bchl synthesis, the whole photosynthetic apparatus, including the membrane structure, is formed in wild-type strains (see Section IV.A, p. 37) and, second, that an intimate relationship exists between the syntheses of Bchl and other constituents of the membrane-bound photosynthetic apparatus. The latter is supported by the following observations: (i) mutual effects of heme and Bchl on their respective biosynthesis pathways have been described (Lascelles, 1978); (ii) synthesis of Bchl, in particular of the light-harvesting Bchl I1 complex, and carotenoid synthesis seem to be correlated (Marrs, 1978; Sistrom, 1978); (iii) fluidity changes in the membrane lipids, resulting from continuous incorporation of proteinassociated Bchl complexes, have been postulated to trigger phospholipid synthesis (Fraley et ul., 1978; Kaplan, 1978; Wraight er ul., 1978b). Because of this obviously central function of Bchl synthesis in the formation and differentiation of the photosynthetic apparatus, we will comprehensively repeat physiologically important regulatory phenomena involved in this pathway before we speculate about processes that might be active on a cellular basis. Inhibition of 6-aminolaevulinate synthase, which is a key enzyme in tetrapyrrole synthesis (Lascelles, 1978), by heme, protoporphyrine, magnesiumprotoporphyrine and ATP has been reported. On the other hand, the enzyme is activated by trisulphides. It has been suggested that trisulphides are involved in the conversion of a low-activity form of 6-aminolaevulinate synthase into a high-activity form (Fig. 9; Hayasaka and Tuboi, 1974; Sandy et ul., 1975; Lascelles, 1978). Generally speaking. regulation of 6-aminolaevulinate synthase activity is subject to end-product control, parameters representing energy metabolism and the redox state of specific effector molecules. In 1962, Sistrom concluded that the cellular Bchl content was largely inversely related to the specific growth rate. Subsequent investigations, however, restricted this relationship to conditions of growth limited by light (Sistrom, 1963). On the basis of this and on the observations that under otherwise optimum conditions, phototrophic growth depends on photo-

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phosphorylation and chemotrophic growth on oxidative phosphorylation (Oelze et al., 1978), it may be concluded that the inverse relationship between growth rate and the cellular Bchl contents are coincident results of the respective state of cellular energy metabolism. In accordance with this, Gest (1972) postulated the cellular energy state to be involved in regulation of Bchl synthesis. Direct determinations of adenine nucleotide cellular pools as well as calculation of the energy charge, however, did not allow clearcut conclusions (Schon, 1969; Schon and Bachofen, 1970). However, MioviE and Gibson ( I 973) reported that in Chromatium vinosum different rates of Bchl synthesis were measurable without any relationship to the levels of ATP or energy charge. The concept that the redox state of an effector molecule might control Bchl synthesis was first postulated by Cohen-Baziare et al. (1957). These authors proposed a constituent of the photochemical electron transport to be the responsible effector. Marrs and Gest (1973b) extended this hypothesis by placing the redox effector aside from, but into some loose contact with, the photochemical electron-transport chain. This contact at the level of cytochrome c2 was proposed to enable light-driven electron flow to indirectly influence Bchl synthesis. Accordingly, at high-light intensities, the redox state of cytochrome c2 becomes positive (the rate-limiting step in photochemical electron transport is postulated to be situated in the section between cytochromes b and c2 (see also Section I1 of this article) which allows draining of electrons off the effector. On the other hand, at low-light intensities, lack of light-induced electron flow keeps cytochrome c2 in a more reduced state, allowing electrons to flow to the effector which in turn activates Bchl synthesis. At this point, it is rather tempting to speculate that the effector molecule might be represented by trisulphides involved in the activation of 6-aminolaevulinate synthase (Fig. 9; Sandy et al., 1975). Alternatively, the redox state of nicotinamide nucleotides was proposed by Sistrom ( 1963) to regulate Bchl synthesis. Schon and Drews (1968) tested the possible involvement of the redox state in the regulation of Bchl formation and did not find any significant indications to support this hypothesis, on the basis of nicotinamide nucleotides or c- and b-type cytochromes. At present, we know that the experimental conditions (illumination of low-aerated cultures) chosen by Schon and Drews (1968) do not necessarily allow Bchl synthesis. Thus, the question is still open to experimental control. We do not want to evaluate the different hypotheses on external effectors in Bchl synthesis, but rather to propose that all of them, in principle, do not exclude each other. This is supported by the following considerations. In photoheterotrophically growing cells, ATP is formed coupled to the photochemical process, whereas reduced nicotinamide nucleotides become available through oxidations of organic substrates (Fig. 17). Both ATP and

G. DREWS AND J. OELZE

74 P865

0000-850

8875

bolites

Biosyntheses

Growth

FIG. 17. Model for regulation of syntheses of Bchl and related proteins to form pigment-protein complexes. The redox state of a factor F (Marrs and Gest, 1973b) which holds the central position can be influenced by (i) the redox state of a constituent of the electron-transport chain (Cohen-Bazire et al., 1957), (ii) the redox state of nicotinamide nucleotides (Sistrom, 1963), (iii) oxygen and (iv) light (which is supposedly mediated by a sensory pigment; Kaplan, 1978). The factor F is postulated to be involved in regulation of enzymes of the Bchl synthetic pathway of which 8-aminolaevulinate (ALA) synthase is a most likely candidate (Lascelles, 1978). The activity of this enzyme has been shown to be regulated by ATP (Fanica-Gaignier and Clement-Metral, 1973b) and it thus becomes a likely target for the regulatory effect postulated to be exerted by energy charge on Bchl formation (Gest, 1972). Further regulation of the synthase activity has been reported by haeme and probably also by precursors of Bchl (Lascelles, 1978). Also, a direct influence of oxygen on enzymes catalysing tetrapyrrole synthesis must be considered (not shown in the scheme; Lascelles, 1978). In those species of phototrophic bacteria able to form a second light-harvesting Bchl (11) complex, the F factor (active) may be involved in signal chains which, via transcription and translation, affect expiession of genes coding for additional Bchl and appropriate protein moieties. Besides their respective regulatory functions, ATP and reduced nicotinamide nucleotides feed, together with metabolites, anabolic pathways like those active in formation of pigments and protein of Bchl-protein complexes, as well as those generally required for growth. For a more detailed description see the text. Factors influencing the state of F are indicated by - - -+, inhibitory effects by ------+ and stimulation by A. ~

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reduced nicotinamide nucleotides are used for various anabolic processes; thus, under otherwise balanced conditions, a surplus in ATP may lead to an accumulation of oxidized nicotinamide nucleotides. Conversely, low ATP levels may lead to increased levels of reduced nicotinamide nucleotides. ATP levels, on the other hand, depend on the efficiency of the cellular photochemical electron-transport systems, and, accordingly, on the respective light intensities that in turn influence the redox state of electron-transport constituents like cytochrome C Z . The model proposed by Marrs and Gest (1973b) (Fig. 17) allows the functioning of all of the three effectors; i.e. ATP (or energy charge) levels and the redox states of electron-transport carriers as well as of nicotinamide nucleotides. In this context, it might be possible that oxygen is indirectly involved in regulation by influencing the redox state of nicotinamide nucleotides. This might apply to cultures growing aerobically in the dark, but not to aerobic cultures growing in the light. In the latter case, light inhibits respiration and thereby the oxidation by oxygen of reduced nicotinamide nucleotides. Consequently, aeration of illuminated cells should alter neither the redox level of nicotinamide nucleotides nor the composition of the adenine nucleotide pool and, therefore, Bchl synthesis should not be affected. This, however, is not the case. Because of this discrepancy, Marrs and Gest (1 973b) suggested that oxygen might directly influence the redox state of the regulating factor (F). Apart from this, it should be kept in mind that through feedback inhibition and ATP levels, 6-aminolaevulinate synthase may be influenced directly in activity (Fanica-Gaignier and Clement-Metral, 1973b; Lascelles, 1978). Regulation of the light-harvesting Bchl I1 complex was proposed to be dependent on the energy state of cells (Lien et al., 1973). Evidence for this has been derived from the following findings: (i) Rp. capsulata (mutant strain Zl) contains higher amounts of light-harvesting Bchl 11 after growth at low temperatures (22 C) than after growth at elevated temperatures (38 ' C )and, as might be expected, growth rates representing energy flux were lower at 22°C than at 38'C (Lien et al., 1973); (ii) during optimal growth under aerobic conditions in the dark, Rp. capsulata forms a photosynthetic apparatus that lacks light-harvesting complex I1 (Lien et al., 1973); (iii) cultures of Rp. sphaeroides grown at high-light intensities have a lower light-harvesting Bchl I1 content compared with cultures grown at low-light intensities (Aagaard and Sistrom, 1972; Takemoto and Huang Kao, 1977). As mentioned above, however, the results may be correlated not only to the cellular energy state but also to the state of redox constituents. Alternatively, Kaplan (1 978) explained differences in the regulation of reaction-centre and light-harvesting Bchl systems on the basis of a repressor postulated to exhibit greater affinity for regulatory sites involved in lightharvesting Bchl synthesis than for those involved in reaction-centre Bchl

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G DREWS AND J OELZE

synthesis. Formation of the repressor, consisting of a co-repressor and an aporepressor, depended largely on the level of co-repressor which, according to Kaplan, can be regulated by several factors like light intensities, carbon source and oxygen partial pressure. A combination of the different hypotheses may be proposed on the basis of identical regulating effectors which, in the cases of reaction-centre and light-harvesting Bchl I production, control enzyme activities and, in the case of light-harvesting Bchl 11, control enzyme formation plus activities. Before going into a more detailed evaluation of this possibility, we shall repeat some crucial observations: (i) in Rs. ruhrum, Bchl is associated with reaction centres and a quantitatively invariable light-harvesting Bchl complex, in addition, Rhodopseudomonas species form a second quantitatively variable light-harvesting Bchl (11) complex (see Sections I11 and IV.A, pp. 9 and 37); (ii) the content of light-harvesting Bchl I1 is higher in cells grown under low- than under high-light intensity and aeration, respectively (see Section IV.B, p. 53); (iii) formation of the photosynthetic apparatus is always started by production of the reaction-centre-light-harvesting Bchl I complex, while the production of light-harvesting I1 complex is always relatively delayed (see Section IV.A, p. 37); (iv) some members of the phototrophic bacteria, as for example Rp. capsulata and Rp. palustris, form the reaction-centre-light-harvesting I complex even under conditions of high aeration, i.e. under typical chemotrophic conditions (see Section IV.B, p. 53). These results suggest that the system synthesizing the reactioncentre and light-harvesting I Bchl is largely constitutive, whereas its activity is regulated immediately by alterations in environmental factors. On the other hand, the system synthesizing light-harvesting Bchl I1 complexes exhibits properties characteristic of inducible or derepressible enzyme systems. Except for special mutants, this type of regulatory device permits, rather economically, formation of light-harvesting I1 complexes only if reaction centres and light-harvesting I complexes are already present. On the basis of present knowledge, the requirements of such a hypothetical enzyme system are largely satisfied by 6-aminolaevulinate synthase and the respective dehydratase of Rs.ruhrum and Rp. sphaeroides. In particular: (i) phototrophically grown cells of Rs. rubrum that lack light-harvesting Bchl 11 exhibit about twice as much 6-aminolaevulinate synthase activity as fully aerated chemotrophic cells, whereas 6-aminolaevulinate dehydratase activities were identical with both types of cultures (Drews and Oelze, 1966); (ii) phototrophically grown cells of Rp. sphaeroides which produce light-harvesting Bchl I1 exhibit up to ten times more 6-aminolaevulinate synthase activity and twice as much dehydratase activity as fully aerated chemotrophically grown cells (Lascelles, 1968); (ii) increases in 6-aminolaevulinate synthase activities after transfer of fully aerated cells of Rp. sphaeroides to conditions of Bchl

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synthesis are attributed primarily to enzyme synthesis de novo (Lascelles, 1978); (iv) at least two different forms of 6-aminolaevulinate synthase have been isolated from phototrophically grown Rp. sphaeroides (Fanica-Gaignier and Clement-Metral, 1973a; Hayasaka and Tuboi, 1974), but in Rs. rubrurn there is no indication for multiple synthases (H. Zilg, unpublished observations). Of course, the regulatory mechanism postulated above needs further experimental proof. On the basis of methods and mutants presently available, however, this should, in principle, be feasible. Biosynthesis and incorporation of Bchl into the membrane depends on the synthesis of proteins, specifically those of reaction centres and lightharvesting Bchl complexes (Sections IV.A,2 and 3, and Section IV.B, pp. 41, 47 and 53). The results reported in this article indicate that polypeptides of reactioncentre and light-harvesting complexes I are co-regulated, whereas regulation of polypeptides of light-harvesting complex I1 is presumably largely independent of reaction-centre complexes. Genes for carotenoid and Bchl are closely linked (Yen and Marrs, 1976). A block in the synthesis of mature Bchl inhibits incorporation of several specific proteins into the intracytoplasmic membrane (Takemoto and Lascelles, 1973; Drews et al., 1976). A genetic replacement of a block in the pigment cluster in the genome of a white mutant of Rp. capsulata by transfer of a small genome segment, restores the synthesis of reaction-centre and light-harvesting I protein complexes. A second transfer restores the additional formation of light-harvesting I1 complex and carotenoids (Drews et al., 1976). Hence, regulatory and/or structural genes for synthesis of these polypeptides seem to be localized in, or close to, the pigment cluster of the genome. Moreover, the intimate relationship between protein and Bchl synthesis is supported by the following findings. Bchl-negative mutants synthesize, at the most, basic levels of such proteins that are associated, under normal conditions, with Bchl. Several mutants have been described which excrete precursors of Bchl into the medium. The precursors have been identified as being bound to proteins, which are, however, not identical with reaction-centre or light-harvesting complex proteins detected in the membrane (Oelze and Drews, 1970c; Drews et al., 1971; Drews, 1974). Furthermore, inhibition of protein synthesis at the level of transcription or translation also inhibits Bchl formation (Bull and Lascelles, 1963; Biedermann et al., 1967). These results raise the question of how synthesis of proteins is co-ordinated with Bchl synthesis and vice versa. Lowering of oxygen tension induced, first of all, formation of reaction centres and light-harvesting Bchl I complexes. It is proposed that a hypothetical factor (Fig. 17) in its reduced state activates the pathway of Bchl synthesis and effects, by a signal chain,

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G. DREWS AND J. OELZE

transcription of genes for polypeptides of pigment complexes. Lascelles (1978) postulated that if more Bchl is produced than can be associated with the proper polypeptide, an intermediate in Bchl synthesis might inhibit 6-aminolaevulinate synthase by a feedback mechanism. On the other hand, polypeptides that cannot assemble with Bchl in the membrane might also be able to affect their own synthesis on the basis of translational control. Thus, both pigments and polypeptides may supposedly effect a fine control of the co-ordinated synthesis of Bchl and their specific polypeptides. According to the regulatory model proposed above (Fig. 17) synthesis of Bchl and the polypeptides of light-harvesting Bchl 11 complexes might also be co-regulated. This means that a hypothetical factor, repressing or derepressing the synthesis of enzymes for Bchl formation, should exhibit the same effect with respect to synthesis of polypeptides in forming the lightharvesting complex 11. This hypothesis would include the possibility that the signal chains between factor F (see Fig. 17) and the effector controlling transcription of genes for enzymes and polypeptides of light-harvesting complex 11, respectively, differed from each other. The experimental data support the idea that syntheses of light-harvesting complex I1 polypeptides and of enzymes for Bchl synthesis might be controlled by a specific regulatory gene which is probably linked to a carotenoid gene (Marrs, 1978). Effectors influencing the activity of enzymes for Bchl synthesis may also influence protein synthesis on a translational basis. It has been reported in Section IV.A.3 (p. 47) that in Rp. capsdata, one polypeptide of lightharvesting I1 complex presumably originates from a larger precursor through limited proteolysis (R. Dierstein and G. Drews, unpublished observations). On the basis of these findings, a fine control of the formation of lightharvesting I1 polypeptides might be due to translational control by one of the products of polypeptide processing.

V. Conclusions It is generally accepted that phototrophic bacteria are recent representatives of an evolutionary ancient group of living organisms. One of the major arguments in favour of this hypothesis is the fact that all members of the phototrophic bacteria perform an anoxygenic photosynthesis. It may be speculated that the acquisition of tetrapyrrole synthesis enabled original forms of these organisms to adapt to an aerobic energy metabolism employing cytochromes in a respiratory chain with oxygen as the electron acceptor. Nowadays, phototrophic bacteria can be subdivided with respect to oxygen tolerance into the obligate and the facultative phototrophic members, with which phototrophy depends largely on the absence of oxygen.

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In this article, it has been demonstrated that the photosynthetic apparatus of several species is localized in intracytoplasmic membranes, whereas the respiratory chain is contained predominantly in the cytoplasmic membrane. However, this cytological compartmentation of electron-transport chains cannot be generalized. There are also species that d o not respire but form photosynthetically active intracytoplasmic membranes (e.g. members of the Chromatiaceae); others contain both the photosynthetic apparatus and the respiratory system in the cytoplasmic membrane (e.g. Rs. tenue and ChloroJEexus)and, moreover, there are obligate phototrophs that contain the photosynthetic apparatus in the cytoplasmic membrane (Chlorobium). Although photosynthetic bacteria exhibit considerable metabolic versatilities, all of them are largely restricted to ecological niches providing anaerobic conditions (Pfennig, 1978). This is because phototrophy, as far as is known, is the principal mode of energy metabolism and oxygen, which inhibits Bchl formation, also inhibits the formation of the photosynthetic apparatus (Section IV, p. 36). The formation of Bchl in general, and the formation of different functional units (i.e. reaction-centre and lightharvesting Bchl complexes) in particular, is rather sensitive towards changes in environmental conditions (Section IV, p. 36). In this context, it has been reported above that conditions affecting photometabolisms of cells like light intensity, temperature and substrate- and oxygen-mediated pool sizes of intermediates also regulate Bchl formation. All of the species investigated so far show higher Bchl cellular levels at low-light intensities, low temperatures, or low-oxygen tensions than at higher values of these parameters. (It is understood that this applies to physiologically relevant ranges only.) In addition, in those species that produce a second light-harvesting Bchl complex or contain light-harvesting bacteria chlorophylls c-e in chlorosomes with high levels of cellular Bchl, also have high proportions of the variable Bchl complexes. Consequently, factors influencing the formation of Bchl and it’s associated proteins also influence the formation and composition of the photosynthetic apparatus (Section IV, p. 36). This indicates that both the cellular contents in photosynthetic units, as approximately determined with competent species on the basis of intracytoplasmic membrane contents, as well as the ratio of light-harvesting Bchl to reaction centre, are subject to variations. On the basis of the present knowledge which, admittedly, rests on investigations with a few selected species, the following principles of cellular differentiation can be noted with respect to the photosynthetic apparatus. Features that are probably also inherent in other representatives are exhibited by Rs. rubrum. In this organism, Bchl is associated with reaction-centre proteins and a quantitatively and qualitatively invariable light-harvesting Bchl-protein unit. Over a relatively broad range of cellular Bchl contents, the amount of photosynthetic units is constant on a chroma-

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tophore protein basis. This infers that within this range, cells vary the amounts of intracytoplasmic membranes under appropriate changes in the culture conditions. Below this range, however, specific Bchl contents of cells and chromatophores are, for the most part, proportional. Apparent rates of photophosphorylation on a reaction-centre basis stay constant as the Bchl contents of membranes stay constant. But, rather unexpectedly, they increase or decrease as the cells reach the region of decreasing or increasing Bchl contents of chomatophores. Comparable dependencies of specific cellular Bchl contents and the relative amounts of the reaction-centre lightharvesting Bchl I (B 875) unit of chromatophores have also been reported for various Rhodopseudomonas species. For members of this group there also exist ranges of Bchl cellular contents within which chromatophores exhibit either variable or constant contents in the invariable reaction-centre lightharvesting Bchl (B 875) complex. This, however, is superimposed by the second variable light-harvesting Bchl complex (B 800-850) which varies in proportion to the cellular Bchl contents. In other words, the formation of the two light-harvesting units (B 875 and B 800-850) follow different kinetics. In contrast to facultative phototrophic bacteria, like various species of Rhodospirillum, Rhodopseudomonas and Chlorojexus, some members of the obligately phototrophic bacteria investigated so far show only small changes in the relative amounts of a variable light-harvesting Bchl complex in response to considerable changes in light intensities. Additionally, Chr. vinosurn also alters the spectral properties along with a maximal two-fold alteration of the amount of the variable light-harvesting Bchl moiety, thereby conferring different efficiencies to the photosynthetic apparatus (Section IV.B, p, 53). Overall, the data reported in this article demonstrate essential similarities as well as specific differences in the responses of various members of the phototrophic bacteria towards changes in the environmental conditions. Information already scattered in the literature and notebooks, however, suggests that the degree of dissimilarity will increase after extending the investigations to other species. We hope that this review not only provides information, but will also stimulate new research activities on the physiology of a group of bacteria that has already contributed a good deal of knowledge towards the understanding of cell differentiation in living systems.

V I. Acknowledgements We thank Mrs. Nahrig for making the line drawings. The work of the

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authors was supported by the Deutsche Forschungsgemeinschaft (Dr 29/22-23; Oe 5513-1 and SFB 46).

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Worden, P. B. and Sistrom, W. R. (1964). Journal of’ Cell Biology 23, 135. Wraight, C. A., Cogdell, R. J. and Chance, B. (1978a). In “The Photosynthetic Bacteria” (R. K . Clayton and W. R. Sistrom, eds.), p. 471. Plenum Press, New York. Wraight, C. A., Lueking, D. R., Fraley, R. T. and Kaplan, S . (1978b). Journal of’ Biological Chemistry 253, 465. Yen, H.-C. and Marrs, B. (1976). Journal of’Bacferiology 126, 619. Yoch, D. C., Carithers, R. P. and Arnon, D. I. (1977). Journal of’Biological Chemistry 252, 7453. Yubisui, T. and Yoneyama, Y. ( 1 972). Archives oj’Biochemistry and Biophysics 150,77. Zankel, K. L. (1978). In “The Photosynthetic Bacteria” (R. K. Clayton and W. R. Sistrom, eds.), p. 341. Plenum, New York. Zannoni, D., Melandri, B. A. and Baccarini-Melandri, A. (1976a). Biochimica et Biophysica Acta 423. 413. Zannoni, D . , Melandri, B. A. and Baccarini-Melandri, A. (l976b). Biochimica et Biophysica Acta 449, 386. Zannoni, D . , Jaspers, P. and Marrs, B. (1978). Archives of’Biochemistry and Biophysics 191, 625. Ziirrer, H., Snozzi, M., Hanselmann, K. and Bachofen, R. (1977). Biochimica et Biophysica Acta 460, 273. Note added in proof: Since this review was submitted, several papers on the function and composition of the bacterial photosynthetic apparatus have been published, including reviews by Blakenship and Parson (1 979). Thornber and Barber (1979) and Olson ( I 980). Much research has been on isolating and characterizing functional subunits, particularly Bchlcontaining complexes, of the bacterial photosynthetic apparatus, and data on B 875 and B 800850 complexes have been reviewed (Cogdell and Thornber, 1980). Research on photochemical reaction centres has mainly concerned investigating functional parameters in artificial systems (Schonfeld et al., 1979; Janzen and Seibert, 1980), and further purification and characterization. Vadeboncoeur et al. (1979) and Rivas et al. (1980) reported molecular weights of 90,000 and 84,000 for the reaction-centre protein from Rhodospirillum ruhrum and Rhodopseudomonas sphaeroides, respectively. Thornber et al. (1 980) found no basic difference in the biochemical composition of isolated reaction centres from Bchl b-containing bacteria compared with Bchl a-containing centres. Bengis-Garber and Gromet-Elhanan (1979) isolated an oligomycin and A”’-dicyclohexylcarbodiimide-sensitive ATPase preparation with only eight different polypeptides rather than the reported 13 (see Section III.B.3, p. 27). In experiments on cytoplasmic and intracytoplasmic membranes, Holmqvist (1979) proposed lack ofcontinuity in Rp. sphaeroides membranes, whereas a high degree of continuity was found from functional and labelling experiments with Rs. ruhrum and Rp. sphaeroides (Oelze and Post, 1980; Francis and Richards, 1980), which supports previous results (Prince et al.. 1975). Intracytoplasmic membranes prepared by cell homogenization contained mostly closed insideout orientated chromatophore vesicles (Oelze, 1978; Elferink et al., 1979; Takemoto and Bachmann, 1979). However, Elferink et al. (1979) isolated cytoplasmic membrane vesicles with more than 75% exhibiting right-side out orientation. Arrangement of the three polypeptides of reaction centres is unresolved (see Section 1II.C. p. 30). Results from labelling membranes of Rs. rubrum suggest that the heavy (H) polypeptide spans the membrane (Bachofen, 1979; Odermatt et al., 1980), but none of the reaction-centre polypeptides was accessible to labelling from the inner or periplasmic space of intracytoplasmic membranes of Rp. sphaeroides (Francis and Richards, 1980). Investigations on the development of photosynthetically active membranes were concerned with the question of the involvement of pre-existing cytoplasmic membranes. Freeze-etch electron micrographs showed that, on adaptation from chemo- to phototrophic conditions, cytoplasmic membranes of Rhodospirillum tenue and Rs. rubrum became homogeneously differentiated by appearance of intramembrane particles of typical sizes (Golecki and Oelze, 1980). No such differentiation was observed in Rp. sphaeroides, supporting the data of Nieder-

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man (see Sections IV.A.2, p. 43 and IV.A.3, p. 47). Accordingly, material of the “upper pigmented band” (of sucrose density gradients) served as a precursor in intracytoplasmic membrane formation. Niederman et al. (1979) suggested that the material of this band arose from specialized sites in the cytoplasmic membrane at which invagination is initiated, rather than from native precursor particles. Rhodopseudomonas capsulata, in contrast to Rp. sphaeroides. contains tubular intracytoplasmic membranes when grown aerobically in the dark. Under growth-limiting conditions, both cytoplasmic and tubular intracytoplasmic membranes of Rp. capsulata incorporated constituents of the photosynthetic apparatus (Dierstein et al., 1981). This agrees with data for Rp. sphaeroides (see Section IV.A.3, p. 47) and suggests that existing intracytoplasmic and cytoplasmic membranes may undergo independent differentiation. Taken together, the data suggest that the extent of involvement of cytoplasmic membranes in intracytoplasmic membrane formation varies according to the species. One hypothesis on regulation of Bchl formation and, consequently, also of formation of photosynthetic apparatus, postulated an inverse relation between rates of energy regeneration (represented by growth rate) and cellular Bchl levels, particularly of the accessory light-harvesting unit B 80&850. Studies with Rp. capsulata grown at various temperatures (see Section IV.C, p. 71) support this hypothesis, but results with Rp. sphaeroides and Rs. rubrum grown at different temperatures do not (Kaiser and Oelze, 1980a. b). REFERENCES

Bachofen, R. (1979). Federution of the European Biochemical Society Letters 107, 409. Bengis-Garter. C. and Gromet-Elhanan, 2. (1979). Biochemistry 18, 3577. Blankenship. R. E. and Parson, W. W. (1979). In “Photosynthesis in Relation to Model Systems” (J. Barber, ed.), p. 71. Elsevier, Amsterdam. Cogdell, R. J . and Thornber, J. P. (1980). Federation of the European Biochemical Society 122, I . Dierstein, R., Schumacher, A. and Drews, G. (1981). Archives of Microbiology 128, 376. Elferink, M. G. L., Hellingwerf, K. J., Michels, P. A. M., Seyen, H. G . and Konings, W. N. (1979). Federation of the European Biochemical Society Letters 107, 300. Francis, G . A. and Richards, W. R. (1980). Biochemistry 19, 5104. Golecki, J. R. and Oelze, J. (1980). Journal ofBacteriology 144, 781. Holmqvist, 0. (1979). Federation of the European Microbiological Society Letters 6, 37. Janzen, A. F. and Seibert, M. (1980). Nature, London 286, 584. Kaiser, I . and Oelze, J . (1980a). Archives of Microbiology 126, 187. Kaiser, I . and Oelze, J. (1980b). Archives of Microbiology 126, 195. Niederman, R. A,, Mallon, D. E. and Parks, L. C. (1979). Biochimicu et Biophysica Acta 555, 210.

Odermatt, E., Snozzi, M. and Bachofen, R. (1980). Biochimica et Biophysica Acta 591, 372. Oelze, J . (1978). Biochimica et Biophysica Act 509, 450. Oelze, J. and Post, E. (1980). Biochimicu et Biophysica Acta 591, 76. Olson, J. M. (1980). Biochimica et Biophysica Acta 594, 33. Prince, R. C., Baccarini-Melandri, A,, Hauska, G. A,, Melandri, B. A. and Crofts, A. R. (1975). Biochimica et Biophysica Acta 387, 212. Rivas, E., Reiss-Husson, F. and le Maire, M. (1980). Biochemistry 19, 2943. Schonfeld, M., Montal, M. and Feher, G. (1979). Proceedingsof the National Academyof Sciences of the United States of America 76, 635 1. Takemoto, J. and Bachmann, R. C . (1979). Archives of Biochemistry and Biophysics 195, 526. Thornber, J. P. and Barber, J . (1979). In “Photosynthesis in Relation to Model Systems” (J. Barber, ed.), p. 27. Elsevier, Amsterdam. Thornber, J . P., Cogdell, R. J., Seftor, R. E. B. and Webster, G. D. (1980). Biochimica et Biophysica Acta 593, 60. Vadeboncoeur, C., Noel, H., Poirier, L., Cloutier, Y.and Gingras, G. (1979). Biochemistr-v 18, 4301.

Physiology of Killer Factor in Yeast HOWARD BUSSEY Department of Biology, McGill University, Montreal, Quebec, Canada H3A IBI

1. Introduction . . . . . . . . . . 11. Encapsidated double-stranded ribonucleic acid plasmids in killer yeast

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.

A. Double-stranded ribonucleic acid . . . . . . B. Capsid . . . . . . . . . . . C. Other fungal double-stranded ribonucleic acid . . . . D. Physiology of plasmid replication . . . . . . E. Mutants in nuclear genes essential for plasmid maintenance and control F. Replication of double-stranded ribonucleic acids . . . . 111. Killer toxin . . . . . . . . . . A. Structure and properties . . . . . . . . B. Toxin synthesis and secretion . . . . . . . C. Physiology of toxin action. . . . . . . . D. Cell-wall receptor for toxin . . . . . . E. The membrane-damaging event . . . . . . . F. Toxin immunity . . . . . . . . G. Other killer toxins . . . . . . . . . H. Ustilago killer system . . . . . . . . I. Action of killer toxins on pathogenic yeasts. . . . . References . . . . . . . . . . .

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93 95 95 98 99 100 101

103 104 104

106 108 109 111 114

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114 116

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117 118

I. Introduction

‘Death opens unknown doors .. .’ John Masefield. Ever since their cloistered discovery at Oxford, by the Catholic priest Mallory Makower (Makower and Bevan, 1963), killer yeasts have appealed to a wider audience than that enjoyed by most esoteric problems of yeast genetics. The sinister name helps of course, but so does the venomous nature of the organism; poisons and their machinations have always attracted attention, in science as in other human endeavours. 93

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FIG. 1. Killer and sensitive phenotypes. An agar plate seeded with a sensitive strain was streaked with cells of, from the top, a superkiller, a killer, and a sensitive strain, and the seeded sensitive strain was left to grow. Toxin diffused from killer strains and left around them a clear zone of inhibition of the seeded sensitive strain. No such zone is seen around the lower sensitive strain. The clear circular inhibition zones a t the bottom of the plate result from a dilution series of cell-free toxin protein spotted onto the seeded plate before incubation. The series from right to left represents a dilution of I , 1 : 50, 1 : 100 and 1 : 500 of a toxin preparation. This serves as a simple assay for the toxin.

Makower’s killer yeast secrete a plasmid-coded protein toxin and they are immune to it, but most strains of Saccharomyces cerevisiae without the plasmid are killed. The plasmid is a linear dsRNA molecule encapsidated in a virus-like particle. Killing by killer yeast is an easily scored, well defined phenotype (see Fig. 1) and its presence in this simple eukaryotic microbe offers a fine model system for studying the molecular biology of the interaction of the plasmid with its host. In this interaction, the plasmid makes use of many cellular processes, some of which are currently of great interest to cell biologists, because they are judged to be problems amenable to solution. These processes include: ( I ) plasmid replication and exclusion phenomena, and integration of replication with cell growth; (2) posttranslation processing and expression of plasmid genes; the killer toxin has to be secreted and the immunity component probably inserted into the plasma membrane of producing cells; (3) the mechanism of action of the membrane-acting toxin on sensitive yeast cells. Research on the molecular biology of these problems is helped by the ease of manipulation of the microbial cell and by the fact that yeast genetics is a well advanced discipline. Although the full potential of the killer system is far from being realized, it is now the best understood fungal virus.

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This review is a personal one, with all of the idiosyncrasies and biases that implies. I will offer my view of the current state of knowledge of killer yeast, and point where future work may be profitable. Although this is a general review, some emphasis is placed on the problems of toxin synthesis and action. Some excellent reviews of killer and related fields exist (Wickner, R. B., 1979; Bruenn, 1980; Buck, 1980) and are cited here when pertinent.

11. Encapsidated Double-Stranded Ribonucleic Acid Plasmids in Killer Yeast A. D O U B L E - S T R A N D E D R I B O N U C L E I C A C I D

Two species of cytoplasmically inherited encapsidated dsRNA molecules occur in (K1) killer yeast strains and both are necessary for the killer phenotype (Somers and Bevan, 1969; Bevan et al., 1973; Mitchell et a[., 1976). The larger species of molecular weight 2.5-3.5 x lo6 (3.8-5.3 kb), called L, is present in most yeast strains, though when present alone the strain is sensitive to killer toxin. A smaller species of molecular weight 1.1-1.7 x lo6 (1.7-2.6 kb), called M, is present in killer strains; it has never been found in the absence of L. A compelling reason for this is the fact that LdsRNA codes for the major coat protein of the M virus-like particle capsid (Bostian et al., 1980b) and behaves as a helper virus. Both L and M molecules are linear when viewed by electron microscopy (Fried and Fink, 1978). The higher molecular weight of the L molecule was calculated by equilibrium sedimentation and calibrated for contour length against G4 DNA. The results suggested that the RNA was in a A-like configuration with a base pair separation of 0.273 nm (2.73 A), whereas the G4 DNA was in a B-like configuration with a base pair separation of 0.33 nm (3.3 A) (Holm et al., 1978). See Bruenn (1980) for a discussion of the problems associated with molecular-weight determinations of dsRNA molecules. The M dsRNA molecule in the presence of a low concentration of salt has a prominent singlestranded “bubble” when viewed by electron microscopy. This bubble is a useful calibration feature and represents a high AU, rich region that is about 200 base pairs in length. The L and M molecules are composed of different base sequences and have little common sequence either by hybridization or by T1 fingerprinting. There is, however, some limited sequence homology at the 3’-termini of L and M, and both 3’-termini show some sequence heterogeneity (Bruenn and Brennan, 1980). Preliminary sequencing data indicate that neither dsRNA is capped at the 5‘-end, both having a pppGp-structure (Bruenn and Keitz, 1976) and are not polyadenylated at the 3’-end.

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TABLE 1. Summary of killer plasmid genotpyes and phenotypes Plasmid genotype

Phenotype

[K 1 L-k]

K+R+

[K 1L-n]

K- R+

[K 1L-01

K-R

[K 1 L-S]

K-R

[K 1 L-i]

K+R t

[K 1L-sk]

K++R+

[K 1 L-b]

K+R+

[K I L-sd]

K+R+

[K 1 L-d] [K I L-k,]

K+R+ Kz+Rz+

Killer, has wildtype plasmid M dsRNA, cells secrete toxin ( K + ) and are immune to it (R+). The K 1 type of plasmid is indicated here unless mentioned. Neutral, has a full-sized M plasmid, cells are defective in active toxin, but active for the immunity component. Some neutrals secrete an inactive toxin, some, possibly defective in processing, do not secrete toxin. Sensitive strain, lacks M plasmid, does not produce toxin, normally sensitive to toxin, unless defective in a nuclear gene product necessary for killing. Suppressive sensitive, contains a deletion in the M plasmid, the defective S plasmid interferes with M plasmid replication and causes loss of [K I L-k]. Suicide strains, defective in immunity, though none are as sensitive as [K 1L-01; produce normal toxin. Can be maintained at pH 6.0 where toxin is inactive. Superkiller, plasmid mutation confers superkiller killer phenotype, e.g. higher plasmid number and more stable toxin in strain T158C (Palfree and Bussey, 1979). A possible explanation for this pleiotiopic phenoiype has been given by Buck I 1980). A plasmid that bypasses the need for most MAK gene products. Normal M plasmid is lost from cells with a mak gene defect. Maintenance of plasmid conditional on the presence of a ski mutation Plasmid dependent on a diploid host. Denotes K z plasmid, distinct toxin and immunity component. K z plasmid is excluded from cells if [KIL-k,] or [KIL-sl].

A study of deletion mutants of the MdsRNA plasmid, which behave as suppressors of the killer character, has revealed some further features of the M dsRNA. Historically, the suppressive sensitive strains carrying M dsRNA deletions provided the first good evidence for killer toxin and immunity component being coded on the M dsRNA. Suppressive sensitive strains with a K - R - phenotype were able to suppress the K + R + phenotype in a cross

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giving rise to K - R - strains retaining the suppressive character (Somers, 1973; Tzen et al., 1974; Vodkin r t al., 1974; Sweeney et al., 1976) and see Table I . These suppressive sensitive strains were shown to be missing the M dsRNA but to have a smaller suppressive or S dsRNA, thus showing a correlation between loss of the K R phenotype and a modified M dsRNA. Suppressive dsRNA species probably behave like defective interfering particles seen in animal virus systems. Defective animal viruses are usually deletions and are thought to act by having a competitive advantage over full-length viral genomes during replication, and are thus preferentially replicated in a situation where replication proteins are in limited supply. Heteroduplex analysis and T 1 ribonuclease fingerprinting of M dsRNA and three SdsRNA species showed that the SdsRNA species arose from M dsRNA by internal deletion and tandem duplications (Fried and Fink, 1978; Bruenn and Kane, 1978; Kane et al., 1979). If the parent M dsRNA of 1.83 kilobases has a sequence ABCDEFGHI, the SdsRNA S3 (0.73 kilobases) is an internal deletion ABCI, the S dsRNA S1 (1.5 kilobases) is a tandem duplication of S3, ABCIABCI and S4 (1.4 kilobases) has the structure of S1 with a small internal deletion of one C giving ABCIABI. Part of the S3 deletion extends to the 200 bp bubble sequence. These SdsRNA species lack a functional genome for toxin and the immunity component but retain the sequence of the replication regions of the M dsRNA. Common features of the suppressive dsRNA species examined are that the parental 3’-termini on both strands are conserved, as is a 0.5-0.6 kilobase core sequence (Kane et al., 1979). Attempts have been made to estimate the number of DNA copies of the L or M dsRNA. Despite earlier reports to the contrary, there is no evidence for such DNA copies. For reviews of the “provirus” story see Bruenn, (1980) and Buck (1980). Upper limits of estimates are less than 0.5 copy per haploid genome for L, and 0.1 copy for M (Wickner and Leibowitz, 1977; Hastie et al., 1978). The structure of the L or M genome has yet to be determined, although it has been shown (Hopper et a/., 1977; Oliver et al., 1977; Bostian et al., 1980a) that both code for proteins (This is discussed further in Sections 1I.B (p. 98) and 1II.B (p. 106). Apart from the suppressive dsRNA, several other M dsRNA mutants have been reported, conferring altered stability of toxin, affecting the immunity to toxin and affecting the replication of the plasmid (see Table 1). Genetic analysis of the M plasmid is a weak area of the system. Because there is no infectious cycle, chemical mutagenesis of virions in vitro cannot be performed. N o systematic work has been done on MdsRNA plasmid mutations, which have in general been isolated as incidentals in other mutant screens. Because of their large copy number per cell (approximately 12) it is perhaps not surprising that they are found relatively infrequently. +

+

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In the hope of learning more about the identity and regulation of immunity component and toxin, we have begun to isolate M dsRNA mutants using diploid strains that have been heat treated so that many cells contain only one dsRNA COPY.Mutagenesis of such strains appears to yield preferentially plasmid rather than chromosomal killer mutants. Strains isolated to date are [KlL-n] and [Kl L-i]. Such mutants should assist structural analysis of the genome in conjunction with further work on the sequence of the dsRNA genome. B. C A P S I D

The isometric virus-like particles (diameter 38-40 mm) can be purified by differential centrifugation. The capsid or coat protein consists of a single major species of molecular weight 88,000 which is coded on the LdsRNA. In killer strains, L and MdsRNA species are separately encapsidated, as particles containing them can be separated on sucrose gradients. The coding of the major capsid protein ScV-PI (Succh. cerevisiue virus protein 1) on LdsRNA was shown (Hopper et ul., 1977) using antibody to ScV-PI to probe for immunoprecipitable in vitro translation products when denatured LdsRNA was used as a message in a wheat-germ translation system. The major immunoprecipitable protein was identical to ScV-PI in size on sodium dodecyl sulphate-polyacrylamide-gel electrophoresis and had a similar tryptic digest peptide map. Correct translation of the ScV-PI from the denatured L genome implies that splicing of RNA does not occur. Further work (Bostian et al., 1980b) showed that the ScV-PI capsid protein was used to encapsidate the MdsRNA. Capsid proteins were obtained for L- and M-containing particles and subjected to partial proteolysis and peptide mapping on sodium dodecyl sulphate-polyacrylamide gels, and shown to be identical by this criterion. A similar conclusion was reached by Harris (1978) on the basis of precipitation of M-containing particles by antibody raised against L virus-like particles. These experiments establish a clear functional relationship betwem L and M, with a helper virus role for L in maintaining M by providing a capsid protein. Oliver et ul. (1977) found three proteins, termed V, B and D, associated with the L dsRNA containing virus-like particles from Sacch. cerevisiae sensitive strain S7. The major protein V had a molecular weight of approximately 75,000 and is probably the coat protein ScV-P1 seen by Hopper et al. (1977). The other two proteins are present in the proportion IOV: 1B: 1D with B 53,000 mol. wt. and D 37,000 mol. wt. No evidence was found for generation of B and D from V, although peptide mapping would be necessary to establish whether these proteins are coded separately from V on the L dsRNA.

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The maximum coding capacity of L, assuming one sense strand and no overlapping genes, would be 230,000 daltons of protein; 165,000 daltons is the upper limit for proteins V, B, and D. This would leave some 52,00045,000 daltons for other proteins possibly a RNA polymerase, which might also be used by M dsRNA. [See Buck (1980) for a discussion of how this estimate could be stretched or shrunk.]

C. O T H E R F U N G A L D O U B L E - S T R A N D E D R I B O N U C L E I C A C I D

Many fungi have virus-like particles containing dsRNA, and the field has been reviewed (Lemke, 1976; Hollings, 1978; Buck, 1980). Among the systems best studied are those of Aspergillus and Penicillium species and the killer strains of Ustilago maydis. Some viruses have undivided genomes, but many are segmented and each dsRNA species is usually separately encapsidated with a common coat protein. None of these virus-like particles shows natural infectivity, most are present in low copy number and, apart from the killer systems, none has an identifiable phenotype. These characteristics make study of the virus-like particles difficult. It has been suggested that all have their killer toxins (Buck, 1977, cited in Hollings, 1978), and certainly the screening for phenotypes, although unsuccessful to date, should be continued. A compelling circumstantial case has been made for some fungal viruses causing fungal disease (Buck, 1980). Yeasts have been screened for the killer phenotype, and killer yeasts in the genus Saccharomyces appear to have dsRNA as the genetic determinant. Young and Yagiu (1978) have examined dsRNA from the Saccharomyces strains of three killer types (K1-K3) differing in their immunity. In each case, the L and M species vary in size on polyacrylamide-gel electrophoresis. The sizes for the LdsRNA were: K,, 2.44 x lo6; K,, 2.54 x lo6; K,, 2.5 x lo6; and for M: K 1 , 1.3 x lo6; K2, 1.0 x lo6; K3, 0.9 x lo6. Some minor RNA species were also found. Curing of all Saccharomyces killers by heat or cycloheximide treatment resulted in sensitive, non-killer producing strains with a loss of M dsRNA, and some increase in the amount of L dsRNA. These observations suggest that, like K 1 , groups K z and K 3 killers have toxin and immunity coded on M dsRNA molecules. It is also interesting that differences in the LdsRNA helper virus are found in Kz and KJ. Other yeast genera contain killer yeasts (Section III.G, p. 114) but they are not curable like Saccharomyces killers and d o not appear to contain dsRNA, suggesting that they may have fewer copies of dsRNA or use some different genomic system. J. S. Kandel (personal communication) has, how-

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ever, found dsRNA in some pathogenic killer yeast. The Ustifago system will be discussed in Section (IILH, p. 116).

D. P H Y S I O L O G Y O F P L A S M I D R E P L I C A T I O N

The yeast virus-like particles are non-infective and non-lytic and so depend on cell growth or cell fusion through mating for their propagation. The number of particles per cell stays constant through many generations, which implies some close integration of particle growth with that of the host (Oliver et af., 1977). The integration process seems better regulated in L than M particles, M being lost far more readily from cells. Yeast strains vary in the number of particles that they maintain per cell, ranging from 100 L and 12 M in some killer strains (Wickner, 1976a) to some 24,000 copies of L in sensitive strain S7 where dsRNA is present at 5% of the level of rRNA (Oliver et al., 1977). The basis for this wide variation is not understood. In strains examined, those without M dsRNA made more LdsRNA, and killer strains cured of M made more LdsRNA, but this effect alone is insufficient to explain the wide variation of LdsRNA in sensitive strains. Strain S7 has been used as a convenient source of dsRNA for studies on the physiology of L in cells. The amount of LdsRNA per cell varies with growth in glucose-containing medium; a burst of synthesis occurs as the culture enters the stationary phase, leading to a three-fold increase in L dsRNA per cell. Presumably some preferential inhibition of synthesis occurs when stationary-phase cultures return to exponential-phase growth. The burst of synthesis on transition from exponential to stationary phase of growth could have been caused by a switch from a fermentative to oxidative metabolism. Consistent with this, cells grown oxidatively on ethanol as a carbon source have elevated levels of LdsRNA compared with those grown on glucose, the difference being about two-fold by stationary phase (Oliver et al., 1977). In further work (Clare and Oliver, 1979), it was demonstrated that the regulation of synthesis of LdsRNA in S7 was interrelated with host protein synthesis. Synthesis and degradation of L dsRNA were measured when cultures were starved for nitrogen, leucine or when protein synthesis was inhibited by cycloheximide. On nitrogen starvation, synthesis of dsRNA continued but there was degradation leading to a decrease in the absolute amount of dsRNA. Complete loss of LdsRNA did not occur even on prolonged nitrogen starvation and, on re-addition of ammonia, the normal amount of L dsRNA was quickly attained, implying some preferential synthesis of L dsRNA as cells resumed growth. Leucine starvation of a leucine-requiring auxotroph of S7 led to a retardation in the

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rate of LdsRNA synthesis but did not lead to degradation of pre-existing L dsRNA molecules. Cycloheximide inhibition also prevented degradation of pre-existing L dsRNA molecules. The leucine and cycloheximide data suggested that protein synthesis was necessary for continued synthesis and degradation of dsRNA molecules to occur. Instability of the M dsRNA in killer strains under certain conditions has been demonstrated; strains can be depleted of M dsRNA by cycloheximide (Fink and Styles, 1972), heat (Wickner, 1974) or 5-fluorouracil treatment (D. T. Rogers, personal communication). The basis of this selective loss of M is not understood, although selective loss of yeast mitochondria1 DNA with ethidium bromide is well known (Goldring el al., 1970) and has little effect on M stability (Al-Aidroos et al., 1973).

E. M U T A N T S I N N U C L E A R G E N E S E S S E N T I A L FOR P L A S M I D MAINTENANCE A N D CONTROL

By exploiting the genetic system of the host Sacch. cerevisiae, and using the simple plate test for the killer phenotype of the MdsRNA plasmid, R. B. Wickner has undertaken the ambitious project of. isolating, mapping and identifying nuclear genes necessary for the M dsRNA to conduct its affairs in (and for?) its host. The system is clearly a powerful model one for the study of the eucaryotic hosts side of the affair. Host genes found code for proteins involved in the maintenance and regulation of the plasmid, and the expression of the M dsRNA-coded proteins (see Section IILB, p. 106). The properties of these mutants have been reviewed (Wickner, 1976a, 1979; Bruenn, 1980) so only a summary will be presented here. 1. Maintenance of Killer (rnak) Genes

These form by far the largest class, with a formal tally of some 29 genes two of which have other known functions (Wickner, 1978). These genes are necessary to maintain the M dsRNA plasmid in haploid yeast cells. Mutants in mak genes lose the plasmid and become [KlL-o] with a K - R - sensitive phenotype. These genes show no clustering and are widely scattered on the yeast genome, some 15 out of 17 chromosomes having one or more of the mak genes. Many of the mak genes are identified through a single allele, and the probable total number of mak genes has been estimated at about 100. One might expect that many of the known rnak genes would be defective in components involved in events common to L and M dsRNA maintenance,

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but this is not the case. Only one gene, mak3, results in a significant decrease in the level of LdsRNA and even here there is no complete loss of the L plasmid (Wickner and Leibowitz, 1979). One killer maintenance gene (petl8), is also necessary for maintenance of mitochondria (Fink and Styles, 1972; Leibowitz and Wickner, 1978). Mutations in some of the other mak genes lead to a pleiotropic phenotype. Thus, makl6 is temperature sensitive for growth, and mak13, 15, 17, 20, 22 and 27 result in slow growth of the mutants independent of the temperature (Wickner and Leibowitz, 1979). But, as Wickner, R. B. (1979) has pointed out, although these mutations are defective in host functions they preferentially affect MdsRNA as the host cells remain viable but M dsRNA is lost from them. One killer maintenance gene (spe2) has a known function, it is the structural gene coding for S-adenosylmethionine decarboxylase, an enzyme involved in spermidine and spermine synthesis (Cohn et al., 1978).

2. Superkiller (ski) Genes Mutation in any of four ski genes confers a superkiller phenotype, and they appear to be involved in regulation of replication of the M dsRNA plasmid, and may define a replication pathway (Toh-e et al., 1978). These recessive mutations lead to overproduction of toxin activity, probably caused by an increased number of M plasmids per cell. The ski mutants have the ability to “suppress” mak mutants such that ski,mak double mutants are able to replicate the M plasmid. A ski1 mutant is able to suppress all mak mutants, except for mak16, and ski2, ski3 and ski4 mutants are able to suppress all mak mutants, except for mak16, mak3, maklO and pet18 (Wickner, R. B., 1979; Toh-e and Wickner, 1980). Wickner has proposed that in some way SKI products prevent use of an alternate pathway of M dsRNA replication. Whether this alternate pathway is that used by LdsRNA is not known. Products of MAK gene acting as negative control elements, and inhibiting SKI products which themselves normally inhibit replication, would also be an explanation consistent with the results. A dominant chromosomal gene (KRB or killer replication bypass) also causes suppression of mak7- and petl8-dependent loss of M dsRNA; KRB is not a suppressor of ochre or amber nonsense mutations (Wickner and Leibowitz, 1977). A further observation (Wickner, 1977) is that deletion of mitochondrial DNA will allow suppression or bypass of the mak 10 mutant. Phenotypic suppression of mak 10 does not occur if mitochondrial protein synthesis is inhibited with chloramphenicol, or if a respiratory deficiency is caused with antimycin A.

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A cytoplasmic mutation, presumably on the M dsRNA can also lead to mak suppression. Mutant [KlL-b] (see Table 1, p. 96) has a suppression pattern similar to ski2, 3 and 4,and plasmid mutant [KlL-sd] is dependent on the presence of a ski mutation. A further mutant [K 1 L-d] can only be maintained in a diploid host cell (Wickner, R. B., 1979). This welter of mutants emphasizes once again the power and weakness of a genetic analysis. Mutants with unforseeable phenotypes arise, but how do you connect the mutants to biochemical processes? Information on the products of these genes will be necessary to order the components identified by genetics into replication pathways.

F. R E P L I C A T I O N O F D O U B L E - S T R A N D E D R I B O N U C L E I C A C I D S

An RNA polymerase was associated with L-containing virus-like particles in a sensitive yeast strain (Herring and Bevan, 1977). This activity has been used in vitro to prepare L single-stranded RNA (Hastie et al., 1978). The product of the polymerase formed in vitro was largely single-stranded RNA that was up to unit length for L, 1.6-1.63 x lo6 molecular weight. Further work (Welsh and Leibowitz, 1978) showed that the polymerase activity was DNA-independent and purified at least 150-fold with the viruslike particles. With a killer strain, approximately full-length single-stranded RNA copies of both L and M were released from purified virions in vitro in the presence of ribonucleotides and magnesium. The polymerase appears, then, to act as a transcriptase. If this pattern of RNA synthesis is related to dsRNA replication, it would favour the reovirus model, where single-stranded transcripts act as templates for a single-stranded RNA polymerase to make dsRNA in a conservative fashion (Silverstein et a/., 1976). A model for replication of L virus-like particles with these features has been proposed (Buck, 1980). Consistent with this, Bevan and Herring (1976) found a dsRNA forming polymerase or replicase activity in a yeast strain containing L particles. The replicase was interpreted to be extending the double-stranded portion of partially replicated molecules, although the activity was not further characterized. In contrast, replication of dsRNA in vitro in the PsV-S virus of Penicillium stoloniferum proceeds by a semiconservative mechanism, with displacement of one strand from parental dsRNA by the RNA strand being newly synthesized (Buck, 1978).

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111. Killer Toxin A.

S T R U C T U R E A N D PROPERTIES

The K 1 killer toxin is a small protein of molecular weight 11,470 (Palfree and Bussey, 1979). It is composed of a single polypeptide chain some 109 amino-acid residues long, and estimates of the size of the active toxin, by gel filtration in urea, were consistent with the monomer being the active species. The toxin has a pl value, by isoelectric focusing, of 4.5, and it is rich in acidic amino-acid residues (2I:i of total) and lacks proline and arginine. Proteolytic digestion of toxin by the V8 protease of Staphylococcus uureus, which cleaves peptide bonds on the carboxy terminal side of acidic amino acids (Drapeau e f d.,1972), yields at least 21 peptides (Bostian et al., 1980a), out of an expected 23 or 24. The toxin is hydrophobic, having insufficient hydrophilic amino-acid residues to expose to the solvent even if a spherical shape for the molecule is assumed (Palfree, 1978; Fisher, 1964). The toxin is active and most stable within the narrow pH window of 4.2-4.6, close to the PI value of the protein and the pK value of the acidic amino-acid residues. Inactivation of toxin at high pH values appears irreversible, and could be the result of a conformational change in the protein to a lower energy state. The protein is heat labile, but this instability can be partly overcome by addition of glycerol (Ouchi et al., 1978), a substance that also stabilizes toxin up to neutral pH value. Toxin from superkiller strain TI 58C is more stable than wild-type killer toxin; the stable phenotype is M plasmid-based (Vodkin et a/., 1974) and probably arises from a missense mutation (Palfree and Bussey 1979). The toxin is also unstable at high ionic strength and in the presence of chaotropic agents, but is stable to antichaotropic agents such as sulphate (Palfree, 1978). At pH 4.6 in 15% glycerol and at 4 C, the toxin remains active indefinitely, and as a routine is assayed on growing cells at pH 4.6 at 18-22 C. The toxin is secreted into the growth medium of producing strains during the exponential phase of growth (Palfree and Bussey, 1979). Toxin can be readily purified in high yield from the culture medium. The procedure involves concentration of the extracellular macromolecules by ultrafiltration, followed by precipitation using poly(ethy1ene glycol), 4 M urea treatment, and fractionation on a glyceryl controlled-pore glass column (Palfree and Bussey, 1979). The urea treatment is necessary to remove toxin from extracellular mannan to which it binds during the concentration process. The tight association of mannan with toxin, which mimics the toxin receptor inter-

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FIG. 2. Sodium dodecyl sulphateepolyacrylamide-gelelectrophoresis of K I and Kz killer proteins of Saccharomyces cerevisiae. A shows properties of K I protein: 1, sensitive strain; 2, isogenic killer strain; 3, purified killer toxin. B shows properties of K, protein: I , K , killer strain, toxin indicated; 2, K, killer strain; 3, isogenic sensitive strain. Note absence of two protein bands, both candidates for the toxin, neither of which corresponds to the K , toxin.

action in sensitive cells (see Section 1II.D p. 109) led to earlier reports that the toxin was a mannoprotein. In fact there is now good evidence that the toxin protein is not glycosylated. The toxin protein isolated by the procedure described is pure by sodium dodecyl sulphate-polyacrylamide-gelelectrophoresis and rechromatography on Sephadex C-25. This protein is missing from isogenic [KlL-o] sensitive strains devoid of the plasmid (Fig. 2). The toxin can be identified in crude extracellular preparations of protein and glycoproteins where it comprises some 5-10% of the protein (Fig. 2). Quantitation of toxin activity recovery during purification shows a yield of 80% of initial activity in the pure preparation. Quantitation of toxin protein using sodium dodecyl sulphate-polyacrylamide gels gives a different story. Only some 10% of total toxin protein is recovered in the active fraction. The controlled-pore glass column appears to separate active and inactive toxin, suggesting that there are two distinct chemical species. Any modification must be minor as both active and inactive toxin are identical in size

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on sodium dodecyl sulphate-polyacrylamide gels, have the same PI value and cross-react to antibodies raised against the other. As activity is retained throughout purification, it is possible that any modification leading to inactive toxin occurs in vivo.

B. T O X I N S Y N T H E S I S A N D S E C R E T I O N

Synthesis of extracellular killer toxin affords a good system for studying the events involved in secretion of a non-glycosylated protein from a eukaryotic cell. A study of the translation products of denatured M dsRNA formed in vitro has provided convincing evidence that the M dsRNA contains the gene for the toxin protein (Bostian et al., 1980a). Purified MdsRNA was denatured and translated in a wheat-germ system using [3 SS]-methionine as a label. Two M dsRNA-dependent proteins were detected after sodium dodecyl sulphate-polyacrylamide-gel electrophoresis and autofluorography, namely a major protein of 32,000 molecular weight (M-PI) and a lessabundant protein of molecular weight 30,000 (M-P2). The total coding capacity of M dsRNA, assuming non-overlapping genes, would be about 70,000 daltons of protein. Only the 32,000 molecular-weight M-P1 protein was immunoprecipitable with antitoxin antibody, and the 11,500 molecular-weight killer toxin competed with M-PI in the immune reaction. The M-PI fraction was shown to contain the killer toxin protein by a comparison of peptides from toxin and M-PI proteins. Of the 21 toxin peptides released by digestion with Staphylococcus aureus V8 protease, all but one was present in digests from M-PI, suggesting that the toxin is present in M-PI in a terminal position. The M-P1 protein may be a readthrough product of the toxin gene formed in vitro, or it may represent an authentic toxin precursor or protoxin formed in vivo. Such a protoxin would have to be processed by proteolytic cleavage, presumably in a system common to other secreted proteins and polypeptide hormones (Blobel and Dobberstein, 1975; Wickner, R. B., 1979). The large size of the 32,000 molecular-weight M-P1 protein prompted the idea (Bostian et al., 1980a) that it might also contain another protein, such as the immunity component. Postransational cleavage of polygenic polypeptides occurs in several viral systems (Racevskis and Koch, 1978; Glanville et d., 1978). An interesting idea is that protein M-P1 is a protoxin, pro-immunity protein with a common membrane-recognition sequence. The M-P1 protein is inserted into the plasma membrane as an integral protein and the amino-terminal group on the toxin protrudes externally and is detached from the remaining immunity protein by a proteolytic nick. Plasmid mutants in the immunity of toxin functions should help in testing this

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idea. For example, plasmid mutants defective in the immunity protein because of nonsense mutations would be expected to show an altered 32,000 molecular-weight M-P1 protein product in vitro in the above scheme. Toxin mutants defective in processing may form an abnormally large unprocessed immunity protein. A candidate for such a mutant would be the cytoplasmic neutral strain N2, K - R + [KlL-n]. This mutant, with normal immunity function, made a 32,000 molecular-weight M-P1 protein in a translation system in vitro (Bostian et al., 1980a) but is one of a class of neutral strains that does not secrete detectable amounts of killer toxin (D. T. Rogers and H. Bussey, unpublished results). Several nuclear genes affect toxin production while still maintaining a wildtype [KlL-k] plasmid (Leibowitz and Wickner, 1976; Wickner and Leibowitz, 1976b). Mutants in two killer-expression genes, k e x l and kex2, secrete much less toxin ( < 5 % ) than KEXl or KEX2 strains, and may be defective in secretion-related processes. Mutants in the kex2 gene on chromosome XIV have a pleiotropic phenotype consistent with defective secretion. Strains with a kex2 mutation are unable to secrete killer toxin or an active a-factor polypeptide pheromone; they fail to undergo spore maturation and show poor growth oncomplex media (Leibowitz and Wickner, 1976; Rogers et al., 1979). An examination of extracellular proteins and glycoproteins from culture filtrates of a kex2 mutant by two-dimensional polyacrylamide-gel electrophoresis indicated that many are abnormal compared with the parent strain, being both larger and more basic. Some proteins and glycoproteins were not secreted by kex2 mutants, and these included the killer toxin. These pleiotrophic alterations cosegregated with the kex2 mutation in genetic crosses, indicating that the mutation was responsible for the phenotype. Attempts to locate a protoxin molecule from kex2 strains by immunoprecipitation with antitoxin-antibody have been unsuccessful, but the low-titre antibody used would make re-examination worthwhile. The kex2 defect is unknown, but is probably in protein processing, e.g. a mutation in a specific processing portease, leaving a recognition sequence on the secreted protein (Rogers et al., 1979). The behaviour of the kex mutants strongly suggests that toxin is secreted from producing cells. In Escherichia coli, plasmid-coded colicin proteins El and E3 do not appear to be secreted by a specific process (Jakes and Model, 1979). Rather they can be induced to leak with intracellular proteins from cells permeabilized with mitomycin C . The kexl mutants show a normal pattern of secreted proteins, except for the absence of the toxin polypeptide. Screening for killer-expression mutants has turned up many alleles of k e x l and kex2 implying that few other genes are likely to be found by the current screening procedures. It is expected that many secretion-related events would be vital, and conditional mutants would have to be screened to obtain a wider repertoire of genes. Novick

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and Scheckman ( 1979) have isolated a temperature-sensitive secretory mutant in Sacch. cerevisiae secl, which accumulates secretory vesicles at the restrictive temperature; mutants of this type should be valuable in testing models of secretion developed in animal cells. The value of a genetic approach to secretion problems has been cogently stated by Beckwith et ul. (1978).

C.

PHYSIOLOGY OF TOXIN ACTION

The killer protein is a potent toxin. Some 6 x lo3 molecules are needed to kill a cell of the sensitive strain S6, whereas 2.8 x lo4 molecules kill a cell of the less sensitive strain S14a. These values, which are upper limits, are based on survival curves with known amounts of toxin (Palfree and Bussey, 1979) corrected for the proportion of active toxin, based on the ability of 35S-labelled toxin to bind to sensitive cells (Bussey et al., 1979b). Expressed as a concentration 0.2 nM toxin or 2.3 ng ml-' will kill a culture of S6 at 2 x lo7 cells ml-' with a multiplicity of unity. Comparable values for protamine sulphate, a lethal protein that indiscriminately kills killer, sensitive and killer-resistant mutants of yeast, would be 100 pg ml-' or 2.6 x 108 molecules per cell, almost four orders of magnitude more than for the killer protein.

1. Effects on Sensitive Cells Growing sensitive cells treated with killer toxin at 20-22 'C at pH 4.7 show a rapid decrease in viability measured by ability to form colonies on agarcontaining medium. Maximum killing is attained by two to three hours depending on the strain (Makower, 1964 cited in Woods, 1966; Bussey, 1972; Skipper and Bussey, 1977). Measurements of metabolic and macromolecular biosynthetic events after toxin addition shows that there is a lag period of about 40 minutes during which no effects are seen, followed by a co-ordinate shut-off of synthesis of macromolecules (Bussey and Sherman, 1973). The biosynthetic shut-off coincides with plasma membrane damage, measured by loss of potassium ions or ATP (Bussey and Sherman, 1973; Skipper and Bussey, 1977). Membrane-damaged cells shrink in volume (Bussey, 1974), but toxin-treated cells do not lyse or contain large pores, as macromolecules are not lost from killed cells. The pattern of inhibition provides few clues as to the primary site of action of the toxin. Although the plasma-membrane damage could account for cessation of macromolecular synthesis, the presence of a lag after cells have bound a lethal dose of toxin allows of many possibilities, including entry

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of the toxin into sensitive cells. At this stage, the primary site of action remains unknown, but several lines of evidence are consistent with the idea that the toxin acts at the plasma membrane. In addition to examining the physiology of intoxicated cultures, another approach to understanding toxin action is to follow the protein along its lethal path. This can be done biochemically, using radioactive toxin, and genetically by isolating mutants from sensitive cells that are resistant to toxin action. A two-stage model for toxin action has emerged from analyses of this kind.

D.

C E L L - W A L L R E C E P T O R FOR T O X I N

The killer toxin acts initially by binding to a receptor on the yeast wall. Evidence for this comes from toxin-resistant mutants (Bussey et al., 1973; Al-Aidroos and Bussey, 1978). Mutants in two unlinked genes (killer resistant) krel and kre2 are resistant to toxin and show a lower binding of toxin to cells than the wildtype. A same-site revertant of a krel mutant reacquired both toxin-binding ability and sensitivity. Binding of ?+labelled toxin to a wall receptor on cells of a sensitive strain and derived resistant mutants provided further information about the receptor (Bussey et al., 1979b). Binding of pure 35S-labelledtoxin to sensitive-cells is a rapid process, shows no lag, is not energy dependent and is complete within three minutes at 20 'C at pH 4.7. A major part of the labelled-toxin binding appeared to be biologically relevant, because it was diminished in resistant mutants krel and kre2. Non-radioactive toxin competed effectively with labelled toxin for binding to receptors on a sensitive strain, but did not compete significantly with binding to the resistant mutant krel, suggesting that toxin binding to krel was non-specific. Specific binding to the receptor of the sensitive strain has been examined quantitatively; there are some 1.1 x lo7 receptor sites per cell with an association constant of 2.9 x 1 0 6 ~ - ' .A Scatchard analysis was consistent with the presence of a single receptor species. Toxin receptors can be solubilized from the yeast wall by digestion with Zymolyase 60,000 (an endoglucanase containing preparation from Arthrohacrer luteus) (Kitamura er a/., 1974). Non-dialysable, soluble receptors can by assayed as they compete with sensitive cells for binding of labelled toxin and also competitively rescue sensitive cells from a toxin-induced death. Similar soluble-receptor extracts from krel strains fail to show significant activity by these competition tests. The receptor molecule appears to be a /3-( 1 +6)-~-glucan. This glucan can be purified from other cell wall glucans and mannan by its differential

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solubility in alkali and dilute acetic acid (Manners et al., 1973). The p-( 1 +6)-glucan is the receptor by the following criteria (H. Bussey and K. Hutchins, unpublished observations). It has the highest specific activity of all yeast cell-wall polysaccharides in the receptor assay. It is lowered in amount in a krel mutant, other polysaccharides are not. In a krel samesite revertant, the level of the glucan is at that of the wild-type concomitant with regaining receptor activity. The glucan from a krel mutant has a much lower proportion of p-( 1 -+6) linkages in the molecule as determined by linkage analysis using the Hakamori procedure (Lindberg, 1972).It seems a reasonable speculation that the krel mutant is partially defective in p-( 1 +6)-glucan synthetase activity, leading to smaller amounts of intrachain p-( 1 +6)-linked glucose residues. Although the cell-wall receptor is necessary for toxin action, it does not appear to be the only component in the killing process. Several lines of evidence suggest that some second receptor probably on the plasma membrane is involved. The kinetics of killing of sensitive cells show that saturation is achieved at 0.2 p~ toxin, a concentration some 50-fold less than that necessary to saturate the cell-wall glucan receptor (Bussey et al., 1979b). Thus, it appears as if some other component (receptor) becomes limiting in the lethal process at concentrations where only a small fraction of the wall receptors are occupied. In strain S14a, 2.8 x lo4 toxin molecules can kill one cell, although the cell has some 1.1 x lo7 receptors. A similar situation occurs in the action of the protein toxins abrin and ricin, where some 3 x lo7 sites/cell are available on the cell surface, although only one toxin molecule is necessary to kill a cell (Achtman et al., 1978). Further evidence for a post-wall receptor step or stage-2 event comes from the observation that the wall receptor-dependent events can be by-passed if sphaeroplasts are treated with toxin. The original abservation (Bussey et al., 1973) was that a krel non-complementing, resistant mutant was resistant as a cell, but sensitive to toxin when converted into a sphaeroplast. A similar observation was made for a mutant resistant to a killer toxin from a sake strain of Sacch. cerevisiae (Imamura rt al., 1975). More recent work indicates that sphaeroplasts from krel and kre2 resistant mutants show toxin sensitivity (Al-Aidroos and Bussey, 1978; Skipper, 1978). It has been found that sphaeroplasts made by the Zymolyase procedure are often more sensitive than those prepared by the glusulase method. This important observation is consistent with the concept of a stage-2 receptor being masked from toxin in whole cells. The wall (or stage 1) receptor would be required to mediate transfer of toxin to the stage-2 receptor in whole cells. In this scheme, sphaeroplasts have the stage-2 receptor exposed directly to toxin, and thus by-pass the need for stage 1. A stage-2 process is also suggested by mutants in a third nuclear gene

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for toxin resistance kre3 (Al-Aidroos and Bussey, 1978; M. A. Ahmad, unpublished results). These mutants bind toxin normally to the wall receptor but are toxin resistant.

E.

THE

MEMBRANE-DAMAGING

EVENT

Interaction of toxin with the plasma membrane, leading to the observed change in permeability of small molecules, remains a major unsolved problem, whose solution would be of value in understanding how extracellular proteins interact with the plasma membrane of target cells. An early observation (Woods, 1966) was that sensitive cells in the exponential phase of growth were more susceptible to killer toxin than cells in the stationary phase of growth. This observation was extended to show that toxin-induced membrane damage was an energy-dependent event (Skipper and Bussey, 1977). Poisons such as 2,4-dinitrophenol, which act on energy-generating processes, prevented toxin-mediated potassium efflux from sensitive cells, but did not prevent toxin binding to the cell-wall receptor. Mere inhibition of cell growth, by use of the DNA synthesis inhibitor hydroxyurea, had no effect on toxin action (Skipper, 1978). A similar but more far reaching set of observations (Kotani et al., 1977) were made on the action on sensitive strains of Sacch. cerevisiae of a killer toxin from a sake strain of Succh. cerevisiae. This toxin has the same immunity as the K 1 toxin (M. J. Leibowitz, cited in Wickner, R. B., 1979), but may not be the same protein. With this sake-yeast toxin, calcium ions were found to prevent toxin-induced membrane damage to sensitive cells, without preventing toxin binding. The non-inhibitory effect of calcium ions enabled the plating of cells in its presence, and allowed a demonstration that calcium ions “rescued” cells that had bound a lethal dose of toxin, preventing both membrane damage and loss of cell viability. In this system, ADP and to a lesser extent ATP and 3’ : 5’-cyclic AMP had the apparent effect of enhancing toxin activity, as these compounds increased growth lag of a culture in the presence of the sake-yeast toxin. These findings are reminiscent of the action of cholera or diphtheria toxins, where toxinmediated ADP-ribosylation of protein results in inhibition of their function (Moss and Vaughan, 1977; Honjo et ul., 1968). Direct attempts to analyse events after cell-wall receptor binding of toxin have used SS-labelled toxin, and are consistent with the concept of the toxin acting at the membrane (H. Bussey, unpublished results). Killer toxin does not appear to be cleaved during the killing process. Autoradiograms of sodium dodecyl sulphate-polyacrylamide gels of toxin recovered from cells killed at low multiplicity show only the 1 1,500 molecular-

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weight band, with a limit of detection of some 300 molecules of toxin per cell, or lx of a lethal dose. Thus the A-B mechanism observed with some toxins, with the B subunit or domain binding to a receptor and permitting an A subunit or domain to insert or pass through the membrane (Olsnes and Pihl, 1978), is not the mechanism used. The toxin remains outside sensitive cells, being accessible to proteases that do not penetrate the yeast plasma membrane. All labelled toxin was recovered from toxin-killed cells by digestion with Zymolyase followed by pronase, proteins known to leave an intact plasma membrane (Bussey et al., 1979a). The limit of sensitivity of these experiments was about 50 molecules per cell. The formal possibility remains that only a small number of molecules kill sensitive cells, as is the case with diphtheria toxin or the abrin or ricin pro-

Glucan receptor Stage I Outside

Plosma membrane

eo,"l

Inside

l.

Stage 2

ATP

Potassium ions; law molecular-weight metabolites

FIG. 3. Simple two-stage model of toxin action. See text for details. T indicates toxin molecule; R membrane receptor. the kre3 gene product. The glucan receptor is pictured with 8-(1 4 6 ) backbone as a heavy line. The exact branching structure is unknown, but involves /I1 +3) ( branch points and 8-(1 +6) side chains (Manners et af.,1973). N o specific interaction of toxin is implied, and molecular dimensions are not to scale.

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teins. Labelled toxin of higher specific activity would be needed to resolve this issue. Attempts to measure toxin binding to the expected relatively small number of membrane receptors have been unsuccessful, and may also require toxin of higher specific activity. The current state of knowledge can be summarized in a two-step model of toxin action (Fig. 3). This model merely focuses attention, and is only one of many that would explain the results. Toxin binds to the cell wall glucan receptor (stage 1) a process that is energy independent and requires krel and kre2 gene products. Stage 2 follows in whole cells, and in it toxin is shown interacting directly with some trans-membrane protein R ( h e 3 gene product) whose normal function is pre-empted to serve as a channel for potassium, ATP and small metabolites to leave the cell. The time taken after addition of toxin to complete the process to stage 2 would be the lag period before membrane damage occurs. Insertion of toxin into the membrane, or toxin-induced covalent modification of a membrane protein, may be energy dependent. Alternatively, efflux of ions and metabolites through the protein pore may require energy. In the sake-yeast toxin system, stage 2 would be stimulated by ADP and inhibited by calcium ions. The immunity protein, perhaps also an integral membrane protein, would prevent toxin interaction with the pore or channel by blocking recognition or some modification reaction. A slightly simpler model would have the toxin inserted into the membrane at stage 2 to form a trans-membrane pore in its own right. In this scheme, kre3 mutants would be defective in some component necessary for toxin to recognize or insert into the membrane. Identification of the components in stage 2 is clearly a key requirement for understanding toxin action. Our present knowledge is similar to that for insulin (Kahn, 1979), and can be whimsically paraphrased as a two-step model in which toxin binds to a receptor and events then occur leading to membrane damage. The killer-toxin system allows both biochemical and genetical techniques to be applied to the problem of how a protein interacts with a membrane. Toxin-resistant mutants should continue to be of use, and conditional mutants or mutants resistant to other toxins, e.g. KZ or K J , may extend the analysis. The availability of cross-linking reagents such as photo-affinity labels offers a strategy for cross-linking of toxin with membrane receptors and may offer a direct way to identify stage-2 components. An alternative biochemical approach is to ‘second guess’ the membrane component where toxin acts, and show that it is modified by toxin in vitro. A plausible candidate would be the vanadate-sensitive plasma-membrane adenosine triphosphatase (Willsky, 1979), though this appears unaffected by toxin both in vitro and in vivo (H. Bussey and R. Theolis, unpublished work). Other likely membrane targets can be imagined, for example, adenylate cyclase.

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F. T O X I N I M M U N I T Y

Toxin-producing strains are immune to toxin action, and an immunity component, presumably a protein, is coded by the M plasmid (see Section 1II.B). Curing of the M plasmid results in loss of both toxin production and toxin immunity. Immunity can be distinguished as a separate activity from toxin production by the behaviour of M mutants. Neutral mutants [K 1 L-n] are phenotypically K - R +,they are defective in active toxin production, but they remain immune. A second class of plasmid mutants, suicides [KlL-i], are phenotypically K+R- and are presumed defective in the immunity component but continue to produce active toxin. Suicide mutants can be kept at pH 6.0 although they remain unstable. A nuclear gene (resistance expressed rexl, is also implicated in immunity, being phenotypically K + R - with a [KlL-k] plasmid. Mutants in this gene have not been well characterized, but could be in any component necessary for immunity expression. Whether rexl is defective in expression of the KZ immunity component is not known. Immunity is plasmid specific. Several yeast plasmids code for killer toxins, for example the K , , K , groups in Sacch. cerevisiae (see also Section III.H, p. 1 16; Naumov and Naumova, 1973; Stumm et al., 1977; Rogers and Bevan, 1978; Young and Yagiu, 1978). In each case, toxins bypass the immunity of plasmids other than their own and kill non-homologous killer strains. This argues for each plasmid coding for a distinct immunity component, and forms the main basis for discriminating between various killer groups in classification schemes. The mechanism of immunity is unknown although in the K1 system the 32,000 molecular-weight protoxin protein formed in vitro may contain the immunity protein as may the in vitro translation product of M dsRNA 30,000 M-P2 (Bostian et al., 1980a; see Section III.B, p. 106). Immunity does not operate a t the cell-wall receptor since killer strains display equivalent amounts of wall receptor to that found in isogenic sensitive strains (Bussey et al., 1979b). Cell wall receptor-defective (krel) killer strains bind less toxin to their walls and so appear to secrete higher amounts of the toxin protein (H. Bussey, unpublished results). Immunity probably occurs at stage 2 of the killing process, and the immunity protein may prevent membrane damage by preventing toxin recognition of a membrane receptor as suggested in Section 1II.E (p. 1 1 1). G. O T H E R K I L L E R T O X I N S

Killer strains are widespread among members of yeast genera (Maule and

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Thomas, 1973; Philliskirk and Young, 1975; Bussey and Skipper, 1975; Stumm et ul., 1977; Rogers and Bevan, 1978; Young and Yagiu, 1978; Kandel and Stern, 1979; Middelbeek et uf., 1980). At least 10 different killer-yeast groups have been identified on the basis of their patterns of killing of other killer strains. Each killer type is immune to its own toxin, so killing of one killer by another probably indicates that both the immunity and toxin proteins are different in each case. This simple picture is complicated by altered resistance patterns of some killers within a group to other toxins. These resistance patterns are best explained by nuclear gene-dependent difference between strains (Young and Yagiu, 1978). Toxins from all yeast-killer groups examined are protease-sensitive heatlabile macromolecules. Most are stable and act only at pH values below 5. All kill Succh. cerevisiue strain A8209B, and appear to be dependent on some component of the K, toxin cell-wall receptor. Receptor-defective K , resistant mutants of gene krel are resistant to all toxins tested (Rogers and Bevan, 1978; Al-Aidroos and Bussey, 1978). Succhuromyces cerevisiue K toxin-resistant mutants in gene kre2 are also defective in the cell-wall receptor, but show a more complex picture being sensitive to all but two toxins. Clearly, more knowledge of the structure of the receptor will be needed to understand these observations. An interesting finding was that Succh. cerevisiue K l resistant mutant kre3, with normal cell-wall receptors, was also resistant to all but two killer groups of toxin. This may suggest a common stage-2 target for many toxins. However, the possibility still exists that yeast toxins from different groups have different modes of action. This would allow experimenters to use them as probes for studying aspects of the molecular biology of the yeast cell, an approach that has been fruitful with colicins and with bacterial proteins toxic to eukaryotic cells. The few toxins examined so far for action on Succh. cerevisiue appear to act like the K , toxin in causing membrane damage. The K 2 toxin from Succh. cerevisiue strain Y1 is very similar to K , in its action (Rogers, 1976), although it appears to be a different protein. Two proteins, one larger and one smaller than the K1 toxin, are missing from extracellular protein extracts of K2 strains when they are cured of the Kz dsRNA plasmid (see Fig. 2, p. 105). The K, toxin or pool efflux-stimulating toxin (PEST) from Torulopsis glubruta also acts at the membrane, but differs from K , in not having an energy-dependent step necessary for membrane damage and in causing leakage of AMP not ATP from sensitive cells (Skipper and Bussey, 1977). Although the kre3 mutant is resistant to K4 toxin, it seems probable that K4 toxin has a target on the cell surface different from K1 toxin. The action of the toxin from Pichiu kluyveri 1002 on Succh. cerevisiue ScF1717 has been examined in detail (Middelbeek et ul., 1980). This toxin

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again shows great similarity to K1 in its action. Sensitive cells bind toxin rapidly, but respond physiologically after a lag of 50-90 minutes when the cells leak potassium ions and ATP and shrink in volume. Co-ordinate with potassium ion and ATP leakage, toxin-treated cells become more permeable to protons, the intracellular pH value falls from 6.3 to 5.1, with an extracellular pH value of 4.3. In addition, intoxicated cells showed a drastic decrease in their ability to transport L-leucine, although this effect was more pronounced at lower leucine concentrations, suggesting that a low-affinity leucine-transport system was less affected than a high-affinity one. The membrane-damaging events seemed dependent on an energy source, being less pronounced when cells were suspended in buffers or solutions of poisons that act on energy-generating metabolism than in rich growth media. Four other toxins from different killer yeast groups have been examined in my laboratory. Sensitive strains of Succh. cerevisiue all show a lag after toxin addition followed by efflux of potassium ions from cellular pools. The killer strains used were Kluyveromyces wickerhamii NCYC 546, Debaryomyces vunrijii NCYC 577, Pichiu vunrijii NCYC 51 1, and K . drosophilanum NCYC 575. H.

U S T I L A G O K I L L E R SYSTEM

Killer strains of Ustilago muydis produce protein toxins that kill sensitive strains of U . muydis and related smuts, but have no effect on Succh. cerevisiue (Puhalla, 1968; Hankin and Puhalla, 1971; Koltin and Day, 1975). Three distinct killer strains occur, namely P1, P4 and P6, and each is immune to its own toxin but is killed by the other two (Koltin and Day, 1976). The specificity of each toxin is also demonstrated by the isolation of three independent classes of nuclear gene-based resistant mutants, each resistant to one toxin type (Puhalla, 1968; Koltin and Day, 1976). The killer phenotypes are inherited cytoplasmically and strains contain viruslike particles (Wood and Bozarth, 1973; Koltin and Day, 1976). The viruslike particles are associated with from five to seven dsRNA species, with a common coat protein. The system is more complex than that of Succh. cerevisiue, and has been well reviewed (Wickner, W., 1979; Buck, 1980). All three toxins are proteins composed of single polypeptides of molecular weight 9,000-11,000. The toxins have been purified to homogeneity by the criteria of sodium dodecyl sulphate-polyacrylamide-gel electrophoresis and isoelectric focusing (Kandel and Koltin, 1978; Levine et al., 1979), although details of the purification schemes and their efficiency have not been published. The toxins appear to be distinct proteins based on different mobilities on non-denaturing gel electrophoresis. In addition toxins of types P1 and P4 do not cross-react with antibodies raised against P6 toxin.

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Assignment of toxins to specific dsRNA segments is still tentative (Koltin, 1977; Koltin et al., 1978). The P6 toxin is missing from extracellular extracts of non-killer mutants derived from P6 that lack the 700,000 molecularweight dsRNA species, indicating that this molecule is necessary for toxin production probably as the toxin genome (Koltin and Kandel, 1978). Complementation tests for toxin production among non-killer mutants in vitro and in vivo are consistent with this interpretation. A possible involvement of small dsRNA molecules, distinct from fragments of the 700,000 molecularweight species, in expression of toxin activity remains uncertain (Koltin and Kandel, 1978). The Ustilago toxins appear to be far more robust proteins than those from yeasts. Toxins from strains P4 and P6 are stable and act over a pH range of 4 9 , have a half-life of 5-7 min at 80 C and can be eluted as active proteins from gels after sodium dodecyl sulphate-polyacrylamide-gel electrophoresis (Kandel and Koltin, 1978). Complementation of extracts from inactive P6 toxin mutants in vitro suggests that more than one identical polypeptide chain may be involved in the native active toxin. All three Ustilago toxins have nuclease activity in vitro against single-stranded RNA and DNA, but not against dsRNA (Levine et al., 1979). The activity against single-stranded DNA is consistent with an endonucleolytic attack, although the nature of the cleavage site and whether the nuclease is active on doublestranded DNA remain unknown. Such observations have obvious relevance to the mode of action of these toxins. More rigorous evidence for association of the nuclease activity with the toxin protein would be useful, as contamination of the preparation by extracellular nuclease remains a possibility. Scrutiny of the homogeneity of the purified toxin by the sensitive silver-staining procedure (Switzer et al., 1979) after electrophoresis or isoelectric focusing would be worthwhile, as would screening for nuclease activity in strains lacking dsRNA. In addition, one would predict a class of inactive toxin mutants also lacking the nuclease activity. In bacteria, colicins with nuclease activity cause specific cessation of DNA or protein synthesis in intoxicated bacteria (Schaller and Nomura, 1976; Senior and Holland, 1971). Studies on the physiology of sensitive Ustilago strains treated with Ustilago toxins have not been reported; this would seem an area worth investigating.

I.

A C T I O N O F KILLER T O X I N S O N P A T H O G E N I C YEASTS

Several studies (Bussey and Skipper, 1976; Rogers and Bevan, 1978; Young and Yagiu, 1978; Kandel and Stern, 1979; Middelbeek et al., 1980) indicate that many pathogenic yeasts including Candida albicans, Torulopsis

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H. BUSSEY

glubruta and Cryptococcus neoformuns are sensitive to killer toxins. The most

comprehensive study is that of Middelbeek et al. (1980), in which of 142 pathogens tested, 116 were sensitive to one or more of seven different killer toxins tested. Such a pattern of killing implies that killer-toxin proteins act as specific antifungal antibiotics. Their antigenicity, and in many cases their lability and pH sensitivity, make them poor candidates for therapeutic antifungal drugs. In view of the fact that therapeutically useful antifungal agents are few in number, perhaps our knowledge of the specific features of pathogenic yeasts revealed by toxin-recognition elements should be further extended. Armed with such knowledge, an obvious approach in principle would be to design antibiotics exploiting the known specificity, for example it might be possible to synthesize stable low molecular-weight analogues of the protein toxins. If the specific toxin-recognition sites are used for other essential metabolic purposes, then analogues retaining site-recognition specificity alone would competitively inhibit the essential function.

REFERENCES

Achtman, M., Jarett, L., Brady, R. O., Collier, R. J., Cuatrecases, P., Dales, S., Helenius, A., Olsnes, S.. Rosenbusch, J. P. and Tomasz, A. (1978). In “Transport of Macromolecules in Cellular Systems” (S. C. Silverstein, ed.), pp. 133-142. Dahlem Konferenzen, Berlin. Al-Aidroos, K. and Bussey, H. (1978). Canadian Journal of Microbiology 24, 228. Al-Aidroos, K., Somers, J. M. and Bussey, H. (1973). Molecular and General Genetics 122, 323. Beckwith, J., Silhavy, T., Inouye, H., Shuman, H., Schwartz, M., Emr, S., Bassford, P. and Brickman, E. (1978). In “Transport of Macromolecules in Cellular Systems” (S. C. Silverstein, ed.), pp. 299-3 14. Dahlem Konferenzen, Berlin. Bevan, E. A. and Herring, A. J. (1976). In “Genetics, Biogenesis and Bioenergetics of Mitochondria” (W. Bandlow, R. J. Schweyen, D. Y. Thomas, K . Wolf and F. Kaudewitz, eds.), p. 153. Walter de Gruyter, Berlin and New York. Bevan, E. A,, Herring, A. J. and Mitchell, D. J. (1973). Nature, London 245, 81. Blobel, G . and Dobberstein, B. (1975). Journal of Cell Biology 67, 835. Bostian, K. A,, Hopper, J. E., Rogers, D. T. and Tipper, D. J. (1980a). Cell 19, 403. Bostian, K . A., Sturgeon, J. A. and Tipper, D. J. (1980b). Journal o/ Bucteriolog~~. 143, 463. Bruenn, J. A. (1 980) Annual Review of Microbiology 34, 49. Bruenn, J. A. and Brennan, V. R. (1980). Cell, 19, 923. Bruenn, J. and Kane, W. (1978). Journal of Virology 26, 762. Bruenn, J. and Keitz, B. (1976). Nucleic Acids Research 3, 2427. and Exploitation” Buck, K. W. (1977). In “Biological Substances-Exploration (D. A. Hems, ed.), p. 121. Wiley, New York. Buck, K . W. (1978). Biochemical and Biophysical Research Communications 84, 639.

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Buck, K. W. (1980). In “The Eukaryotic Microbial Cell” (G. W. Gooday, D. Lloyd and A . P. J. Trinci, eds.), Society for General Microbiology Symposium 30, pp. 329-375. Cambridge University Press. Bussey, H. (1972). Nature New Biology 325, 73. Bussey, H. (1974). Journal of’ General Microbiology 82, 171. Bussey, H. and Sherman, D. (1973). Biochimica et Biophysica Acta 298, 868. Bussey, H. and Skipper, N. (1975). Journal sf’ Bacteriology 124, 476. Bussey, H. and Skipper, N. (1976). Antimicrobiology Agents and Chemotherapy 9, 352. Bussey, H., Sherman, D. and Somers, J. M. (1973). Journal of’ Bacteriology 113, 1193. Bussey, H. Saville, D., Chevallier, M. R. and Rank, G. H. (1979a). Biochimica et Biophysica Acta 553, 185. Bussey, H., Saville, D., Hutchins, K. and Palfree, R. G . E. (1979b). Journal of Bacteriology 140, 888. Clare, J . J. and Oliver, S. G . (1979). Molecular and General Genetics 171. 161. Cohn, M. S., Tabor, C. W. and Wickner, R. B. (1978). Journal of’ Biological Chemistry 253, 5225. Drapeau, G. R., Boily, Y. and Houmard, J. (1972) Journal of’ Biological Chemistry 241. 6720. Fink, G . R. and Styles, C. A. (1972). Proceedings of’ the National Academy o f Sciences of’ the United States of’ America 69, 2846. Fisher, H. F. (1964). Proceedings of’ the National Academy of’ Sciences of the United States of’ America 51, 1285. Fried, H. M. and Fink, G. R. (1978). Proceedings of’ the National Academy of’ Sciences of’ the United States of’ America 15, 4224. Glanville, M., Lachmi, B., Smith, A. E. and Kaarinanen, L. (1978). Biochemica et Biophysica Acta 518, 497. Goldring, E. S., Grossman, L. I., Krupnick, D., Cryer, D. R. and Marmur, J . (1970). Journal of’ Molecular Biology 52, 323. Hankin, L. and Puhalla, J. E. (1971). Phytopathology 61, 50. Harris, M. S. (1978). Microbios 21, 161. Hastie, N. D., Brennan, V. and Bruenn, J. A. (1978). Journal of’ Virology 28, 1002. Herring, A. J. and Bevan, E. A. (1977). Nature, London 268, 464. Hollings, M. (1978). Advances in Virus Research 22, 1. Holm, C. A,, Oliver, S. G., Newman, A. M., Holland, L. E., McLauglin, C. S.. Wagner, E. K. and Warner, R. C. (1978). Journal of’ Biological Chemistry 253, 8332. Honjo, T.. Nishizuka, Y.. Hayaishi. 0. and Kato, I. (1968). Journal sf’ Biological Chemistry 243, 3553. Hopper, J. E., Bostian, D. A,, Rowe. L. B. and Tipper, D. J. (1977). Journal of’ Biological Chemistry 252, 9010. Imamura, T., Kawamoto, M. and Takaoka, Y. (1975). Journal of’ Fermentation Technology 53, 41 7. Jakes. K. S. and Model, P. (1979). Journal of Bacteriology 138, 770. Kahn, C. R. (1979). Trends in Biochemical Sciences 4, N263. Kandel. J. and Koltin, Y. (1978). Experimental Mycology 2, 270. Kandel, J . S. and Stern, T. A. (1979). Antimicrobial Agents and Chemotherapy 15, 568. Kane, W. P., Pietras, D. F. and Bruenn, J. A. (1979). Journal o f Virology 32, 692.

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Kitamura, K., Kaneko, T. and Yamamoto, Y. (1974). Journul of'Gencw1and Applied Microbiology 20, 32 3. Koltin, Y. (1977). Genetics 86, 527. Koltin, Y. and Day, P. R. (1975). Applied Microbiology 30, 694. Koltin, Y. and Day, P. R. (1976). Proceedings oftlie National Academy cd' Sciences of' the United States of' America 73, 594. Koltin, Y. and Kandel, J. S. (1978). Genetics 88, 267. Koltin, Y., Mayer, 1. and Steinlauf, R. (1978). Moleculur und Ganeral Genetics 166, 181.

Kotani, H., Shinmyo, A. and Enatsu, T. (1977). Journal of' Bucteriology 129, 640. Lemke, P. A. (1976). Annual Review of' Microbiology 30, 105. Leibowitz, M. J. and Wickner, R. B. (1976). Proceedings o f t h e Nationul Academy of' Sciences of' the United States of' America 73, 206 1. Leibowitz, M. J. and Wickner, R. B. (1978). Moleculur and General Genetics 165, 115. Levine, R., Koltin, Y . and Kandel, J. (1979). Nucleic A d s Research 6, 3717. Lindberg, B. (1 972). Methods in Enzymology 28, 178-1 95. Makower, M. (1964). D.Phil. Thesis: Oxford University, England. Makower, M. and Bevan, E. A. (1963). Proceedings of' the International Congress o f Genetics X I 1. 202. Manners, D. J., Masson. A. J., Patterson, J. C., Bjorndal. H. and Lindberg, B. (1973). Biochemicul Journal 135, 3 1. Maule, A. P. and Thomas, P. D. (1973). Journal qf'the Institute' of'Brewing 79, 137. Middelbeek, E. J., Hermans, J . M . H., Stumm, C., and Muytjens, H. L. (1980). Antonie vun Lreuwenhoek (In press). Mitchell, D. J., Herring, A. J. and Bevan, E. A. (1976). Heredit.v 37, 129. Moss, J . and Vaughan, M. (1977). Journal of Biological Chemistry 252, 2455. Naumov, G. I. and Naumova, T. I. (1973). Geiietiku 9, 140. Novick, P. and Schekman, R. (1979). Proceedings qf'the National Academy of'Sciences of' the United States of' America 76. 1858. Oliver, S. G., McCready, S. J., Holm, C., Sutherland, P. A., McLaughlin, C. S. and Cox, B. S. (1977). Journal of Bacteriology 130, 1303. Olsnes, S. and Pihl, A. (1978). Trends in Biochemical Sciences 3, 7. Ouchi, K., Kawase, N., Nakano, S. and Akiyama, H. (1978). Agricultural Biology and Chemistry 42. 1. Palfree, R. G. E. (1978). Ph.D. Thesis: McGill University, Montreal. Palfree, R. G. E. and Bussey, H. (1979) Europeun Journal of' Biochemistry 93, 487. Philliskirk, G. and Young, T. W. (1975). Antonic. van Leeuwenhoek 41, 147. Puhalla, J . E. (1968). Genetics 60, 461. Racevskis, J. and Koch, G. (1978). Virology 87, 354. Rogers, D. T. (1976). Ph.D. Thesis: Queen Mary College, University of London. Rogers, D. T. and Bevan, E. A. (1978). Journal of General Microbiology 105, 199. Rogers. D. T., Saville, D. and Bussey, H. (1979). Biochemical and Biophvsicd Research Communications 90, 187. Schaller, K . and Nomura, M. (1976). Proceedings ofthe National Academy ofsciences of the United States of America 73, 3989. Senior, B. W. and Holland, I. B. (1971). Proceedings of the National Academy of Sciences of the United States of' America 68, 959.

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Silverstein, S., Christman, J. K. and Acs, G . (1976). Annual Revien qf Biochemistry 45, 376. Skipper, N. A. (1978). Ph.D Thesis, McGill University, Montreal, Canada. Skipper, N. and Bussey, H. (1977). Journal qfBacteriology 129, 668. Somers, J. M. (1973). Genetics 74, 571. Somers, J. M. and Bevan, E. A. (1969). Genetics Research 13, 71. Stumm, C., Hermans, J. M. H., Middelbeck, E. J., Croes, A. E. and De Vries, G. J . M. L. (1977). Antonie van Leeuwenhoek Journal of Microbiology and Serology 43, 125. Sweeney, T. K., Tate, A. and Fink, G . R. (1976). Genetics 84, 27. Switzer, R. C., Merril, C. R. and Shifrin, S. (1979). Analytical Biochemistry 98, 231. Toh-e, A. and Wickner, R. B. (1979). Genetics 91, 673. Toh-e, A. and Wickner, R . B. (1980). Proceedings of the National Academy of Sciences of the United States of America 77, 527. Toh-e, A., Guerry, P. and Wickner, R. B. (1978). Journal of Bacteriology 136, 1002. Tzen, J. C., Somers, J . M. and,Mitchell, D. J. (1974). Heredity 33, 132. Vodkin, M., Katterman, F. and Fink, G . R. (1974). Journal of Bacteriology 117, 681. Welsh, J. D. and Leibowitz, M. J. (1978). 9th International Conference on Yeast Genetics and Molecular Biology, Rochester, New York. Abstracts page 101. Wickner, R. B. (1974). Journal ofBacteriology 117, 1356. Wickner, R. B. (1976a). Bacteriological Reviews 40, 757. Wickner, R. B. (1976b). Generics 82, 273. Wickner, R. B. (1971). Genetics 87, 441. Wickner, R. B. (1978). Genetics 88, 419. Wickner, R. B. (1979). Plasmid 2, 303. Wickner, R. B. and Leibowitz, M. J. (1976a). Journal of Molecular Biology 105, 427. Wickner, R. B. and Leibowitz, M. J. (1976b). Genetics 82, 429. Wickner, R. B. and Leibowitz, M. J . (1977). Genetics 87, 453. Wickner, R. B. and Leibowitz, M. J. (1979). Journal qf Bacteriology 140, 154. Wickner, W. (1979). Annual Review of Biochemistry 48, 23. Willsky, G . R. (1979). The Journal of Biological Chemistry 254, 3326. Wood, H. A. and Bozarth, R. F. (1973). Phytopathology 63, 604. Woods, D. R. (1966). D.Phil. Thesis, Oxford University, England. Young, T. W. and Yagiu, M. (1978). Antonie van Leeuwenhoek Journal of Microbiology and Serology 44, 59, Note added in proof: Two recent findings are relevant to the mechanism of action of the killer toxin. Bruce Kagan (personal communication) has found that purified KI toxin will insert in vitru into a phospholipid bilayer and form a pore. It is likely, as in the case of colicin K (Schlein et al., 1978), that this is the mechanism of toxin action in vivu. Partially purified K I toxin inhibits the uptake of leucine and histidine and the cotransport of protons, and also glucoseinduced proton pumping from sensitive cells (De la Pena et al., 1980). These effects are seen within 10 minutes of toxin addition, and occur without a lag after toxin binding to target cells. These results taken together suggest that the toxin forms a plasma-membrane pore which causes disruption of the cellular electrochemical potential. In an interesting development (Gunge et al., 1981) have found a doubie-stranded DNA plasmid associated with the killer character of a strain of Kluyveromyces lactis. The K. l a d s killer strain lacks detectable dsRNA, but has two linear dsDNA plasmids which cosegregate cytoplasmically with the killer phenotype in a genetic cross of the strain with a non-killer.

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The D N A plasmids are linear, by electron microscopy and by the mapping of restriction fragments. The smaller of the plasmids G k 1 1 is lost by heat shock or ethidium bromide treatment, procedures which lead to the loss of the killer phenotype. This plasmid system may prove useful both for solving problems of killer plasmid molecular biology, and as a cloning vehicle for recombinant D N A in yeast. REFERENCES

Schlein, S. J., Kagan, B. L. and Finkelstein, A. (1978). Nafure, London 276, 159. De La Pena, P. Barros, F.. Gascon, S., Ramos, S. and Lazo, P. S. (1980). Biochemical and Biophysical Research Communications %, 544. Gunge, N., Tamaru, A,. Ozawa, F. and Sakaguchi, K. (1981). Journal qf Bacferiology 145, 382.

Regulation of Glucose Metabolism in Growing Yeast Cells A. FIECHTER, G. F. FUHRMANN" and 0. KAPPELI Swiss Federal Institute of Technology, ETH-Honggerberg, CH-8093 Zurich, Switzerland and "Institute of Pharmacology and Toxicology, Philipps University, Lahnberge 0-3550Marburgl Lahn, Germany

I. Introduction . . . . . 11. Growth . . . A. Control of growth . . . B. Physiology of growth . . . 111. Molecular background of regulation . A. Crabtree effect . . . . B. Pasteur effect: Sols model . C. Energetical considerations . . IV. Sugar transport . . . . A. Introduction . . . . B. Transport systems . . . V. Conclusions . . . . . VI. Acknowledgements . . . References . . . . .

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123 125 125 132 142 142 153 157 159 159 162 176 177 177

I. Introduction Metabolism of yeasts was investigated in the era of classical biochemistry. Such investigations stamped this discipline to a considerable extent. As a result, important contributions to our general knowledge of central metabolic pathways emerged from these studies and, consequently, yeast technology has been developing to an appreciable degree (see Cook, 1958; Rose and Harrison, 1969, 1970, 1971). These studies have contributed not just to the advancement of the numerous existing yeast technologies but also to the 123

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unity of biochemistry. After the development of the old Vienna process for production of baker’s yeast, this cell type became something like a true eukaryotic model system for biochemical investigations due to its easy accessibility . In terms of DNA content, yeast cells are close to the prokaryotes (Escherichiu coli: 4 x 10 l z mg, 2.4 x lo9 mol. wt) and carry five times more DNA than E . coli, but are true eukaryotes with approximately half the amount of DNA of a typical fungus like Nrurospora sp. or Aspergillus niduluns (eight times that of E. coli) or less than 0.5% of humans (one thousand times that of E . coli). Nevertheless, structure and function resemble typically those of eukaryotic cells and, as a unicellular system, offer an excellent experimental access to metabolic or cytological problems. In view of the progress made in chemostat methods, it can be foreseen that yeast cells also will represent an excellent tool for research in developmental biology. Warburg (1926) used yeast cells extensively for development of the manometric measurement of gas exchange in active biological material (the Warburg technique), and he was able to demonstrate important parallels between glucose-affected yeast cells and tumour cells of vertebrates. This famous work on “Stoffwechsel der Tumoren” appeared in 1926. Furthermore, decisive progress was made in the 1920s and 1930s on the mechanism and enzymology of the glycolytic pathways and their regulation, making use of yeast cells. However, an attempt to give a full explanation of the effects of oxygen on the glycolytic sequence, first named in honour of Pasteur by Warburg, turned out to be unsuccessful. During the last decades, several theories have been developed to explain glycolytic regulation by oxygen, sometimes creating more confusion than clarity. The problem could not be satisfactorily solved until Sols and others published their relevant enzymic work on phosphofructokinase. In the early 1970s, Krebs (1972) stated that most relevant aspects of this regulatory phenomenon have appropriate explanations. This statement, however, was not unanimously accepted. Racker (1976) came to the conclusion that no unique mechanism underlying glycolytic control exists. He emphasized that a variety of different situations are encountered in microbial or animal cells undergoing different experimental treatments. Glycolysis in growing cells operates quite differently from that in resting cells, and is affected by respiratory products. Consequently, respiration as energy-producing and cell synthesis as energy-consuming activities are parts of a very complex control loop. The Pasteur regulation was originally considered simply as an effect of oxygen. Since, according to the Sols model, respiration is involved, glucose, acting on tricarboxylic acid (TCA) cycle as well as other nutrients (acting on growth), plays an important role (Stickland, 1956). ~

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The so called “glucose-effect’’ observed in a large number of yeast species was first classified simply as a phenomenon of catabolite repression. However, the classical interpretation of this mechanism is unsuitable for giving a complete explanation of observations made during repression and derepression. Additional mechanisms, such as “catabolite inactivations” and sugar transport, may be involved in the control of what was designated in the past as “fermentation”. It is the aim of the following sections to give an up-to-date picture of the co-ordinated control of central metabolic pathways in yeast, when glucose serves as a carbon and energy source. Efficient experimental methods for investigating growing cells are mandatory. Chemostat techniques, including pulse and shift experiments, are increasingly adopted. Combined with a good analytical arsenal, like automatic gas analyses, turnover rates, mass balances and adherent enzyme activities can be assessed in a consistent manner. On the basis of recent data from various yeasts, the classical terms for various regulatory phenomena and expressions will be clarified and, finally, recent knowledge on sugar transport in yeast will be summarized. The model of Peterkofsky (1977) for co-ordination of sugar transport in prokaryotes with catabolite repression is discussed in relation to eukaryotes. We hope that evaluation of the facts given may stimulate new endeavours with an interesting class of organisms that has contributed so much to our present knowledge of metabolism and its regulation.

11. Growth A. C O N T R O L O F G R O W T H

In biological experiments, a response of a cell culture has to be analysed in relation to the environment. There is an exchange between the environment and the cells (e.g. substrate and solute uptake), and environmental parameters may influence the behaviour of cells. In regulatory studies, it is therefore of great importance to know the precise reason for a given cellular reaction. The question of whether one is dealing with external restriction (i.e. restrictions originating from the environment) or with intrinsic limitations or metabolic regulation (inherent to the organism) is a matter of considerable significance. At the beginning of a regulatory study, the environmental factors need to be determined and optimized with respect to the planned experiments.

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1. Physicochemical Factors

Culture techniques. Historically, the shake flask was used first for production of cell material. The medium was usually a very complex one containing inorganic and organic components of unknown composition. Few parameters were controlled, usually only temperature and pH value. It is obvious that data frcm these early experiments had only limited value. With advances in technology, cells began to be cultivated in bioreactors which offered much better possibilities for the surveillance of biological processes. In batch or static cultures, pH value, temperature, agitation and aeration are easily controlled, thus increasing the reliability of experiments. Since nutrients are added at the beginning of a batch culture, and then are used by the cells, the environmental conditions are changing continually with time thereby complicating reproducibility. This problem was overcome with the introduction of continuous culture. By this technique, it is possible to induce a particular regulatory state in

C e l l yield (Y,; g dry mass (1009 substrate-'))

FIG. 1. Correlation of oxygen demand with biomass formation by Sacchuromjws cerevisiue. Oxygen is a stringent effector of synthesis of all mass in obligate and facultative yeast types. From Dellweg et a/. (1977). 0 indicates responses in batch in continuous culture. culture,

GLUCOSE METABOLISM IN GROWING YEAST CELLS

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a culture and maintain it for an indefinite time in a constant environment. Consequently, reproducibility is much better since the cell population is standardized. The continuous-culture technique is also suitable for dynamic studies since changes induced by shifts or pulses can be monitored as a function of time (compare Fig. 9, p. 151). The advantage of continuous-culture technique is very well demonstrated in investigations on the regulation of glucose metabolism in yeast. In a batch culture, with glucose as the carbon source, it is not possible to obtain glucosederepressed growth with Saccharomyces cerevisiae. Derepression does not set in before complete depletion of the medium of glucose. In this situation, ethanol is the substrate for derepressed growth. In continuous culture, however, glucose-derepressed growth with simultaneous use of glucose as the substrate is possible (see Fig. 2, p. 128; Fig. 3 , p. 130). b. Mass transfer. Considering the problem of mass transfer, one has to take into account the fact that there exists a certain demand of nutrients by cells. A bioreactor has to provide this nutrient flow from the medium to the cells. Rates of uptake of glucose, for example, are considerable (Just, 1940). Musfeld (1942) calculated the rate as lo7 molecules of glucose cell-' s-'. Schatzmann (1975) observed a maximum glucose uptake rate of approximately 20 mmol g- h- I under anaerobic conditions (see Fig. 4, p. 134). Even more problematical is an adequate transfer of oxygen due to the properties of oxygen, such as its low solubility in water. Dellweg et al. (1977) calculated a maximum oxygen demand of 25 mmol g-l dry cell weight (Fig. 1). In regulatory studies, especially where oxygen plays a regulatory role (Wimpenny, 1969) as it does in yeasts, an adequate performance by the bioreactor with respect to mixing has to be ensured. c. Nutritional requirements. The development of defined media is the basis for any regulatory experiment, a fact that has quite often been overlooked. Nutritional limitations may change the physiology of the cells drastically (Fig. 2) and lead to faulty conclusions. There are two basic approaches for development of a defined medium. The first one was used by Fiechter (1966) for devising a defined yeast medium with carbon limitation. This method is based on a mass balance established after elemental analysis of cell material (Sperber, 1945; White, 1954). The resulting purely mineralic medium with glucose as the carbon source needs to be supplemented with yeast growth factors (meso-inositol, pantothenic acid, biotin and the vitamins of the B complex). In the absence of vitamins, growth is scarce, yields are decreased and steady states in continuous culture are difficult to maintain. The medium as described by Fiechter (1966) and revised by Schatzmann (1975) has the following composition (mg gglucose): (NH,),SO,, 200; (NH,),HPO,, 64; HCI, 30; MG S0;7 H,O, 15; CaCI.4 H,O, 10; FeC1;6 H,O, 0.5; ZnSO;7 H,O, 0.3; MnSO;2 H,O,

A. FIECHTER, G . F. FUHRMANN AND 0. KAPPELI

128

I00

0

(a) 0

0

0

:/o

-15

0

1

80

0

A

0

(L

0

5 al

60

m.0

0. I

0.2 Dilution rate ( h - ' )

0.3 Dc

8l

5e 12 c

8 u

2

W

4

DR Dilution rate ( h - ' )

FIG. 2. Growth of Schizosaccharomyces pombe on the Wickerham medium (a, 1% glucose) and the Dw medium (b, 2% glucose). indicates biomass concentration X, V ethanol concentration A, respiratory quotient, 0carbon recovery, and A glucose concentration. The limitation of the Wickerham medium is expressed in the absence of glucose-derepressed growth and a lower yield in biomass. From Brandli (1980).

0.6: CUSO;~H,O, 0.08 meso-inositol, 2; calcium pantothenate, 1; nicotinic acid, 0.2; pyridoxine HC1, 0.05; biotin, 0.001 (for details, see Schatzmann, 1975). For supplements of growth factors (Mor, 1972) and trace elements compare also Suomalainen and Oura (1971). The second method is based on the establishment of yield coefficients for

C

f

GLUCOSE METABOLISM IN GROWING YEAST CELLS

129

individual elements as described by Pirt (1975) and Aiba et al. (1973). For the determination of essential and growth-stimulating components, a method applying pulse techniques was reported by Kuhn (1979) and Kuhn et al. (1979). Final testing of a medium is done by performing an X-S diagram. In a continuous culture with a stable dilution rate and a constant nutrient concentration, the concentration of the carbon source is increased stepwise. As long as the medium is carbon limited, the biomass concentration is a linear function of the concentration of the carbon source. As soon as another nutrient in the medium becomes limiting, the biomass concentration levels off. By this method, it is possible to determine the maximum concentration of the carbon source up to which the medium is carbon limited. Brandli (1980) demonstrated that the Wickerham (1951) medium which TABLE 1: Composition of the Wickerham (1951) medium which contains an ironcopper limitation and the newly developed synthetic D, medium for aerobic growth of Schizosaccharomyces pombe.

Component

Composition of Wickerham (1951) D, Medium medium (g I-')

17.8 5.7 1'.2

(NH4)2S04 KH2PO4 M&S04*7HzO NaCl CaCI2.6 H 2 0 ZnS04-7 H 2 0 FeC13.6 H 2 0 MnS04mH20 CuS04.5 HzO H3B04 KI (NH4)6M07024*4 H2O C0C12.6 HzO d-Biotin Calcium panthothenate Nicotinic acid Meso-Inositol Thiamin hydrochloride Pyridoxine hydrochloride L- Asparagine Sodium citrate (EDTA) Ortho-Phosphoric acid (conc.) Glucose

-

0.04 0.5 0.1 1.o

.-

0.05

1 .o 1.o 10.0 1 .o 1.o 1.5 & I - ' -

10.0 g I - '

(mg I - ' ) 150.0 30.0 100.0 (FeS04.7 H20) 32.0 0.79 15.0 2.0 5.0 (Na2Mo04*2HzO) 5.6 0. I 20.0 15.0 160.0 4.0 10.0 ~

0.5 g I - ' (0.1 g I - ' ) 0.75 ml I - ' 0 4 0 . 0 & 1-

130

A. FIECHTER, G. F. FUHRMANN A N D 0. KAPPELI

TYPE I Candido sp.

Glucose - insensitive

: I

Mono-auxie Oxygen li mito tion

+

Time

I

,

,,D, Dc.0.6 Dilution rate (h-')

TYPE 2 Saccharomyces sp.

ikGlucose-sensitive

Diauxie

11 II

m

D

T i me

TYPE 3

n

Schizosaccharomyces sp.

Glucose - sensitive

Secondary mono-auxie

I

'X

/

J

-'

Time

FIG. 3. Growth patterns of various yeast types in batch and chemostat cultures. In the batch-culture experiments, Type 1 represents the growth behaviour of a glucose insensitive strain (e.g. Trichosporon sp; Janshekar, 1979). Exponential growth characteristics occur until complete depletion of the limiting medium component. There is no formation of a metabolic intermediate. Minor diauxic growth is observed in some strains under oxygen limitation and under very high concentration of glucose (e.g. Cundidu tropicalis; Knopfel, 1972). Type 2 is obtained with glucose-sensitive strains

GLUCOSE METABOLISM I N GROWING YEAST CELLS

131

is widely used for Schizosaccharomyces pombe contains a hidden ironxopper limitation for this organism. Figure 2 shows growth of Schizosacch. pombe in a chemostat, at different dilution rates, with the two media. With the Wickerham medium, derepressed growth was not detected. Ethanol was formed at low dilution rates. With the newly developed D, medium (Table I), derepressed growth on glucose was observed up to a maximum dilution rate of 0.14 hK1. It is noteworthy that this limitation is not detectable in batch cultures, since growth of Schizosacch. pombe in static cultures is qualitatively identical on both media. 2. Growth patterns of typical regulatory yeast types

For a long time, two main categories of yeasts were differentiated on the basis of their sensitivity to free glucose. Yeasts in the first group are glucose sensitive. Their respiration is repressed in the presence of small concentrations of the free sugar, ethanol accumulates to large concentrations under strong repression, formation of biomass (yield) is drastically decreased and they can be grown anaerobically in supplemented media. They are known as “fermenting yeast” or “Garungshefen”, and were recognized by release of ethanol. Consequently, differentiation between aerobic and anaerobic “fermentation”, respectively, had to be made. This group is represented by strains from the genera Saccharomyces, Schizosaccharomyces, Debaryomyces, Torufopsis and others. Baker’s yeast and brewery yeast (bottom) are typical glucose-sensitive yeasts, but “fermenting” grape juice may contain many other types. It will be shown that the underlying metabolic regulation is far from being uniform. (e.g. Saccharomyces cerevisiae) under conditions of maximum oxygen supply. No limitation of glucose uptake is detectable. The first exponential phase is affected by glucose. Owing to repression, ethanol as an intermediate is formed and serves as a carbon source during the second phase (after release from glucose repression). Type 3 represents the behaviour of a glucose-sensitive yeast strain (e.g. Schizosaccharomyces pornbe) growing on glucose but not on ethanol. Only one exponential growth phase is obtained. Ethanol, accumulated during the first phase, is completely oxidized (absence of glyoxylic acid shunt; Flury, 1973). Plots of biomass and substrate concentration against dilution (rate are shown in chemostat experiments for comparison. They show the regulatory patterns as they occur in function of dilution rates. Section I represents a regulatory state of decreasing yield (depression). Accumulation of storage carbohydrate is considerable. There is extensive budding in a part of the cell population (Kuenzi, 1970). No ethanol formation. The respiration quotient is less than unity. Section I1 shows derepression with a full yield and no ethanol formation. RQ = 1 energy charge at maximum. Section I11 shows the phase of glucose repression. The specific substrate uptake rate is above the critical value. Ethanol formation and lowered yield are consequences of glucose repression. The repiratory quotient is greater than unity.

132

A. FIECHTER, G. F FUHRMANN AND 0. KAPPELI

A second category of strains is insensitive to free glucose. These types show relatively fast growth and high yields of biomass under unrestricted oxygen supply. Ethanol is not released. They cannot be grown in the absence of oxygen. Typical representatives are found among the genera Cundidu, Rhodotorula, Trichosporon, Pichia, Torulopsis and Hansenula. Classical data from taxonomic work sometimes may be misleading in view of the absence of ethanol formation because rigorous testing under strong glucose repression together with oxygen limitation have not always been carried out. Under such conditions, numerous strains of Cundidu, for instance, show a repressed metabolism. As Fungi Imperfecti, Candidu spp. are very heterogeneous in many respects. This second category of organisms are known as “respirative yeast” or “Atmungshefen”, and are best represented by the industrially important “fodder yeasts”. As already indicated, glucose is not necessarily the only effector of this particular metabolic behaviour. Oxygen may interfere in some cases, and proper testing under precisely defined conditions for growth and respiration are a prerequisite for regulatory investigations. On this basis, various growth patterns can be detected both in batch-culture and chemostat experiments. Figure 3 shows that, with an excess oxygen supply, growth on glucose induces typical growth behaviour for a given strain. Insensitive yeasts show exponential growth kinetics towards the exhaustion of the carbon source (i.e. mono-auxie). Diauxic growth results with repressible strains like Succh. cerevisiue which is different from the classical diauxie with two carbon sources (i.e. glucose and lactose) both present in the medium at the beginning of the experiment (Monod, 1942). The glucose-sensitive yeast is strongly repressed and forced to accumulate ethanol (in the first growth phase). After release from repression, ethanol acts as a carbon source during the second phase. Secondary mono-auxie is obtained as with Schizosacch. pombe without assimilation of the excreted ethanol (Fig. 3). This type of regulation reflects the lack of a glyoxylic acid bypass (Flury, 1973).

B. P H Y S I O L O G Y O F G R O W T H

1. Regulatory Definitions

The phenomena associated with metabolic control in yeast and other microbes are very complex. The nomenclature of these regulatory phenomena appears sometimes rather confusing. The term “fermentation” in particular has become very unspecific and has quite different meanings to the micro-

GLUCOSE METABOLISM IN GROWING YEAST CELLS

133

biologist or biochemist, the engineer and the industrialist. In fact, it is only tolerable as an expression to the practioneer and therefore should be dropped as a scientific notion. Regulatory results are preferably classified according to their effects. In this article, only the expressions appearing in Table 2 are used. Accordingly, growth, resting (active) cells and respiratory regulation can be clearly differentiated.

2 . Anaerobiosis

Data on anaerobic metabolism are frequently reported from natural “fermentation” substrates like brewing wort, fruit juices, whole fruits submitted to an alcoholic process, molasses or other residual material from natural products. Even in chemically more defined systems, dependence of ethanol formation on growth has rarely been taken into consideration where mass balances or kinetics were studied. . Ample ethanol formation occurs with non-growing cells particularly in wort and molasses. However, cells in such a suspension do not grow fully, but die after a certain time and data from such experiments may differ greatly. TABLE 2. Definitions of some terms commonly used to describe regulation of glucose metabolism in yeast Term Growing cells

Definition

Proliferating cells expressed in the formation of biomass with simultaneous assimilation of a carbon source present in adequate concentration Resting cells Non-proliferating cells, no formation of biomass because of incomplete nutritional requirements (mainly starvation of a nitrogen source) Aerobiosis Growth under aerated conditions Anaero biosis Growth under complete oxygen exclusion Pasteur effect Inhibition of the glycolytic pathway in the presence of oxygen (manifested as inhibition of ethanol formation) Custers effect Negative Pasteur effect, “inhibition of alcoholic fermentation (Custers, 1940) in the absence of gaseous oxygen and a stimulation of the process in the presence of oxygen” (Wiken, 1968) Crabtree or glucose Repression of respiratory activity by glucose under aerobic effect conditions and subsequent deregulation of glycolysis with formation of ethanol Assimilation Incorporation of substrate Repression Relative decrease of enzyme formation irrespective of the underlying mechanism Relative increase of enzyme formation irrespective of the Derepression underlying mechanism

134

A. FIECHTER, G . F. FUHRMANN A N D 0.KAPPELI

The main difficulties in anaerobic chemostat studies are correct supplementation of media and complete exclusion of oxygen. Complete exclusion of oxygen in anaerobic studies is an experimental prerequisite. Careful p u ification of an inert displacement gas, and preventing re-entrance of oxygen through glass joints or tubings of peristaltic pumps, permits the experimenter to maintain residual oxygen concentrations below a critical level for respiration. Anaerobic growth can be achieved only with glucose-sensitive yeast strains. Attempts to grow the strictly respiratory types anaerobically have failed. Addition of ergosterol and unsaturated fatty acids are essential (Andreasen and Stier, 1953, 1954). Careful re-examination of growth of Sacch. cerevisiae and Sacch. uvarum (= carfsbergensis)by Schatzmann (1975) revealed that other growth factors, like nicotinic acid, are needed as well. This indicates that molecular oxygen is involved in several biosynthetic

Dilution rate ( h - ' )

I

Dilution rate (h-')

FIG. 4. Anaerobic growth of Saccharomyces cerevisiae, a glucose-sensitiveyeast. (a) Yield of biomass is roughly one-fifth compared to aerobiosis. Major by products glycerol (0)and pyruvate (A)respectively. (b) Specific rates of are ethanol carbon dioxide production (Qcoz;a), ethanol production (QA;v ) and glucose uptake (Qs; A) and biomass and substrate (0)concentrations are shown. Note the characteristic rise of residual glucose at dilution rates above 0.1 h - I .

(a),

(m)

GLUCOSE METABOLISM IN GROWING YEAST CELLS

135

pathways. It is also known that a constant percentage of esters of ergosterol precursors seem to be unable to be metabolized to ergosterol because, once esterified, the fatty acids d o not appear to be metabolized during starvation conditions (Bailey and Parks, 1976). Anaerobic growth of Succh. cerevisiue follows closely the model of Monod (1942). Experimental data for biomass concentration (x) and substrate concentration (S) are in good agreement with the theoretically expected values (Fig. 4). The kinetic parameters were: pmax,0.34 h - l , K,, 39 mM; and Y,, 0.1. Thus, only about 10% of the glucose was converted into biomass. The rest of the carbon feed appears in three exudates, namely ethanol, glycerol and pyruvate (Fig. 4). Interestingly, release of pyruvate is observed only when the dilution rate is above 0.04 h- and is increased with increasing growth rate. Glycerol is released in an anaerobic chemostat only (see also Section II.B.3, p. 138). Cells regenerate some NADH by transforming the hydrogen to dihydroxyacetone. According to Oura (1977), it is probable that glycerol is not an essential building block for continuing anabolic sequences. Rather, it reflects the redox-balance in such cases when acetaldehyde is removed (second form of “fermentation” of Neuberg) or acetate is formed from acetaldehyde by oxidation (third form of Neuberg). Oura (1977) postulates direct correlation of the redox-balance and glycerol formation (a NADH-oxidizing step). He was unable to demonstrate glycerol excretion in all cases where other ways for removing excessive NADH could be used by the cell, e.g. the presence of reducible substances like acetaldehyde or a-0x0 acids, formaldehyde or even small amounts of oxygen (initiating respiration). Formation of cell mass is related to glycerol excretion according to the relation of 1.3-1.5 mol NADH formed per 100 g of biomass from glucose (Lagunas and Gancedo, 1973; Oura, 1974). The observation that glycerol formation is three to four times greater in the early stages of batch growth (during consumption of the first third of sugar) indicates a relationship to a glucose effect rather than to ethanol formation as such. Succinate may also be present as it always accumulates under conditions of hypoxia or in impaired respiration mutants according to a postulated reductive function of the TCA cycle leading from oxaloacetate to succinate (Lupiariez et ul., 1974). Formation of other byproducts in small amounts has been reported since the days of Pasteur (Oura, 1977). However, their concentrations appear to be very low and strongly dependent on the media used. Release of succinate under anaerobic conditions agrees with the observations made by Schatzmann (1975). He was able to demonstrate that TCA-cycle enzymes are active even under anaerobic conditions (Fig. 5). The findings of Schatzmann (1975) are in accordance with the view of Oura, that glycerol formation is the mechanism for removing the otherwise accumulating reducing power generated by the TCA cycle. It also explains the anaerobic preparation of

136

A. FIECHTER, G . F. FUHRMANN AND 0. KAPPELI

building blocks for the biosynthetic pathways. The TCA cycle, even diminished in its overall activity, generates monomers and energy similar to the aerobic situation. However, energy is not transformed to ATP but stored in the form of glycerol. Regulation of the TCA-cycle enzymes by glucose under anaerobic and aerobic conditions will be discussed in greater detail in Section 1II.A (p. 142).

3. Aevo biosis

Free glucose as a regulatory effector has been known for a long time, and its effects on metabolism in micro-organisms are widespread (see Table 3). Catabolite repression is historically referred to as the glucose effect. In yeasts, glucose is the main regulatory substrate under aerobic conditions. Growth of a glucose-sensitive yeast' is governed by glucose regulation, and leads to the characteristic growth pattern with repression of respiration and accumulation of ethanol. A typical representative of this yeast type is Succh. cerevisiae. There are, however, yeasts that do not fit this ordinary pattern. Trichosporon cutaneum, for example, does not form ethanol under conditions where glucose repression is observed in other yeasts. a. Glucose-insensitiveyensts. As shown in Section II.A.2 (p. 131), there are yeasts that do not exhibit the growth characteristics of glucose-sensitive yeasts, i.e. no diauxi'c growth in static culture, and no formation of ethanol

15

I

0

Dilution r a t e ( h - ' )

FIG. 5. Regulatory effects of glucose under anaerobiosis in Succharomyces cerevisiae. Effect of dilution rate on activities of citrate synthase (u),malate dehydrogenase (0)and succinate cytochrome c oxidoreductase (A) during anaerobic growth of Succharon~ycescerevisiue. Increasing repression of citrate synthetase, malate dehydrogenase and succinate cytochrome c oxidoreductase at supercritical feed rates of glucose (2.44 mmol g- h - I ) (Schatzmann, 1975).

TABLE 3. Glucose effects on enzyme synthesis by micro-organisms. The examples given show that primarily the metabolic sequences of respiration are hit by glucose (TCA cycle, glyoxylic acid shunt and electron-transfer chain). Glycolytic sequence is less affected (Paigen and Williams, 1970) Enzymes

Organisms

References

Condensing enzyme

Sacch. cerevisiae E. coli Candida tropicalis Sacch. sp., Bac. sub t ilis E. coli Candida tropicalis Sacch. sp.

Polakis and Bartley (1965) Gray et al. (1966) Knopfel (1 972) Gorts (1967); Hanson and Cox (1967)

Aconitase

Isocitrate dehydrogenase NAD+-bound E. coli NADP+-bound B. subtilis Staph. aureus a-Oxoglutarate Staph. aureus dehydrogenase E. coli Succinate Sacch. sp. dehydrogenase E. coli Staph. aureus B. subtilis Fumarase Sacch. sp. E. coli Staph. aureus B. subtilis Malate Sacch. cerevisiae dehydrogenase Sacch. cerevisiae Schizosacch. pombe Candida tropicalis Isocytrate lyase E. coli Sacch. cerevisiae Rhizopus nigricans Hydrogenomonas sp. Schizosacch. pombe Candida tropicalis Malate synthase Sacch. cerevisiae Hydrogenomonas sp. Schizosacch. pombe Candida tropicalis Cytochromes Sacch. cerevisiae Staph. aureus Salmonella typhimurium NADH: Sacch. cerevisiae cytochrome Candida tropicalis Schizosacch. pombe Succinate: Sacch. cerevisiae cytochrome c-oxidoreductase

Polakis and Bartley (1965) Knopfel (1972) MacQuillen and Halvorson ( 1 962) Halpern et a/. (1 964); Gray et al. ( I 966) Hanson et al. (1963, 1964) Gershanovich and Burd (1 964) Gershanovich and Burd (1964) Amarasingham and Davis (1965) MacQuillan and Haivorson (1 962) Gale (1943) Gershanovich and Burd (1964) Hanson et al. (1963, 1964) Polakis and Bartley (1965) Halpern et al. (1964) Strasters and Winkler (1963) Hanson et al. (1963, 1964) Polakis and Bartley (1965) Mor (1972) Flury (1973) Knopfel (1972) Kornberg (1966) Polakis and Bartley (1965) Wegener and Romano (1964) Schlegel and Truper (1 966) Flury (1973) Knopfel (1972) Polakis and Bartley (1965) Schlegel and Truper (1966) Flury (1973) Knopfel (1972) Strittmatter (1957) Strasters and Winkler (1963) Richmond and Maalere (1962) Zimmerli (1970) Knopfel (1972) Flury (1973) Zimmerli (1 970)

Logarithm of respiratory quotient

fz 5 :. 4 2 3 "

U

0 c

0

300

600

900 Time (min)

1200

1500

500r50r 0

r

6

e -9 100

p

10

;

P 0

0 Dilution rate (h-')

FIG. 6. Growth of Saccharomyces cerevisiae on a defined medium containing 5% glucose under excessive aeration. From Fiechter (1966). (a) describes diauxic growth behaviour in batch culture. The substrate of the second growth phase (ethanol) is formed during the first phase under the conditions -of glucose repression. Effects of oxygen can be excluded during the excessive aeration. Changes in biomass

GLUCOSE METABOLISM IN GROWING YEAST CELLS

139

and a resulting drop in yield of biomass in continuous culture at a distinct dilution rate. Little is known about the physiology of these organisms with respect to glucose repression. Trichosporon cutaneum, an example of this yeast type, was used in our laboratory for production of single-cell protein from molasses (Janshekar, 1979). The main features of its growth physiology in continuous culture were: (i) lowering of the maximum yield of biomass from oxygen (Y;;"") at high substrate concentrations, i.e. a 14 and 35% decrease in the Y;;"" value at sugar concentrations of 5 and lo%, respectively, compared with the value at 0.5% sugar concentration; (ii) decrease of the maximum dilution rate (Dmax)from approximately 0.50 h-' to 0.31 h-' with a change in sugar concentration from 0.5 to 10%; (iii) a 23% decrease in the maximum yield of biomass from sugar (YY)at 10% sugar compared with that at a sugar concentration of 0.5%. From these data, it was concluded that excess sugar may affect values for Y;;"", D,,, and Y y , i.e. that even in this yeast there is a certain regulatory effect of sugars (most probably glucose) which needs to be elucidated. b. Glucose-sensitive yeasts. Growth of Sacch. cerevisiae, a typical glucosesensitive yeast in a static culture, is characterized by two phases. In the first growth phase, formation of biomass is paralleled by release of ethanol (Fig. 6). Since these experiments were conducted in a bioreactor under controlled conditions, limiting oxygen supply can be excluded as the reason for this growth behaviour. Prevention of endoxidation of substrate is consequently due to a glucose repression. It is characterized by a repressed respiration as indicated by the high values for the respiratory quotient and an accumulation of ethanol. In the second growth phase, ethanol formed under glucose repression serves as the substrate. Derepression of respiration begins as soon as the medium is depleted of glucose. The respiratory quotient drops as a consequence. It is noteworthy that, in static cultures, derepressed growth with glucose as the substrate cannot be achieved. In continuous culture, the growth rate is controlled by the rate of substrate feeding. Three main regions are distinguishable in Fig. 6 (compare also Fig. 3). Region 1: l o w substrate feed and consequently low growth rate. Derepressed growth with an RQ value close to unity. There is a decreased yield due to a high proportion of maintenance energy in the overall metabolic activity. A broad spectrum in population segregation is observed (Kuenzi, 1970). Region 2 shows an elevated supply rate below the critical value where glucose repression sets in, thereby showing respirative growth. Maximum

(a),glucose concentration (o), ethanol concentration (A) and respiratory quotient

(A)are shown. (b) describes growth in chemostat culture. Repression is occurring above a critical feed rate corresponding to approximately 2.5 mmol glucose (g dry matter)- * h- I . Symbols for biomass, glucose and ethanol concentrations are as in (a). A,describes changes in Qco~values and in QO2 values respectively.

A FIECHTER, G. F FUHRMANN A N D 0. KAPPELI

140

possible yields and maximum biosynthetic activity as expressed in maximum productivities are observed. Segregation of cell population is less pronounced. Most cells are able to bud after a short individual lag (von Meyenburg, 1969). Region 3 indicates feed rate and growth rate above the critical value, showing glucose repression and decreasing respiration rates. There is a marked increase in Q,,, values with increasing growth rates. Ethanol accumulation and subsequent drop in biomass concentration are due to the drop in yield of biomass on substrate ( Y J .The population consists mainly of two classes, namely mother cells with one or several bud scars and daughter cells with only a birth scar (Beran, 1969). By using continuous culture, it is possible to grow glucose-sensitive yeasts under derepressed conditions. It is difficult to find definite values for culture parameters at which repression sets in. Schatzmann ( 1 975) related the onset of repression to a critical rate of glycolysis. He observed that repression starts at a flux of approximately 2.44 mmol g- 'h- l . The validity of this hypothesis was, however, contested by Karrer (1978). He found that the critical dilution rate (DR)at which glucose repression starts is strongly dependent on the glucose concentration in the inlet medium (So). In chemostat experiments with different So concentrations, he found the following relations:

so (g 1 - l ) DR

@-'I

5 0.32

10 0.3 1

30 0.24

50 0.2

Since the glycolytic flux is theoretically independent of So values at a given dilution rate, the observations made by Karrer (1978) complicates the picture. The hypothesis of a constant glycolytic flux at the onset of repression is also difficult to sustain in view of the results of Brandli (1980) referred to in Section 1I.A (p. 127). The Fe-Cu-limitation (see Table 1) on the Wickerham medium induces repressed growth at very low dilution rates where the glycolytic flux is far below that observed at the onset of repression in the nonlimited medium D, (see Fig. 2, 128). This fact indicates that the onset of repression reveals a limiting capacity of cellular metabolism which may arbitrarily be introduced by any limitation and leads to drastic physiological changes. Any current speculation about the limiting capacity are premature, but possible bottlenecks may exist in energy-yielding reactions, in the capacity of the cells to produce building blocks for biosynthetic pathways or at the transcriptional level. There are marked differences in the metabolism of glucose under anaerobic, aerobic glucose-derepressed and aerobic glucose-repressed conditions in S a d . cerevisiae. They concern (i) the amount of substrate carbon converted into biomass and carbon dioxide respectively, and (ii) the amount and nature of products. Table 4 shows a survey of the carbon-balances under different culture conditions and reflects metabolic changes due to the absence of

141

GLUCOSE METABOLISM IN GROWING YEAST CELLS

oxygen and to the effect of glucose, respectively. It is especially remarkable that glycerol is excreted only under anaerobiosis. Formation of glycerol as a consequence of a lack of hydrogen acceptors was discussed in Section 1I.B (p. 132). Further, it should be pointed out that the amount of glucose converted int.0 ethanol is nearly as effective under aerobic glucose-repressed conditions as it is under anaerobic conditions. It shows the potential for aerobic ethanol production by yeasts. TABLE 4. Carbon balance in the absence and presence of oxygen during growth of Succhurornyces cerevisiae in a defined medium. From Schatzmann (1975). For minor components like succinate, fuse1 oils, acetate, acetaldehyde, butylene glycol, see Oura (1977) Percentage of totally metabolized carbon found in Carbon dioxide

Ethanol

Glucose

Pyruvate

12 55

32.5 45

46 0

9.5 0

g o .2

23

38

39

0

g o .1

Cultural conditions Biomass Anaerobiosis Aerobiosis (derepression) Aerobiosis (repression)

0

c. Intermediary yeast type. Besides the two yeast types discussed, there are intermediary types. With respect to glucose sensitivity, yeasts of the genus Candida (e.g. C. tropicalis or C. utilis) show a weak susceptibility to glucose, if any at all. Repression and subsequent formation of ethanol is observed with high concentrations of substrate only (So > 75 g 1-l). With such concentrations, it is difficult to determine the reason for ethanol release. External parameters (especially oxygen-transfer limitations) may influence the physiology of these yeasts leading to erroneous conclusions. The effect of transfer limitations needs to be investigated in more detail in these yeasts before the question of glucose sensitivity can be answered definitely. On the other side of the spectrum there exist yeasts with a very prominent glucose sensitivity. A yeast of this type is Schizosaccharomyces pombe. The critical dilutions rate (D,) for this yeast is 0.14 h- (see Fig. 2, p. 128), which is significantly lower than that of Sacch. cerevisiae (minimum DR value observed, 0.20 h - '). There are probably more highly sensitive yeast strains, such as the brewer's yeasts (e.g. Sacch. carlsbergensis). The extreme on this side of the spectrum are the po-mutants which lack mitochondria1 DNA and are glucose-sensitive under anaerobic conditions (see Schatzmann, 1975; Weibel, 1973).

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A. FIECHTER, G. F. FUHRMANN A N D 0. KAPPELI

111. Molecular Background of Regulation A. C R A B T R E E E F F E C T

1. Degree of Regulation of’ Susceptible Enzymes in Saccharomyces cerevisiae

and Candida tropicalis Regulation of metabolism by glucose, which is the basis for the growth physiology discussed in Section 11, is analogous to the observations of Crabtree (1929) with tumour cells. Crabtree (1929) investigated several types of tumour cells with respect to respiration rate and glycolysis rate under aerobic conditions. In his studies, he found “excessive fermentation”, i.e. the glycolysis rate was excessive compared with the corresponding respiration rate. It was concluded that glucose or one of its metabolites acted as a repressor of respiration since replacement of glucose by xylose did not show repression of respiration. Since the manometric techniques of Warburg had been applied, the data published by Crabtree (1929) were typical for resting cells. In view of the definitions given in Section 1I.B (p. 132), his data cannot be directly compared with those from growing and metabolically active cells. TABLE 5 . Examples of the extent of the glucose effect in Succhurornyces cerrvisiae. Enzyme activities (nmol of substrate converted min - I mg - 1) are those in the supernatant of crude cell extracts after centrifugation at 3500 g for 10 min. The value R : D is the ratio of activities in extracts of repressed to derepressed cells.

Metabolic pathway TCA cycle TCA cycle TCA cycle

Glyoxylic acid shunt Respiratory chain

Enzyme Citrate synthese Aconitase Fumarase Malate dehydrogenase

Defined medium (%glucose)

Activity under repression (nmol min- I mg- I )

1

100

1 : 12 Knopfel(l972)

20 50 500 350

1 : 35 1 : 10 1 :22 1 : 16

2 2 20 5

R :D

Reference

Knopfel(l972) Knopfel(1972) Beckand von Meyenburg (1968) 1 : 100 Beck and 1 : 200 von Meyenburg (1968) 1 : 5 Zimmerli (1975) 1: 10 Knopfel(1972)

GLUCOSE METABOLISM I N GROWING YEAST CELLS

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At the molecular level, several enzymes are affected by glucose repression in micro-organisms (compare Table 3, p. 137). The metabolic pathways mainly concerned are the TCA cycle, the glyoxylic acid shunt as well as the mitochondria1 electron-transfer chain. Interestingly, the glucose effect cannot be attributed to a specific step in one of these metabolic sequences, but is obviously expressed in a multitude of enzymes. Thus, the primary observations related to as the Crabtree effect are relatively unspecific. Transferred to yeast, it means that the respiratory pathways are repressed in the presence of glucose leading to a decrease in the specific oxygen uptake rate and to accumulation of ethanol. The question of which enzymes are affected in yeasts then arises. A reasonable account of the situation was obtained by measuring enzyme activities in crude cell extracts of yeasts harvested at different physiological states from a chemostat culture. Although this method yields a very general picture, the differences observed in certain enzyme activities under derepressed and repressed conditions allowed some conclusions to be made regarding the enzymes affected by glucose regulation. As already observed for other micro-organisms, the oxidative pathways were the main target of regulation (Table 5). A considerable repressive effect of glucose can be detected in the activity of the enzymes of these sequences. The span of repression is relatively wide, and ranges from a 5- to a 200-fold decrease in activity under repressed conditions compared with glucose derepression. It can be concluded that glucose regulation in yeasts also includes an extensive number of enzymes and probably is based on a rather complicated regulatory situation in terms of underlying mechanisms. It seems that catabolite repression represents only one of the mechanisms involved and it may be accompanied by other regulatory mechanisms, specific controls of protein synthesis, and modification and turnover of enzymes. An interesting aspect is the comparison of enzyme activities in yeasts which show a different sensitivity towards glucose as revealed in their growth physiology. For this purpose, Candida tropicalis, an intermediary yeast type, was investigated. As shown in Table 6, the differences in growth physiology are also expressed on the enzymic level in that (i) corresponding enzymes in C. tropicalis are repressed to a lower degree by glucose and (ii) repression occurs significantly only at elevated concentrations of glucose (5% and above). It must be emphasized that the regulation observed in C. tropicalis may not be a glucose effect at all but rather an oxygen limitation occurring with the high concentrations of glucose. Furthermore, it is remarkable that the activities of several enzymes undergoing glucose repression are significantly higher when acetate is the carbon source compared with activities under conditions of glucose derepression. Assimilation of acetate seems to induce synthesis especially of enzymes of the TCA cycle and the glyoxylic

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144

TABLE 6. Examples of the extent of regulatory effects in Cundida tropicalis. A indicates acetate, G glucose, R repressed and D derepressed. Glucose repression is generally obtained only in the more concentrated media. Limitation of oxygen supply in diluted media results in similar phenomena (Knopfel, 1972).

Metabolic pathway

Enzyme

TCA cycle

Citrate synthese

TCA cycle

Aconitase

TCA cycle

Fumarase

TCA cycle GIyoxylic acid shunt Glyoxylic acid shunt

Malate dehydrogenase

Respiratory chain Succinate: cytochrome c oxidoreductase

Concentration Activity and of substrate in repression Ratio medium (%) (nmol ml-I mg-I) R : D

G1 G5 GI G3 A2 G1 G5 A2 GI G5 A2 GI G5 A2 G5 GI G5 A2 GI G5 A2

500 350 250 400 1200 220 50 700 1 1,000 8,500 26,000 2 3 390 5 90 30 70 45 25 45

1 : 1.5 1.5

1:1 1 : 1.5 D 1 : 1.25 1 :20 D 1:1

1 : 2.5 D 1:25 1.50

D 1 :25 1:l 1:5 D 1:l 1:5 D

acid shunt which is in accordance with the main purpose of these pathways, namely to produce building blocks for biomass formation out of C z units.

2 . Is there a Crabtree effect under Anaerobic Conditions?

Sticking strictly to the definitions given in Table 3, a Crabtree effect under anaerobic conditions cannot be defined. If the molecular events as described in the previous section are taken into account, a more diverse picture has to be considered. As already mentioned in Section II.B.2 (p. 133), TCA-cycle enzymes were found to be active under anaerobic conditions by Schatzmann (1975) and others. On this basis, there is some evidence that some sort of glucose regulation takes place under anaerobic conditions. Arguments for a glucose effect at anaerobic conditions are as follows.

GLUCOSE METABOLISM IN GROWING YEAST CELLS

145

(i) A relatively steep decrease in malate dehydrogenase and succinate: cytochrome c oxidoreductase activities at a dilution rate of approximately 0.05 h- in the anaerobic chemostat. The glycolytic flux at the onset of the decrease in activity was 2.44 mmol g- 'h- which is equal to the flux observed at the beginning of glucose repression under aerobic conditions.

X (nm)

X (nm)

FIG. 7. Effects of oxygen (a) and glucose repression (b) on cytochromes uu3, b, pigment P, in Saccharomyces cerevisiue. From Schatzmann (1975). (a) shows reconstitution of pigments in anaerobically grown (glucose derepressed) cells (a), and b h , after 4, 8, 14, 60, 75, 120, 230 min of aeration. (b) shows glucose repression of cytochromes in aerobically grown cells. a indicates anaerobic (repressed) cells; M, 95, 89, 0% of repression. cc,,

(ii) Differences in the kinetics of respiratory adaptation (Schatzmann, 1975). After 8-10 min aeration, the oxygen-uptake rate of anaerobic derepressed cells was approximately 10% of that of fully adapted cells, whereas in anaerobic repressed cells only about 2 to 3% of the final value was achieved. (iii) Differences in the cytochrome spectrum of anaerobic glucosederepressed and -repressed cells. Glucose-derepressed cells show a broad absorption band with a maximum at 587 nm (Fig. 7a). Schatzmann (1975) attributed this peak tentatively to coproporphyrin, which is accumulated by anaerobically growing cells (Lascelles, 1964; Porra et af., 1973). Synthesis

FIG. 8. Electron micrographs of freeze-etched cells of Saccharomyces cerevisiae. On p. 146 are derepressed cells grown aerobically (a) or anaerobically (b). Both types show the presence of large pellets of storage carbohydrate in the cytoplasm, and elongated mitochondria ( > 1 pm long). The elongated mitochondria show very fine cristal structures in aerobically grown cells. Cristae did not develop during anaerobic growth although there are cristallinic inclusions in the matrix (see arrows). (c) shows

repressed cells grown aerobically, and (d) anaerobically. Both types display rather small cytoplasmic pellets and globular mitochondria ( < 1 pm long). Well developed cristae are not visible in cells grown in the absence of oxygen. M indicates mitochondrion, N nucleus, and V vacuole. From Schatzmann (1975) and Yotsuyanagi (1962).

148

A FIECHTER, G F FUHRMANN A N D 0 KAPPELI

of coproporphyrin seems to be repressed by glucose since anaerobic glucoserepressed cells do not show the peak at 587 nm (Fig. 7b). (iv) The morphology of the mitochondria has some characteristic features which can be attributed to glucose repression independently of the presence or absence of oxygen (Fig. 8). The mitochondria are smaller in repressed cells, their form is more spherical and the fracture face of the bilayer of the envelope is different from that in glucose-derepressed cells. Accumulation of an unidentified cristalline substance was observed in the matrix of mitochondria only in deprepressed anaerobic cells. TABLE 7. Combined effects of oxygen and glucose respiration on growing Saccharomyces cerevisiae. From Schatzmann (1975) Glucose feed Oxygen status Present

Parameter studied Glucose uptake Glycolysis Respiration Products Growth rate

Absent

Supercritical Controlled; correlated to concentration N o deregulation Repression (aerobic Crabtree effect) Biomass (reduced); ethanol, carbon dioxide R Q > I); DR < D < D,,, High

Storage carbohydrates

Lowered

Glucose uptake

Correlated to glucose; concentration controlled No regulation No oxygen uptake; repression of enzymes (anaerobic Crabtree effect) Biomass (low); ethanol (high); glycerol; pyruvate (low); carbon dioxide

Glycolysis Respiration Products

Growth rate

High

Storage carbohydrates

Low

Gi:b)

i

Subcritical

N o limitation Regulated by oxygen (Pasteur effect) Derepression Biomass (Y, = 0.5); carbon dioxide (RQ = 1) Low (dilution rate below maximum) High

No control No regulation No oxygen uptake; derepression of enzymes Biomass (low); ethanol (high); glycerol; pyruvate (trans); carbon dioxide Low High

GLUCOSE METABOLISM IN GROWING YEAST CELLS

149

These observations made with anaerobic cells indicate that there is a regulatory effect of glucose at the molecular level in the cells. Consequently, there are four regulatory types of yeasts with respect to oxygen and glucose regulation. Aerobically and anaerobically growing yeasts can both be either repressed or derepressed (Table 7). 3. Proposed Mechanisms of Regulation

a. Catabolite repression model of Peterkofsky. Current views of the underlying mechanisms of the catabolite repression concentrate on the regulation of transcription by cAMP combining with a cAMP receptor protein to initiate synthesis of mRNA. Negative regulation, for example few cAMP molecules-high gene-product level, is relatively rare. Regulation of adenylate cyclase, which catalyses synthesis of CAMP, is considered decisive in contrast to regulation of the phosphodiesterase or loss of cAMP through excretion. An extensive treatment of the situation in typical prokaryotes is given by Pastan and Adhya (1976). Regulation of adenylate cyclase is now much better understood since its association with sugar transport systems has been demonstrated (Peterkofsky, 1977). In this complex system, the nonphosphorylated form of the sugar acts on the phosphotransferase system which in turn is phosphoenolpyruvate (PEP)-dependent acting on enzyme I of the system. For a review of the PEP-phosphotransferase system see, for example, Postma and Roseman (1976). The presence of glucose gives rise to a rather complicated mechanism in low adenylate cyclase activity preventing initiation of mRNA. It must be noted that the main portion of the glucose flux is not associated in this regulatory step. Release from repression occurs only after free glucose has been used up, an observation which is also valid for yeasts (e.g. Fig. 6a). The potential applicability of the model of Peterkofsky (1977) to eukaryotes seems to be very unlikely. It is shown in Section IV (p. 159) that sugar transport in yeast is not mediated by the PEP system. It is admitted, however, that information on key pool concentrations of PEP or AMP and enzyme activities like adenylate cyclase or cAMP phosphodiesterase (Londesborough, 1978) are not available for yeast and that kinetics of repression-depression of respiratory enzymes indicate a rather complex regulation. At present, no information is available on a potential control of cAMP by interaction of the sugar-transport system with adenylate cyclase or cAMP phosphodiesterase. The primary effector of this reaction chain therefore remains unknown and it is not deducible from the data given in Section IV (p. 159) how the hexose- or pentose-transport systems could be connected with cAMP synthesis. Clearly, any model for cAMP control in yeasts has

150

A. FIECHTER, G . F. FUHRMANN AND 0. KAPPELI

to include an explanation for the various modes of sugar transport observed in yeasts or the lack of catabolite repression in the glucose-insensitive cell types. Whatever the speculation about the potential role of sugar-uptake mechanism(s) on the regulatory mechanisms of transcription might be, the basic observations of catabolite repression combined with other mechanisms remain. It is hoped that the important problem of these regulatory mechanisms can be solved soon. b. Catabolite inactivation. In yeast, glucose effects on respiratory pathways display a number of phenomena concerning kinetics during derepression, repression or pulsing of the sugar. High as well as low time-constants are included indicating that catabolite repression is not the only governing regulatory mechanism. Holzer (1 976), in his review of fast regulatory responses to glucose of some key enzymes of respiration, coined the term catabolite inactivation for this type of control. This is a rather complex control mechanism which differs from catabolite repression (Entian, 1977). It has been demonstrated that the cytoplasmic isoenzyme of malate dehydrogenase from Sacch. cerevisiae is inactivated by a proteolytic process (Hagele et al., 1978). Today, the control principle of catabolite inactivation is understood as a mechanism that acts at the level of protein turnover. As true catalysts, proteolytic enzymes are involved in degradation and synthesis of proteins. Their activity in protein synthesis has been demonstrated by Isowa et al. (1977). There are endo-, exo- or endoexo proteinases in yeasts. They differ in their specificity and can split protein at only one or a few peptide bond(s). So far, seven proteolytic enzymes have been extensively described including swine-, thiol-, carboxyl- and metalloproteinases (Meussdoerfer, 1978). Molecular weights range from 32,000 to 640,000 and these “digestive” enzymes are primarily located in vacuoles of yeasts. The specificity of the proteinases is controlled by a number of mechanisms, They are localized, in general, in lysosomes of mammalian cells and display a broad spectrum of activity. Some group-specific membrane-bound proteinases have been shown to be involved in transport mechanisms. Some of them exert defined signal functions allowing proper transport control of polypeptides across the membrane. Such membrane-bound proteinases have not been found in yeasts so far but, nevertheless, may be present. Four proteinases (A, B, carboxypeptidase Y, aminoaminpeptidase) out of the seven known yeast enzymes are localized in vacuoles. A rather complex mechanism seems to govern the release of enzymes and metabolites stored in vacuoles. This organelle is dependent on the cell cycle, during which it fragments into a number of small vacuoles before the daughter cell is formed causing release of proteinases and amino acids.

12

-

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c

.-

0

-

-

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77

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.-

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E

0

- 0

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4 4-.

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I

60

.-.-.-.-.-.-. I

I20

I I80

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FIG. 9. Effect of a pulse of glucose (27$ w/v) on some properties of a chemostat culture of Saccharomyes cerevisiae growing under glucose-derepressed conditions at a dilution rate of 0.10 h - * . A indicates glucose concentration, V ethanol concentrabiomass concentration, tion, respiratory quotient, 0 Qo, values. and 0 Qcoi values.

152

A. FIECHTER, G . F. FUHRMANN AND 0. KAPPELI

Specifity in proteolytic action is also seen in the different properties of the target proteins or enzymes. Turnover of a given enzyme is very specific. Key enzymes in a metabolic sequence mostly have a very high turnover, due to the. action of regulatory proteinases. In general, proteolytic degradation is high in enzymes with high molecular-weight subunits and an isoelectric point in the acid range. Also, peptide bonds between hydrophobic amino acids are preferentially attacked, but coenzymes exert a stabilizing effect against enzymic degradation. Stabilizing effects against proteolysis by corresponding coenzymes have been reported by Katsunoma et al. (1972) and Holzer et al. (1973). Proteolysis, although an exergonic process, is nevertheless affected by the energy regime of the cell. Lowering the ATP concentration affects this activity; if it is less pronounced, e.g. lowered by 25-50%, only degradation is enhanced. This may explain the pronounced proteolysis observed during starvation conditions. Proteinases in vacuoles of Sacch. cerevisiae undergo glucose repression as reported by Hansen et al. (1977) and Frey and Rohm (1977), whereas carboxypeptidase S or the cytoplasmic aminopeptidase are independent of the glucose concentration of the medium (Wolf and Ehmann, 1978). These authors also demonstrated the dependence of the activities of carboxypeptidases Y and S on the nitrogen source. Availability of carbon and nitrogen sources is decisive in determining rates of synthesis and degradation, and this controls the operating mechanisms which are different from catabolite repression (Holzer, 1976). Inactivation of proteinases, one of many processes effected by glucose or nitrogen sources, plays a prominent role. So far two different ways have been recognized. The first consists in a change in conformation of the active centre where activation occurs by limited proteolysis. The second one is blocking of the active centre after formation of a protein-inhibitor complex. Several inhibitors of proteinases from yeast have been described. These act on proteinases A (IA2, IA3) and B (IB1, IB2). They are small molecules of 7600 and 8500 molecular weight respectively and are located in the cytoplasm. A fifth cytoplasmic inhibitor (1') has a molecular weight of 23,800 and reacts specifically with carboxypeptidase Y . The inhibitors undergo hydrolysis by proteinases more or less specifically. Proteinase B degrades IA2 and IA3, and proteinase A is able to hydrolyse IB1 and IB2. Inhibitor Ic is degraded by both proteinases. Characteristics of the inhibitors is given by Meussdoerfer (1 978). These most interesting findings represent a sound basis for the future explanation of phenomena observed in growth experiments. However, much work will be necessary to arrive at a proper interpretation of the many data in earlier publications. Too many metabolic steps are normally involved in regulation of growth under excess of carbon source (batch) or carbon

GLUCOSE METABOLISM IN GROWING YEAST CELLS

153

limitation (chemostat). Also, strain dependence, as well as carbon source specificity under aerobic and anaerobic conditions, are responsible for the bewildering picture emerging. TABLE 8. Glucose uptake rates in resting cells of Saccharomyces cerevisiae. From Lynen et ul. (1 959). Glucose uptake is drastically decreased in resting cells compared with growing cells (see Table 9) Glucose uptake rates (mmol g- * h - I ) Anaerobically

Aerobically

3.085 1.945

1.950 0.343 0.356 0.699 1.251

Uptake of glucose Degradation by “fermentation” Degradation by “respiration” Total glucose degradation Assimilation (calculated)

B. P A S ? E U K h F F h C I

-

1.945 1.145

:

SOLS MOUbL

In 1867, Pasteur made the important observation that “fermentation is inhibited in the presence of oxygen”. This regulatory phenomonon was later named to his honour as the “Pasteur effect”. It indicates that uptake of sugar by respiring cells is lower than in “fermenting” cells as demonstrated by Lynen et ul. (1959). As shown in Table 8, the decrease of glucose-uptake rates in resting cells of Succh. cerevisiue can attain considerable values. In growing cells, however, glucose-uptake is much less affected (Table 9) and Pasteur regulation appears significantly only on galactose. The data indicate a rather complex situation, including regulatory effects of transport, glucose repression and assimilation. Current views on regulation of the Embden-Meyerhof-Parnas pathway are summarized in the model of Sols (1968a). It must be mentioned, however, that controversies contine to exist on observations made in particular cases. Consensus is more or less reached by defining the observable effects, e.g. inhibition of glycolysis in the presence of oxygen. Confusion may occur, however, if unfounded generalizations are made. [Racker (1976) first expressed this opinion when discussing the comprehensive review by Krebs (1972). The type of cells, uptake mechanism and the choice of the experimental conditions are decisive for demonstrating the observations shown in the example from resting and growing cells of yeast which in the latter case are nearly free of effects by oxygen.] Even differences in defining the

A. FIECHTER, G. F. FUHRMANN AND 0. KAPPELI

154

effects-for definitions, see for example, Wenner (1979)-may have led to confusions in the past. However, a central role for regulation of phosphofructokinase by allostenic inhibition is now generally accepted as the major regulatory step in control of glycolysis. An explanation for the observation that no significant substrate accumulation (fructose 6-phosphate) takes place is given by the existing feedback reaction of fructose 6-phosphate on the hexokinase step involved in uptake of the original substrate. This is basically the explanation in a large number of cell types and metabolic situations. Numerous effectors acting on phosphofructokinase including its main inhibitors ATP, citrate, or Mg2+ and activators like AMP (e.g. Tejwani, 1978) are known. A powerful activator of phosphofructokinase is the ammonium ion which interferes strongly in cells kept under conditions of growth. In yeast, the Pasteur effect has been a standing topic for decennia. Excellent scientists like Warburg, Meyerhof, Burk, Lynen, Lipman and others have 0 . 8 0 ~( a )

i; 25

I 25

Rate of uptake of glucose (mmol g-' h - ' I

Rate of uptake of glucose ( m m o l g-' h-l)

FIG. 10. Plots showing the degree of repression as an effector of glycolytic regulation and partition of assimilation and dissimilation in Saccharornyces cerevisiue. (a) shows a plot of the Pasteur quotient (m) and the degree of repression (A) against rate of glucose uptake. (b) shows a plot of percentage assimilatory activity against rate of glucose uptake for cells grown aerobically (0)or anaerobically (m). From Schatzmann (1975).

155

GLUCOSE METABOLISM IN GROWING YEAST CELLS

contributed to a solution of the problem. Lynen (1941) and Johnson (1941) have developed a model suggesting that the available inorganic phosphate becomes limiting in the presence of oxygen due to the high demand of the respiratory chain for phosphorylation of ADP. The resulting inorganic phosphate limitation at the level of glycolysis would therefore represent the final effector, thereby explaining widespread regulatory phenomenon. It must be stated, however, that regulation of the flux through the glycolytic pathway can be hardly explained by simple limitation of inorganic phosphate. First of all, inhibition in the presence of oxygen is very variable (Sols, 196Xb), and has never been proved to be solely dependent on an actual inorganic phosphate limitation. Indeed, the absence of a Pasteur effect has been observed in several tissues like striated muscle, erythrocytes, intestinal mucosa, renal medulla and even in malignant tumours (Krebs, 1972; Ramaiah, 1974). After a long period of efforts, the inorganic-phosphate theory has been developed and finally established (Lynen, 1963). Careful experimental treatment of the problem has shown, however, that the Lynen data describe only the situation of resting cells (Table 8). Growing cells are glycolytically regulated to a very minor extent at best. Under conditions of respiratory repression when ATP regeneration is drastically decreasing, release from allosteric control is considerable (Fig. 10). The maximum effect in growing cells of Sacch. cerevisiue is surprisingly low (about 15%) when grown on glucose due to their strong repressive effect. TABLE 9. The Pasteur effect in growing and resting cells of Succhuromyces cerevisiue. From Schatzmann (1975). Resting cells were obtained from defined glucosecontaining media devoid of nitrogen. Qs indicates rate of glucose uptake, Qs, rate of glucose assimilation and Qsgrate of glycolysis. The Pasteur quotient is the ratio of the Qsgvalues for cells grown aerobically and anaerobically. Value for Resting cells Growing in a glucose-containing medium: aerobically anaerobically Growing cells Growing in a glucose-containing medium: aerobically anaerobically Growing cells Growing in a galactose-containing medium: aerobically anaerobically

Pasteur quotient

Qs

Qs.

Qsg

2.5 4.6

0.2 0.15

2'3 4.45

18.3 18.9

0.5 0.3

17.8 18.6

0,96

4.5 6.4

0.4 0.2

4'1 6.2

0.66

0.51

156

A. FIECHTER, G. F FUHRMANN A N D 0. KAPPELI

Galactose (Table 9) has a very inefficient transport system and, consequently, prevents respiratory repression due to its inefficiency in acting on the control system for CAMP formation. Thus, this sugar has been used for a long time in studies on derepressed yeast cells. A Pasteur effect of nearly 50'x in galactose-grown cells is considerable even if it does not reach the value observed in resting cells. Consequently, this difference between galactose-grown and resting cells is fundamental and can be attributed only to the operation of other regulatory mechanisms in the latter. Rates of sugar uptake are easy to determine in a growing cell system by chemostat methods. Gradual release from Pasteur effect (PQ) is correlated to glucose repression or respiration according to the model of Sols. In growing cells of yeast, Pasteur effect is relatively low even under complete derepression. Significant assimilatory activity (accumulation of storage carbohydrates) is obtained only during aerobiosis, it is low under repression and is absent during anaerobiosis. Uptake of glucose is important in the Sols model which demonstrated a secondary regulatory site in the presence of oxygen. Owing to the fact that accumulation of down-stream metabolites of glucose is insignificant, uptake of the sugar must be limited. In growing cells (Fiechter, 1978) of Succh. cerevisiue, regulation of hexokinase in the presence of oxygen is negligible. Modest alterations at a high level of activity may not constitute a real regulatory effect. This, together with the observation of unlimited glucose uptake control on the first glycolytic steps, seems to be very unlikely. The question which then arises is how the cell gets rid of the carbohydrates taken up in large quantities. Under repressional conditions, the flux of carbon skeletons is poorly regulated and carbon atoms taken up in the form of glucose are found entirely in ethanol, carbon dioxide and biomass. In the case of derepression, the situation becomes more complicated. Ethanol is no longer excreted but the yield of biomass is increased by up to 50% of the sugar taken up. Allosteric inhibition takes place. As shown by Kiienzi (1970), synthesis of trehalose and glycogen is enhanced during derepression. This report indicated considerable accumulation of storage carbohydrates under conditions of derepression (see also Gbra et ul., 1979). Finally, an observation made by Schatzmann (1975) has to be mentioned concerning the situation in Succh. cerevisiue. In contrast to other systems, citrate can be neglected as an inhibitor of phosphofructokinase. But ATP, as shown by the data from Weibel (1973) (see Section 111, p. 159) is present in high concentrations under conditions of derepression and appears as the prominent effector of the target enzyme.

GLUCOSE METABOLISM IN GROWING YEAST CELLS

157

C. E N E R G E T I C A L C O N S I D E R A T I O N S

Generation of energy is the basis for growth (Payne, 1970). A close relationship between production of heat and metabolism is generally observed in living matter. Microbes display the highest generation of specific heat (Forrest, 1969). For example, E. coli produces 1000 cal g - * , Drosophila melanogaster 100, and man 10. Microbial production of ATP is not easy to determine. It can be calculated for strict anaerobes, where the sites of generation can be located in the general scheme of metabolism. The net gain in anaerobic systems is one mol of ATP per mol of glucose (generated in the glycolytic sequence). Under aerobic conditions, additional ATP is produced in mitochondria. Reducing power from the TCA cycle is transferred to the energy-rich phosphate bonds by the electron-transfer chain involving molecular oxygen as the terminal hydrogen acceptor. The P : 0 ratio, the coupling factor of ATP generation and hydrogen transfer, however, is not constant but may vary within a wide range (0.5-3.0). Nevertheless, the ratio is considered to be a biological constant for given circumstances which is significant for the relationship between catabolism and anabolism, provided that energetic coupling is strict. Unfortunately, extensive studies on mitochondria in connection with the anabolic activity of the cell are rare. It is the aim of this section of the review to present evidence that catabolic kinetics are independent of cell synthesis, and that energy production in glucose-sensitive yeast strains has a strong control on the metabolic flux. Many attempts have been made to determine the biomass: ATP ratio. The average value for YATpis considered to be 10.3 f 0.3 (e.g. Forrest, 1969) embracing a wide span of microbes and substrates. For Sacch. cerevisiae, Dellweg et al. (1977) calculated a value of 9 for YATpbased on the assumption that 28 mol of ATP per mol of glucose are generated. Von Meyenburg (1969a), developing a system of equations to obtain P : O ratios based on metabolic reactions, obtained a value of 1.1 and consequently a constant value of 12 for YATP.A further attempt to obtain a consistent answer to the problem was made by Oura (1972). Dellweg et al. (1977) extracted mitochondria during his study. He first assayed the oxygen demand for biomass synthesis and found a characteristic dependence on cell yield (Y,) with a maximum value of 23.8 mmol g - ' (Fig. 1, p. 126).This is in accordance with earlier data (von Meyenburg, 1969a; Knopfel, 1972; Weibel, 1973; Schatzmann, 1975) showing that yield of biomass and oxygen-uptakes rates are strictly correlated in both glucosesensitive and glucose-insensitive yeast strains. More recently, uncoupling of oxygen uptake and cell yield has been observed in alkane-grown Saccharomy-

158

A FIECHTER, G F FUHRMANN AND 0 KAPPELI

copsis lipolytica ( = Candida tropicalis) and the question arises whether mitochondrial organization in the two yeasts is comparable. It is noteworthy that Dellweg and his coworkers (1977) with their essentially intact mitochondrial preparations, contributed to an understanding of the reaction kinetics of oxygen. They found Michaelis-Menten kinetics in the reaction of oxygen with mitochondria (NADH as substrate, K,,, 1.65 Fmol 1 - I ) and pure cytochrome oxidase ( K , 1.73 pmol 1 - I ) . They concluded that transport limitation does not operate at the mitochondrial barrier, and that the kinetics is governed by cytochrome oxidase. With whole cells, oxygenuptake kinetics deviate drastically from those of mitochondria, and seem to be a mixture of Michaelis-Menten and diffusion kinetics. Experiments of this type show up some of the difficulties encountered in studying the relationship between oxygen uptake and energy formation in glucosesensitive yeasts. Measurement of the content of ATP in whole cells is equally unsuitable for direct determination of the P :0 ratio because it yields pool concentrations representing the difference between production and consumption of the nucleotide. However, data of this kind show that production of energy-rich metabolites is a regulatory effect. Glucose-sensitive yeasts, like Sacch. cerevisiae, show a clear-cut uncoupling of catabolism and energy generation. Energy charge (EC) plays an important role in energetical considerations (Atkinson and Walton, 1967). It is defined as: C = - [ATP] [f ADP] [ATP] + [ADP] + [AMP]Weibel(1973) was able to demonstrate a strong correlation between growth, oxygen uptake and EC in Sacch. cerevisiae (Fig. 11). The energy charge decreases similarly to biomass with increasing glucose repression and reaches levels of nearly 0.9 under full derepression. Yield of biomass is highest at highest values for energy charge and, consequently, also productivity (biomass produced per gram per hour = D X) at highest energy production (EC D). Onset of glucose repression affects energy production similarly to the activity of respiratory enzymes. Specific oxygen-uptake rates behave very similarly to energy charge suggesting a strict coupling of ATP formation and hydrogen transport. Nevertheless, P :0 ratios cannot be calculated from such data, because the adenylate values represent concentrations and do not relate to the generationxonsumption of energy. Careful interpretation is also indicated in view of the basically different situation in Candida tropicalis (Weibel, 1973) a strain with a low susceptibility to free glucose. Here, ATP levels are constant in parallel with cell yield, whereas oxygen-uptake rates increase proportionally with productivity. A similarity between Sacch. cerevisiae and C. tropicalis exists insofar as ATP levels (and correspondingly those of ADP and AMP) run parallel with yield of biomass, whereas oxygen-

+

GLUCOSE METABOLISM IN GROWING YEAST CELLS

159

+ al +

14u 0, F al V

2

0'

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3

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FIG. 11. Energy charge (--0 - -), Qo, value (-; mmol g - ' hk'), biomass g l-l), and contents of ATP (-@---; pmol & - I ) , ADP concentration (--.--; (-Ap; pmol g - l ) and A M P (-m-; pmol g - ' ) in Succharomyces cerevisiue as a function of dilution rate. The cells were grown aerobically in a chemostat with a defined medium. The arrow indicates the onset of repression. From Weibel (1973).

uptake rates correspond to productivity (XD), and, therefore, no proper scheme for calculating ATP generation under aerobiosis can be developed. In most cases, assumptions such as a constant value for the P : O ratio in specific growth rate and YATphave to be made (see also von Meyenburg, 1970). However, as long as the molecular basis of regulation in these different yeast strains cannot be revealed, all attempts to model ATP generation during growth must necessarily stay empirical. High values for energy charge are in good agreement with current views on regulation of synthesis of trehalose (Kiienzi, 1970; Panek and Mattoon, 1977) and glycogen (Rothman and Cabib, 1970) as well as with repression-derepression during cell cycle (von Meyenburg, 1969a; Kiienzi, 1970).

IV. Sugar Transport A. I N T R O D U C T I O N

The plasma membrane of the yeast cell is the limiting barrier for penetration of sugars from the outside into the cell interior. Despite an extensive surface area in relation to cell mass, physical diffusion of sugars through the plasma

160

A FIECHTER, G F FUHRMANN AND 0 KAPPELI

membrane is generally low. This is mainly because of the insolubility of hydrophilic sugars in the hydrophobic membrane phase and the limited size of pores in the plasma membrane. From a determination of sugar distribution volumes Conway and Downey (1950) found that galactose and arabinose enter the cell-wall space, but are not distributed in one hour to an appreciable extent in the cell water. However, addition of the same concentration of glucose, mannose or fructose to the suspension of Succh. cerevisiue resulted in their total uptake in 3 to 5 minutes (Rothstein, 1954). Therefore, a highly specific mechanism for uptake of metabolizable sugars such as glucose, mannose and fructose has been postulated in contrast to the nonmetabolizable sugars galactose, arabinose and sorbose. Burger et ul. (1958, 1959) and Cirillo (1959, 1961) re-investigated the question of penetration of non-metabolizable sugars into Succh. cerevisiae. By using more sensitive methods such as the volume-distribution technique these authors found penetration of D-galactose, L-sorbose, D-xylose, and a-methyl-D-glucose into the cells. Glucose inhibited their entrance and, when added after equilibration, displaced the non-metabolizable sugars from the cell. These observations strongly indicated a specific transport system for sugars with graded affinities. The characteristics of the most important sugar specificity for transport and the competitive inhibition exhibited between transported sugars have been investigated extensively by Kotyk (1967) and Cirillo (1968a). Heredia et ul. (1968) estimated an acceleration factor of lo6 and more for penetration of glucose at low concentrations in Succh. cerevisiue. This means that glucose enters the yeast cell at least a million times faster than it would be expected by assumption of physical diffusion. In spite of this high rate of glucose penetration, little or no free glucose can normally be detected within the intracellular space (Rothstein, 1954; Sols, 1968a). There are two possible interpretations for this observation. In the first it is assumed that glucose will be transported in a chemically unchanged form through the plasma membrane and that it is metabolized as fast as it enters the cell. In this situation, where transport of glucose is the limiting factor for metabolism, the nature of the glucose transport with respect to the driving force is difficult to determine. An active-transport process coupled to metabolism, or a facilitated diffusion process with the concentration gradient in the inward direction maintained by continuous glycolytic removal of glucose might be operative. A continuous glycolytic removal is in accordance with the high concentration of hexokinase in Succh. cerevisiue, which phosphorylates glucose nearly as fast as it enters, and there are rather minor regulatory effects on the enzymes of the glycolytic pathway even under aerobic and anaerobic growth conditions (von Meyenburg, 1969~;Knopfel, 1972; Schatzmann, 1975; Fiechter, 1978). This result is in agreement with measurements of glucose consumption by growing Succh. cerevisiue by these

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authors, and a recent investigation of Lagunas (1979) with three diflerent strains of Succh. cerevisiue in which no significant difference in glucose consumption under aerobic and anaerobic growth conditions was detected. Since only in starved cells is an inhibition of sugar consumption rate by anaerobiosis noticeable, the importance and generalization of the Pasteur effect in yeast has been doubted. In the second interpretation, a hypothetical group-translocation mechanism for glucose transport in Sacch. cerevisiue has been postulated (Rothstein, 1954; Van Steveninck and Booij, 1964; Van Steveninck and Rothstein, 1965; Dreierkauf and Booij, 1968; Van Steveninck, 1969). This mechanism involves phosphorylation of glucose on the outside of the plasma membrane and transport of phosphorylated glucose into the cell. Polyphosphates, as phosphate donors outside the plasma membrane, were assumed to be essential for this active transport. However, the experimental evidence given for such a reaction could not be confirmed (Fuhrmann and Rothstein, 1968; Fuhrmann, 1973, 1977a). Also, no evidence for a phosphoenolpyruvate phosphotransferase system of the type found in numerous bacteria (Roseman, 1969) could be detected in Succh. cerevisiue by Meredith and Romano (1977). However, the appearance of 2-deoxy-~-glucosein a phosphorylated form inside the cell has been discussed by these authors in terms of a possible group-translocation mechanism for transport of this sugar and, by analogy, for D-glucose. Meredith and Romano (1977) stated that if there is a group-translocation system for phosphorylated glucose the nature of the transport and the phosphorylating system is yet to be determined. Further, the argument for rapid intracellular phosphorylation of the sugar by hexokinase could not be ruled out. In contrast to the above observations with 2-deoxy-~-glucose,Kotyk and Michaljanikova (1974) observed, with ~ - ' ~ C - g l u c o safter e pulse-labelling in Succh. cerevisiae, a transitory appearance of free glucose inside the cell. This result is in favour of the first interpretation. In spite of the high level of activity in research on sugar transport in yeast, the nature of the carriers involved is still unknown. There is little doubt that proteins with the ability to bind sugars are engaged in the transport mechanism, but the molecular mechanisms discussed are still hypothetical and even controversial. From a functional point of view, the plasma membrane is an important control system in regulation of transport and metabolism. This is not so obvious from studies of the constitutive glucose carrier in Succh. cerevisiae and similar yeasts, but becomes evident by the fact that carriers of mono- and disaccharides are inducible. Here, regulation is achieved by synthesis of a new plasma-membrane component through the stimulus of an inducer, under the ultimate control of the genetic apparatus. As a further regulatory mechanism, catabolite inactivation, as proposed

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by Holzer (1976), has to be mentioned. Thus, addition of glucose to yeast cells causes not only catabolite repression, as it has been described extensively in the regulation of glucose metabolism, but in addition inactivation of inducible sugar-transport systems by proteolysis. Metabolic regulation is also very important in active sugar-transport processes. Hofer and Kotyk (1968) found an extremely tight coupling of monosaccharide transport in the obligatory aerobic yeast R . glutinis with energy-producing reactions. Attempts t o show a passive facilitated diffusion in the absence of metabolism, as in Sacch. cerevisiae, have been without success (Hofer, 1971). Thus, a remarkable difference seems to exist between glucose transport in obligatory aerobic and facultative aerobic yeasts. Since it is now possible to prepare plasma-membrane vesicles (Fuhrmann et al., 1976; Scarborough, 1975), a great deal of emphasis is now placed on the chemistry of the plasma membrane, particularly of the membrane proteins. The most important advantage of these wall-less vesicles is that structure and function can be examined without interference from cellular metabolism. The aim of this section of the review is to inform the reader on current knowledge of monosaccharide and disaccharide transport in different yeasts, including obligatory and facultative aerobic cells. B. T R A N S P O R T S Y S T E M S

Sugar transport through the plasma membrane can be achieved by three different processes; namely physical diffusion through aqueous pores, facilitated diffusion, and active transport. The last two processes might be constitutive or inducible by substrates. 1. Physical Dfiusiun

Physical diffusion through aqueous pores in the plasma membrane is dependent on the size and shape of the diffusible molecule. In Sacch. cerevisiae, the size of the pores has been estimated with a series of inert probing molecules (Scherrer et al., 1974). The uptake-exclusion threshold for the plasma membrane corresponds to a monodisperse ethylene glycol with a molecular weight of 110 and an Einstein-Stokes hydrodynamic radius of 0.42 nm. The threshold for the cell-wall porosity was determined to be at a molecular weight of 760 and an Einstein-Stokes radius of 0.89 nm. The dependence of the shape of the molecules is obvious from the fact that ringstructured monosaccharides are excluded from penetrating the pores in the plasma membrane, whereas D-ribose, occurring in 10% as a linear sugar,

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enters into Sacch. cerevisiae and R. glutinis by diffusion (Horak and Kotyk, 1969). Also acyclic polyols like erythrisol, xylitol, ribitol, D-arabinitol, mannitol, sorbitol, and galactinol are taken up by Sacch. cerevisiae by simple diffusion (Canh et a)., 1975).

2. Facilitated Dgjusion Facilitated diffusion of monosaccharides into yeast has been described particularly in Sacch. cerevisiae, which is the only species of the genus Succharomyces investigated in sufficient detail (reviewed by Suomalainen and Oura, 1971; Kotyk, 1973). Facilitated diffusion has also been demonstrated by Fuhrmann et al. (1 976) in plasma-membrane vesicles derived from Succh. cerevisiae. Some authors discuss or report, in addition to facilitated diffusion, the presence of active transport of monosaccharides in Sacch. cerevisiue (Van Steveninck, 1972; Brocklehurst et al., 1976; Meredith and Romano, 1977). Neurospora crassa, which has been grown in a medium containing a high concentration of glucose, transports glucose via a low-affinity facilitated-diffusion system, whereas cells grown in a medium containing little or no glucose transport glucose via a high-affinity active-transport system (Schulte and Scarborough, 1975). Physical and facilitated diffusion do not require metabolic energy. Both processes lead to equilibration of sugar concentrations outside and inside the cell. In facilitated diffusion, it may be the carrier-substrate complex that undergoes diffusion, either translational or rotational, in response to a gradient of the complex within the plasma membrane. Therefore, both processes are ultimately the result of diffusion. They can, however, be distinguished by measurements of the rates of substrate transport. There are four criteria defining facilitated diffusion. a. Saturation. The characteristic of a physical diffusion process is a direct proportionality of transport rate to sugar concentration, whereas in facilitated diffusion the system becomes saturated with increasing sugar concentration, resulting in a maximum rate of transfer. In order to explain the difficulties of determining maximum rates ( V,,,) and dissociation constants of the carrier substrate complex (Kc,) in a living yeast cell, a conventional carrier equation (Wilbrandt, 1954) for facilitated diffusion of sugar is shown:

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In equation (I), it is assumed that the rate of reaction between sugar and carrier is high compared with movement of carrier through the membrane. In this simple equation, net flux (v,,) is the difference between influx (vln) and outflux (v,,,) of sugar obeying Michaelis-Menten-type kinetics. Whereas in a living yeast cell, the sugar concentration outside the cell (So) is easy to measure, this is nearly impossible for the inside concentration (S,). With a non-metabolizable sugar, the inside concentration cannot be accurately estimated because the size of the compartment or of the compartments is unknown and the sugar might be bound rather than in a free form. In addition to this, Spoerl et ul. (1975) described changes in insidecompartment permeability and binding of sugar in dependence of metabolism. When a metabolizable sugar is transported, the inside concentration might be negligible, if the sugar is removed by metabolism as fast as it enters. However, the possibility of adjustment to the glycolytic turnover by maintaining a steady state between entry and phosphorylation, with an efflux of the amount in excess of what can be handled through glycolytic transformation, cannot be excluded (Serrano and Delo Fuente, 1974). According to this hypothesis, an adjustable net transport of sugar would take place, and the authors suggested that in a steady state between net flux and utilization, the experimental K,,, values deduced from saturation curves at variable concentrations of external sugar should bear little connection with the dissociation constant of the carrier-sugar complex. Cirillo (1968a) solved the above-mentioned difficulties in a very elegant way by measuring the initial uptake velocity of a low- and a very low-affinity sugar in the presence and in the absence of an inhibitory sugar. Under such conditions, the inside concentration ( S , ) and consequently the outflux (v,,,) become negligible. The parameters K,, and Vmdx, can now be easily determined from the influx equation: V,, = V,,,, (SJS0 K c s , ) , for example by using the standard Lineweaver-Burk plot. From the inhibitory effect, Cirillo (1 968a) calculated the dissociation constant of the “carrierinhibitor complex” (KJ of low-and high-affinity sugars. However, measuring the initial velocity of sugar uptake under conditions of negligible outflux (v,,,) is practically impossible in the case of high-affinity sugars. The inside concentration (S,) rises in a few seconds to significant values (Serrano and Dela Fuente, 1974; Kotyk and Michaljanicovi, 1974), because of efficient transport and the small size of the cellular compartment, which is probably much less than 100 pm3. Under these circumstances, the outflux (v,,,) is not negligible and values calculated from the influx equation, as it has been done by many authors, are erroneous. b. Spec@city of transport. The second criterion of facilitated diffusion is the specificity for the sugar transported. For example, Succh. cerevisiue transports D-glucose, D-fructose and D-mannose as well as a number of

+

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structurally related compounds, which might be metabolizable or nonmetabolizable, by a common constitutive system (Cirillo, 1968a). Kotyk (1967) divided sugars on the basis of their stereospecificity into two groups each with its own carrier. The specific constitutive carrier system prefers the alpha-anomers over the beta-anomers of sugar (Ehwald et al., 1973). This property has not been recognized in sugar transport so far. In addition to these, a third carrier is formed as a result of D-galactose induction or through induction by gratuitous inducers such as D-fucose or L-arabinose (Kotyk, 1967; Cirillo, 1968b). The structural gene for the galactose-transport system, G a 2, has been localized on chromosome XI1 (Douglas and Hawthorne, 1964; Mortimer and Hawthorne, 1966). In contrast to other inducible transport systems, which will be discussed later, galactose transport occurs by facilitated diffusion. In Sacch. cerevisiae, there is sufficient experimental evidence that galactose transport before and after induction occurs exclusively by facilitated diffusion (Cirillo, 1968b; Kuo et ul., 1970; Kuo and Cirillo, 1970; Kotyk and Michaljanicova, 1974; Wilson, 1974) and not as reported by Van Steveninck (1972) after induction by transport-associated phosphorylation of galactose. The rates of net transport of galactose measured by Van Steveninck (1972) were analysed in induced and non-induced cells only with consideration of the influx (v,,) and not for the outflux ( V J . Since there is already measurable free galactose inside the cell (Kuo and Cirillo, 1970; Kotyk and Michaljanicova, 1974), especially in non-induced cells, the values calculated by Van Steveninck (1972) for the half-saturation constants and V,,, by neglecting the outflux in induced and non-induced Sacch. cerevisiae are artifacts. The availability of mutants that were deficient in galactokinase and galactose 1-phosphate uridyltransferase was especially helpful in clarification of the galactose-transport system in Sacch. cerevisiae (Kuo et al., 1970; Kuo and Cirillo, 1970). c. SpeciJicity of inhibition. The third criterion for facilitated diffusion is inhibition by small concentrations of specific inhibitors indicating that the substrate receptor site occupies only a small fraction of the plasma-membrane surface. Sugars with the same stereospecific grouping compete for the same transport site. Despite a similar pattern of selectivity in facilitated diffusion of sugars into Sacch. cerevisiae to that described for human erythrocytes (Cirillo, 1968a), there is no similarity with respect to primary inhibitors interfering with the transport mechanism. For example, phlorizin and polyphloretin displayed a substrate-competitive action in facilitated diffusion of sugar in human erythrocytes (Wilbrandt and Rosenberg, 196l), however, such an effect could not be observed in Sacch. cerevisiae (G. F. Fuhrmann, unpublished results). Also, thiol-inhibitors such as p-chloromercuribenzosulphonate (G. F. Fuhrmann, unpublished results) and cytochalasin A (Lampen et ul., 1973) are without effect in Succh. cerevisiue.

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Uranyl ions in low concentration inhibit sugar transport in Sacch. cerevisiae (Rothstein, 1965). The cation does not penetrate the plasma membrane, but it does bind to phosphoryl and carboxyl groups on the outside of the plasma membrane. The inhibition process is reversible, and the inhibition for glucose transport has been shown to be non-competitive (Rothstein, 1954). In addition to sugar transport, uranyl ions have been reported to inhibit urea, glycerol, vitamin and amino acid uptake in Sacch. cerevisiae (Cirillo and Wilkins, 1964; Maxwell et al., 1971; Kotyk, 1973). There are also other heavy-metal ions like thorium with rather non-specific effects on plasma-membrane transport (Passow et al., 1961). Van Steveninck (1966) described an effect of nickelous ions on carbohydrate transport in Succh. cerevisiae which is similar to the effect of uranyl ions with no inhibition cjf cellular sugar metabolism. In contrast, the inhibitory effect of nickelous ions on glucose uptake could only be shown if they were transported into the cell (Fuhrmann and Rothstein, 1968; Fuhrmann, 1973). Once nickelous ions are within the cell, the inhibition can be modified by a number offactcrs such as the presence of ethanol or acetaldehyde, removal of ethanol by flushing with gas, the glucose concentration and aerobic or anaerobic conditions. These modifications can be explained by assuming that the predominant effect of nickelous ions is to inhibit the enzyme alcohol dehydrogenase. A primary effect of nickelous ions on the transport mechanism could be ruled out by use of plasma-membrane vesicles (Fuhrmann, i 977b). Whereas uranyl ions inhibited glucose uptake in vesicles, nickelous ions were without effect. Another non-specific inhibitor like uranyl ions seems to be methylphenidate (Spoerl and Doyle, 1968; Spoerl, 1971). This compound affected passage of a number of different compounds, like sugars and glycine, in to and out of Sacch. cerevisiae. Membrane injury in the sense of increased porosity or leakage could be excluded with the concentrations used to block transport. In high concentration, however, disruption of the plasma membrane occurred. d. Counter-transport and competitive acceleration. A fourth criterion of facilitated diffusion is the interdependence of translocation of different substrates resulting in the two important phenomena of counter transport and of competitive acceleration (Wilbrandt, 1973). The mechanism as such is not capable of uphill transport, but the important property of coupling transport of different substrates enables the system to transport sugars uphill similar to a molecular pump. If one sugar, for example, is at equilibrium distribution and a second sugar is added to one side of the membrane, transport across the membrane of the second sugar will cause a transient uphill transport of the first sugar. This phenomenon of counter transport has been observed in sugar transport in Succh. cerevisiae cells (Cirillo, 1961;

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Kotyk, 1973). Also, in plasma-membrane vesicles prepared from Sacch. cerevisiae, counter transport of sugars and inhibition of the counter transport by uranyl ions have been demonstrated (Fuhrmann et al., 1976). These properties of facilitated diffusion in Sacch. cerevisiae strongly suggest that the plasma membrane contains proteins capable of reversibly binding specific sugars and transporting them across the membrane. e. Attempts to demonstrate involvement qf plasma membrane proteins in facilitated diiTusion ojsugars. By incubating Sacch. cerevisiae in the presence of cycloheximide, an inhibitor of protein synthesis on the 80s ribosonies of eukaryotes, the apparent half-lives of sugar carriers have been determined (Alonso and Kotyk, 1978). That of the constitutive glucose carrier was difficult to estimate; the decay of the constitutive system was practically not measurable. The half-life for the inducible galactose carrier was found to be 2.2 hours. Decay of the galactose carrier was also affected by several agents. Ethanol especially showed a somewhat protective effect, whereas glucose or fructose in the presence of cycloheximide demonstrated a faster decay than with cycloheximide alone. The presence of chloramphenicol counteracted the effect of cycloheximide. In addition, it was found that induction of the galactose carrier was not under the control of a mitochondria1 factor and took place in p - mutants. Matern and Holzer (1977) found that cycloheximide prevented completely recovery of the galactose carrier on further incubation of Sacch. cerevisiue in a galactose-containing glucose-free medium. The effects of cycloheximide can be taken as evidence that protein synthesis is necessary for resynthesis of the galactose carrier. Galactose metabolism in Sacch. cerevisiae involves induction of a facilitated diffusion system for galactose and the enzymes of galactose catabolism. After addition of glucose to galactose-induced cells, the induced system decreases faster than can be explained by catabolite repression of synthesis of the system and by dilution by other newly synthesized proteins. Therefore, the effect was named by Holzer (1976) “catabolite inactivation”. The mechanism of inactivation is probably selective proteolysis. After inactivation by glucose in galactose-induced Sacch. cerevisiae, Matern and Holzer (1977) found no effect on the activities of the enzymes galactokinase, galactose 1-phosphate uridyltransferase, UDP-galactose-4-epimerase and phosphoglucomutase, which channel galactose into the glucose-degradation pathway. This strongly points to a selective effect on galactose transport. The kinetic data given by these authors to explain the effect on galactose transport are rather insufficient, because the measured net flux of galactose has been analysed only with consideration of the influx (vin)and not the outflux (v,,,). If catabolite inactivation of galactose transport does operate, this would be a further example for catabolite inactivation, which has previously been demonstrated for maltose permease, cytoplasmic malate dehydrogenase,

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fructose bisphosphatase, phosphoenolpyruvate carboxykinase and a-isopropylmalate synthase (Holzer, 1976). Attempts have been made to isolate sugar-binding proteins from Sacch. cerevisiae (Horak and Kotyk, 1973) and Schizosucch. pombe (Bhandari and Hayashibe, 1977). In contrast to bacterial systems where simple osmotic shock (Neu and Heppel, 1965) has been successfully employed for solubilization of these proteins, extraction with a non-ionic detergent like Tween 80 was necessary with yeast plasma membranes. After purification of the binding proteins by column chromatography, a glucose-binding protein capable of binding glucose, xylose, arabinose and sorbose was obtained from Sacch. cerevisiae. With a similar procedure in Schizosacch. pomhe, two glucose- and one mannose-specific binding proteins were isolated. Instead of a protein with considerable amounts of phospholipid and a molecular weight of 7000-8000 in Sacch. cerevisiae, more pure proteins with molecular weights of 190,000,220,000 and 180,000 and an appreciable amount of carbohydrate were isolated. These differences are remarkable, especially when one considers that similar techniques have been used for isolation. The results clearly demonstrate differences in these yeasts provided that breakdown of high molecular-weight binding proteins has not occurred during the isolation procedure. Plasma-membrane vesicles prepared from Sacch. cerevisiae are suitable for investigation of mutation effects on transport (Fuhrmann et a/., 1976). Plasma-membrane vesicles derived from cells before induction of the galactose carrier show a low uptake rate for galactose. This result is in agreement with the observation on intact Succh. cerevisiae deficient in the structural gene for galactose transport (De Robichon-Szulmajster, 196 I). After induction of the galactose pathway in intact cells and subsequent preparation of plasma-membrane vesicles, the rate of uptake of galactose into vesicles was twice as high as in control preparations without galactose induction. Since the uptake measurements were done under ice-bath conditions and glycolysis was not detectable in vesicles, the increase in rate of galactose uptake can only be attributed to the plasma membrane. Investigations of plasmamembrane proteins by sodium dodecyl sulphate-polyacrylamide-gel electrophoresis before and after galactose induction showed rather minor changes in the protein pattern with no additional protein band. Only a slight but significant increase of a 200,000 molecular-weight protein was detectable after induction (Fuhrmann, 1976). Attempts to identify components of the galactose-transport system by the more sensitive double-labelling technique are under investigation.

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3. Active 7ransport of Sugars Active-transport processes across the yeast plasma membrane share the characteristic properties of saturability, substrate specificity and specific inhibition with facilitated-diffusion mechanisms. By application of other criteria, active transport can be separated from facilitated diffusion. The first criterion for an active transport is that transport of the sugar can occur uphill against a concentration gradient. However, the precise magnitude of a sugar-concentration gradient is not easy to measure. As already pointed out for the facilitated-diffusion process, the size of the compartment on the inside of the plasma membrane cannot be accurately estimated. The sugar under question might be bound and not in a free form, the distribution in other compartments like the vacuole is not known, trapping of a sugar in a third compartment cannot be excluded and, finally, the rate of removal of a sugar by metabolism might be difficult to measure. The second criterion of an active-transport process is its dependence on metabolic energy. If sugar transport depends on respiration, glycolysis, highenergy compound hydrolysis, or is secondarily coupled to an ion gradient, which is primarily dependent on metabolic energy, then it may be considered to be an active process. In their normal energy-requiring functions these processes are unidirectional. This behaviour is in contrast to passive facilitated diffusion of sugars, which may be in either direction, depending on the concentration of free sugar at both sides of the plasma membrane. Active transport of monosaccharides and disaccharides has been reported to occur in several yeasts. There are two mechanisms of active transport which have been mainly discussed in different yeasts and even in the same yeast strain (Jaspers and Van Steveninck, 1977). The oldest hypothesis (Rothstein, 1954) predicts a group translocation mechanism for assuring unidirectional sugar transport. The sugar is thought to be phosphorylated on the outside of the plasma membrane or within the plasma membrane (Kulaev, 1975; Umnov et al., 1976; Jaspers and Van Steveninck, 1977; Meredith and Romano, 1977) and translocated in the phosphorylated form, thus preventing outflux via the same carrier. Such a mechanism does not fulfil the strict definition of an active transport, which requires the sugar to be transported uphill unaltered; however, the consequences are the same. There is clearcut experimental evidence against the phosphorylation hypothesis as proposed by Van Steveninck (1 970, 1972) with a-methlyglycoside and galactose transport into Sacch. cerevisiae (Cirillo, 1968b; Kuo et al., 1970; Kuo and Cirillo, 1970; Kotyk and Michaljanieova, 1974; Brocklehurst et al., 1977). The phosphorylation hypothesis for constitutive hexose transport has not yet been excluded definitely in Sacch. cerevisiae (Meredith

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and Romano, 1977) and in Sacch.fragilis (Jaspers and Van Steveninck, 1977). The second mechanism that has been postulated for active sugar transport in several yeasts is a proton-symport model. The first experimental evidence for this model in micro-organisms was provided by West (1970) in E. coli. It has been shown that bacterial sugar transport can be accomplished by chemiosmotic coupling, as originally developed for oxidative and photosynthetic phosphorylation (Mitchell, 1966). The chemiosmotic hypothesis proposes that electron transport generates an electrochemical gradient of protons across the membrane, composed of an outside-to-inside oriented proton gradient and an inside-negative membrane potential. This gradient, which is called a proton-motive gradient, can be generated by either oxidation or ATP hydrolysis. A membrane potential, negative on the inside, can also be achieved by an appropriate gradient for permeable cations or anions across the membrane. West and Mitchell (1973) demonstrated in E. coli that fluxes of lactose and of protons were found to be strictly coupled with a stoicheiometry of 1 : 1. Neutralization of the charge inside the cell was accomplished by an equivalent outflow of potassium (West and Mitchell, 1972). Similar models have been predicted for sugar and amino-acid transport in several yeasts. In Neurospora crassa, there is evidence that the primary transport system is the plasma-membrane ATPase which pumps protons out of the cell, thereby generating an electrochemical gradient that can drive transport processes (Slayman et al., 1973; Scarborough, 1976; Bowman and Slayman, 1977). The question as to whether the plasma-membrane ATPase in Sacch. cerevisiae and other yeasts is also a proton pump cannot yet be answered positively. In contrast to the mitochondria1 ATPase, phosphorylated intermediates have been found as components of the plasma-membrane ATPase in Sacch. cerevisiae (Fuhrmann, 1977a, b; Willsky, 1979; Serrano, 1980), pointing to similarities in the metal-ion pumps. This ATPase might belong to a new class of this enzyme showing phosphorylated intermediates and pumping protons (Willsky, 1979). a. Active sugar transport in obligatory aerobic yeasts. It is noteworthy that, in yeasts which cannot be grown in the absence of oxygen, monosaccharides can only be accumulated by active transport. An exception to this rule is the fungus Neurospora crassa. The most extensive studies have been done with Rhodotorula glutinis (Kotyk and Hofer, 1965; Hofer and Kotyk, 1968). This yeast has been reported to accumulate a wide variety of metabolizable and non-metabolizable monosaccharides against considerable concentration gradients. Also polyols are actively transported by this organism (Kloppel and Hofer, 1976). Whereas the alditols are transported by means of the constitutive monosaccharide carrier, a second carrier was found to be inducible for pentitols. Induction was shown to be dependent on protein synthesis de novo. Transport

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of sugars in R . glutinis is strictly coupled to metabolic energy and is completely inhibited by metabolic inhibitors, uncouplers or anaerobiosis (Hofer, 1971). Attempts to demonstrate a passive facilitated diffusion in the absence of metabolism have been unsuccessful (Hofer and Kotyk, 1968; Hofer, 1971). The uptake of monosaccharides is coupled to an influx of protons, which exhibited a stoicheiometry of 1 : 1 (Misra and Hofer, 1975; Hofer and Misra, 1978). Also an inside-negative membrane potential dependent on pH value has been measured. After addition of transportable monosaccharides, the membrane potential was depolarized. This fact was taken as evidence for a proton-sugar symport. Since all agents which depressed the membrane potential inhibited monosaccharide transport, it was assumed that the plasma-membrane potential provides energy for active sugar transport in R . glutinis (Hauer and Hofer, 1978). The inhibitory action of the polyene antibiotic nystatin was shown to be restricted to an interaction with the plasma membrane. By this, oxygen consumption was decreased and the proton permeability of the plasma membrane enhanced. Consequently, the pH gradient and the plasma-membrane potential collapsed and active sugar transport ceased (von Hedenstrom and Hofer. 1979). Kinetic analysis of the constitutive monosaccharide transport suggests that at least more than one carrier is involved in active transport (Alcorn and Griffin, 1978). Transport of disaccharides in R . glutinix is accomplished by splitting the disaccharides into monosaccharides in the cell wall space after which the monosaccharides are taken up by the constitutive carrier (Janda, 1975). The strictly aerobic yeast Cundidu purupsilosis transports monosaccharides by an active process, which is inhibited by uncouplers but not by iodoacetamide (Kotyk and Michaljanitova, 1978). This transport seems not to involve a stoicheiometric proton symport. 'The apparent half life of the carrier was estimated to be 3.5-4 hours. Active monosaccharide transport of this type has also been described in Cundidu guillermondii (Miersch, 1977), Cundidu hevcvwijkii (Deak and Kotyk, 1968) and Torulttpsis cundidu (Haskovec and Kotyk, 1973). The strictly aerobic fungus N . crussu has been extensively investigated. Klingmuller ( 1967) observed a fructose-induced or glucose-repressed active uptake of sugars. In addition to this active glucose-transport system in N . crussa, Scarborough (1970) demonstrated in glucose-grown cells a lowaffinity facilitated-diffusion process for glucose similar to that in Sacch. cerevisiue, whereas in fructose-grown cells or in cells grown in a medium lacking glucose the active-transport system with a high affinity was responsible for sugar transport. This high-affinity system was repressed by high concentrations of glucose, changing to the facilitated-diffusion process with low affinity (Scarborough, 1970, 1971). In this respect, N . crussu is different from R. glutinis and other obligatory aerobic yeasts in which no facilitated-

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diffusion process for sugar has been observed. The energy that can drive the active sugar transport was thought to be a proton gradient produced by action of the plasma-membrane ATPase which pumps protons out of the cell, thereby generating an electrochemical gradient (Slayman et al., 1973). To establish this hypothesis, Slayman and Slayman (1974) used intracellular microelectrodes to measure the effects of glucose transport on membrane voltage and membrane resistance in N . crassa. A sudden activation of highaffinity glucose transport by glucose addition produced a dramatic depolarization of the membrane potential, followed by a slow spontaneous recovery. A saturation curve was obtained by plotting the depolarization peak against the initially applied glucose concentration. The half-saturation constant derived by this technique was 42 PM, which was in good agreement with that for the high-affinity glucose transport of ~ O P Mwith , a range of 3 0 - 5 0 ~ (Schneider ~ and Wiley, 197 1). Furthermore, the depolarization paralleled the behaviour of the active glucose-transport system in respect to its substrate specificity and in its absence from glucose-repressed cells. In accordance with a proton-linked sugar uptake, the suspension medium became alkaline after addition of a non-metabolizable sugar which was actively transported into the cells. However, the decrease in alkalization and the partial recovery of voltage during sustained sugar addition are not well understood. Therefore, a time-dependent shutdown of a potassium leakage channel has been discussed. The electrophysiological evidence for an electrogenic pump in N . crassa could be supported by studies in plasma-membrane vesicles derived from intact cells (Scarborough, 1976). These plasma-membrane vesicles were mainly inside-out oriented with the plasma-membrane ATPase facing the suspension medium. When the ATPase catalysed ATP hydrolysis, an insidepositive membrane potential was generated in the vesicles, which is in accordance with the opposite potential in intact cells. There is now considerable evidence that the primary transport system in the plasma membrane of N . crassa is the plasma-membrane ATPase with the properties of an electrogenic proton pump (Bowman and Slayman, 1977; Bowman et al., 1978). There is hope that secondary coupling of active sugar transport to a proton gradient can be demonstrated in plasma-membrane vesicles to elucidate the molecular transport mechanism. b. Active transport ofsugars in facultative aerobic yeasts. In 1964, Okada and Halvorson (l964a, b) described a genetic control of facilitated diffusion and active transport of a-thioethyl-D-glucoside in Sacch. cerevisiue. In glucosegrown cells, only facilitated diffusion was observed, whereas in cells induced by a-thioethyl-D-glucoside or a-methylglucoside the investigated a-thioethylD-glucoside was transported actively against a concentration gradient. The inducible active transport with a high affinity for the non-metabolizable

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a-thioethyl-D-glucoside was inhibited by azide, p-chloromercuribenzoate and partially by arsenate, arsenite and iodoacetic acid. The constitutive facilitated-diffusion system with a low affinity was not affected by these inhibitors. Other inducible active a-glucoside transport systems (maltose) were found to be independent. Seaston et a/. (1973) demonstrated in Sacch. carlsbergensis, which had been grown with maltose, that uptake of maltose or a-methylglucoside was accompanied with proton uptake. With increasing maltose concentration, the rate of proton uptake showed saturation kinetics with a half-saturation constant of 4 mM similar to that of maltose transport. The stoicheiometry was 2-3 protons absorbed for each maltose molecule taken up. Whereas with maltose, a-methylglucoside and sucrose protons were absorbed, this was not so with glucose, galactose or 2-deoxyglucose. Uptake of protons with sugar was accompanied by a potassium efflux, which was thought to be necessary to neutralize proton uptake into the cell. These results were discussed by the authors assuming a proton-symport model. If the cells were grown on glucose instead on maltose, proton uptake with sugars could not be observed. Also, Sacch. Jragilis grown on lactose demonstrated the phenomenon of proton uptake with this sugar but, if the cells were grown on glucose, lactose transport was not induced. Different phenotypes for lactose utilization, uptake and enzyme induction have been investigated in Kluyveromyces and Saccharomyces species (Algeri et a/., 1978). Absorption of protons with a-methylglucoside and a-thioethylglucoside in Sacch. cerevisiae has been observed by Brocklehurst et al. (1977). Cells grown with maltose absorbed about 1 equivalent of protons with each equivalent of a-methylglucoside, and 1 equivalent of potassium left the cells. This stoicheiometry was taken as strong evidence for the proton-symport model. Also, maltose and a-thioethylglucoside caused an initially accelerated uptake of protons and efflux of potassium across the plasma membrane. This effect was not inhibited by antimycin and iodoacetamide as shown also by Seaston et a/. (1973), but was inhibited by dinitrophenol and by raising the pH value. The statement that the a-methylglucoside and the maltose transport systems are distinct entities (Okada and Halverson, 196413) has been doubted. The authors discuss the probability that glucose and sorbose may be substrates of the a-methylglucoside transport system. Energy requirement in the form of a proton gradient has also been found by Serrano (1977) for maltose transport in Sacch. cerevisiae. The transport was inhibited as shown above by uncouplers, and no inhibition was observed with antimycin and deoxyglucose, which lowered the ATP content 50- 100-fold. One proton is cotransported with every maltose molecule. Electroneutrality during maltose transport and proton uptake can be maintained by potassium efflux or by entry of a permeable anion. In addition to the

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uncouplers azide, dinitrophenol and carbonylcyanide nz-chlorophenylhydrazone, sodium fluoride and p-chloromercuriphenylsulphonic acid also inhibited maltose transport strongly. Serrano (1977) claims that ATP is not involved in transport of maltose, but the possibility of generation of an electrochemical gradient by a proton pump driven by ATP (Riemersma and Alsbach, 1974) is not excluded. For short time periods, the proton gradient was thought to be maintained in the absence of ATP at the expense of the diffusion potential of potassium, which would leave the cells much faster than anions. When the electrochemical gradient is abolished by uncouplers, transport of maltose is not able to function even when a concentration gradient of maltose is present. Okada and Halverson (1964b) observed that trehalose inhibited uptake of a-thioethylglucoside in Succh. cerevisiue, and Alonso and Kotyk (1978) found that trehalose interfered with uptake of a-methyl-D-glucoside and maltose. The storage sugar trehalose will not be metabolized immediately when added to Succh. cerevisiue suspended in water or growth medium, but it supports growth after a lag phase of about 10 hours. Kotyk and Michaljanicovi (1979) demonstrated that the sugar was transported into the cells by at least two transport systems. Transport was considered to be active, with a pH optimum at 5.5 similar to other disaccharide transport systems, but in contrast no significant proton uptake was observed with the sugar transport. However, there are also reports that certain yeast strains take up a-glucosides without accompanying protons (Eddy, 1978). Dinitrophenol caused only a minor retardation of trehalose transport. The observation that there was a highly active transport of trehalose and maltose immediately after growth of the cells on glucose is difficult to understand. After growth on glucose, trehalose transport decayed, but could be reactivated by several a-glucosides, glucose or ethanol. However, this phenomenon could be observed only under aerobic conditions. Comparative analysis supported the existence of four carriers for a-methyl-D-glucoside, four for maltose and two for trehalose in Succh. cerevisiae after trehalose induction. Various a-glucosides should share the same carrier, so that the total number of a-glucoside transport systems is five. In an earlier publication, Alonso and Kotyk (1978) reported the halflives of sugar-transport proteins in Succh. cerevisiue. In these determinations, the decrease in transport capacity was measured after a-glucoside induction by cycloheximide. In comparison to the relatively long half-life of the inducible galactose carrier (2-2 hours), that of the inducible maltose carrier was 1.2 hours. An even shorter half-life (0.8 hour) was found for the inducible a-methyl-D-glucoside carrier. The shortest half-life was observed for the trehalose carrier with about 0.3 hour. In this respect, a possible effect of cycloheximide on either resynthesis of transport proteins or synthesis of specific proteases degrading the transport proteins has been discussed. Kotyk

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and Michaljanicbva (1979) found an activation of protease activity with cycloheximide and glucose. This observation favours the latter possibility and adds a further type of regulation, namely that by “enzyme inactivation”, to those proposed by Holzer (1976).

C. C O N C L U D I N G R E M A R K S

From a theoretical standpoint, mechanisms of sugar transport in yeasts can be divided into four groups. The first group is constitutive facilitated transport, the second group inducible facilitated transport, the third group constitutive active transport and the fourth group inducible active transport of sugars. It has been shown that there is experimental evidence for the existence of the four groups in Sacch. cerevisiur. In the obligatory aerobic yeast R. glutinis, only the presence of the last two groups for active sugar transport has been shown, whereas the obligatory aerobic fungus N . crassa demonstrates facilitated diffusion transport and induction of active sugar transport. Regulation of transport by metabolism is very obvious for the inducible sugar carriers, the biosynthesis of which can be modified by inducers. The mechanism of catabolite repression and of catabolite inactivation has been shown and proposed to be instrumental for another type of regulation. In contrast to bacterial sugar transport which is inducible, the time of induction in genela1 is longer, and increase in transport rate is about ten-fold rather than a thousand-fold as in bacteria. Further, there is experimental evidence that the inducible carriers are distinct entities, whereas in bacteria the same carrier for active transport is also thought to be functional in facilitated diffusion in the absence of energy. Another important difference is that a coordinate all-or-none induction and repression, as for example in the galactose metabolic system, does not appear to take place in yeasts. The structural genes required for such a system are rather scattered among several chromosomes and they are not regulated in a simple coordinated manner. A tight coupling of metabolism and active sugar transport has been shown in R. glutinis. Regulation by metabolism in this case becomes very prominent, in contrast to the constitutive facilitated diffusion for glucose transport in Sacch. cerevisiue, which seems only to be rate determining by its equilibration capability in metabolically depleted cells. The models discussed so far for the sugar-transport systems are the phosphotransferase and the proton-symport model. There is no experimental evidence for a phosphotransferase system like that in bacteria, but a different phosphotransferase system in yeast has not been completely excluded. There is, however, a considerable amount of evidence for a proton-symport sugar

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transport in several yeasts as well as in N . crassa. Difficulties arise in separation of different transport mechanisms, for example, ion transport, sugar transport or amino-acid transport, and by interference with general metabolism in intact cells. The possibility of using vesicles derived from plasma membranes, which are suitable for membrane and transport studies, is a great advance. Metabolism is negligible in vesicles and interference with transport systems can be diminished considerably. By creating ionic gradients, the mechanism of coupling can be studied and the importance of the plasmamembrane ATPase can be investigated in detail. Further, the power of genetic manipulation and effects on the plasma membrane can be elucidated more directly in vesicles than in intact organisms.

V. Conclusions Growth of yeasts on sugars is rather well documented. There remain, however, several problems with respect to the molecular basis of regulation. In this review, two distinct groups of yeasts were discussed, namely glucoseinsensitive and glucose-sensitive yeasts. It is important to compare both yeast types and to determine whether there exist principal regulatory differences or whether there are distinct steps in the enzyme pattern which lead to the differences in the response toward glucose. The reason for glucose repression is still obscure. The model of Peterkofsky as outlined in this review represents a reasonable working hypothesis, but needs to be verified in the yeast system. The phenomenological similarities (i.e. ethanol formation, diminished respiration, drop in yield) of the cells when other limitations are applied need to be investigated with respect to similarities or dissimilarities in the underlying molecular events. The aim of such studies is the elucidation of where the regulation takes place and whether there exists a uniform background for all observations. After learning where regulation takes place, the regulatory mechanisms need to be investigated. In eukaryotes an extension of the bacterial mechanisms is probably necessary. The discovery of catabolite inactivation for regulation of enzyme activity gives rise to such speculations. The differences discussed in the transport systems indicate that there are possibly also differences in internal metabolite concentrations which in turn may be expressed in the different regulatory responses. These observations call for an extension of regulatory studies to an investigation of transport systems with respect to glucose-insensitivity and glucose-sensitivity respectively. The current status of the research on glucose metabolism in yeasts has

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reached a point where new approaches are needed for elucidation of the different regulatory phenomana. The conventional methods that were applied to bacteria lead to a certain stagnation. We think that with advanced cultural techniques and improved analytical procedures new opportunities will lead to a better understanding of the events in yeasts growing on carbohydrate substrates.

VI. Acknowledgements Our own experiments reviewed in this article were supported by the Schweizerische Nationalfonds zur Forderung der wissenschaftlichen Forschung Project Nos. 4025, 3.1.68;3.628.71, 3.3590.74, 3.184.77, 3.465.79 and by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg, Western Germany.

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Slayman, C. 1.and Slayman, C. W. (1974). Proceedings ofthe National Academy of Sciences of’the United States of’ America 71, 1935. Slayman, C. L., Long, W. S. and Lu, C. Y.-H. (1973). Journal of’Membrane Biology 14, 305. Sols, A. (1968a). In “Aspects of Yeast Metabolism” (A. K. Mill and H. A. Krebs, eds.), p. 47. Blackwell Scientific Publication, Oxford. Sols, A. (1968b). In “Reflexions on Biochemistry” (A. Kornberg, B. L. Hovecker, 1.Corundella and J . Oro, eds.), p. 199. Pergamon Press, New York. Sperber, E. (1945). Arkiv Kemi. Mineral. Geologi 21, 17. Spoerl, E. (1971). Journal of’Bacteriology 105, 1168. Spoerl, E. and Doyle, R. J. (1968). Journal of’ Bacteriology 96, 744. Spoerl, E., Benedict, S. H., Lowery, S. N., Williams, J. P. and Zahand, J. P. (1975). Journal of Membrane Biology 20, 3 19. Steveninck, J. Van (1966). Biochimica et Biophysica Acta 126, 154. Steveninck, J. Van (1969). Archives of’ Biochemistry and Biophysics 130, 244. Steveninck, J. Van (1970). Biochimica et Biophysica Acta 203, 376. Steveninck, J. Van (1972). Biochimica et Biophysica Acta 274, 575. Steveninck, J. Van and Booij, H. 1.(1964). Journal of’General Physiology 48, 43. Steveninck, J. Van and Rothstein, A. (1965). Journal of’General Physiology 49, 235. Stickland, 1.H. (1956). Biochemical Journal 64,498. Strasters, K. C . and Winkler, K . C. (1963). Journal of General Microbiology 33, 213. Strittmatter, C. F. (1957). Journal uf General Microbiology 16, 169 Suomalainen, H. and Oura, E. (1971). In “The Yeasts”, vol. 11, p. 3 (A. H. Rose and J. S. Harrison, eds.). Academic Press, London and New York. Tejwani, G. A. (1978). Trends in Biochemical Sciences 3, 30. Umnov, A. M., Stebliak, A. G . ,Unmova, N . S., Mansurova, S. E. and Kulaev, I. S . (1976). Mikrob~ulugii~ 44,414. Warburg, 0. (1926). “Ueber den Stoffwechsel der Tumoren”. Springer, Berlin and New York. Wegener, W. S. and Romano, A. H. (1964). Journal of’Bacteriology 87, 156. Weibel, E. K . (1973). Zur Energetik von Saccharomyces cerevisiae. Thesis No. 5155, ETH Zurich. Wenner, C. E. (1979). Methods in Enzymology 55, 289. West, I. C. (1970). Biochemical and Biophysical Research Communications 41, 655. West, I . C. and Mitchell, P. (1972). Journal of’ Bioenergetics 3, 445. West, I. C. and Mitchell, P. (1973). Biochemical Journal 132, 587. White, J. (1954). “Yeast Technology” p. 127. Academic Press, London. Wickerham, L. J. (1951). Technical Bulletin 1029, United States Department of Agriculture, Washington D.C. Wiken, T. 0. (1968). In “Aspects of Yeast Metabolism” (A. K. Mills and H. Krebs, eds.), p. 122. Blackwell Scientific Press, Oxford. Wilbrandt, W. (1954). Symposium of the Society for Experimental Biology 8, 136. Wilbrandt, W. (1973). In “Biomembranes” (F. Kreuzer and J. F. G . Slegers, eds.) 3, p. 79. Plenum Press, New York. Wilbrandt, W. and Rosenberg, Th. (1961). Pharmacological Reviews 13, 109. Willsky, G. R. (1979). Journal of Biological Chemistry 254, 3326. Wilson, D. B. (1974). Journal of’Biologica1 Chemistry 249, 553. Wimpenny, J . W. T. (1969). In “Microbial Growth”, 19th Symposium of the Society of Genetical Microbiology, p. 161. Wolf, D. and Ehmann, C. (1978). Federation of’ the Europeun Biochemical Socierjs Letters 91, 59.

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Yotsuyanagi, Y. ( 1962). Jourtiul of' Ultrustruc~rure~ RescwcA 7. I2 I . Zimmerli. A . (1970). Diplomarbeit. Swiss Federal Institute of Technology. Zurich. Zimmerli. A . ( 1975). Zur Lokalisation der Malat-Dehydrogenase in S[,/ii-o.c.ac.c,/iarom , ~ w . rp o n i h ~ Thesis . N o 5469, ETH Zurich. Note added in proof: Recent results in our laboratory indicate that the glycolytic flux remains the most reliable indicator for the beginning of repression and. hence, that repression starts at a particular dilution rate independent of the initial glucose concentration ofthe medium (cf. p. 140). Deviation from this principle may occur due to physiochemical effects or even originate from the experimental set up. Starting from lower dilution rates. M . Rieger and 0. Kippeli (unpublished results) found that the critical dilution rate has to be approached in very fine steps in order to avoid glucose pulses which trigger repression prematurely. When 11, uab approached from dilution rates higher than D,, a hysteretic behaviour was obscrved leading to an apparently lower D, value. Furthermore. the amount of air entering the reactor may influence the metabolism of the cells which most probably is a carbon dioxide effect. h e liminary results indicate that inappropriate gassing-out of the carbon dioxide affects the critical dilution rate D,. Two relevant review articles have been published since the inanuscript u a s prepared. Both arc related to the phenomenon of catabolite inactivation described on p. ISO. They provide il very detailed picture of the present knowledge.

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Unity and Diversity in Some Bacterial Citric Acid-Cycle Enzymes* P. D. J. WEITZMAN Department of Biochemistry, University of Bath, Bath, England I . A view of the citric acid cycle . 11. Citrate synthase

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A. Energy controls . . . . B. Biosynthetic controls. , . C. Molecular-size patterns . D. Allosterism: kinetic and molecular features . , E. Enzyme characteristics as an aid to bacterial taxonomy F. Exceptions to the enzyme patterns . . G . Mutants: dysfunction as a clue to function . . Succinate thiokinase . A. Molecular-size patterns . . B. Nucleotide-specificity patterns . Isocitrate dehydrogenase Pyruvate and a-oxoglutarate dehydrogenases , . Malate dehydrogenase , . . Multipoint control of the cycle . Evolutionary aspects . . . Concluding remarks . Acknowledgement . . . . . . References , . . . .

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185 191 192 198 202 204 207 209 211 218 219 220 223 221 230 231 234 231 238 238

1. A View of the Citric Acid Cycle

The crucial discovery by Krebs and Johnson (1937) that the repertoire of cellular metabolism included the formation of citrate from oxaloacetate and *This review is dedicated to the memory of a dear friend and colleague Dr. Bill Ferdinand, 193&1980. 185

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pyruvate converted the already known oxidative reaction sequence from citrate to oxaloacetate into a cyclic pathway and clearly linked it with the pathway of carbohydrate metabolism. The citric acid cycle was soon accepted as the terminal pathway for the oxidation of foodstuffs in all respiring animal tissues. The scheme of the cycle originally proposed still holds good, though it required the discovery of coenzyme A and the elucidation of the roles of acetyl-CoA and succinyl-CoA as intermediates for the cycle to be elaborated to the expanded form in which we now know it (Fig. I). In addition to its role as an energy-yielding catabolic pathway, Krebs et al. (1952) later suggested that the cycle may also serve an anabolic function in providing the acet vl-CoA

i’

rnalate

4

I

isocijrate

f u rn$r a t e

t

succinate

a -0xoglutarate /

FIG. 1, The citric acid cycle.

carbon skeletons of cell constituents. and subsequent investigations by various workers have amply confirmed this second role. We now commonly refer to the “dual role” of the citric acid cycle and we shall later consider the complexities of metabolic regulation posed thereby. Application of the procedures successfully adopted with animal tissues to demonstrate the occurrence of the citric acid cycle in micro-organisms were initially unsuccessful and were hampered by the permeability barriers towards metabolites and inhibitors which micro-organisms possess to a more marked extent than do animal tissues. The problems encountered and the ways in which the cycle was finally established as the major pathway of terminal respiration in micro-organisms have been reviewed (Kornberg, 1959; Krebs and Lowenstein, 1960). As noted above, the citric acid-cycle pathway serves to append a sequence of oxidative reactions onto the pathway of carbohydrate catabolism. It may

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be profitable to pause and consider the functions of, and connection between, these two major metabolic pathways in a rather unconventional way which will emphasize the key role of certain citric acid-cycle enzymes and will alert us to their potential as sites for the operation of regulatory mechanisms. Both glucose and citric acid are compounds containing six carbon atoms per molecule. Their respective molecular formulae are C6H ,O, and C,H,O,. Citric acid thus has four hydrogen atoms less and one oxygen atom more than glucose, a difference which is equatable with three oxidations, one C,

(glucose)

I I I

(pyruvic acid)

C,

C, (pyruvic acid)

(oxaloacetic acid)

C4

C,

Ch

(acetyl-CoA)

(citric acid)

FIG. 2. Scheme for the conversion of glucose into citric acid.

adding an oxygen atom and two removing two hydrogen atoms each. The metabolic equivalents may be identified as three dehydrogenase-generated NADH molecules, two being formed in glycolysis at the glyceraldehyde 3-phosphate dehydrogenase step (each half of the original glucose molecule being acted on by this enzyme) and the third being produced by pyruvate dehydrogenase acting only on one of the two fragments formed from glucose. The conversation of glucose into citric acid may be very simply represented by the scheme shown in Figure 2. The glycolytic breakdown of glucose to pyruvic acid can provide energy in the form of ATP as well as precursors for various biosynthetic routes. Two molecules of the 3-carbon end-product pyruvic acid can then join up to reform a 6-carbon compound, not directly, but by the loss of carbon

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P D. J. WEITZMAN

dioxide from one molecule of pyruvate and the capture of carbon dioxide by the other. The decarboxylation and carboxylation reactions are catalysed, respectively, by pyruvate dehydrogenase and pyruvate carboxylase t o give acetyl-CoA(C,) and oxaloacetic acid (C,). These two products can then be condensed to give citric acid by the action of citrate synthase. Thus the carbon atoms originally present in glucose may be brought together again and regrouped into a completely different 6-carbon compound, thereby making possible an entirely new round of chemical transformations. These latter are the reactions of the citric acid cycle whereby the oxidative metabolism of citrate can provide both energy and biosynthetic intermediates such as a-oxoglutarate and succinyl-CoA. The regrouping of the C2 and C, compounds into citric acid would therefore be expected to be related to the cellular demands for energy, a-oxoglutarate and succinyl-CoA. Since these physiological demands differ between organisms, the controls exerted over the formation of citric acid are likely to be diverse and to reflect different “metabolic life-styles”. These regulatory differences would, moreover, be expected to reside in differences in molecular structure of the enzyme catalysing the condensation, i.e. citrate synthase. The capacity of citrate synthase to regroup the products of carbohydrate breakdown into a form conferring new metabolic possibilities clearly places this enzyme at a critical and central position in the metabolic map. Of course, the entire citric acid cycle is at the very core of cellular metabolism. Although the chemical natures of the intermediates of the cycle and their interconversions are fixed, the multifunctional role of the cycle and the different emphasis that diverse organisms may place on its metabolic functions could well be reflected in differences in the properties of other enzymes in the cycle, in addition to citrate synthase. For the purposes of the present article, it may be instructive to consider the reactions of the citric acid cycle in a much simplified form (Fig. 3). This scheme focuses attention on certain essential features of the pathway and emphasizes four distinct phases of the overall sequence of reactions. Phase (a) is the ”condensation” phase which effects the entry of carbon, in the form of acetyl units (C2), into the cycle. This is followed by two successive “cleavage” phases, (b) and (c), each resulting in the loss of a carbon atom by oxidative decarboxylation, first of isocitrate (C,) to a-oxoglutarate (C,) and then to succinyl-CoA (Cg). In bacteria, these two decarboxylations are accompanied by the formation of NADPH and NADH respectively. In a sense, the pathway might terminate at this point. Two carbon atoms, equivalent to the pair initially introduced, have been removed by oxidation, reduced nicotinamide nucleotides have been produced-both NADPH for biosynthesis and NADH for reoxidation coupled to ATP production-together with the biosynthetic materials a-oxoglutarate and

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FIG. 3. Simplified scheme for the citric acid cycle. For details, see the text.

succinyl-CoA. However, by rearrangement of C t (succinyl-CoA) to the C, starting compound (oxaloacetate), a complete cycle is put into operation and a new round of chemical conversions may be undertaken. By this device it is possible for the pathway to oxidize, and hence derive free energy from, a supply of C, “fuel” without running out of the requisite “carrier” compound C,. This final “rearrangement” phase (d) involves no addition or loss of carbon atoms but consists of four reactions which prepare the 4-carbon compound for re-entry into phase (a). Phases (a), (b) and (c) therefore make up what might be called an “outward” section which fulfils the biosynthetic functions of the cycle, while phase (d) constitutes a “return” section which permits the whole sequence to act catalytically in the total oxidation of acetyl-CoA. The initial enzymes of these two sections are, respectively, citrate synthase and succinate thiokinase. It may be significant that both these enzymes act on an acyl-CoA substrate and, in addition to their synthetic functions, serve to regenerate free CoA-SH. This is required as a substrate for the two metabolically preceding enzymes, pyruvate dehydrogenase and a-oxoglutarate dehydrogenase, and its regeneration from acylated derivatives is obligatory for the maintenance of metabolite flux round the cycle. This review will concentrate heavily on aspects of citrate synthase and succinate thiokinase. The emphasis will be on the variations displayed by the enzymes from different organisms around a common catalytic theme. Insofar as diversity has been detected in other enzymes of the cycle, some discussion will also be devoted to isocitrate dehydrogenase, to the multi-

190

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enzyme complexes pyruvate dehydrogenase and a-oxoglutarate dehydrogenase and to malate dehydrogenase. Thus all four phases of the cycle shown in Fig. 3 will be dealt with. The considerations advanced above suggest that it is at the level of regulation that a major portion of enzyme diversity is likely to be encountered. Particular attention will be paid here to variations in control behaviour but this will be restricted to the control of the activity, not the synthesis, of the enzymes. This follows from the aim of the review which is to explore the unity and diversity shown by the citric acid-cycle enzymes themselves, not their synthetic machinery. There are, however, various reports in the literature concerned with the factors influencing bacterial contents of the citric acid-cycle enzymes (e.g. Amarasingham and Davis, 1965; Gray rt al., 1966; Hanson and Cox, 1967; Flechtner and Hanson, 1969; Eidels and Preiss, 1970; Fortnagel, 1970; Peeters Pt ul., 1970; Charles, 1971; Ng and Dawes, 1973: Colby and Zatman, 1975a; Ohne, 1975; Hebeler and Morse. 1976; Dawes, 1978). In connection with srudies on the regulation of enzyme activity, it seems appropriate to interject some comments on the problems of assessing the significance in vivo of observations in vitro. In the case of straightforward biosynthetic pathways, the operation of feedback inhibition of the activity of an early enzyme in the pathway may be revealed by studying mutant organisms lacking one of the enzymes later in the pathway and therefore unable to synthesize the regulatory end product. The mutant may overproduce and excrete that intermediate which immediately precedes the enzymic lesion. Provision of the end product in the growth medium will be necessary for growth to occur and may also be observed to prevent the accumulation of the intermediate metabolite. In this way, it is possible to "observe" feedback controls operating in vivo. It may also be possible to obtain mutants that produce a modified form of what, in the wild-type parental strain, appears to be a regulatory enzyme, the mutant form lacking sensitivity to the regulatory metabolite. Such mutants would then be expected to overproduce the biosynthetic end product (see e.g. Moyed, 1961; Cohen, 1969). This type of approach may be useful in probing controls exercised over the biosynthetic functions of the citric acid cycle (see Section II.G, p. 21 1 ) but it is not appropriate to the examination of the regulation of its energyproducing role. Thus the production of ATP is a highly complex and composite process and one cannot make mutants that lack the ability to carry out this process and compensate them by providing ATP in the growth medium. Similarly, one cannot supply NADH for uptake by cells from the external medium. The physiological significance of regulatory effects exerted by metabolic "energy signals", i.e. the adenine and nicotinamide nucleotides,

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is therefore difficult (or impossible) to assess and, in the absence of direct experimental demonstration, one must adopt a rather more circumspect approach. Arguments rest on the accumulation of circumstantial evidence which is then viewed from a frankly teleological stance (Atkinson, 1968). Nevertheless, conclusions are inevitably points of view; however attractive these may appear, they must be accepted not with finality but with a good measure of caution and open-mindedness.

11. Citrate Synthase

Citrate synthase occurs in virtually all living cells though its apparent absence from some bacteria has been reported, for example, Lucrohucillus plariturum and Streptococcus jaeculis (Weitzman and Jones, 1968) and Gemellu huemoljvans (Weitzman and Jones, 1975). The enzyme catalyses the reaction: Acetyl-CoA + Oxaloacetate + H,O

+ Citrate

+ CoA-SH

The key metabolic position of the enzyme has resulted in many investigations aimed at probing its structure, catalytic function and regulation and some previous reviews are recommended for their coverage of topics omitted here (Spector, 1972; Srere. 1972; Weitzman and Danson, 1976). One source of diversity amongst bacterial citrate synthases stems from the stereochemistry of the enzyme-catalysed reaction (see Srere, 1972). Whereas almost all citrate synthases have the si stereospecificity, the enzyme from only a few strictly anaerobic bacteria has the converse re stereospecificity (Gottschalk and Barker, 1966, 1967; Stern and Bambers, 1966; Gottschalk and Dittbrenner, 1970). On the basis of the observation that intermediates of the citric acid cycle do not normally accumulate, Krebs and Lowenstein (1960) concluded that the rate-limiting step of the cycle must be that catalysed by citrate synthase. This proposition is in line with the considerations advanced above, which view citrate synthase as playing a crucial initiating role in the cycle and which therefore lead to the expectation of various regulatory effects. Indeed, citrate synthases from a wide range of organisms have been examined and from in vilro observations of the modification of their enzymic activities various presumptive regulatory effects have been deduced. Before discussing the variety of functional and structural behaviour displayed by the enzyme, a few comments on the assay of its activity may be appropriate. Indeed it is the availability of very convenient assay procedures that has so greatly facilitated the many investigations which have been

P D J WEITZMAN

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undertaken. In early studies, two ultra-violet spectrophotometric methods were employed, one based on coupling with malate dehydrogenase (Ochoa, 1955) and measurement at 340 nm, and the other relying o n following the decrease in absorbance at 233 nm consequent o n the consumption of acetyl-CoA (Srere and Kosicki, 1971). Neither of these assays is well suited to crude extracts, as these inevitably contain high concentrations of materials which absorb at ultra-violet wavelengths and frequently also exhibit N A D H oxidase activity. Nor are these methods appropriate when it is desired to examine the effects of A T P o r N A D H on enzymic activity. The introduction by Ellman (1959) of the chromogenic reagent D T N B (5,5'-dithiobis(2-nitrobenzoate)) provided a much more convenient means of assaying citrate synthase activity (Srere et a/., 1963). D T N B reacts rapidly, nonenzymically and stoicheiometrically with the free thiol group of CoA-SH to form a mixed disulphide and to liberate the yeilow thionitrobelizoate anion which absorbs strongly at 412 nm ( E =~ 13.600 I.mol-'.crn-I). Thus the formation of CoA-SH in the course of the citrate synthase reaction may be monitored colorimetrically and continuously. This simple assay readily permits the determination of enzymic activity and of the effects of metabolite inhibitors o r activators and hence makes it possible to examine the properties of the citrate synthases of large numbers of organisms. The reactivity of D T N B towards thiol groups also means, however, that i t can attack thiol groups on the enzymes themselves with the possibility ofinactivation o r desensitization towards effectors. To overcome this, another assay method was devised based on the polarographic signal produced at the dropping mercury electrode by CoA-SH but not by its S-acyl derivatives (Weitzman, 1966a, 1969, 1976). This, too, is a convenient, sensitive and continuous method by which the rate of formation of CoA-SH may be monitored directly, and it can readily be employed with crude cell extracts and in the presence of a variety of possible regulatory metabolites.

A.

ENERGY CONTROLS

I . Iriliihition h!. A TP mid Otlitlr Nuclrotides Hathaway and Atkinson (1965) first discovered a direct inhibitory effect of adenine nucleotides on citrate synthase from Suc.r.liurnm!,c.escercvisiue. The inhibition was competitive with respect to acetyl-CoA. The order of effectiveness of the inhibitors was A T P > A D P > AMP, and it was suggested that it might be the relative, rather than the absolute, effects of the three nucleotides which were of metabolic significance. Since the citric acid cycle functions

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193

to produce energy in the form of ATP, inhibition of the initial enzyme of the cycle by ATP constitutes a highly plausible end-product feedback control process governing oxidative energy metabolism. When the ATP level drops, i.e. ATP is degraded to ADP or AMP, the inhibition of citrate synthase would be diminished as the latter nucleotides are less effective inhibitors of the enzyme. As a result, citrate synthase would be relatively more active and the ensuing increase in overall cycle activity would serve to re-establish the ATP level. The pattern of relative effects of the three adenine nucleotides as inhibitors of citrate synthase, together with similar responses displayed by other enzymes, contributed to the development by Atkinson of the "energy charge" concept (Atkinson, 1968). The finding of ATP inhibition of yeast citrate synthase was followed by numerous reports of a similar inhibition of citrate synthases from a very wide range of organisms, including animals, plants, fungi and bacteria (see Weitzman and Danson, 1976). Bacteria whose citrate synthases have been reported to be inhibited by ATP are listed in Table 1. Indeed, the inhibition of citrate synthase by ATP is now cited in several textbooks of biochemistry as an established control feature in the citric acid cycle, although we shall shortly consider the uncertainty that attends this claim.

TABLE I , Bacterial citrate synthases showing inhibition by ATP Organism

Reference _ .

~~~

Swissa and Benziman (1976) Kleber and Tauchert (1974) Flechtner and Hanson (1970) Tanaka and Hanson (1975) Higa and Cazzulo ( 1976) Flechtner and Hanson (1969) Shiio PI NI. (1977) Jangaard er (11. (1968); Srere ( 1968) Flechtner and Hanson (1970) Flechtner and Hanson (1970) Borriss and Ohmann ( 1972) Weitzman (1978) Taylor (1970) Colby and Zatman (l975b) Lucas and Weitzman ( 1975) Lucas and Weitzman (1975) Taylor (1973); Lucas and Weitzman (1975) Lucas and Weitzman (1975) Taylor (1973); Lucas and Weitzman (1977)

194

P. D. J. WEITZMAN

Some of the citrate synthases which are sensitive to adenine nucleotides have also been examined for their response to nicotinamide nucleotides (Kosicki and Lee, 1966; Lee and Kosicki, 1967; Srere er a/., 1973; Danson et al., 1979). These too have been found to act as weak inhibitors, their order of effectiveness being NADPH > N A D H > N A D P + > N A D + , and like the adenine nucleotides they act in competition with the substrate acetyl-CoA. All of the adenine and nicotinamide nucleotides share common structural features with acetyl-CoA and it is therefore quite possible that their inhibitory action stems from their competition for the acetyl-CoA binding site on the enzyme. Direct kinetic evidence for such “isosteric” inhibition has been adduced from multiple-inhibition studies using ATP, NADH and the substrate analogue bromoacetyl-CoA on both mammalian and bacterial citrate synthases (Harford and Weitzman, 1975). Moreover, the order of inhibitory effectiveness of the nucleotides stated above suggests that electrostatic interactions may make an important contribution to the nucleotideenzyme binding (Srere and Matsuoka, 1972; Srere et d., 1973). The more negative charge resulting from increased phosphate content in the adenine nucleotides appears to produce a stronger inhibitor. Likewise, the additional phosphate group in the reduced and oxidized forms of NADP, compared with those of NAD, confers greater inhibitory effectiveness, whereas the presence of a positively charged nicotinamide ring in the oxidized form of both nucleotides makes these less effective than their reduced counterparts. Consistent with the idea that the negative charge on the nucleotide (and on acetyl-CoA itself) is important, are the effects of positively charged divalent metal ions, such as Mg2+,on the behaviour of the enzyme. These cations inhibit enzymic activity and decrease, or abolish, the inhibition by nucleotides (Kosicki and Lee, 1966; Lee and Kosicki, 1967); they probably act by binding ionically, to the phosphate groups on acetyl-CoA or the nucleotides, thereby weakening their electrostatic interaction with the active site on the enzyme. Further evidence for the view that the sensitivity of citrate synthases to non-specific nucleotide inhibition results from structural similarities between the inhibitors and acetyl-CoA has been gained from a comparison of the apparent affinities of various citrate synthases for both ATP and acetyl-CoA. A measure of the enzymic affinity for ATP is represented by the value of K , obtainable from inhibition studies, whereas the affinity for acetyl-CoA may be approximately indicated by the K,, value for this substrate. As values ranging from 2 PM to 400 PM have been reported for this K,,,, suggesting a wide spectrum of affinities, it was of interest to see whether this was reflected in a similar spread of K, values. Weitzman et at. (1981a) compared these two parameters for a large number of citrate synthases using both their own results and data derived from the literature. A linear plot of

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195

K , verses K , was obtained, indicating a very close relationship between the interaction of citrate synthases with acetyl-CoA and ATP. The simplest conclusion to be drawn from the above findings is that ATP and related nucleotides act as inhibitors of citrate synthase by binding to that portion of the active site of the enzyme that is normally occupied by acetyl-CoA. This is supported, in a negative sense, by the fact that no citrate synthase has been desensitized to A T P inhibition with retention of catalytic activity. Although we are now accustomed to think in terms of specific, ullosrrric, sites on a regulatory enzyme to which “signal” metabolites may bind and thereby exert some modulation of activity there is, of course, no reason why regulation should not be exerted by effectors acting at the active site itself. However, one of the less attractive features of the ATP inhibition of citrate synthase is that the effect is largely overcome by Mg2 ions. As the intracellular A T P is thought to exist predominantly in a magnesium-chelated form, this casts some doubt on the operation in v i v o of this inhibition. Weitzman and Hewson (1973) examined citrate synthase in cells of Succhurornyes cerevisiur made permeable to substrates by treatment with toluene. The enzyme activity measured in situ appeared not to be affected by ATP. Similar studies on toluene-treated rat liver mitochondria (Matlib rt d., 1978) showed that this citrate synthase was also markedly less sensitive to A T P in siru. These observations raise the possibility that the condition of the enzyme within the cell alters the properties observable in virro. Finally, the appeal of A T P inhibition ofcitrate synthase as a plausible feedback control mechanism is diminished by the finding that inhibition is observed even with citrate synthases from organisms in which the citric acid cycle plays a biosynthetic, and not a n energy-producing, role, for example cyanobacteria (Taylor, 1973; Lucas and Weitzman, 1975, 1977) and a strictly autotrophic Thiohacillus sp. (Taylor, 1970). When citrate synthase from Eschc>richiuc d i was tested for sensitivity to ATP, the enzyme was found to be only weakly inhibited under the particular assay conditions employed (Weitzman, 1966b). In line with the argument advanced above that ATP acts isosterically with acetyl-CoA and that their binding affinities for the enzyme are related, this relatively weak effect of ATP is consistent with the rather high K,,, value for acetyl-CoA (approximately 400 PM) displayed by citrate synthase of E. c d i . In the presence of K C I , the K,,, value is lowered and the response to A T P is enhanced (Srere, 1968). Similar effects are produced by lowering the p H (Weitzman and Danson, 1976). However, the relative insensitivity of the enzyme from E . coli to A T P prompted the search for a n alternative metabolite which might prove a more effective regulator of this bacterial enzyme. +

196

P. D.J. WEITZMAN

2 . Inhibition by NADH Referring back to Figure 3 and the attendant discussion (p. 188), it will be recalled that the cycle may be thought of in two halves-an “outward” section and a “return” section. It is therefore noteworthy that one of the end products of the “outward” section is NADH (produced in the final reaction of this half-sequence, i.e. the oxidation of a-oxoglutarate to succinyl-CoA), and one of the end products of the “return” section is also NADH (produced in the oxidation of malate to oxaloacetate). I t is through the reoxidation of these molecules of NADH, coupled to the phosphorylation of ADP to ATP, that the free energy conserving role of the citric acid cycle is largely fulfilled. NADH thus qualifies as an end product of the cycle and as a “precursor” of ATP, and might therefore be well suited to exert a controlling influence over the activity of the cycle. I t was found that NADH is a powerful and specific inhibitor of citrate synthase from E. coli (Weitzman, 1966b). The inhibition was competitive with respect to acetyl-CoA, and no inhibition was produced by N A D + , NADP+ or NADPH. The specificity of NADH inhibition for the enzyme from E. coli was underlined by the finding that neither yeast nor mammalian citrate synthase was sensitive to inhibition by NADH, whereas both the latter enzymes were considerably more sensitive to ATP inhibition than was the enzyme from E. coli. It was therefore suggested that although eukaryotic citrate synthases might be regulated by ATP, in bacteria it is NADH that acts as a feedback regulator of energy production (Weitzman, 1966b). Examination of the citrate synthase of another bacterium, Acinrtobuctrr calcouceticus, showed that it, too, was inhibited by NADH but there were two significant differences from the E. coli enzyme. First, there was a kinetic distinction; the enzyme from E. coli displayed a hyperbolic dependence of inhibition on NADH concentration, whereas with the enzyme from A . culcoucrticus this dependence was sigmoidal. Secondly, the inhibition by NADH could be completely overcome by low levels of AMP in the case of citrate synthase from A . calcoaceticus, but not with the enzyme from E. coli. These differences prompted a survey of citrate synthases from a large range of bacterial genera (Weitzman and Jones, 1968). I t was hoped that such a survey would show whether or not NADH inhibition is a general property of bacterial citrate synthases and would also reveal the incidence of AMP reactivation. The results of this survey, together with additional observations in my laboratory and by other investigators, are presented in Table 2. A quite remarkable pattern emerged from these studies which showed that bacteria fall into distinct groups based on the regulatory properties of their citrate synthases. Classification into two major groups may be made on the

TABLE 2. Regulatory patterns among bacterial citrate synthases NADH inhibition N o AMP reactivation

AMP reactivation anitratus

Aeromonas formicans Arizona arizonae Erwinia uredovora Escherichia coli Hafnia alvei Klebsiella (Aerobacter) aerogenes pneumoniae Pasteurella‘oseudotuberculosis rettgeri Proteus vulgaris anatum Salmonella cholerae-suis typhimurium Serratia marcescens Thiobacillus A2

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AMlb 3A2‘ denitrijcansd Pseudomonas aeruginosa Juorescens ovalis syringae Rhodopseudomonas capsulata suhaeroides Rhodospirillum rubrum Vibrio tyrosinaticus Xanthomonas hyacinthi bacterium 5B 1 (facultative methylotroph)’ ‘

1

N o NADH inhibition Achromobacter liquejaciens* atrocyaneus Arthrobacter globijormis nicotianae cereus megaterium Bacillus polymyxa‘ stearothermophilus subtilis

I

1

Brevibacteriurn

{EZ

Cellulomonas cellasea Clostridium acidi-urici equi

Haemophilus vaginalis* Kurthia zopfii Microbacterium thermosphactum luteus Micrococcus sp,

{

r

phlei rhodochrous smegmatis Nocardia corallina farcinica Pseudornonas iodinum* Staphylococcus aureus ~. somaliensis Streptomyces viridochrornogens Mycobacterium

{

{

michiganense References Weitzrnan (1980); “Senior and Dawes (1971); hAnthony and Taylor (1975); ‘Colby and Zatman (1975b); “Weitzman and Kinghorn (1981b); (‘Tanakaand Hanson (1975). *See Section I1.E. p. 207.

198

P. D J WEITZMAN

basis ofwhether or not thecitratesynthaseisinhibited by NADH. The NADHsensitive group contains the Gram-negative bacteria, whereas the NADH-insensitive group consists of the Gram-positive bacteria. Thus NADH inhibition of citrate synthase is not a general bacterial feature but is restricted to Gram-negative organisms. Within the NADH-sensitive group, a further division may be made between those citrate synthases that show relief of NADH inhibition by AMP and those that d o not. This subdivision coincides with clear metabolic differences between organisms. The citrate synthases that are reactivated by AMP come from strictly aerobic bacteria, whereas those that are insensitive to AMP come from facultatively anaerobic organisms. Weitzman and Jones (1968) proposed a rationale for this regulatory difference based on the different energy-generating metabolic pathways employed by these two classes of bacteria. The strict aerobes are absolutely dependent on the citric acid cycle for energy production and it is therefore desirable that they should regulate the first enzyme of the cycle, citrate synthase, in response to the energy state of the cell. The facultative anaerobes, on the other hand, can generate energy by fermentation, using the citric acid cycle in a biosynthetic mode only. in such a situation, regulation of citrate synthase by positive effector action of AMP would be a pointless mechanism; instead, such facultatively anaerobic organisms require (and possess) glycolytic enzymes responsive to energy signals. As for the distinction between the Gram-negative and Gram-positive bacteria, no rationale suggests itself for the striking difference between the regulatory properties of their citrate synthases. Inhibition by NADH is certainly an attractive and plausible feedback mechanism for the regulation of the citric acid cycle, but why it should occur in Gram-negative bacteria and not in other organisms is a mystery. Perhaps the answer lies in differences in metabolic organization between the two classes of organisms. It is also worth noting that the insensitivity to NADH of their citrate synthases groups the Gram-positive bacteria together with all eukaryotic organisms so far tested. We shall return to this point later.

B . BIOSYNTHETIC CONTROLS

So far the discussion has focused on the role of the citric acid cycle in energy production and the ways in which this is reflected in the control of citrate synthase. Consideration must now be given to the biosynthetic function of the cycle. The two major biosynthetic starting materials generated by the cycle are a-oxoglutarate and succinyl-CoA, necessary for amino acid and porphyrin biosynthesis. In the scheme of 'Fig. 3, these are compounds C, and Cx. When the pathway functions as a complete cycle, these com-

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199

pounds are intermediates rather than end products. However, there are situations in which the a-oxoglutarate dehydrogenase enzyme complex, linking C, and C:, is absent, thus rendering the pathway incomplete. Under such circumstances, a-oxoglutarate and succinyl-CoA have the status of end products rather than intermediates, and we must consider how this might be reflected in new regulatory sensitivities. ace t y I-CoA

oxaloacetate

citrate

J

malate

i

isocitrate

fu marate \

J

succinate

succinyl-CoA

FIG. 4. Branched non-cyclic pathway operating anaerobically

Mention was made earlier of the use by facultative anaerobes of the citric acid cycle in a strictly biosynthetic mode. Under these circumstances, a branched non-cyclic pathway is required to meet the biosynthetic demands for both a-oxoglutarate and succinyl-CoA (Amarasingham and Davis, 1965) (see Fig. 4). In fact with such organisms, for example E . coli, this split pathway probably operates even under aerobic conditions when glucose is the growth substrate and glycolysis the energy-yielding pathway (Amarasingham and Davis, 1965). It is clear that a-oxoglutarate may now be considered the end product of a short sequence of reactions initiated by citrate synthase acting in a purely biosynthetic capacity. The formation of succinyl-CoA does not depend on the activity of citrate synthase, but is achieved by a reversed set of reactions leading from oxaloacetate. I t might thus be anticipated that a-oxoglutarate would have a feedback effect on citrate synthase.

200

P D . J . WEITZMAN

TABLE 3. Inhibition of citrate synthases by a-oxoglutarate. From Weitzman and Dunmore (lY6Ya)

The inhibition of citrate synthase by a-oxoglutarate was first reported by Wright c'f ul. ( 1 967). Weitzman and Dunmore (1969a) confirmed this observation and extended the examination of the effect of a-oxoglutarate on citrate synthase to a wide range of organisms (Table 3). They found that the inhibition by a-oxoglutarate appeared to be restricted to citrate synthases from Gram-negative facultatively anaerobic bacteria; citrate synthases from Gram-negative aerobes, Gram-positive bacteria and eukaryotes showed no effect. a-Oxoglutarate inhibition of citrate synthase is thus a typical case of end-product inhibition of the first enzyme of a biosynthetic pathway and is consistent with the operation of the split pathway of Fig. 4. That inhibition is apparent only in organisms which employ this split pathway lends credence to the view that i r is a control process of physiological significance and extends the diversity of design of citrate synthase to fit the metabolic life-style of the host organism. Taylor (1970) reported aoxoglutarate inhibition of citrate synthase from strictly autotrophic Gramnegative thiobacilli, although these organisms are strictly aerobic. However, these organisms also lack ~~-oxoglutarate dehydrogenase and hence have a-oxoglutarate as a biosynthetic end product of citrate synthase activity. I t may be significant that facultatively autotrophic thiobacilli d o contain a-oxoglutarate dehydrogenase and have citrate synthases that are not inhibited by a-oxoglutarate (Taylor, 1970). a-Oxoglutarate inhibition has also been reported with citrate synthases from the Gram-positive facultative anaerobes Bucillus p o I j m j - . u i and B . muwrutis (Tanaka and Hanson, 1975) and the strict anaerobe Closrrirliimi ucidi-uric; (Gottschalk and Dittbrenner, 1970). I t may therefore be that the effect is not restricted to Gram-

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201

negative bacteria, but is rather a feature of all bacteria that contain an incomplete cycle lacking a-oxoglutarate dehydrogenase.

2 . Inhibition by a-Oxoglutarate and Succinyl-CoA Yet another situation occurs in the cyanobacteria (blue-green bacteria). These Gram-negative bacteria do not contain the a-oxoglutarate dehydrogenase complex and do not use the citric acid cycle for energy production. Inhibition of citrate synthase by NADH would thus be a purposeless mechanism and, indeed, has been found not to operate (Taylor, 1973; Lucas and Weitzman, 1975). The absence of a-oxoglutarate dehydrogenase again makes a-oxoglutarate the end product of citrate synthase action, and cyanobacterial citrate synthases are inhibited by this metabolite (Taylor, 1973; Lucas and Weitzman, 1975). However, in cyanobacteria the formation of succinyl-CoA is believed to occur not by the split pathway of Fig. 4 but rather through the operation of the glyoxylate cycle (Pearce et al., 1969; Lucas, 1974) as shown in Fig. 5. The succinate formed on cleavage of isocitrate by isocitrate lyase may be converted into succinyl-CoA by CoA transfer from acetoacetyl-CoA (Pearce et al., 1969) or directly by succinate thiokinase (Weitzman and Kinghorn, 1980). Thus, according to the scheme of Fig. 5, both a-oxoglutarate and succinyl-CoA are end products of the initial action of citrate synthase, and one might therefore anticipate that succinyl-CoA would be an additional feedback inhibitor of this type of citrate synthase. Lucas and Weitzman (1977) found that succinyl-CoA is indeed acet y I -Co A

i

isocitrate

2 -oxoglutarate

succinate

\,

succinyl-CoA

1

FIG. 5. Modified citric acid cycle operative in cyanobacteria.

202

P. D. J. WEITZMAN

an inhibitor of cyanobacterial citrate synthases; the inhibition is exerted competitively with respect to acetyl-CoA, whereas the inhibition by a-oxoglutarate is competitive with respect to the other substrate, oxaloacetate. No succinyl-CoA inhibition of the citrate synthases of E. coli or A . calcoaceticus was observed, suggesting that the response of cyanobacterial citrate synthases is a specific one and relates to the particular metabolic pathway operative in these organisms. The three schemes shown in Fig 1, 4 and 5 represent the normal citric acid cycle and two variations on it, and it is encouraging to observe that the control of the citrate synthase step through inhibition, respectively, by NADH, a-oxoglutarate, and a-oxoglutarate plus succinyl-CoA is consistent with the diverse functions of this key enzyme in the three metabolic schemes.

C.

MOLECULAR-SIZE PATTERNS

From the foregoing discussion, there emerges quite clearly a picture of the Gram-negative bacterial citrate synthases as functionally more complex than their counterparts from Gram-positive bacteria and eukaryotes. The range of regulatory responses displayed by the citrate synthases from Gramnegative bacteria is clearly absent from the enzyme from other organisms. Moreover, as will be seen in Section I1.D (p. 204), many of the regulatory properties appear to operate via allosteric mechanisms which we invariably associate with the presence of multiple subunits and subtle interactions. This greater functional complexity might well reside in a comparably greater structural complexity, rather like the classic example of myoglobin and haemoglobin. Both of these proteins perform a similar oxygen-carrying function but only the larger haemoglobin molecule exhibits allosteric control properties. Weitzman and Dunmore ( 1969b) explored this possibility at the gross level of molecular size, having noted that the citrate synthase of A . calcoaceticus had a molecular weight of around 250,000, whereas the enzyme from pig heart had been reported to have a molecular weight under 100,000. The molecular sizes of a variety of citrate synthases were estimated by gel filtration of partially purified enzyme preparations on Sephadex G-200 with catalase and lactate dehydrogenase as marker proteins. All the citrate synthases examined fell into two groups-“large” and “small”. The “large” enzymes had a molecular weight of around 250,000 and were eluted from the column of Sephadex G-200 ahead of catalase. The “small” enzymes had a molecular weight of around 100,000 and were eluted after lactate dehydrogenase. Moreover, the “large” citrate synthases were, without exception, all derived from Gram-negative bacteria, whereas

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203

TABLE 4. Molecular sizes of bacterial citrate synthases. From Weitzman (1980)

‘‘Small” citrate synthases A r t h r o h u c ~ t rgloh;/ornii.s ~ Bac~illus.strurothi~rmophilus Buc illus sub t ilis Brevihwtrrium Javuni Brcvihuc.tcrium lincws

the enzymes from Gram-positive bacteria (and eukaryotes) were all of the “small” type. Additional bacterial citrate synthases have subsequently been examined and the data obtained have confirmed this scheme correlating the molecular size of the enzyme and the Gram staining reaction of the source organism (Table 4). These results substantiate the proposal that the more complex regulatory properties of Gram-negative bacterial citrate synthases are linked to a more complex molecular structure and direct the search for an understanding of citrate synthase regulation to a proper knowledge of the quaternary structure and subunit interactions with the enzyme. The question of the precise subunit compositions of the “small” and “large” citrate synthases is not yet unequivocally settled. Mammalian citrate synthase (“small”) has clearly been shown to be a dimer of similar subunits (Wu and Yang, 1970; Singh et a/., 1970; Moriyama and Srere, 1971; Wiegand et a/., 1979). N o subunit studies have so far been conducted on Gram-positive bacterial citrate synthases, but their very similar molecular weights to that of the mammalian enzyme suggests that they, too, are probably dimeric. In the case of the “large” citrate synthases, the enzymes from Acinetohacter calcoaceticus and Azotohacter vinelandii have been suggested to be tetrameric (Johnson and Hanson, 1974); the enzyme from E . coli has been reported to be a tetramer (Wright and Sanwal. 1971; Danson and Weitzman, 1973) but Tong and Duckworth (1975) have presented evidence for a hexameric structure. Recent studies in my laboratory have also furnished evidence for a hexameric structure in the “large” citrate syn-

204

P. D. J. WEITZMAN

thases. Further studies are needed to resolve these conflicting values and to extend the analysis of subunit composition to a larger number of both "small" and "large" bacterial citrate synthases. I t may transpire that the subunits of the different types of citrate synthase will be found to resemble each other closely and that relatively minor differences underlie the different aggregation states encountered (see also Section 1I.G. p. 21 1).

D.

ALLOSTERISM: KINETIC A N D MOLECULAR FEATURES

The two groups of NADH-sensitive citrate synthases show differences in their kinetic behaviour with respect to substrate and NADH. Facultative anaerobes, for example E. c d i , exhibit a hyperbolic dependence of inhibition on NADH concentration (Weitzman, 1966b), whereas a sigmoidal dependence is shown by the citrate synthases of aerobic bacteria (Weitzman, 1967: Eidels and Preiss, 1970; Borriss and Ohmann. 1972; Kleber and Tauchert. 1974; Massarini ot d., 1976; Higa e l ul., 1978); the latter also show a sigmoidal dependence of reactivation on AMP concentration. (Note that the studies reported by Weitzman (1967) were subsequently found to relate to Acinetohacter culcoucc~ric.usand not to E. coli-see Weitzman and Danson, 1976.) With the enzyme from E. coli. inhibition by NADH is competitive with respect to acetyl-CoA, and both competitive and noncompetitive inhibition have been observed with the citrate synthases of aerobic bacteria. The dependence of activity on acetyl-CoA concentration is sigmoidal in the E. c d i group of citrate synthases (Faloona and Srere, 1969; Wright and Sanwal, 1971) but is hyperbolic with enzymes from aerobic bacteria (Eidels and Preiss, 1970; Flechtner and Hanson, 1970; Borriss and Ohmann, 1972; Johnson and Hanson, 1974). It is thus apparent that the different citrate synthases display the characteristics of both the "K" and "V" systems described by Monod rt d . (1965) for allosteric regulatory enzymes. Further, more direct, kinetic evidence for the allosteric nature of the NADH inhibition has been obtained by multiple-inhibition analysis (Harford and Weitzman, 1975). Thus with citrate synthase from Pseudoomonas iirrugitiosu the results indicated that the sites of interaction of the enzyme with acetyl-CoA and NADH were distinct, i.e. NADH does not bind at the active site. When. however. the enzyme from Ps.ucwginosa was desensitized to NADH by chemical treatment (see below) a much weaker response to NADH could be observed: this was no longer a specific inhibition but appeared to be a manifestation of the non-specific general nucleotide inhibition displayed by all citrate synthases, and was isosteric. These kinetic indications of allosteric sites have been confirmed by the

BACTERIAL CITRIC ACID-CYCLE ENZYMES

205

phenomenon of desensitization of the enzymes towards NADH inhibition, without destruction of the catalytic activity. Various treatments have been found to be effective in desensitization. Weitzman ( 1 9 6 6 ~first ) showed that alkaline pH values or the presence of 0 . 2 KCI ~ both overcome the NADH inhibition of the enzyme from E. c d i , and similar findings have been reported with other citrate synthases (Rowe and Weitzman, 1969; Flechtner and Hanson, 1970; Taylor, 1970; Kleber and Tauchert, 1974; Massarini et al., 1976; Weitzman et ul., 1 9 8 1 ~ )Senior . and Dawes (1971) found that the citrate synthase of Azotohacter beijerinckii lost its sensitivity to NADH inhibition in the presence of DTNB, and desensitization with DTNB has been observed with several other bacterial citrate synthases (Weitzman and Danson, 1976; Danson and Weitzman, 1977). These observations implicate thiol groups, either directly or indirectly, in the overall response of the enzyme to NADH. Desensitization by treatment with diethyl pyrocarbonate (Danson and Weitzman, 1973; Iredale and Weitzman, 1981) has likewise implicated histidine residues in the response to NADH of the citrate synthases from E. c d i , A. cal(miceticus and Ps. aeruginosu and photo-oxidation in the presence of the photosensitive dye methylene blue has also been shown to produce desensitization to NADH and to support the indication of histidine involvement (Danson and Weitzman, 1973; Weitzman et al., 1974). The distinct nature of the NADH interaction site has also been shown by immunodesensitization. Rabbit antiserum prepared against purified citrate synthase from A . calcoaceticus exerted a differential effect on the NADH inhibition and activity of the enzyme. Measurements made with increasing amounts of antiserum showed that the sensitivity to NADH was abolished prior to the abolition of the enzymic activity (Weitzman et al., 1981~).This suggests that the antiserum contained antibodies directed against the allosteric NADH site as well as those specific for the active site. The inhibition of activity produced by allosteric interaction of the enzyme with NADH is likely to result from conformational changes in the tertiary and quaternary structure of the enzyme; this has been confirmed in several ways. One simple method of detecting likely conformational changes rests on an examination of thermal inactivation; the temperature range over which loss of activity occurs and the kinetics of inactivation at a fixed temperature can be determined both in the absence and presence of the metabolite effector. Structural changes induced by the latter may well result in an alteration of the thermal inactivation characteristics. The value of such studies in diagnosing conformational changes has been reviewed by Citri (1973). The citrate synthases of both A . calcoaceticus and Ps. ueruginosa are greatly protected against thermal inactivation in the presence of either NADH or AMP (both of which are effectors of these enzymes). However, when similar experiments were performed on the desensitized enzymes (followingtreatment

206

P.

D.J. WEITZMAN

with diethyl pyrocarbonate or DTNB) no protective effects were produced (Iredale and Weitzman, 1981; Weitzman et ul., 1981e). These results are consistent with the view that NADH and AMP modulate enzymic activity by inducing conformational rearrangements within the enzyme molecule; the prevention of such rearrangements by appropriate chemical modification leads to loss of functional sensitivity to the effectors. Rowe and Weitzman (1969) used the direct approach of examining citrate synthase molecules in the electron microscope in the hope that the conformational changes between one state of the enzyme (active) and the other (inactive) might be sufficiently gross to be directly visible. The enzyme was purified from A . culcoucrticus and was examined in the electron microscope by negative-contrast staining with uranyl acetate or ammonium molybdate. The mean diameter of the enzyme molecules was found to increase in the presence of NADH. When measurements were made in 0 . 2 ~ KCl, no such increase was produced by NADH. A minority of the enzyme molecules (about 10%) showed a definite three-fold symmetry and were probably favourably orientated for visualization of substructure. Superimposition of micrographic images derived in the absence and presence of NADH rendered the difference between these two states of the enzyme readily apparent to the unaided eye and suggested that, in the presence of NADH, the enzyme subunits move apart from each other. These negativecontrast studies were complemented by examination of the enzyme by metalshadowing. Again the results indicated a "swelling" of the enzyme in the presence of NADH, and a "contraction" back to the original dimensions in the additional presence of AMP. These structural changes parallel the activity changes associated with the presence of the effectors, i.e. NADH converts the enzyme into an inactive form and AMP produces reversal into the active form. That the observed changes are true reflections of effectorinduced structural alterations, and not artefacts of the electron microscopic examination, was supported by the apparent specificity of NADH in producing the size changes; neither N A D + nor NADPH caused any change in molecular diameter. Further support was obtained from analytical ultracentrifugation studies. NADH produced a retardation in the sedimentation rate of the enzyme whereas NADPH was without effect. In the presence of both NADH and AMP the sedimentation rate was the same as that of the enzyme alone. These hydrodynamic changes are again consistent with effector-induced swelling and contraction of the enzyme. On the basis that regulatory effects depend on conformational flexibility, it should be possible to modify the enzyme chemically so as to "lock" the subunits into a less flexible condition and thereby destroy regulatory sensitivity. Bifunctional cross-linking reagents should be capable of achieving the appropriate modification. This approach has been used successfully by

BACTERIAL CITRIC ACID-CYCLE ENZYMES

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Enns and Chan (1978) who modified aspartate transcarbamylase from E. coli with the bifunctional reagent tartaryl diazide and stabilized the enzyme in a conformational form having high substrate affinity. The modification was accompanied by desensitization towards the allosteric effectors ATP and CTP. Some preliminary experiments on citrate synthase from A . c~alcouceticus using the bis-imidate cross-linking agent dimethyl adipimidate have suggested that it is possible to “freeze” the conformation of the enzyme and thereby desensitize it to NADH (P.D.J. Weitzman and H. A. Kinghorn, unpublished observations). It will be interesting to examine cross-linked citrate synthase by electron microscopy and to see if the swelling/ contraction changes are abolished. Mitchell and Weitzman (1981) treated citrate synthase from A . calcoaceficus with the cleavable cross-linking agent dithiobis (succinimidyl propionate). The modified enzyme was still inhibited by NADH and reactivated by AMP, but the sigmoidal dependences of these two effects on the concentrations of NADH and AMP were transformed to non-cooperative hyperbolic forms. Cleavage of the disulphide bonds in the cross-links with dithiothreitol restored both sigmoidal inhibition and reactivation kinetics. These results suggest that the conformational constraints imposed by the cross-linking prevent cooperative homotropic interactions between the effector sites but still permit the rearrangements that lead to modulation of activity, and emphasize the molecular complexity of this superficially straightforward regulatory process. I t is likely that the a-oxoglutarate inhibition of certain bacterial citrate synthases as well as the a-oxoglutarate and succinyl-CoA inhibitions of the cyanobacterial enzyme are also allosteric processes, as shown by both desensitization and kinetic studies (Wright et ul., 1967; Weitzman and Dunmore, 1969a; Taylor, 1970, 1973; Danson and Weitzman, 1973; Lucas and Weitzman, 1977). It was noted in Section I that the particular role and metabolic location of the citrate synthase reaction prompts anticipation of diverse control processes with concomitant diversity in molecular structure of the enzyme. We now see that this anticipation is amply fulfilled by the observations of specific and subtle design features in the structures of different citrate synthases which confer functional distinctiveness appropriate to the metabolic life-styles of the organisms.

E.

E N Z Y M E C H A R A C T E R I S T I C S AS A N A I D TO B A C T E R I A L T A X O N O M Y

The patterns of regulatory function and molecular size that have emerged from the study of diverse bacterial citrate synthases clearly have some potential as aids to taxonomic studies. Thus there are organisms that give an

208

P . D. J. WEITZMAN

equivocal response to the Gram stain and whose taxonomic classification has been the subject of some dispute. Where the Gram reaction is not clear-cut, resolution of the uncertainty may be achieved by electron microscopic examination of thin sections of the cell wall, the wall structures typical of Gram-negative and Gram-positive bacteria being sufficiently distinctive. However, interpretation of such electron micrographs is also sometimes ambiguous. Thus with Haemophilus vaginalis the Gram stain is not unequivocal and electron micrographs of the cell wall have been interpreted by different workers as being indicative of both a Gram-positive structure (Reyn et a/.. 1966) and a Gram-negative one (Criswell er al., 1972). Moreover, the technique of electron microscopy may not always be available to bacteriologists and is, in any case, a relatively laborious procedure requiring specialized skills for both execution and interpretation. By comparison, some straightforward enzymic tests are simple, quick and inexpensive to perform and therefore constitute an attractive taxonomic tool. Weitzman and Jones (1975) used the properties of citrate synthase in an examination of several bacteria over which there has been some taxonomic uncertainty. The citrate synthases of Achromobucter liquejaciens, Haemophilus vaginalis and Pseudomonus iodinum were each characteristic of Grampositive bacteria, being of the “small” type and insensitive to inhibition by NADH. These three organisms give mixed negative/positive Gram stains and Weitzman and Jones ( 1975) summarized the controversy surrounding their proper classification. The weight of evidence seems to indicate Gram-positive status for these bacteria and the clear-cut citrate synthase results offer additional support to these conclusions. Conversely, the sizes and regulatory sensitivities of the citrate synthases from Cellulomonas rossica and Corynebacterium nephridii showed these organisms to be Gram-negative aerobes and not Gram-positive, as originally proposed. The Gram-negative Aeromonas jormicans metabolically resembles E. coli though it is a polarly flagellated rod and has been grouped with the pseudomonads (Colwell and Liston, 1961). Its citrate synthase was found to be “large”, inhibited by NADH, but not reactivated by AMP; these properties clearly indicate a similarity to the enterobacteria and not to the pseudomonads. The properties of citrate synthase may even provide a first clue to misclassification. In the course of an investigation of the genus Brevihacterium, Jones and Weitzman ( 1974) examined Brevibucterium leucinophagum (ATCC 13809). This organism was described by Kinney and Werkman (1960) as a Gram-positive bacterium which frequently shows Gram-negative staining after 12 hours. However, Jones and Weitzman (1974) found the citrate synthase to be of the “large” type and to be inhibited by NADH and reactivated by AMP, i.e. properties unambiguously indicative of a Gramnegative aerobic organism. These results suggested an error in the original

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209

classification of B . leucinophugum and prompted a thorough examination, including electron microscopy studies. The organism was shown definitely to be Gram-negative and classification within the genus Acinrtobacter was proposed. In view of the potential usefulness of citrate synthase characterization for bacterial identification and classification it seemed desirable to speed up the enzyme tests so that they might be suitable for adoption in bacteriological laboratories. Earlier. it had been shown that the activity and regulatory properties of citrate synthase may be examined in a suspension of bacterial cells rendered permeable to substrates and effectors of the reaction by treatment with toluene (Weitzman, 1973). I t thus seemed possible to extend this in situ examination to bacterial colonies on an agar plate, thereby obviating the necessity to grow up bacteria in liquid medium and prepare a suitable extract. Harford et ul. (1976) showed that this "plate" method allows very rapid examination of bacterial citrate synthases and yields results in total agreement with those obtained by the conventional procedure of assaying the enzyme spectrophotometrically in cell-free sonic extracts. These workers also devised a miniaturized method for molecular-size assessment which permits rapid assignment of a particular citrate synthase to the "large" or "small" group. These rapid micro-methods may find further application in the future.

F.

EXCEPTIONS TO THE ENZYME PATTERNS

The patterns of diversity displayed by bacterial citrate synthases with respect to regulatory function and molecular structure, and their correlation with bacterial groupings, are, like most other biological patterns, not without TABLE 5. Gram-negative bacteria whose citrate synthases are not inhibited by NADH Organism

Reference

~

Ac~>Iohuc,Irr .ylinuni Hulohucti,riuni c,utiruhruni Hulohwt~~riuni licrlohiurn Hulohuc~trriumsulinurium Tlicwnus uyuuticus TI1iohui~illusden it r ijicuns Tliiohucillus tieupolitunus Tlliohuc,illus no vc4lus obligate and restricted facultative methylotrophs

1

Swissa and Benziman (1976) Cazzulo ( 1973) Weitzman and Danson (1976) Weitzman and Kinghorn ( 198 1 a) Weitzman (1978) Taylor ( 1970) Colby and Zatman (1975b)

21 0

P D J WElTZMAN

their exceptions. Several Gram-negative bacteria apparently produce citrate synthases that are not inhibited by NADH and which, in some cases, are of the “small” type, generally characteristic of Gram-positive organisms. These anomalous bacteria are listed in Table 5. Swissa and Benziman (1976) found that the enzyme from Acetobacter xylinum was not inhibited by NADH although its molecular weight is similar to that of other “large” citrate synthases. Experiments in the author’s laboratory have suggested that other Acetobacter species may also produce this anomalous type of citrate synthase. I t was at first thought possible that the Acetobacter enzyme is exquisitely susceptible to desensitization, but exploration of this possibility has produced no supportive experimental evidence. In fact the incidence of a “large” NADH-insensitive citrate synthase amongst Gram-negative bacteria may be more widespread and may have to be reckoned with as a distinct sub-group of the enzyme. Thus, preliminary investigations on a number of species of Branhamella, Kingella, Moraxella and Neissrria have also shown the presence of a “large”, but NADHinsensitive, citrate synthase (P. D. J. Weitzman and K. Bervre, unpublished observations) and further studies will be required to ascertain whether the occurrence of this enzyme can be correlated with the particular metabolic mode of these organisms. We shall return to a further consideration of this citrate synthase variant in Section II.G, p. 214. Rather different are the citrate synthases of Halobacterium spp. and Thermus aquaticus. These enzymes are unaffected by NADH and are of the “small” type (Cazzulo, 1973; Weitzman and Danson, 1976; Weitzman 1978). Although both types of organism are Gram-negative, they have adapted to a rather extreme mode of existence; the halobacteria grow only in the presence of very high (about 4111) salt concentrations (Larsen, 1973) and T . aquaticus has an optimum growth temperature of 70 C (Brock and Freeze, 1969). It may be that under these extreme, disruptive conditions the smaller enzyme molecule is better able to maintain its native structure than the larger, more polymeric, form. As the “large” type of citrate synthase appears to be necessary for NADH sensitivity it may be that loss of NADH inhibition is a consequence of the adaptive modification of the enzyme in order to retain catalytic activity under extreme conditions. Since the NADH inhibition of citrate synthase is always overcome by elevated salt concentration (see Section II.D, p. 205) Cazzulo (1973) has speculated that the adaptation of halobacteria from a pseudomonad ancestry to halophilic life rendered the NADH inhibition of its citrate synthase physiologically useless; the inhibition sensitivity, together with the requisite subunit structure, was therefore lost in the course of evolution. Of the thiobacilli, Thiobacillus denitrificans and T . neapolitunus are both strictly autotrophic and lack the enzyme a-oxoglutarate dehydrogenase

BACTERIAL CITRIC ACID-CYCLE ENZYMES

21 1

(Taylor et d., 1969; Kelly, 1967). The insensitivity of their citrate synthases to NADH (Taylor, 1970) may be considered as consistent with the absence of a complete oxidative citric acid cycle. Taylor (1070) found that the citrate synthase of the facultative autotroph Thiohacillus A2 was inhibited by NADH whereas the enzyme from T. novellus, also a facultative autotroph, was unaffected. These facultative autotrophs possess a complete citric acid cycle and so the absence of NADH inhibition in the case of T. novrllus is not readily explicable. The sensitivity of the enzyme from Thiohacillus A2, but not that from T. novellus, to NADH may contribute to the better heterotrophic potential of the former organism (Taylor and Hoare, 1969). Unfortunately no determinations of molecular size have yet been reported for the citrate synthases of the thiobacilli, so that it is not known whether their insensitivity to NADH is associated with the presence of the “small” enzyme or whether they represent further examples of the “large”, insensitive enzyme form. Colby and Zatman (1975b) examined citrate synthase from several Gramnegative methylotrophs. Inhibition by NADH and reactivation by AMP was observed with the enzyme from two typical facultative methylotrophs, consistent with the NADH-generating role of the cycle when these organisms grow on non-C, compounds. Similar results were obtained by Anthony and Taylor (1975) with the facultative methylotroph Pseudomonus AM 1. On the other hand, the cycle does not fulfil an energy-yielding role in obligate and restricted facultative methylotrophs and NADH was found not to inhibit the citrate synthases of organisms representative of these classes. N o information is available on the molecular sizes of these insensitive enzymes. Two other apparent exceptions have been reported but these have been refuted by other workers. Flechtner and Hanson (1970) claimed that the citrate synthase of Rhodospirillum rubrum was insensitive to NADH, but Eidels and Preiss (1970) and Massarini et a/. (1976) found that NADH does inhibit this enzyme. Similarly, the report by Johnson and Hanson (1974) that the enzyme from Acinrtobacter unitratus was unaffected by NADH could not be confirmed (Weitzman and Danson, 1976).

G.

M U T A N T S : D Y S F U N C T I O N AS A C L U E T O F U N C T I O N

Bacterial mutants either deficient in citrate synthase activity or containing an active but modified form of the enzyme are potentially valuable tools for studies of the physiological role of the enzyme and its apparent regulation. Several workers have isolated and studied mutants devoid of citrate synthase. Gilvarg and Davis (1956) isolated such mutants from E. coli W and Aerobacter aerogenes after mutation by ultra-violet irradiation followed

21 2

P. D. J. WEITZMAN

by penicillin enrichment and selection for glutamate auxotrophs. These mutants displayed a nutritional requirement for glutamate or a-oxoglutarate, had virtually completely lost the ability to oxidize acetate and showed greatly decreased ability to oxidize glucose to carbon dioxide. These results constituted important evidence for the operation of the citric acid cycle in bacteria. Ashworth et al. (1965) isolated a citrate synthase-deficient mutant of E. coli K 12 by mutagenesis with N-methyl-N’-nitro-N-nitrosoguanidine followed by selection for glutamate auxotrophs; by using the mutant in recombination experiments they were able to map the position of the citrate synthase gene within the E. coli chromosome. Carls and Hanson (1971) obtained various citric acid-cycle mutants of Bacillus subtilis by heating spores and then plating them out on nutrient-agar containing calcium carbonate. Mutants lacking citric acid-cycle enzymes accumulated organic acids which resulted in the formation of halos around mutant colonies by solubilizing the calcium carbonate. Among such mutants, some were deficient in citrate synthase. Lakshmi and Helling ( 1976) isolated citrate synthase-deficient mutants of E. coli K12 as double mutants also lacking isocitrate dehydrogenase. They found that E. coli mutants lacking isocitrate dehydrogenase grew very slowly on medium containing glucose plus glutamate, but were overgrown by other mutants found, additionally, to lack citrate synthase. It was suggested that isocitrate dehydrogenase-deficient mutants produce a build-up of citrate or isocitrate which inhibits growth; mutants additionally devoid of citrate synthase cannot produce such an accumulation and thus avoid this growth inhibition. Beatty ef al. (1977) investigated a mutant of Rhodopsrudomonas c.ap.suluta isolated after N-methyl-N’-nitro-N-nitrosoguanidine mutagenesis for its inability to grow on minimal malate plus NH,fmedium without added glutamate, and found that it contained virtually no citrate synthase or isocitrate dehydrogenase. These enzymic defects incapacitate the mutant for a-oxoglutarate synthesis and thus explain the growth requirement for glutamate. On the basis of the frequencies with which prototrophic recombinants and spontaneous revertants were obtained, it was concluded that a single-site mutation was responsible for the loss of both enzyme activities. It was suggested that the mutation may inactivate a regulatory element essential for expression of both enzymes or may be a polar mutation in an operon containing the structural genes for both enzymes. To complement chemical modification studies aimed at identifying particular groups in the enzyme which participate in its catalytic and regulatory functions (Danson and Weitzman, 1973, 1977) the mutant approach has been employed in studies of enzyme structure and function. The aim has been to isolate mutant organisms producing variant forms of citrate synthase with genetically modified regulatory properties. Studies on such mutant enzymes may yield information on the molecular basis of the regulatory sensitivities

21 3

BACTERIAL CITRIC ACID-CYCLE ENZYMES

while examination of any physiological dysfunction in the mutants arising from alterations to the enzyme may throw some light on the normal operation in vivo of regulatory effects. A two-stage mutation strategy was adopted. First, citrate synthase-deficient mutants were produced and, secondly, revertants were selected which had regained citrate synthase activity. Such revertants would arise from the occurrence of a second mutation in the citrate synthase gene which would effectively compensate for the initial (inactivating) mutation. The citrate synthase of such a revertant strain would probably have a slightly altered amino acid sequence compared with that of the enzyme from the parent organism and it was hoped that these structural changes might lead to changes in the molecular and regulatory properties of the enzyme. The examination of a number of revertants was facilitated by use of the rapid plate-scanning method (Harford et al., 1976) referred to earlier. When this approach was applied to E. coli (Danson et al., 1979) several revertants were isolated which appeared to have citrate synthases less sensitive to the inhibitors NADH and a-oxoglutarate than was the enzyme from the original, wild-type strain. In one particular revertant, the enzyme appeared completely unaffected by these inhibitors and a detailed investigation of this mutant and its variant citrate synthase was therefore undertaken. The enzyme was partially purified and it was confirmed that even 2 m NADH ~ produced no inhibition of activity whereas, under similar assay conditions, as little as 0.1 mM NADH caused almost total inhibition of the wild-type enzyme. Similarly, the mutant enzyme was only weakly responsive to aoxoglutarate, the K , being more than 50 times greater than for the wildtype enzyme. In addition to these regulatory differences, the mutant enzyme showed marked kinetic differences from the wild-type enzyme. Whereas the latter displayed sigmoidal dependences of rate on the concentration of both substrates, the mutant enzyme showed a hyperbolic dependence in each case. TABLE 6. Comparison of some properties of citrate synthases from wild-type Escherichiu coli, mutant Escherichiu coli and pig heart. From Danson et ul. ( 1979). Property

Wild-type E. coli

Mutant E . coli

Molecular weight (approx.) Substrate dependences Acetyl-CoA K , or [S],), (PM) Oxaloacetate K,,, or [S],, (VM) Inhihition by 1 mM a-oxoglutarate" Inhibition by 0.1 mM NADHb (7;)

230,000 Sigmoid 400 55 75 90

100,000 100,000 Hyperbolic Hyperbolic 11 7 10 10

(yo)

"Determined at an oxaloacetate concentration of twice the K,,, value. Determined at an acetyl-CoA concentration of half the K, value.

Pig heart

10

14

0

0

214

P D. J. WEITZMAN

Marked decreases in the two K,,, values were also observed with the mutant enzyme. Indeed, the properties of the mutant enzyme were very similar to those of a typical Gram-positive bacterial, or eukaryotic, citrate synthase. This resemblance extended also to the demonstration of sensitivity of the mutant enzyme to isosteric inhibition by adenine nucleotides-a sensitivity that is probably related to the very much lower K, value for acetyl-CoA of the mutant enzyme. Finally, determination of the molecular weight of the mutant enzyme by a combination of active-enzyme centrifugation and gel filtration gave a value of 100,000. The mutant enzyme is thus both structurally and functionally closely similar to the “small” type of citrate synthases, and Table 6 summarizes the properties of the mutant enzyme in comparison with those from wild-type E. coli and pig heart. The comparison is striking and shows that the mutational events have resulted in the conversion of a “large” citrate synthase into a “small” form with all the associated catalytic and regulatory alterations. That the mutant was still a strain of E. coli was confirmed by Gram staining and electron micrographic examination, various diagnostic tests for E. coli, and by retention of auxotrophic requirements characteristic of the original strain. There still remained the possibility that the “small” enzyme of the mutant was a product of a different gene from that coding for the normal “large” enzyme of E. coli. However, gene mapping by conjugation and phage transduction led to the conclusion that the gene yielding the “small” mutant enzyme is a modified form of the gene coding for the ‘‘large’’ enzyme in the wild-type strain. Consequently it appears that relatively minor genetic alterations can result in the conversion of a Gram-negative bacterial type of citrate synthase to the Gram-positive (and eukaryotic) type and this raises the possibility that Nature’s own diversity of citrate synthases may have been achieved by minor genetic changes. This mutant of E. coli has also provided useful information on the likely physiological significance of the a-oxoglutarate inhibition of citrate synthase from wild-type E. coli. When streaked onto a glucose minimal medium agar plate lawned with a glutamate auxotrophic strain of E. coli, the mutant appeared to overproduce a compound capable ofcross-feeding the auxotroph. Wild-type E. coli, on the other hand, does not cross-feed the auxotroph. These observations suggest that the mutant, but not the wild-type, overproduces glutamate, or a closely related metabolite, and this may well be the result of loss of feedback control of citrate synthase by a-oxoglutarate. Feedback control in the wild-type E. coli may therefore be a significant process governing the synthesis of a-oxoglutarate and glutamate. In other studies of revertants from citrate synthase deficiency, Harford and Weitzman (1978) found that three distinct types of E. coli revertant citrate synthases could be identified. One type was “large” with kinetic and

BACTERIAL CITRIC ACID-CYCLE ENZYMES

21 5

regulatory properties very similar to those of the enzyme of wild-type E. coli. A second type was also “large” but otherwise had properties like those

of “small” citrate synthases. The third type, of which the mutant described above is representative, was “small” and had properties typical of the “small” enzymes. It will be recalled that some naturally occurring citrate synthases are also “large” though they lack the functional properties normally associated with the “large” enzyme; Acetohacter xylinum is an organism containing such a variant (Swissa and Benziman, 1976). Thus, starting from a single citrate synthase-deficient mutant of E. coli, a family of mutants can be isolated containing citrate synthases resembling those occurring naturally in diverse bacteria, for example, E. coli, Acetobacter sp. and Gram-positive bacteria. The obvious genetic relatedness of these mutant enzymes, despite their molecular and functional differences, re-emphasizes the possibilities that the natural enzymic diversity results from minor genetic changes and that the amino acid sequences of the different enzymes may well prove to have extensive homology. Mutant citrate synthases have also been obtained from the Gram-negative aerobe Acinetobacter calcoaceticus (Weitzman et al., 1978). A similar strategy was employed to that described above for E. coli. However, the isolation of citrate synthase-deficient mutants of A . calcoaceticus proved a problem because of its insensitivity to penicillin and the consequent ineffectiveness of the penicillin enrichment procedure for mutant isolation. This led to the devising of a novel method for obtaining citrate synthase-deficient organisms (Harford and Weitzman, 1980) relying on the long-known lethal effect of fluoroacetate which is believed to be exerted after its conversion via citrate synthase, into fluorocitrate (Peters, 1957). Mutants devoid ofcitrate synthase cannot produce fluorocitrate and should therefore be resistant to the toxic effect of fluoroacetate. Selection for resistance to fluoroacetate following mutagenesis with ethylmethanesulphonate proved effective for isolating A . calcoaceticus mutants devoid of citrate synthase and hence requiring glutamate for growth. Auxotrophic strains of Acinetobacter have been shown to be competent for genetic transformation to prototrophy (Juni and Janik, 1969; Juni, 1972). Weitzman et al. (1978) found that citrate synthasedeficient mutants of A . calcoaceticus may be transformed to prototrophy by crude preparations of DNA not only from wild-type A . calcoaceticus but also from Ps. aeruginosa. Two classes of transformant citrate synthases were obtained after using DNA from the latter organism. One class consisted of “large” enzymes which showed inhibition by NADH and reactivation by AMP and were completely inactivated by antiserum raised against purified enzyme from wild-type A . calcoaceticus. The other class was of “small” enzymes which were insensitive to NADH and were unaffected by the antiserum. These, and other properties, showed that the two groups of

21 6

P. D. J. WEITZMAN

transformant citrate synthases clearly resemble, on the one hand, the wildtype enzyme and, on the other, the Gram-positive bacterial enzyme. Thus, once again, gene mutability resulting in conversion from a Gramnegative bacterial enzyme into a Gram-positive bacterial type has been demonstrated. The results with both E. coli and A . calcoaceticus citrate synthase variants underline the importance of subunit association and the “large” enzyme structure for the expression of regulatory sensitivity towards NADH, AMP and a-oxoglutarate. The “conversion” from “large” into “small” form suggests that both forms may have rather similar subunits, but that minor, though subtle, changes in the structure of those subunits may crucially affect the extent of their polymerization as well as their functional cooperation. Future comparative studies on these enzyme variants should yield valuable information on the molecular basis of the subunit assembly and of the regulatory behaviour. Although the diversity of naturally occurring citrate synthases discussed in earlier sections makes the comparative study of such enzymes attractive for probing structure--functionrelationships, the generation, from the same initial organism, of variant forms that mimic in function and gross structure the diverse forms occurring naturally offers an even more attractive system for investigating such relationships. The mutational events leading to the artificial variants are likely to have caused much smaller alterations in the primary sequence of the enzyme than occur between the different enzymes of naturally occurring organisms. Thus the normally encountered complications of evolutionary divergence between natural variants may be overcome and clearer identification of crucial residues or structural features may more readily be achieved. Some quite novel findings have recently been made in studies on a mutant of Ps. arruginosa. The mutant was originally isolated by Skinner and Clarke (1968) for its inability to grow on acetamide or acetate and was found to have very low citrate synthase activity (about 7% of that of the wild type). Solomon and Weitzman (1981) investigated this mutant, initially in the hope that it might be possible to use it as a citrate synthase-deficient organism from which revertants might be produced; it was anticipated that enzyme variants might thereby be obtained with Ps. aeruginosa as had been produced for E. coli and A . calcoaceticus. In the event, however, it was found that the citrate synthase present in low amounts in the Ps. aeruginosa mutant was itself different from the wild-type enzyme in being “small”. I t thus appeared that the Ps aeruginosa mutant as isolated by Skinner and Clarke (1968) was affected not only in its level of production of citrate synthase but that some mutation in the structural gene for the enzyme had occurred resulting in loss of the normal “large” type of enzyme and production of the “small”. Like other “small” citrate synthases, this mutant enzyme showed

B A C T E R I A L CITRIC A C I D - C Y C L E E N Z Y M E S

21 7

no inhibition by N A D H . When gel-filtration elution profiles were examined it was observed that, although the major portion of the enzynls behaved

like the “small” form. a minor peak of activity emerged from the column in the position expected for the “large” enzyme. I t was though( possible that the two forms might arise from a n association dissociation equilibrium. but when fractions corresponding to each form were re-run on a second gelfiltration column n o sign of a redistribution between the two forms was observed. The possibility ofcontamination o f t h e mutant culture with another organism was also made unlikely by repeated single-colony isolation of the mutant with retention of this “double enzjme” phenomenon. Moreover, all single colonies tested were Gram-negative. These observations suggest that both forms of the enzyme are present in the mutant bacterium in contradiction to the general rule that organisms contain only one form or the other. The occurrence of two forms of citrate synthase has also been reported by M a rini and Cazzulo (1975) in a marine pseudomonad. These workers suggested that the “small” enzyme might be derived from the “large” form by dissociation. and dialysis of the “large” enzyme against dilute phosphate buffer was shown to lead to dissociation to the “small” form with loss of regulatory properties. In the case of the mutant from Ps. urruKinosu, n o evidence of a similar dissociation was found but, as with the marine pseudomonad, the activity of the “large” enzyme was stimulated several-fold by low concentrations of AMP, as well as being inhibited by N A D H and reactivated by A M P . Solomon and Weitzman (1981) examined the proportions of “large” and “small” citrate synthase at different stages of growth of a batch culture of the P.Y.ucruginosu mutant, and uncovered a quite remarkable situation. In early exponential phase the enzyme was present predominantly in the “large” form. I n later exponential phase and into stationary phase this ”large” citrate synthase disappeared and wan replaced by the ”small” form of the enzyme. The changes were readily apparent on following the extent of A M P stimulation of the enzymic activity in extracts of cells harvested at different stages (the ”small” enzyme was completely unaffected by A M P ) and were confirmed by gel filtration of these extracts. These novel findings would appear to hold considerable potential for further studies on the citrate synthase system. The apparent changeover in viw from one form of the enzyme to the other suggests that this is in response to changing physiological demands on the enzyme. Thus it may be that during the exponential phase of batch culture the fast rate of growth of the cells and the consequent demands for energy and biosynthesis imposed on the citrate synthase are best served by the construction and action of the ”large” enzyme with its attendant regulatory features. The rather different demands made by the decreased, or zero, growth rate as the culture

21 8

P D J WEITZMAN

goes into stationary phase may, on the other hand, be satisfactorily met by the presence of the “small” (unregulated) enzyme. This is, of course, entirely speculative and the reasons for the occurrence of the changeover in this mutant, but not in the wild-type organism, will need to be explored. Another example of the change in vivo of an enzyme between two molecular size forms is that of aspartate transcarbamylase from Citrobucter jreundii (Coleman and Jones, 1971). In early exponential phase, only a small form of the enzyme (molecular weight about 93,000) was present, but from midexponential phase increasing amounts of a large form of the enzyme (molecular weight about 250,000) were produced until, in stationary phase, only the large form was present. The two forms differed in their response to ATP, the small enzyme being inhibited and the large enzyme being activated. This type of enzyme conversion in viw at the level of oligomeric structure in response to physiological factors may constitute an additional facet of the complex phenomenon of enzyme regulation, and suitable investigation may reveal other examples of its occurrence. In the case of citrate synthase and the Ps. ueruginosu mutant the presence, and conversion in vivo, of the two forms may be a valuable aid to studies on structure-function relationships among the “large” and “small” citrate synthases. I t is intriguing that the “large” and “small” forms now appear to occur in three different sets of conditions: (a) different types of organisms; (b) different mutant forms of an organism; and (c) different physiological states of a single organism. Exploration of the molecular basis of the interconversion taking place in the latter circumstances may provide clues to the identity of the molecular differences inherent in the enzyme diversity of the former cases.

111. Succinate Thiokinase

Succinate thivkinase (succinyl-CoA synthetase) catalyses the only reaction of the citric acid cycle in which a nucleoside triphosphate is produced. The reaction is one of substrate-level phosphorylation of a nucleoside diphosphate, the energy being provided by cleavage of the thioester succinyl-CoA: Succinyl-CoA + N D P + P, = Succinate + NTP + CoA-SH N D P and NTP represent nucleoside di- and triphosphate respectively. Based on the apparent specificity which they display for these nucleotides, two distinct classes of succinate thiokinases have been identified-one from animal sources which utilizes G D P and is designated EC 6.2.1.4 and one

BACTERIAL CITRIC ACID-CYCLE ENZYMES

21 9

from plant and bacterial sources which utilizes ADP and is classified as EC 6.2.1.5. We shall see shortly, however, that many bacterial succinate thiokinases appear to be able to utilize more than one nucleotide as substrate. The complexity of the reaction catalysed and the participation of a phosphorylated form of the enzyme molecule have resulted in considerable attention being given to the structure and mechanism of action of this enzyme. Moreover, the reversibility of the reaction and its capacity to generate succinyl-CoA for biosynthetic purposes at the expense of ATP (or GTP) confer on the enzyme both a catabolic and an anabolic role. The reader is referred to two review articles (Nishimura and Grinnell, 1972; Bridger, 1974) for discussion of general properties of this enzyme not covered here.

A. M O L E C U L A R - S I Z E P A T T E R N S

The succinate thiokinases from pig heart and E. coli have been investigated most thoroughly and one striking difference to emerge is in their molecular weights. The enzyme from E. coli has a molecular weight of 140,00~150,000 whereas the enzyme from pig heart is approximately half this size with a molecular weight of 70,000-75,000, Furthermore, the enzyme from E. coli is a tetramer made up of two different types of subunit (azpz), whereas the enzyme from pig heart is a dimer of ap structure. Kelly and Cha (1977) found the molecular weights of succinate thiokinases from three other Gram-negative bacteria to be around 155,000, i.e. very close to that of the enzyme from E. coli. This led to the implicit assumption that all bacterial succinate thiokinases resemble that of E. coli. However, the molecular-size difference between the bacterial and eukaryotic enzymes is very reminiscent of the differences between E. coli and eukaryotic citrate synthases and prompted a survey of the molecular sizes of a range of succinate thiokinases (Weitzman and Kinghorn, 1978). As with citrate synthases, this was performed by gel filtration through Sephadex G-200 using lactate dehydrogenase (molecular weight about 140,000) as a marker protein. These studies were facilitated by the development of a polarographic assay for succinate thiokinase rather similar to that used with citrate synthase; the formation of CoA-SH from succinyl-CoA is monitored directly and continuously with a dropping mercury electrode. Consistent with the molecular weights quoted above, the succinate thiokinase from E. cwli was eluted from the Sephadex column slightly ahead of lactate dehydrogenase whereas the enzyme obtained from pig heart emerged considerably later. All the other organisms tested had succinate thiokinases that conformed to one of these two types, i.e. “large” or “small”, and the striking result was that only Gram-negative bacteria produced the “large” enzyme, Gram-positive bac-

220

P. D J WEITZMAN

teria and eukaryotes producing only the “small” enzyme. There is thus a remarkable correlation between the incidence of “large” and “small” succinate thiokinases and that of “large” and “small” citrate synthases, and Weitzman and Kinghorn (1978) speculated that there might be some evolutionary link between the two enzymes. Clearly, all bacterial succinate thiokinases d o not resemble that of E . r d i ; rather, the succinate thiokinases from Gram-positive bacteria resemble, at least in molecular size, the enzymes from eukaryotes. T o complement the data on citrate synthase from cyanobacteria, Weitzman and Kinghorn ( 1980) examined succinate thiokinase from these Gram-negative organisms. It has been reported that succinate thiokinase is absent from cyanobacteria (Pearce (’/ u/., 1969). However, by use of the polarographic assay method which is considerably more sensitive than other assays used for succinate thiokinase activity, and which is not affected by the high light absorbance of extracts (Weitzman, 1976) such as are produced from cyanobacteria. Weitzman and Kinghorn ( 1980) demonstrated the presence of succinate thiokinase activity in each of several cyanobacteria tested, and in every case the enzyme proved to be of the “large” type. I t will be recalled that, with citrate synthase, Thrrmus uyuuricus and Hulohuctc~riumspp. exhibited anomalous behaviour in that, although Gramnegative, they produced a ”small” enzyme. Weitzman and Kinghorn ( 1 981a) therefore examined the succinate thiokinases of these organisms and found them to be “large” and thus to conform to the pattern of correlation of enzyme molecular size with the Gram staining character of the bacteria. I t thus appears that the “large” succinate thiokinase is an even more rigidly conserved feature of Gram-negative bacteria than is the “large” citrate synthase, and characterization of succinate thiokinase is thus a potentially useful tool in bacterial classification (Weitzman er d., 1981b). A summary of the molecular-size pattern of succinate thiokinases from diverse organisms is presented in Table 7.

B.

NUCLEOTIDE-SPECIFICITY PATTERNS

The nucleotide specificity of succinate thiokinase from a variety of sources has been examined. The mammalian enzyme was specific for guanine and inosine nucleotides (Sanadi et al., 1956) although an adenine nucleotidelinked thiokinase of animal origin has been reported (Hansford, 1973). On the other hand, the enzyme isolated from plant sources appears to be specific for the adenine nucleotides (Kaufman and Alivisatos, 1955; Nandi and Waygood. 1965; Palmer and Wedding, 1966; Bush, 1969; Wider and Tigier, 1971: Fluhr and Harel, 1975) as was also reported for the enzyme from

BACTERIAL CITRIC ACID-CYCLE ENZYMES

22 1

TABLE 7. Molecular sizes of succinate thiokinases. From Weitzman (1980) ~~

“Small” enzyme Bacillus niegutcriuni Bucillus steurotlii~rmopliilus Brevibuctc~riuniliniws Corytiehacteriuni ruhruni

Baker’s yeast Cauliflower mitochondria Wheat germ Pig heart Pig liver

E. coli (Smith et a/., 1957; Gibson et al., 1967). As with molecular size, so with nucleotide specificity has it been suggested that bacterial succinate thiokinases might generally resemble that of E . coii and be specific for ADP/ATP (Bridger cf ul., 1969). However, the enzyme from Rhodopseudomonu.~sphueroidcs has been found to utilize ATP, G T P and ITP (Burnham. 1963) and further examination of the enzyme from E. mli showed that G T P and ITP can also serve as substrates, albeit less effectively than ATP (Murakami et a/., 1972). Several other bacterial succinate thiokinases were examined by Kelly and Cha (1977) and shown to be active with both adenine and guanine nucleotides with varying relative effectiveness. In view of the molecular-size differences among diverse bacterial thiokinases discussed above, Weitzman and Jaskowska-Hodges ( 1981) undertook a more extensive study of the nucleotide ‘preferences’ of the enzyme from a range of bacteria drawn from diverse taxonomic groups. Again the polarographic assay method proved a particular advantage as it can be used even at very high nucleotide concentrations, whereas the 235 nm spectrophotometric method (Cha, 1969) is subject to interference even at moderate nucleotide concentration. Several classes of succinate thiokinase, distinguished by their nucleotide substrate specificities, were shown to exist in different groups of bacteria. Approximate K,,, values were determined for ADP and G D P and the

P. D. J. WEITZMAN

222

bacteria were classified into four groups along the following lines. The succinate thiokinases of one group of bacteria, exemplified by A . calcoaceticus, showed a very high K, for ADP (about l m ~ but ) a much lower K , for G D P (about 0.02 mM). The enzyme of another group, exemplified by Ps. aeruginosa, showed similar and low K, values ( < 0.05 mM) for both ADP and GDP. E. coli is typical of a third group whose succinate thiokinase exhibited a low K,,,for ADP (about 0.01mM) but a significantly higher K,,, for G D P (about 0.lmM). Finally, a fourth group of bacteria had succinate thiokinases which operated with ADP ( K , < 0.1 mM) but which showed no activity with GDP. These groups of bacteria are listed in Table 8. These findings confirmed previous reports (Burnham, 1963; Murakami et al., 1972; Kelly and Cha, 1977) that some bacterial thiokinases are active with both adenine and guanine nucleotides and thus clearly repudiate the belief that the bacterial enzyme is specific for adenine nucleotides.

TABLE 8. Nucleotide specificity patterns of bacterial succinate thiokinases. From Weitzman and Jaskowska-Hodges (1981) ~

Group

Organism

K , ADP

K, G D P

I

Acinetohacter anitratus Acinetohacter calcoaceticus Bordeteh bronchiseptic,a Brevihacterium Ieucinophagum Chromohacterium violaceum Mimu polymorpha Xanthomonas hyacinthi

Very high

Low

Low

Low

111

IV

Alcaligenes jaecalis Arizona arizonae Cellulomonas cellasea Escher ichia coli Klehsiella (Aerohacter) ac~rogcwes Serratia murceswns Arthrobacter simpler Bacillus megaterium Bacillus stearothermophilus Brevihacterium linens Kurthia zopfii

1

High

Low

N o activity

223

BACTERIAL CITRIC ACID-CYCLE ENZYMES

Examination of Table 8 shows that the classification of bacteria according to the nucleotide substrates utilized by their succinate thiokinase has led to some grouping together of bacteria with established taxonomic relatedness. Groups I, I1 and I11 are all Gram-negative bacteria, whereas Group IV is comprised of Gram-positive bacteria. The enterobacteria are collected into Group 111 whereas the pseudomonads and related genera come together in Group 11. The organisms in Group I share several additional very distinctive features of some other citric acid-cycle enzymes (pyruvate, isocitrate and a-oxoglutarate dehydrogenases), as we shall see later, which are not displayed by the bacteria in the other three groups. These results suggest that there are enzyme patterns based on substrate diversity which distinguish groupings of bacteria previously identified by other taxonomic criteria. I t remains to be seen whether the examination of even more bacteria will support this view and also whether it will be possible to relate the patterns of nucleotide specificity of their succinate thiokinases to other physiological differences between the various organisms.

1V. Isocitrate Dehydrogenase lsocitrate dehydrogenase catalyses the first oxidative decarboxylation reaction of the citric acid cycle (phase (b) of Fig. 3): Isocitrate

+ NAD(P)+ -+

a-Oxoglutarate

+ NAD(P)H + H + CO, +

In eukaryotes the enzyme is present in both an NAD- and an NADP-linked form. The NAD-linked enzyme is confined to the mitochondria and displays kinetic and regulatory properties (sigmoidal substrate dependence, activation by AMP or ADP) suggestive of a key controlling role in the energy-yielding function of the cycle (Atkinson. 1966; Plaut, 1970). The NADP-linked isocitrate dehydrogenase of eukaryotes has not been identified as a regulatory enzyme. A few bacteria also contain both NAD- and NADP-linked isocitrate dehydrogenases, e.g. Acetobacter peroxyduns (Hathaway and Atkinson, 1963), Acetobacter aceti (Greenfield and Claus. 1969), Xanthomonas pruni (Ragland ct al., 1966), some thiobacilli (Matin and Rittenberg, 1971), Hydrogenomonus eutropha (Glaeser and Schlegel, 1972) and several methylotrophs (Colby and Zatman. 1975a). There have also been some reports of bacteria apparently containing only NAD-linked isocitrate dehydrogenase activity, e.g. Streptococcus hovis (Burchall et al., 1964), Acetobacter suboxydans (Greenfield and Claus, 1969) and Thiobucillus thiooxiduns (Hampton and Hanson, 1969; Matin and Rittenberg, 1971). However, the kind of regulatory

224

P. D. J. WEITZMAN

properties exhibited by the NAD-linked isocitrate dehydrogenases of eukaryotes have not been observed with any of these bacterial NAD-linked enzymes. The majority of bacteria appear to contain only NADP-linked isocitrate dehydrogenase. This difference from the eukaryotic situation is presumably a reflection of the absence of mitochondria from prokaryotes and hence of compartmentation between the energy-generating and biosynthetic roles of the isocitrate dehydrogenase reaction. Inhibition of isocitrate dehydrogenase by ATP has been observed with the enzyme from several bacterial sources, e.g. Sulmonc~llatyphimuriuvli (Marr and Weber, 1968). P.s.,fluorrscrtis and T. thioo.uidms (Hampton and Hanson, 19691, H~~~rogrtioriiotius eutroplicr (Glaeser and Schlegel, 1972) and Acinctohuctrr cdcoucx~ticus(Kleber and Aurich, 1976) and it has been suggested that this effect may contribute to the energy control of the citric acid cycle in these organisms. The activation of the NADP-linked enzyme of Hydroget1omonu.s eufropliu by AMP and ADP (Glaeser and Schlegel, 1972) may also be a component of such control. Another, and more widespread, inhibitory effect which has been observed is that produced by glyoxylate plus oxaloacetate. Low concentrations of mixtures of glyoxylate and oxaloacetate have been found to exert a concerted inhibition of isocitrate dehydrogenases from a variety of organisms (Shiio and Ozaki, 1968; Hampton and Hanson. 1969; Marr and Weber, 1969, 1971; Barrera and Jurtshuk. 1970; Charles, 1970; Glaeser and Schlegel, 1972; Self of ul., 1973; Ingebretsen, 1976) but the physiological significance of this effect is doubtful. In the course of examining isocitrate dehydrogenase from Acinetohucter litwifi (cdcoucrricus)Weitzman (unpublished observations) found that with extracts of acetate-grown organisms the activity appeared to increase during the course of the assay, whereas normal linear rates were obtained with extracts of organisms grown in nutrient broth. Pursuit of the cause of this effect revealed that the activation resulted from the glyoxylate produced by the action of isocitrate lyase on the isocitrate substrate; the lyase was present in high concentration in acetate-grown cells but not in cells grown in nutrient broth. This finding subsequently led to the discovery of two isoenzymes of isocitrate dehydrogenase in A . culcouceticus which could be separated by ion-exchange chromatography. gel filtration or zonal ultracentrifugation (Self and Weitzman, 1970, 1972). Both isoenzymes were found to be specific for NADP+ but to differ in kinetic characteristics, pH dependence, thermal and urea inactivation and molecular size. The molecular weight of the small isoenzyme is around 100,000 and that of the large isoenzyme is in the region of 300,000. The molecular weights of a few other bacterial NADP-linked isocitrate dehydrogenases have also been examined and found to be in the broad region of 100,000. e.g. Azotohactrr vincdundii

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225

about 80.000 (Chung and Franzen, 1969; Barrera and Jurtshuk, 1970), Brrcil1u.s .~te~~sotlic~sniopliilu,s 92,500 (Howard and Becker, 1970). Rliodopseudomonas .sphuesoide.s 105,000 (Chung and Braginski, 1972), Sulmonellu tj*phiniuriurn 102,000 (Marr and Weber, 1973) and E . coli 83,000 (Burke ('t d.,1974). On the other hand, the much higher molecular weight (about 300,000) of the large isoenzyme of A . culcouc~e~ticusis similar to that of NAD-linked isocitrate dehydrogenase from higher organisms (Sanwal and Stachow, 1965: Cox and Davies. 1969; Giorgio c't ul., 1970: Barnes c>t d.. 1971). The glyoxylate stimulation of A . c~ulcoucc~~icus isocitrate dehydrogenase activity was found to reside only in the large isoenzyme which was activated six-fold by 0.1 mM glyoxylate. The smaller isoenzyme was completely unaffected by glyoxylate. Pyruvate was also found to be an activator and of similar potency to glyoxylate (Self et ul., 1973). The stimulation of activity was completely reversible (e.g. on enzymic removal of the glyoxylate or pyruvate) and involved both an increase in V,,, and a reduction in substrate K , values, particularly for N A D P + . Other studies on the isocitrate dehydrogenase of A . culcouceticus showed that stimulation of enzyme activity could also be brought about by A M P or A D P and that these nucleotide effects were again specifically exerted on the larger isoenzyme (Parker and Weitzman, 1970; Self et ul., 1973). Again, maximum stimulation by A M P was five- to six-fold (at 1 mM AMP); A D P was a less effective activator and ATP produced no change in activity. This nucleotide activation was primarily an effect on the V,,, value of the enzyme. The dissimilarity between the structures of glyoxylate and pyruvate on the one hand and of A M P and A D P on the other suggested that although both types of compound are functionally analogous in producing stimulation of enzymic activity they may well interact with the enzyme at physically distinct sites. A search for any evidence of interaction between the two effects showed that they were quite independent and additive. Thus in the presence of a concentration of A M P that produces maximum stimulation, the addition of increasing concentrations of glyoxylate o r pyruvate elicited further stimulation of activity quantitatively similar to that observed in the absence of AMP. Selective desensitization by urea provided supporting evidence for the non-identity of the two regulatory sites. The larger isoenzyme of isocitrate dehydrogenase in A . culcouceticus is therefore sensitive to two distinct types of activation and it is significant that, as in eukaryotic cells, it is the large enzyme form which displays the regulatory properties. Once again it is likely that the stimulation of activity is associated with conformational rearrangements within the enzyme and the presence of a more complex quaternary structure in the larger isoenzyme may offer more scope for subtle effects.

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P. D. J. WEITZMAN

Consideration of the possible metabolic significance of these activations leads to the following suggestions (Self et ul., 1973). The AMP/ADP stimulation suggests a mechanism of control over the energy-producing function of the citric acid cycle, and the dependence of enzyme activity on “energy charge” shows the expected relationship (see also Section VII, p. 231). Isocitrate stands at a metabolic branch-point, and may be metabolized to aoxoglutarate by isocitrate dehydrogenase or to glyoxylate and succinate by isocitrate lyase. A balance must be achieved between these alternatives. The stimulation of isocitrate dehydrogenase by glyoxylate, itself a product of the other branch, might contribute to the maintenance of such a balance, achieving an appropriate partitioning of isocitrate between the two pathways. The stimulation by pyruvate, on the other hand, may represent an example of ”precursor” (Sanwal, 1970) or “feed-forward” (Shen and Atkinson, 1970) activation, whereby a metabolite stimulates an enzyme further ahead of it in a metabolic pathway. These activation effects and the associated occurrence of the two isocitrate dehydrogenase isoenzymes have been observed with various strains of Acinrtohactrr (Self and Weitzman, 1972; Kleber r t ul., 1974) and also with a few other species of Gram-negative aerobic bacteria, e.g. Borcktrllu hronchisrptica, Brrvihucteriurn frucinophagurn, Chromohuctrriuni violuc.curn and Xanthornonas hpucinrhi (Weitzman et al., I98 Id). Kornberg (1970) drew attention to the fact that although the citric acid cycle serves two purposes there is no evidence that E. c d i contains more than one type of enzyme for any of the component steps. The demonstration of isocitrate dehydrogenase isoenzymes in these other organisms (and the incidence of both NAD- and NADP-linked forms of the enzyme in some bacteria cited above) further emphasizes the diversity of the cycle among bacteria. Moreover. the existence of some hitherto undisclosed measure of metabolic compartmentation is hinted at. Some interesting observations were made by Bennett and Holms (1975) on the changing levels of isocitrate dehydrogenase activity in E. c d i associated with changes in the nature of the growth medium. During growth on acetate as sole carbon source the level of isocitrate dehydrogenase was depressed compared with that in glucose-grown cells. When the organisms were removed from acetate medium or when compounds were added which abolished their dependence on glyoxylate cycle activity (e.g. pyruvate or glucose) there was a rapid three- to four-fold increase in isocitrate dehydrogenase activity: transfer back to acetate medium resulted in a decrease in activity to the previous level. The activity changes appeared to depend neither on protein synthesis or degradation. nor on the action of freely dissociable metabolite effectors, and it was speculated that covalent modification of the enzyme might be involved. The phenomenon was observed in several strains

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of E. coli as well as in Klehsiellu um)genes, Sulnionellu typvpkimurium and Serrutiu niurcescens and would seem to be a plausible mechanism for adjusting the activity of isocitrate dehydrogenase at the isocitrate branchpoint commensurate with the competing demand for isocitrate by the glyoxylate cycle. Recently, Garnak and Reeves (1979) have presented evidence that phosphorylation of isocitrate dehydrogenase in vivo takes place during adaptation of E. coli to acetate utilization, and it may be that an enzyme-mediated phosphorylation/dephosphorylationmechanism is responsible for the controlled changes in the specific activity of isocitrate dehydrogenase which are a feature of such adaptation, though Nimmo and Holms (1980) have cautioned against such a conclusion.

V. Pyruvate and a-Oxoglutarate Dehydrogenases

a-Oxoglutarate dehydrogenase catalyses the second oxidative decarboxylation reaction of the cycle (phase (c) of Fig. 3) whereas pyruvate dehydrogenase catalyses an analogous reaction that serves a “feeder” role and provides the acetyl-CoA fuel for the cycle. Pyruvate dehydrogenase may therefore be accorded associate membership of the citric acid cycle. I t is appropriate here to consider some aspects of these two enzymes. The overall reactions catalysed are:

U - 0 x 0 acid + CoA-SH

+ N A D + +Acyl-CoA + NADH + H + C 0 2 +

where the U-0x0 acid and acyl-CoA pair is either pyruvate and acetyl-CoA or a-oxoglutarate and succinyl-CoA. The two enzymes are in fact multienzyme complexes, each consisting of three types of enzyme: a decarboxylase ( E I ), a dihydrolipoyl acyltransferase (E2) and dihydrolipoyl dehydrogenase (E3). The E2 component constitutes the structural core of the complex to which the El and E3 components are bound non-covalently. A very considerable amount of information has been accumulated concerning the structures of these complexes, their catalytic mechanisms and modes of regulation, and the subject has been reviewed by Reed (1974) and Perham (1975). The activity of pyruvate dehydrogenase from mammalian sources appears to be controlled by covalent modification. Two regulatory enzymes- a kinase and a phosphatase-are bound to the pyruvate dehydrogenase complex and function, respectively, to phosphorylate or dephosphorylate the E 1 component enzyme with concomitant inactivation or reactivation (Reed, 1974). However, pyruvate dehydrogenase from prokaryotes does not appear to be subject to this type of regulation. Although most of the work on pyruvate arid u-oxoglutarate dehydro-

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genases has been conducted with the complexes from mammalian sources or E. coli, some studies have been carried out on complexes from other bacteria-Acetobacter xylinum (Kornfeld et al., 1977, 1978; De Kok et al., 1980), Acinetobacter calcoaceticus (Weitzman, 1972; Parker and Weitzman, 1973; Hall and Weitzman, 1977: Jaskowska-Hodges and Weitzman, 198 l), Azotobucter vinelandii (Bresters et al., 1975a, b; Grande et al., 1975), Bacillus stearothermophilus (Henderson et al., 1979), B. subtilis (Hoch and Coukoulis, 1978; Visser et al., 1980), Pseudomonas aeruginosa (Jeyaseelan et al., 1980), Rhodospirillum rubrum (Liideritz and Klemme, 1977), Salmonella typhimurium (Langley and Guest, 1974), Srreptococcusfuecatis (Yamazaki et al., 1977), whilst Weitzman et al. (198 Id) have surveyed a range of bacteria for the regulatory sensitivity of the two a-0x0 acid dehydrogenases. These various studies have revealed some of the regulatory complexity displayed by the two enzymes. Inhibition by NADH of both pyruvate and a-oxoglutarate dehydrogenases from E. coli was first reported by Hansen and Henning ( 1966)and has also been observed with pyruvate dehydrogenases from A. calcoaceticus (Jaskowska-Hodges and Weitzman, 1981), A z . vinelandii (Bresters et al., 1975b) and R . rubrum (Liideritz and Klemme, 1977), and with a-oxoglutarate dehydrogenases from Ac. xylinum (Kornfeld et al., 1977) and A. calcoaceticus (Weitzman 1972; Parker and Weitzman, 1973). Inhibition by the acyl-CoA product of the overall reaction has also been observed, i.e. by acetyl-CoA of the pyruvate dehydrogenases from A . calcoaceticus (Jaskowska-Hodges and Weitzman, I98 l), Az. vinelundii (Bresters et a/., 1975b), E. coli (Hansen and Henning, 1966; Schwartz and Reed, 1970; Shen and Atkinson, 1970; Bisswanger and Henning, 1971) and R. rubrum (Liideritz and Klemme, 1977), and by succinyl-CoA of the a-oxoglutarate dehydrogenase from Ac. .uq'linum (Kornfeld et al., 1977). Consistent with the energy-yielding role of the two enzyme complexes are the observations that AMP may stimulate enzymic activity. This has been observed with pyruvate dehydrogenases from A. calcoaceticus (JaskowskaHodges and Weitzman, 1981), A z . vinelandii (Bresters et ul., 1975b) and E. coli (Schwartz and Reed, 1968) and with a-oxoglutarate dehydrogenases from A. calcoaceticus (Weitzman, 1972; Parker and Weitzman, 1973) and Ac. xylinum (Kornfeld et al., 1977). Weitzman Pt a/. (1981d) found that AMP stimulated both pyruvate and a-oxoglutarate dehydrogenases from several strains of Acinetobacter as well as from Bordetella bronchiseptica, Brevibacterium leucinophagum, Chromobacterium violaceum and Xanthomonas hyacinthi, whereas various other bacteria did not show this behaviour. The significance of the incidence of these regulatory effects will be considered in Section VII, p. 231. Inhibition of the complexes by NADH is thought to be due to inhibition of the dihydrolipoamide dehydrogenase (E3) component. However, in the

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case of a-oxoglutarate dehydrogenase from A . c.ci1couc~rtic~u.s kinetic evidence suggests that N A D H additionally inhibits the El component, i.e. acts as an allosteric end-product inhibitor of the short sequence of reactions catalysed by the overall complex (Parker and Weitzman, 1973: Hall and Weitzman, 1977). Moreover. the N A D H inhibition of E l is overcome by AMP, consistent with the view that A M P acts at E l : this was confirmed by direct measurements of the effect of A M P o n the activity of the E l component. The kinetic effect of A M P stimulation of this complex is a marked decrease in the apparent K,,, value for a-oxoglutarate: in the presence o f 0 . 2 mM A M P the K,, value decreases from 2.5 mM to 0.27 mM. I t is interesting that this lower value resembles that of the enzyme from E. coli which is not affected by A M P . I t is also noteworthy that the regulatory sensitivities of U - O X O glutarate dehydrogenase resemble those of citrate synthase; in E. c d i N A D H inhibits both enzymes and A M P is without effect, whereas in A . culc~ouc~c.iicus N A D H inhibits both enzymes and A M P overcomes this inhibition. The effects of A M P on the a-oxoglutarate dehydrogenase of A c . qVinuni and the pyruvate dehydrogenase of A:. riticdcmdii also result in lowering the K,,, value for the a-0x0 acid substrate and are believed to be exerted by interaction with the El components (Kornfeld et d., 1977, 1978; Bresters P I d., 1975b). A novel system of functional connection of active sites o r “intramolecular coupling” has recently been discovered in the pyruvate dehydrogenase complex from E. c d i (Bates rt d.. 1977; Danson rt d., 1978) and was subsequently demonstrated also in the a-oxoglutarate dehydrogenase complex of E. c d i (Collins and Reed, 1977). By such coupling, acyl groups may be transferred rapidly between lipoic acid residues of neighbouring E2 polypeptide chains within one molecule of complex, so that a single El unit can service a number of E2 chains. The a-0x0 acid dehydrogenase complexes contain multiple copies of each of the three component enzymes. The activesite coupling process may be a device to permit enzymic connection between the decarboxylation of a molecule of U-0x0 acid on a particular El unit and the availability of a molecule of C o A at a unit of E2 physically removed from that of E l . Thus even when substrates are present in low concentration the overall reaction may proceed at a relatively substantial rate (Danson ~ J I d.,1978). I t will be interesting to see if this coupling process occurs in other bacterial a-0x0 acid dehydrogenases and perhaps also to examine the interaction of the coupling with other regulatory mechanisms operating in these complexes. Studies on the structure and symmetry of pyruvate dehydrogenase from Bucilhrs .st~~uI.otIi~~I.t?iopIiiIuS have recently been reported (Henderson C I d., 1979). Most interestingly this bacterial complex was found to resemble closely the pyruvate dehydrogenase from eukaryotic mitochondria and to contrast significantly with the enzyme from E. c d i . Indeed, Henderson et d.(1979)

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P. D. J. WEITZMAN

drew attention to an apparent pattern of structural diversity among pyruvate dehydrogenases from a variety of sources. The enzymes from Gram-negative bacteria have octahedral symmetry and high molecular-weight subunits for El and E2 whereas those of Gram-positive bacteria and eukaryotes have icosahedral symmetry and smaller subunits. The B. stearothermophilus type of EI/E2 subunits has also been found in B. culdolyticus, B. culdotenax and B. polymyxa (J. Visser. personal communication) and in B. subtilis (Visser e f ul., 1980), while the subunits from Ps. aeruginosu (Jeyaseelan et a/., 1980) and Ps. Juorescens (J. Visser, personal communication) resemble those of the E. coli enzyme. This pattern of the distinctiveness of Gram-negative bacterial enzymes and the similarity between Gram-positive bacterial and eukaryotic enzymes is strikingly similar to that earlier established for citrate synthase and succinate thiokinase (Sections ll.C and 1II.A) and the uniformity of these patterns may offer clues to evolutionary connections (Section VIII). Clearly the study of the structures and of the catalytic and regulatory properties of a-0x0 acid dehydrogenases from a larger number of bacterial species will permit useful comparisons to be made and may reveal significant patterns and diversities. The use of affinity chromatography (Visser et ul., 1978a. b) may facilitate the purification of these bacterial complexes.

VI. Malate Dehydrogenase

Malate dehydrogenase catalyses the final step in the citric acid cycle, resulting in the regeneration of oxaloacetate: Malate + N A D +

= Oxaloacetate

+ NADH + H i

Brief mention of this enzyme is made here in view of the differences in molecular size which have been reported for the enzyme from different sources. Murphey rt a/. (1967b) surveyed a wide range of animals, plants and micro-organisms, examining the sizes of their malate dehydrogenases by gel filtration. Most of the enzymes were found to have a molecular weight in the region of 60,000, but a number of Bacillus species and a few other bacteria had a substantially larger enzyme, with molecular weight nearly 120,000. N o organism was found to contain both forms of the enzyme. Examination of the subunit structures of purified malate dehydrogenases from B. suhtilis and E. coli (Murphey et a/., 1967a) showed the enzyme from Bucillus to be a tetramer of four identical subunits, and that from E. coli to be a dimer of identical units. Sundaram 41 ul. (19x0) examined malate dehydrogenases from mesophilic, moderately thermophilic and extremely

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thermophilic bacteria. All appeared to be made up of subunits of molecular weight 32,000-36,000 and the dimeric and tetramic forms of the enzyme were distributed in each of the three classes of bacteria. Although the occurrence of ”small” and “large” forms of malate dehydrogenase appears similar to the situation encountered with citrate synthase and succinate thiokinase, no correlation between the molecular size of this enzyme and the Gram-staining reaction of the source organism may be made in the case of malate dehydrogenase. It may well be that all Bucrllus species elaborate the ”large” form of malate dehydrogenase, but other Gram-positive bacteria contain the “small” form. Moreover. the “large” enzyme is not restricted to the genus Buc~illus.N o special regulatory properties have been attributed to the “large” enzyme and it is interesting that the incidence of the “large” malate dehydrogenase among certain Gram-positive bacteria contrasts with the occurrence of “large” forms of citrate synthase and succinate thiokinase only in Gram-negative bacteria.

VI1. Multipoint Control of the Cycle

Consideration was given earlier to the multi-functional role of the citric acid cycle in both energy-yielding and biosynthetic metabolism. The competing claims of energy production and biosynthesis on the various intermediates of the cycle are illustrated in Fig. 6. This emphasizes that pyruvate (C.3), acetyl-CoA (C2),oxaloacetate (C4), isocitrate ( C o ) .a-oxoglutarate (C,) and succinyl-CoA (C:) all occupy branch-point positions in bacterial metabolism: at each point there is a choice between continued metabolism via the cycle or withdrawal into biosynthetic pathways. The enzymes that act to maintain the metabolite flow into and round the cycle are pyruvate dehydrogenase, citrate synthase, isocitrate dehydrogenase. a-oxoglutarate dehydrogenase and succinate thiokinase. I t is clear that the regulation ofa multi-branched system such as this is unlikely to operate at a single point. I n this article attention has already been drawn to various apparent regulatory properties of several enzymes of the cycle, and other factors (e.g. kinetic. it7 v;w organisation) may also exert a controlling influence. Srere ( 1971)asked the question: “What enzymes of the cycle are not controlled?” and answered “Probably none”, stressing the desirability of viewing “regulation as a pervasive factor in metabolism operating at every enzyme step”. Conscious of both the need for multiple control points and the regulatory diversity displayed by different organisms, it might be anticipated that some organisms would explicitly display a set of regulatory sensitivities of the five enzymes acting at the cycle branch-points, interpretable in terms of the competition between free energy

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c,-

-- >

FIG. 6. Biosynthetic branch-points in the citric acid cycle. synthetic reactions.

---+

indicate bio-

conservation and biosynthesis. Such a system of “multipoint control” is evident in the genus Acinrfohucfrr. Studies on the enzymes from these organisms (referred to in previous sections) have shown the following.

(i) Pyruvate dehydrogenase is stimulated by AMP by means of a marked decrease in the K,,, value for pyruvate; hence AMP promotes the channelling of pyruvate through the pyruvate dehydrogenase reaction sequence. (ii) AMP acts as a stimulatory effector of citrate synthase, counteracting the inhibitory influence of NADH; hence AMP promotes the metabolism of both oxaloacetate and acetyl-CoA to yield citrate. (iii) There are two distinct isocitrate dehydrogenases, one of which is strongly stimulated by AMP; hence AMP may promote the oxidative decarboxylation of isocitrate over its “biosynthetic” cleavage by isocitrate lyase. (iv) a-Oxoglutarate dehydrogenase is stimulated by AMP in a similar manner to pyruvate dehydrogenase; the K , value for a-oxoglutarate is greatly diminished and hence AMP directs a-oxoglutarate into the dehydrogenase reaction. (v) The dependence of succinate thiokinase activity on the concentration of the substrate ADP is strikingly different in Acinrtobacter from the situation

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in other bacteria: a very much higher K,, value for A D P is exhibited and this may allow control of the activity of the enzyme by variation in the concentration of A D P within the physiological range, whereas other bacterial succinate thiokinases with much lower values of K,,, for A D P may not be sensitive to this kinetic control. Thus all five branch-point enzymes in Acirwtohcrctcr show a response to adenylate control which is in the physiologically “right direction”. By stimulating the activity of these cycle enzymes, the “low-energy” signals A M P and A D P favour metabolic flux round the cycle against biosynthetic withdrawals, promote the ultimate generation of A T P and hence contribute to the maintenance of a balanced energy state in the cell. The adenylate control of the five enzymes may also be observed by examining the dependence of their activities on “energy charge” (Atkinson. 1968). The response of each of the enzymes to energy charge follows the form expected for the regulation of enzymes involved in energy regeneration. This novel “multipoint” control of the cycle is illustrated in Fig. 7. The system of regulatory responses has been found in several Acinctohuctcr

c,---3

1

c,

- - - 3

FIG. 7. Multipoint control of the citric acid cycle. -+ indicate biosynthetic reactions and --t r c x t i o n ~stimulated at lob energy charge. ~~

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P. D . J. WEITZMAN

species, B o r d ~ ~ t r l lhroticiiiseplicu. u Brrvihuctc~riutii Iruc,itiopliLiguni, C‘hroniohuctoriutn \?iolutwniand Xuntiiot?iotiu.shjwitirhi. It is interesting that the five components of multipoint control occur together and it would seem a reasonable speculation that their concerted operation in \~ivocontributes to the regulation of energy metabolism in these organisms.

VIII. Evolutionary Aspects The marked patterns of enzyme diversity which have been discussed in this article appear to have some bearing on the natural relationships between different bacteria. Tracing similarities o r differences in these “marker” enzymes between bacteria and higher organisms may additionally provide some speculative clues to the evolutionary connections between prokaryotes and eukaryotes. The endosymbiotic theory of the evolutionary origin of eukaryotic cells views the organelles of such cells as having evolved from free-living prokaryotic forms (Margulis, 1970). Thus mitochondria may have evolved from organisms having some resemblance to present-day aerobic bacteria. I t is therefore appropriate to comment on the comparative properties of some of the citric acid-cycle enzymes in bacteria and mitochondria. The striking division of both citrate synthases and succinate thiokinases into “large” and “small” molecular size forms (Sections ll.C and 1II.A) parallels the division between Gram-negative bacteria on the one hand and Grampositive bacteria and eukaryotes on the other. Since these two enzymes are located only in the mitochondria ofeukaryotes, we may conclude that mitochondria resemble Gram-positive, rather than Gram-negative. bacteria. In view of such enzymic similarity it was suggested that there might be a n evolutionary connection between mitochondria and Gram-positive bacteria (Weitzman and Jones, 1968). Comparison of the structures of pyruvate dehydrogenase and its subunits from bacteria and mitochondria (Section V) have also led to the suggestion that the mitochondria1 ancestor had Grampositive characteristics (Henderson et ul., 1979). John and Whatley (1975, 1977) have proposed a close resemblance between Paracoccus dmitriJicans and mitochondria, based largely o n a comparison of their electron-transport apparatus and function. In view of the Gramnegative status of P. cienirr$cuti,s, Weitzman and Kinghorn ( 1 98 1b) examined the citrate synthase and succinate thiokinase of this bacterium to see whether the enzymes possessed anomalous properties resembling those of the mitochondrial enzymes. In fact both enzymes proved to be of the typically Gramnegative type and not to resemble the eukaryotic forms. Superficially these findings might be construed as evidence against the ancestral relationship

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between P. c/~witr.Ijicutisand mitochondria. However, we already know (Section II.G, p. 213) that Gram-negative bacteria may. by mutation, give rise to strains which, though Gram-negative. produce a Gram-positive ( o r eukaryotic) type of enzyme. Clearly the identity of the mitochondria1 ancestral symbiont remains uncertain and perhaps is destined ever to be so. Nevertheless the accumulating observed simiiarities between mitochondria and Gram-positive bacteria, and the contrasts with Gram-negative bacteria. should be borne in mind when considering organelle evolution. The chloroplasts of eukaryotic cells may likewise have had a prokaryotic origin and such ancestral forms might be related to blue-green bacteria, o r cyanophytes (Margulis, 1970) o r to prochlorophytes (Lewin. 1980). Both citrate synthase and succinate thiokinase from blue~-greenbacteria are of the typically Gram-negative type (Lucas and Weitzman, 1975, 1977; Weitzman and Kinghorn, 1980) and Proclilor.oii, the type genus of the Prochlorophyta (Lewin, 1977) has also been found to contain a Gram-negative bacterial form o f succinate thiokinase (P. Weitzman, unpublished observations). I t is t h u s interesting that preliminary investigations have shown the presence of a “large”, Gram-negative prokaryotic, type of succinate thiokinase in the chloroplasts of higher plants (Weitzman, 1979). These very restricted enzyme comparisons tempt one to suggest that mitochondria have a Gram-positive bacterial type of ancestor whereas chloroplasts derive from a Gram-negative origin, though this may well be a n oversimplistic or naive proposal. While speculating on the possible evolutionary implications of observations on enzyme structures we may go yet further and enquire of the evolution of the citric acid cycle itself. Examination of the chemical reactions constituting the cycle reveals a definite pattern of duplication which has not previously been emphasized. Consider the following scheme of five reactions: U-0x0 acid

1 oxidize, decarboxylate and form an acyl-CoA

1 “spend” free energy

1

introduce a C = C double bond

1

add H,O across the double bond

1 oxidize, decarboxylate and form an (1-0x0 acid The reactions of the citric acid cycle may be divided into two groups, each of which follows this sequence of steps. Thus, starting with pyruvate, acetyl-

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tunlarate

f

31

citrate

3 J

FIG. 8. Cyclic sequence o f reactions starting with pyruvate

CoA is tirst produced. its free energy is "spent" i n driving the condensation K i t h oxaloacetate to t'orm citrate. a double bond is introduced with the formation of c,i.s-aconitate. hydration across this bond produces isocitrate and oxidative decarboxylation yields (1-oxoglutarate. Carrying out a similar sequence of reactions on {L-oxoglutarate leads first to succinyl-CoA, "spending" its free energy achieves the phosphorylation of a nucleosidc diphosphate to the triphosphate. introduction of a double bond yields fumarate from succinate. hydration produces malate. whose oxidative decarboxylation regenerates the original U-OXO acid. pyruvate. T h u s a cyclic set of reactions based on a two-fold passage through the same sequence could lead from, and back to. pyruvate with the consumption of a molecule o f oxaloacetatc per turn (Fig. X). The enzymes catalysing these reactions may then be grouped into five pairs, one from each "half" of the cycle: pyruvate dehydrogenase and U-0x0glutarate dehydrngenase. citrate synt hase and succinate thiokinase, aconitase and s iicci n ii t e d e h yd roge ti a se, acon i t a se ii nd fu ma ra se. i soci t rat e de h y d r ogenase and "malic" enzyme. Aconitase features twice, as the same protein appears to catalysc the overall reaction citrate -+c.i.s-aconitate+ isocitrate. When juxtaposed in this way. several siniilarities appear within pairs. Thus i t is well documented that pyruvate and a-oxoglutarate dehydrogenases are closely related multienzyme complexes bringing about strictly analogous reactions on pyruvate o r a-oxoglutarate and having a similar multiplicity and assembly of subunits: indeed, the E3 components of the two complexes are probably identical (Langley and Guest, 1974: Hoch and Coukoulis, 1978:

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Perham rid.,1978). Citrate synthase and succinate thiokinase catalyse rather different reactions but the present article has emphasized some markedly parallel structural patterns encountered for these two enzymes in different organisms such that there may be further structural similarities between them (Weitzman and Kinghorn, 1978). The pairing of aconitase with succinate dehydrogenase may at first sight appear a total mismatch. However, recent work has shown aconitase to be an iron-sulphur protein (Gawron o t al., 1974: Suzuki c t a/., 1976a, b: Ruzicka and Beinert, 1978); its comparison with the complex iron-sulphur protein succinate dehydrogenase is thus perhaps not so outrageous. Might it then be that the two halves of the citric acid cycle bear an evolutionary relationship to each other and that the enzyme pairs may have evolved by a process of gene duplication and diversification'? Further comparative investigations on the enzyme pairs and a search for homologies in their amino acid sequences may throw some light on this possibility. Of course. the scheme of Fig. 8 is not quite the form in which we recognize the citric acid cycle. Although the malate -,pyruvate step, catalysed by "malic" enzyme, does occur, the introduction of an oxidative, but nondecarboxylating, reaction would instead convert malate into oxaloacetate and thus provide a cyclic sequence of reactions that constitute the established citric acid -cycle pathway .

IX. Concluding Remarks This review started with Krebs' discovery of the citric acid cycle in 1937. Now, more than 40 years later, the discovery is "middle-aged" or, relative to the pace of biochemical revelation, even old. The cycle's textbook respectability and its thoroughly established central role in cellular metabolism may suggest that all is known and little new remains to be uncovered. It is hoped that this review has at least helped to emphasize how incorrect such an attitude would be. It is perhaps more correct to say that our understanding of the cycle is only superficial and still in its infancy. Its integrated action as a multienzyme system, its organization within the cell, the regulatory controls maintaining balanced metabolite flux and accommodating both energetic and biosynthetic demands, the structure-function relationships in the individual enzymes and the diversity displayed by the enzymes from different organisms are just some of the aspects Qf the CyC\t ~'ooutw b ' n much femalns io b e disclosed. For a middle-aged or old discovery there lies an extremely youthful vista of a lengthy life ahead.

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X. Acknowledgement S u p p o r t f r o m t h e Science Research C o u n c i l for w o r k in t h e a u t h o r ' s laboratory is gratefully acknowledged.

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Weitzman, P. D. J. and Dunmore, P. ( 19694. FiJdiwtiori of' Europcwn Bioc~limiicul Soc~ic~tic~s Lcttos 3, 265. Weitzman. P. D. J. and Dunmore. P. (1969b). Biocliiniica c t Biopliysicu Acrrr 171, 198. Weitzman. P. D. J . and Hewson. J. K . (1973). Fi~tlerutionof Europiwi Biochcmicul Soc~ic~tics Lettc~rs36. 227. Weitzman, P. D. J. and Jaskowska-Hodges. H. (198 I ). Fklrrutiori (!I Europiwi Bioclicwiic,ul SocieticJs Lctters, in press. Weitzman. P. D. J. and Jones, D. (196X). NuturP, London 219. 270. Weitzman. P. D . J . and Jones, D . (1975). Journul of Gcwrrul Micwbiology 89, 187. Weitzman, P. D. J . and Kinghorn. H. A. (197X). F & w / i o n o/'Europcwri Eiocheniicul Socirties Lrttcrs 88. 255. Weitzman. P. D. J . and Kinghorn. H. A. ( 19x0). Fi&ration of'Europiwn Bioc~licwiicd Socic.tic.s Lct/cr.s 114. 225. Weitzman, P. D. J. and Kinghorn, H . A. (1981a). Journal of' General Microbiology, in press. Weitzman. P. D. J . and Kinghorn. H . A . (1981b). Biochmiccrl mid Biopliysicul Rcscurc~liCo/,i/)iutiic.utioti.s.in press. Weitzman, P. D . J.. Ward, B . A. and Rann. D. L. (1974). F d i w i t i o n of'Europcwi Bioclicwiic~crlSoc,ierirs Let tors 43. 9 7. Weitzman, P. D. J.. Kinghorn, H . A.. Beecroft, L. J. and Harford, S. (1978). Bind i c w i i c d Soc,io t j ' Treinsuct i o n s 6, 4 36. Weitzman, P. D. J., Danson, M . J . and Harford, S . (1981a). Fi&rrrtion o/'Europiwi Bioc~htwiiculSoi~ic~tic~s Lettws. in press. Weitzman. P. D. J., Kinghorn. 1-1. A . and Jones. D. (19Xlb).J o u r n d of Gcncwl Mic.robiolosqj..in press. Weitzman. P. D. J . . Kinghorn, H. A . and Sidey. C. ( 1 9 x 1 ~ )Biocli~wiic~ul . urid Biophj.sical Rtwcrrch Communicutions. in press. Weitzman, P. D. J . , Parker, M. G. and Jaskowska-Hodges. H. (19Xld). Journul o f ' GencrciI Mic,robiologj*,in press. Weitzman. P. D. J . , Solomon, M . and Hewson. J. K. (19Xle). Bioc~hc~niic~ul und BiopIij~.vicdRcsrcrrcA Conimun icu t ions, in press . Wider. E. A . and Tigier. H. A . (1971). E n z j m h g i u 41, 217. Wiegand. G., Kukla. D.. Scholze, H.. Jones, T. A . and Huber. R. (1979). Europcm JournuI of Bioclicniistrj~93, 4 1. Wright, J. A. and Sanwal, B. D. (1971). Journul qf' Biologicul Chmiistry 246, 1689. Wright. J. A., Maeba. P. and Sanwal, B. D. (1967). Biochcwiic,crl und Biophjsic,ul Resrurcli C'on~r~iutiic~ution.s 29, 34. Wu. J. Y. and Yang, J. T. ( 1970). Joirrriril of B i o l o ~ i c ~Clwniistrj, d 245. 21 2. Yamazaki, A,, Nishimura. Y. and Kamihara, T. (1977). Federution of' European Biochmiic,ul Socict ies Lrt t i w 74. 62.

Author Index

A

Aurich, H.. 224. 226, 240. 241 Austin, L. A., 22, 23, 24, 25, 81, 88 Ayengar, P., 220, 242

Aagaard, J., 5, 6, 18, 21, 42, 54, 55. 57, 58, 61, 66, 75, 81 Abbach, E. J. J., 174, 181 Ackerson, L. C., 6, 87 Acs, G . , 103, I21 Actman, M., 110, 118 Adams, M. W. W., 30, 81 Adhya, S., 149, 181 Afzelius, B. A,, 14. 31, 86 Aiba, S., 129, 177 Akiyama, H., 104, I20 Akiyama, S., 237, 243 Al-Aidroos, K . , 101, 109, 110. 111, 115.

B Baccarini-Melandri, A,, 5,6,8,27.28,33, 81, 85, 86, 88, Y1

I18

Alberte, R. S., 2, 90 Albrecht. O., 49, 88 Alcorn, M. E.. 171, 177 Aleem, M. J . H., 9, 86, 190, 242 Algeri. A,. 173, 177 Alivisatos, S. G . A,, 220, 240 Allen, K. E., 172, 177 Alonso, A,. 167, 174, 177 Amarasingham, C. R.. 137,177, 190, 199, 238

Amesz, J., 19, 88 Andreasen, A. A,, 134, 177 Anthony, C., 197, 21 I. 238 Arnaud, M., 137, 179 Arnheim, K. 40, 84 Arnon, D. I., 29, 82, YI Artmen, M., 137, 179 Ashworth, J. M., 212, 238 Atkinson, D. E., 158, 177, 191, 192, 193, 223, 225. 226, 228. 233, 238, 240, 242

Bachofen, R., 32, 33. 44, 73, 8Y, 91 Bailey, R . B., 135, 177 Baltcheffsky, H., 27. 28. 29, 83, 84 Baltscheffsky. M., 5 , 27. 28. 29, XI, 84 Bambers, G., 191, 243 Barker, H. A,, 191, 23Y Barnaby. C., 230. 241 Barnes, L. D., 225, 238 Barnes, R., 145, I81 Barrera. C . R., 224, 225, 238 Barrett, J., 29, 81 Bartley, W., 137, 181 Bartsch, R. G., 6, 29. 39. 81 Bashford, C. L.. 6. 83 Bassford, P., 108 118 Bates, D. L., 229, 238 Bearden, A. J., 30, 86 Beatty, J . T., 212, 238 Beck, C.. 142. 177 Becker, R. R., 225, 240 Beckwith, J., 108 118 Beecroft, L. J., 215. 244 Beinert, H., 237, 242 Benedict, S. H., 164, 182 Ben-Hayyim, G.. 7, 31, 85 Bennett, P. M., 226, 238 Benziman, M., 193, 209. 210, 215, 228, 229, 239, 241, 243 Beran, K., 140, 177

245

246

AUTHOR INDEX

Berg, W. H., van der, 6, 83 Betz, H., 152, 179 Bevan, E. A., 93, 95, 103, 114, 115, 117, 118, 119, 120, 121 Bhandari, H. C., 168, 177 Biebl, H., 19, 57, 81 Biedermann, M., 14, 37, 38, 42, 51, 52, 54, 55, 63, 11, 81, 87 Binder, A,, 27, 88 Birch-Anderson, A,, 208, 242 Birrell, G. B., 31, 32, 81 Bisswanger, H., 228, 238 Bjorndal, H., 110, 112, 120 Black, S. H., 208, 239 Blaurock, A. E., 52, 81 Blicharsko, J., 137, 179 Blobel, G., 45, 82, 106, 118 Boatman, E. S., 13, 81 Boehm, C., 162, 163, 167, 168, 178 Boesman-Finkelstein, M., 195, 241 Bohm, S., 14, 40, 87 Boily, Y., 104, 119 Boll, M., 51, 81 Bolt, 22, 23 Bolton, J . R., 6, 81 Bonner, H. S., 6, 83 Booij, H. L., 161, 178, 182 Borriss, R., 193, 204, 238 Bose, S. K . , 8, 81 Bostian, D. A,, 97, 98, 119 Bostian, K . A,, 95. 97. 98. 104. 106, 107. 114, 118 Boucher. F., 18, 81 Boucher, L. J., 21, 85 Bowman, B. J., 170, 172, 177 Boyce, C. O., 19, 81 Boyer, P. D., 221, 238 Bozarth, R. F., 116, 121 Brady, R. O., 110, I18 Braginski, J. E., 225, 239 Brandli, E., 128, 129, 140. 177 Branton, D., 10, 81 Brautigan, D. L., 29, 89 Brazil, H., 192, 243 Brennan, V., 91, 119 Brennan, V. R., 95, 118 Bresters, T. W., 228. 229, 238. 239 Breton, J . , 35, 82, 88, 90 Brickman, E., 108, 118 Bridger, W. A,, 219, 221, 238

Britton, G., 2. 90 Broch-Due, M . , 62, 82 Brock, T. D.. 210, 238 Brocklehurst, R., 163, 169, 173. 177 Bronn, W. K., 126, 127, 157. 158, 178 Brooks, G . C.. 203, 242 Brown, A. E., 41, 82 Brown, J. P., 237, 242 Bruenn, J., 95, 91, 118 Bruenn, J . A,, 95, 97, 101, 118, 119 Brune, D. C., 13, 19. 88 Buchanan, B. B.. 8, 82 Buck, K . W., 95,96,91,99, 103, 116, 118, I19 Biihler, R., 14, 16, 34, 84 Bull, M. J . , 77, 82 Bullivant, S., 10, 81 Burchall, J. J . , 223, 238 Burd, G. I., 131, 178 Burger, M., 160, 177, 178 Burke, W. F., 225, 238 Burnham, B. F., 221, 222, 238 Burns, D. D., 31, 82 Bush, L. P., 220 238 Bussey, H., 96, 101, 104, 107, 108, 109, 110, 1 1 1 , 112, 114, 115, 117. 118, 119, 120, 121

C Cabib, E., 159, 181 Canh, D. S., 163, 178 Carithers, R. P., 29, 82, 91 Carls, R . A., 212, 238 Carmeli, C., 7, 27, 28, 31, 33, 67, 84, 85 Carr, N . G., I , 82, 201, 220, 242 Castenholz, R. W., 12, 35, 62, 88 Cazzulo, J. J., 193, 204, 205, 209. 210, 21 I. 217, 238, 240, 241 Cellarius, R . A,, 13, 63, 82, 88 Cha, S., 219, 221, 222, 238, 240 Chan, W. W. C., 207, 239 Chance, B., 5 , 6. 45, 83, Y l Chaney, T. H., 26, 85 Chang. C. N., 45, 82 Changeux, J . P., 204, 241 Charles, A. M., 190, 224, 238, 239 Chevallier. M . R.. 112, I I Y Christensen, M . S., 165, 169, 180

247

AUTHOR INDEX

Christman, J . K . , 103, I21 Chung, A. E.. 225, 23Y Cirillo, V. P., 160, 164, 165, 166. 169, 178, 180 Citri, N . , 205, 23Y Clare. J . J . , 100, IIY Clarke, P. H., 216, 242 Clarkson, T. W.. 166, 181 Claus, G. W . . 223. 240

Clayton. B. J., 19, 23, 24, 31. 82 Clayton, R. K., 4, 6, 18, 19, 23. 24, 31, 82. YO

Clement-Metral, J . D.. 74, 75. 77, 83 Coedell, R. J . . 5. 18,21,22,23.24,25, 35, 40, 45, 58, 82, 86, YO, Y I Cohen, G. N., 190, 23Y Cohen-Bazire, G., 2, 10, 12, 38, 40, 53, 54. 55, 57. 58,61, 71, 73, 74, 82. 84, XY

Cohn, M. S., 102, IIY Colby. J . , 190, 193, 197, 209, 211. 223, 234,

Coleman, M . S., 218. 230 Collier, R. J., 110, 118 Collins. J . H., 229 23Y Collins. M . L. P.. 43. 51. 82 Colwell, R . R., 208. 23Y Conelly, J . L.. 41, 82 Conti, S. F., 12, 35, 48. 53, 62, 85, YO Conway. E. J.. 160, 178 Cook, A. H., 123 178 Coukoulis, H. J.. 228, 236. 240 Cox, B. S.. 97, 98, 100, 120 Cox, D. P., 137, 17Y. 190, 240 Cox, G. F., 225. 23Y Crabtree, H. G., 142, 178 Crawford. I . P., 29, 83 Criswell. B. S.. 208, 23Y Croes. A. E., 114, 1 1 5. 121 Crofts, A. R., 8, 22. 24. 25, 33. 40,82. 88 Cruden, D. L., 13, 26, 35, 82 Cryer, D. R., 101 l l Y Cuatrecases. P.,110 118 Cuendet. P. A,, 21. 23. 24. 32, K2 Cusanovich, M. A,. 7, 83 Custers. M. T. J.. 133, 178

D Dales, S., 110, I18 Danson, M . J., 191. 193, 194, 195, 203, 204. 205, 209, 210, 211, 212, 229, 238, 23Y, 243, 244

Davies, D. D., 225. 235, Davies, R . C., 37, 38. 72, 73, 83, 8Y Davies, B. D., 44, 8Y, 137, 177, 190. 199. 2 1 I , 238. 23Y Davis. K A., 29. 83, 85 Dawes, E. A., 190. 197, 205, 23Y, 242 Day, P. R., 116, 120 De Abreu. R. A,. 228, 238. 239 Deak, T.. 171, 178 De Boer, W . E., 10, 83 De Kok, A., 228, 229, 238. 23Y De la Fuente, G., 160, 164. 17Y, 181 Dellweg, H.. 126, 127, 157, 158, 178 del Valle-Tascon, S . , 7, 83, 84, 88 De Vries, G. J . M . L., 114, 115, 121 Dierstein. R., 38, 39. 44, 54. 65, 70, 77. 83

Diezel, W., 226, 241 Dittbrenner. S., 191, 200. 23Y Dobberstein, B.. 106. I18 Doi, M., 8, YO Doorley. P. F., 18, 33, 84 Dorrestein, R., 19, 61. YO Dose, K., 28, 8Y Dougherty, R. C., 21. 85 Douglas, H . C., 13. 81, 165. 178 Downey, M.. 160 178 Doyle, R . J., 166. I82 Drapeau, G. R., 104, IIY Dreierkauf. F. A,, 161. 178 Drews, G 2. 4, 8, 10. 12, 13, 14, 16, 18, 19. 21, 22, 24, 25, 26. 30, 31. 32. 33, 34. 35, 36. 37. 38, 39, 40, 41, 42. 43. 44, 46. 47, 48, 50, 51, 52, 54. 55. 56, 57, 58, 59, 60, 61. 62, 63. 64, 65, 66, 67, 70. 7 3 , 76. 71. XI. 82. 84. 85. 86. 87. 8Y. YO, Y l Duckworth, H. W., 203. 243 Dundas. I. E. D., 2. 83 Dunmore. P., 200. 202, 207. 244 Dutton. P. L., 5, 6. 7. 33. 83. 88 Duysens, L. N . M.. 7. 9, 83. YO

.

248

AUTHOR INDEX

E Eddy. A . A., 163, 169, 173, 174, 177, 178, 181 Eggleston, L. V., 186. 241 Ehmann, C., 152, 182 Ehwald, R., 165, 178 Eidels, L., 190, 204, 21 I , 239 Eiserling, F. A,, 47, 82 Eley, J. H., 9, 86 Ellman, G . L., 192, 239 Elsasser, S., 152, 179 Emr, S., 108, 118 Enatsu, T., 1 I I , 120 Enns, C. A,, 207, 23Y Entian. K-D., 150, 178 Erokhin, Y. E., 24. 86 Evans, M. C. W., 8, 82 Even-Shoshan, A,, 137, 174 Everse, J., 230 242

Fortnagel, P., 190, 23Y Fowler, C . F., 13, 26, 84 Fox, G . E., 2, YO Fraker, P. J., 23, 24, 84 Fraley, R. T., 32, 41, 44, 46, 47, 72, 84, 86, 91 Frank, I . R., 221, 242 Franzen, J. S., 225. 239 Freeze, H., 210 238 Frenkel, A-W., 4, 84 Freund-Molbert, E., 5 I , 52. 54, 55. 87 Frey, J.. 152, 178 Fridberg, I., 33, 85 Fried, H. M., 95, 97, I I Y Friederich, U., 129. 180 Fuhrmann.G. F.. 161, 162, 163, 166, 167, 168, 170, 178 Fujimoto, S., 237, 243 Fukuda, H., 237. 243 Fuller, R. C., 12, 13, 14, 19, 26, 31, 35, 62, 81, 84, 85, 8Y

F Fakoussa. R. M . , 8. 9. 73, 87 Faloona, G . R., 204, 239 Fanica-Gaignier. M., 74, 75, 77, 83 Feher, G., 6, 18, 19, 31. 32. 33. 39, 44. 83, 86, 88 Feick, R., 18, 22, 24, 25, 33, 34. 40, 83. 87 Feldman, N., 7, 8, 83 Ferguson, R., 152. 17Y Ferrero, I., 173, 177 Fersht, A. R., 229, 23Y Fiechter, A., 127, 129, 138, 156, 160, 178. 180 Finch, J . T., 228, 229, 234. 240 Fink, G . R., 95, 97, 101, 102. 104. IIY, 121 Firsow, N . N., 24, 34, 58, 60. 66. 84 Fisher, H. F.. 104, 119 Fisher, R. R., 30, 84. 8S Fjerdingen, B. S., 62, 82 Flechtner, V. R., 190, 193, 204, 205, 2 I I , 239 Fleming, J.. 225, 23Y Fluhr, R., 220, 239 Flury, U., 131. 132, 137, 178 Forrest, W. W.. 157, 178

G Gale. E. F.. 137, 178 Galla, H. J., 49, 88 Gancedo, J. M., 135, 180 Garcia, A,, 62, 84 Garcia. A . F., 6, 19. 41, 42, 84, 88 Gardner, D., 163. 169, 173, 177 Gardner, H. L., 208, 23Y Garnak, M.. 227, 23Y Gawron, O., 237, 23Y Gepshtein, A., 27, 28, 67, 84 Gerhardt, Ph., 162, 181 Gershanovich. V. N., 137, 178 Gest, H . , 6, 8,9, 24, 28, 34, 42.48. 53. 58. 61, 66, 67, 68, 73, 74, 75. 81, 84, 86, YO, 2 12, 238 Gibbon, J . A,, 26, YO Gibson, D. M., 220, 242 Gibson, J.. 73, 86, 221. 234, Gibson, K. D., 4, 14. 43, 48, 51. 52. 53. 84. 87 Giesbrecht, P., 13, 54, 83, 84 Gilula, N . B., 10, 81 Gilvarg, C., 21 I , 23Y Gimenez-Gallego, G., 7, 83, 84

AUTHOR INDEX

Gingras. G., 18, 81, YO Giorgio, N. A,, 225, 239 Gitlitz, P. H., 30, 84 Glaeser, H., 223, 224, 239 Glanville, M.. 106, 119 Gobel, F., 54, 57, 84 Gofdheer, J . C., 40, 84 Gogel. G. E.. 23, 24, YO Gogotov, I. N., 30, 84 Goldring, E. S., 101, I19 Golecki, J. R., 10, 12, 13, 14, 16. 21. 26, 31, 34, 35, 36, 44, 47, 51, 52, 62, 84, 85. 87, 89. 90 Gonen, L.. 192, 243 Goodwin. T. W., 2, 90 Gorchien, A,, 38, 40, 44, 47, 48, 51, 53, 83, 84

Goring, H., 165, 178 Gorts, C. P. M., 137, 178 Gottschalk, G., 191, 200, 23Y Govindjee, R., 8, 9, 84 Grande, H. J.. 228, 239 Gray, C. F., 137, 178 Gray, C. T., 190. 239 Grba, S., 156. 178 Greenfield. S., 223, 240 Griffin, C. C.. 171, 177 Griffith. M., 21, 40. 84, 89 Griffith, 0. H., 31, 32, 81 Grinnell, F.. 219, 242 Gromet-Elhanan, Z., 6. 7. 8, 27, 28, 83, 88

Grondelle, R., van, 7, 9, 83. 90 Grossman, L. I., 101, 119 Guerry, P., 102. I21 Guest, J. R., 228, 230, 236, 240. 241 Guillory, R . J., 30, 84 Gunsalus, I. C., 221, 239, 242 Gurin, S., 186, 241

H Haddock, B. A,, 27, 28, 84 Hagele, E., 150, 179 Hale, G., 229, 238 Hall, D. O., 30, 81 Hall, E. R., 228, 229, 240 Hall, R. L., 18, 33, 84 Halpern, Y. S., 137, 179

249

Halvorson, H. O., 137, 172, 173, 174, I7Y. 180

Hampton, M. L., 223, 224, 240 Hankin, L., 116, 119 Hanselmann, K . , 32, 33, 44, 91 Hansen, R., 152, 179 Hansen, R . G., 228, 240 Hansford, R. G., 220, 240 Hanson, R. S., 137, 152, 179, 190, 193, 197,200,203,204,205.21I , 212,223. 224, 238, 239, 240, 243 Hardy, S. J. S., 45, 88 Harel, E., 220, 239 Harford. S.. 194, 204, 209, 213. 214, 215, 239, 240, 244

Harris, M. S., 98, 119 Harrison, J. S., 123, 181 Harrison, R. A,, 237, 242 Hartman, G.. 153, 180 Hartman. W., 49, 88 Hartmeier, W., 126, 127, 157, 158,

I 78

Hasilik, A,. 152, 17Y Haskovec, C., 171, 179 Hastie, N. D., 97, 119 Hateti, Y., 29, 83, 84 Hathaway, J . A,, 192, 223. 240 Hauer, R.. 171, 179 Hauska, G., 8, 81 Hauska. G. A,, 8, 33, 88 Hawthorne, D. C., 165, 179, 180 Hayaishi, O., 1 1 I , 119 Hayashibe, M., 168, 177 Hayasaka, S., 38, 72, 77, 85 Hebeler, B. H., 190, 240 Hedenstrom, M., von. 171, 179 Hejmova, L., 160, 177, 178 Helenius, A., 110. 118 Helling, R. B., 212, 241 Henderson, C. E., 228, 229, 234, 240 Henning, U., 228, 238, 240 Heppel, L. A., 168, 180 Heredia, C. F., 160, 179 Heriot, K., 30, 85 Hermans, J. M. H., 114, 115, 117, 118, 120, I21

Herring, A. J., 95, 103, 118, 119, 120 Hewson, J. K., 195, 244 Hga, A. I., 193, 204, 205, 21 I , 240, 241 Higuchi, M., 38, 85

250

AUTHOR INDEX

Hinze, H., 152, 17Y Ho. E., 44, 46, 88 Hoare, D. S., 26, YO, 21 I , 243 Hoare, S. L., 21 I . 243 Hoch, J. A,, 228, 236, 240 Hockman, A,, 7, 31, 33, 85 Hofer, M., 162, 170, 171, 17Y, 180 Holland, I . B., 117, I20 Holland, L. E., 95, 119 Hollings, M., 99, 119 Holm, C., 97, 98, 100, I20 Holm, C. A,, 95, 119 Holmes, N. G.. 43, 85 Holmes, W. H., 226, 238 Holms, W. H., 227, 242 Holt, S . , 14, 16. 54, 90 Holt, S. C., 12, 13, 14, 35, 62, 85 Holzer, H., 1SO, 152. 162, 167, 168, 175, 17Y. 180

Honjo, T., 111, I l Y Hooper. E. A,, 229, 238 Hopper, J. E., 97. 98, 104, 106, 107, 114, 118,11Y

Horiik, J., 163, 168. 178, 179 Horio, T., 29, 81 Houmard, J., 104, I I Y Howard, R . L., 225, 240 Huang. J. W., 23, 24, 48, 51, 85 Huang, Kao, M . Y. C. 61, 75, YO Huber, R., 203. 244 Hudewentz, A,, 8, 9, 73, 87 Huigen, A,, 228, 230, 243 Humphrey, A. E., 129, 177 Hunter, C. N., 29. 41, 42, 43, 81, 85 Hurlbert, R. E., 13, 14, 85 Hutchins, K., 108, 109, 110, 114. I I Y

I Ichikawa, T., 150, 179 Ichimura. S.. 62, YO Imamura, T., 110, 1IY Ingebretsen, 0. C., 224, 240 Ingledew, W. J., 29, 35. 85 Inkson, C., 173, 181 Inouye, H., 108, 118 Iredale, S. E., 205, 206, 240 Irschik, H., 9, 39, 51, 53, 55, 56, 58, 64, 8.5

Isaacson, R. A., 6, 18, 19, 87 Ishikawa, M., 237, 243 Isowa, Y., 150, 17Y

J Jackson, J. B., 6. 7, 83 Jacob, M., 220, 242 Jacobs, E., 30, 85 Jaeger, K., 61, 83 Jakes, K. S., 107, llY Janda. S., 171, 179 Jangaard, N . 0.. 193, 240 Janik, A,, 215, 240 Jank-Ladwig, R., 10, 71, 8Y Janshekar, H., 130, 139, 17Y Jarrett. L., 110, 118 Jaskowska-Hodges, H., 221, 222, 226, 228, 240, 244

Jaspers, H . T.. 169, 170, 17Y Jaspers, P., 5, 7, Y l Jeyaseelan, K., 228, 230, 240 Johanson, R. A., 225, 238 Johansson, B. C.. 27. 2X, 29.84. 212,238 John, P., 234. 240 Johnson, D. E., 203, 204. 21 I , 240 Johnson. M., 155, 179 Johnson, W. A,, 185. 241 Jolchine, G., 6, 19, 27, 2d, 85 Jones, C. W., 27, 28, 84 Jones, D., 191, 196. 198, 208, 209, 213, 220, 234, 240. 244

Jones, M. E., 218, 23Y Jones, 0. T. G., 5 , 7, 8, 29, 33, 37, 41. 42, 43, 66, 81, 82. 85, 88. 145, 181

Jones, T. A,. 203, 244 Josefsson, L. G., 45, 88 Juni, E., 215, 240 Jurtshuk, P., 224, 225, 238 Just, F., 127, 179

K Kaarinanen, L., 106, l l Y Kahn, C. R., 113, I19 Kakuno, T., 29, 30, 81, 85 Kamen. M. D.. 7. 8. 29. 30, 41. 42. 81, 83, 84, 85, 87, YO

AUTHOR INDEX

Kamihara, T., 228, 244 Kandel, J., 116, 117. 119, 120 Kandel, J. S., 115, 117, 119, 120 Kane, W., 97, 118 Kane, W . P., 97, IlY Kaneko. T.. 109, I20 Kaplan, N. O., 30, 85. 230, 241, 242 Kaplan, S., 23, 24, 32, 41. 44, 46, 47. 48, 49, 53, 54, 61, 72, 74, 75, 84, 85, 86, 91 Karnovsky. M. J., 10, 81 Karrer, D.. 140, 179 Kataoka, M., 14, 90 Kato, I., 111, 119 Katsunama, T.. 152, 17Y Katterman, F., 97, 104, 121 Katz. E., 19, 61, 85, YO Katz, J. J . , 21, 85 Kaufman. S., 220, 240 Kawamoto, M., 110, 119 Kawasaki, T., 223, 242 Kawase, N., 104, 120 Ke, B., 26, 85 Keister, D. L., 5, 7, 30, 53, 64, 85 Keitz, B., 95, 118 Kelly, C. J., 219, 221, 222. 240 Kelly, D. P., 21 I , 240 Kennedy, M. C., 237, 239 Kennel, S. J., 29, 85 Kerber, N. L., 6, 19, 88 Kerr. M. A , , 18, 24, 82 Kester, H., 228, 230, 243 Kikuchi, G., 38, 85 King, M . T., 8, 30, 39, 67, 85 Kinghorn, H. A., 197,201,205,206,209, 215, 219, 220, 234, 235, 237, 244 Kinney, R. W., 208, 240 Kitamura, K., 109, I20 Kitto, G. B., 230, 242 Kleber, H-P., 193,204,205,224,226,240, 24 I Kleinig, H . , 44, 51, 8 7 Kleinzeller, A,, 160, 177, 178 Klemme, J. H . , 8, 27, 28, 30, 85. 228, 241 Klingmuller, W., 171, 179 Kloppel, R., 170, 179 Knaff, D. B., 9, 85 Knobloch, K., 9, 86 Knopfel, H. P., 130, 137, 142. 144, 157, 160, 179

251

Koch, G., 106, I20 Koltin, Y ., 116, 117. IIY. 120 Kondratieva, E. N., 30, 84 Konings, W. N., 14, 31, 86 Kornberg,H. L., 137. 17Y. 186,212.226. 238, 241 Kornfeld, S., 228, 229, 239, 241 Kosakowski, M. H., 48, 53, 86 Kosicki, G. W., 192, 194, 241, 243 Kosobutskaya, L. M., 19, 86 Kotani. H., 1 1 I , I20 Kotyk, A,. 160, 161. 162, 163, 164, 165, 166, 167. 168, 169,170. 171, 174, 177, 178, 179 Krasna. A. I . , 30, 84 Krasnovsky, A. A,, 19, 86 Krebs,H.A., 124, 153, 155. 17Y, 185. 186. 191, 241 Krinsky, N , I . , 40, 86 Kronenberg, G. H. M., 68, 86 Kruczek, J., 10, 47, 8 7 Krupnick, D., 101, IIY Kuehn. G. D., 225, 238 Kuenzi, M.. 131, 139, 156, 159, 180 Kuhn, H. J., 129, 180 Kukla. D., 203, 244 Kulaev, I. S . , 169, 180, 182 Kunau, W. H., 52, YO Kunisawa, R., 2, 10, 12, 38. 54, 55, 57, 82, 89 Kuo, S. C., 165, 169, 180 Kurita, H., 150, 179

L Lachmi, B., 106. 119 Ladwig, R., 21, 39, 43, 44, 54, 77, 83, 89 Lagunas, R., 135, 161, 180 Lakshmi, T. M., 212, 241 La Monica, R. F., 8, 86 Lampe, H. H.. 14, 31, 43, 47, 51, 54, 65. 86 Lampen, J. O., 165, 180 Langan, J. J., 40, 43, 44, 66. 67, 87 Langley, D., 228, 236, 241 Lapage, S. P., 208, 242 Larsen, H., 210, 241 Lascelles, J., 13, 37, 38, 39,44,46, 47.48, 66, 72, 74, 75. 76, 77, 78, 82, 86, YO, 145, 180

252

AUTHOR INDEX

Lavergne. J., 34, 43, 66. 88 Leach. C. K.. 201. 220, 242 Lee. L. P. K.. 194. 241 Leibowitz, M. J., 97. 102. 103. 107, 120. I21 Lemke. P. A,. 99. 120 Lester, R. L., 48. 53. YO Leutiger, I.. 21. 39, 44, 77, 83 Levine. R., I 16, 117. 120 Lewin, R . A,, 2. 86, YO. 235. 241 Liaaen-Jensen, S., 40. 86 Lien, S.. 24. 34, 42, 58, 61, 66. 67, 68, 75, 86 Lin, F. J., 230, 241 Lindberg, B., 110. 112. I20 Liras, P., 165. 180 Liston, J . , 208, 23Y Liu, M. S., 190, 242 Loach. P. A., 23, 24, 29. 8Y. YO Lommen, M. A . J.. 14, 86 Londesborough. H., 149, I80 London, L., 162, 181 Long, W . S.. 170, 172. 187 Low, H., 14, 31, 86 Lowenstein, J . M.. 186. 19 I . 223. 241. 242 Lowery. S. N.. 164. 182 Lu. C . Y-H., 170, 172, 182 Lucas. C., 193, 195, 201, 207, 235. 241 Liicke, F-K., 27. 28. 86 Liideritz, R., 30, 86, 228, 241 Lueking. D. R.. 32. 41. 44, 46. 47. 72, 84 Lupiafiez. J. A,, 135. 180 Lynen, F., 153, 155, 180

M

Mansurova, S. E.. 169. 182 Margoliash, E., 29, 8Y Margulis, L., 234. 235, 241 Marmur. J.. 101, I I Y Marr. A . G., 13, 14, 54, 85 Marr, J. J. 224. 225, 241 Marrs, B., 5. 7. 8. 9, 39. 73. 74. 75. 77, 78, 86. 91 Marrs, B. L., 8, 39. 72, 86 Marx. R.. 37, 38, 42, 51, 63, 77, 81 Massarini. E.,204,205,21 I , 217,240,241 Masson. A-J., 110, 112, 120 Matern, H., 167, 180 Matin, A,, 223. 241 Matlib, M. A , , 195. 241 Matsuoka. Y., 194, 243 Mattoon, J., 159, 181 Maudinas, B.. 10. 86 Maule, A. P., 114, 120 Mauzerall, D. C., 6. 18, YO Maxwell. W. A,, 166, I80 Mayer, H., 2, 83 Mayer, I., 117, 120 Mayor, F., 135, 180 McCready. S. J., 97. 98, 100. I20 McLauglin. C . S., 95. 97, 98, 100, I I Y , I20 Mechler, B.. 18, 23, 26, 34, 62, 68, 69, 86 Mecke, D.. 150, 17Y Melandri. B. A , , 5, 6, 8, 27, 28, 33, 81, 85,86, 88, Y I Meredith, S. A,, 161. 163. 169, 180 Merril. C. R., 117, 121 Metzler, R., 166, 180 Meussdoerfer, F., 150, 152, 180 Meyenburg, H. K., von, 142, 177 Meyenburg, K. von, 140, 157, 159, 160,

Maaloe, 0.. 137, 1x1 Machado, A,. 135. 180 MacQuillan. A . M., 137. 180 Maeba. P.. 200, 207, 244 Magrum, L. J.. 2. YO Mainzer, S. E., 172, 177 Makower. M., 93, 108, 120 Malkin, R., 30, 86 Mallon, D. E.. 40, 43. 44, 66, 67. 83. 87 Mandel, M.. 2. 8Y Manners, D. J.. 110. 112, 120

Michaljanicova, D., 161, 164, 165, 169, 171, 174, 179 Michels, P. A . M . , 14. 31, 86 Middlebeck, E. J., 114, 115, 117, 118, 120. I21 Midgley, M., 31, 82 Miersch, J., 171. 180 Millis, N . F., 129. 177 Milner, Y., 228, 229, 23Y, 241 Minton, M . J., 53, 64, 85 Miovic. M. L.. 73, 86

180

253

AUTHOR INDEX

Misra, P. C., 171, 179, 180 Mitchell, C. G., 207. 241 Mitchell, D. J., 95, 97, 118, 120, 121 Mitchell, P., 31, 89, 170, 180, 182 Mitchell, T., 221, 222, 241 Mitsui, T.. 14, 90 Miyazaki, T., 35, 86 Model, P., 45, 82, 107, 119 Mollenhauer, H., 62, 84 Monger, T. G., 6, 34, 40, 86 Monod, J., 132, 135, 180, 204, 241 Moor, H., 10, 81 Mor, J-R., 128, 137, 180 Mori. K.. 150, 179 Morita. S., 26, 35, 86, 90 Moriyama, T., 203, 241 Morse, S. A., 190 240 Mortimer, R. K., 165, 180 Moskalenko, A. A,, 24, 86 Moss, J., 1 1 I , 120 Mossmann, M. R., 137, 178, 190, 239 Moya, I., 34, 43, 66, 88 Moyed, H. S., 190, 241 Moyle, J.. 31, 89 Muhlethaler, K., 10, 81, 87 Mukherjee, A., 194, 243 Muller, H. W., 28, 89 Murakami, K., 221. 222, 241 Murphey, W. H., 230, 241, 242 Musfeld, H.. 127, 180 Muytjens. H. L., 115, 117, 118, 120

N Nakano, S., 104, I20 Nakao, Y., 237, 243 Nandi, D. L., 220, 242 Naumov, G. I., 114, 120 Naumova, T. I., 114, I20 Neff, J., 150, 179 Nelter, K . F., 153, 180 Neu, H. C., 168, 180 Neuberger, A,, 37, 38, 40, 72, 73, 83, 84, 87. 89 Newman, A. M., 95, 119 Ng, F. M. W., 190, 242 Nicolson, H. M., 48, 89 Niederman, R. A., 4, 14, 18,33,40,43,44,

48, 51, 52, 53, 66, 67, 82. 84, 85, 87. 88, 223, 238 Nieth, K-F., 18, 33, 34, 42, 43, 87 Niklowitz, W., 4, 87 Nimmo, H. G., 227, 242 Nishimura, J. S., 219, 221, 222. 241, 242 Nishimura, M., 8, 30, 55, 64, 87, 89, 90 Nishimura, Y., 228, 244 Nishizuka, Y., 1 1 I , 119 Nomura, M., 117, 120 Norris, J., 21, 85 Northcote, D. H., 10, 81 Nothmann, D. L., 212, 238 Novick, P., 107, 120 Nugent, N. A., 13, 26, 84 Nunez de Castro, I., 135, 180

U

Ochoa, S., 192, 242 Oelze, J., 2, 4, 8, 9, 10, 13, 14, 16, 18, 21, 23, 26, 28, 31, 32, 33, 34, 35, 38, 40, 42, 44, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 58, 62, 63, 64, 68, 69, 73, 76, 77, 83, 84, 85, 86, 87, 90 Oesterhelt, D., 2, 87 Ohmann, E., 193, 204, 238 Ohmori, M., 150, 179 Ohne, M., 190, 242 Okada, H., 172, 173, 174, 180 Okamura, M. Y., 6, 18, 19, 31, 32, 33, 39, 44, 83, 86, 88 Oliver, S. G., 95, 97, 98, 100, IIY. I20 Olson, J. M., 13, 19, 26, 35, 87 Olsnes, S., 100, 112, 118, 120 Oren, R., 28, 84, 88 Ormerod, J. G., 62, 82 Ouchi, K., 104, 120 Oura, E., 128, 135, 141, 156, 157, 163, 180, 181, 182 Oyewole, S. H., 19, 81 Ozaki, H., 193, 224, 242 P

Packer, L., 10, 81 Pahlke. W.. 14. 40. 42. 63. 87 Paigen, K., 137, 181

254

AUTHOR INDEX

Paillotin, G., 35, 88, 90 Palfree, R. G. E., 96, 104, 108, 109, 110, 114, 119, 120 Palmer, J. M., 220, 242 Panek, A., 159, 181 Pardee, A. B., 4, 89 Parker, M. G., 224, 225, 226. 228. 229, 242, 244

Parks, L. C., 4, 14, 48, 51, 52, 88 Parks, L. W., 135, 177 Parson, W. W., 5, 6, 18, 24. 34, 39, 40, 82. 86, 88, YO

Passow, H . , 166, 181 Pastan, I., 149, 181 Pasteur, L., 153, 181 Patterson, J. C., 110, 112, 120 Payne, W., 157, 181 Pearce, J . , 201, 220, 242 Peeters, T. L., 190 242 Perham, B. N., 227, 228, 229, 234, 237, 238, 239, 240, 242

Peterkofsky, A,, 125, 149. 181 Peters, G. A., 13, 63, 82, 88 Peters, R. A,, 215. 242 Petty, K. M., 6, 83 Pfennig, N., 2, 3, 10. 12, 13, 40, 57, 79, 81, 82, 88. 90 Philliskirk, G., I 1 5, 120 Philosoph, S., 27, 88 Picorel, R., 7, 88 Pierson, B. K . , 12, 35, 62, 88 Pietras, D. F., 97, 119 Pihl, A,, 112, 120 Pinder, P. B.. 5, 7, 89 Pirt, S. J.. 129, 181 Plaut, G. W. E., 223, 225, 23Y, 242 Plewis, K. M., 41, 85 Polakis, E. S . , 137, 181 Porra, R. J., 145, 181 Postma, P. W., 149, 181 Pradel, J., 34, 43, 66, 88 Preiss, J., 190, 204, 2 1 I , 239 Prince, R. C., 4, 6, 8, 13, 19, 29, 33, 35, 83, 85, 88

Pucheu. N. L., 6, 19, 88 Pudek, M. R., 37, 88 Puglisi, P. P., 173, 177 Puhalla, J . E., 116, 119, 120

R Racevskis, J., 106. 120 Racker, E., 124, 153, 181 Ragland, T. E., 223, 242 Ramaiah, A,, 155, 181 Ramaley, R. E., 221, 238 Ramirez, J. M., 7, 83, 84, 88 Randall, L. L., 45, 88 Rank, G. H., 112, 119 Rann, D. L., 205. 244 Rauner, R. A,. 237, 239 Raveed, D., 14, 31, 32, 33, 51, 88 Reed, D. W., 14, 31, 32, 33, 51, 88 Reed, L. J., 227, 228, 229, 239, 242 Reeves, H. C., 225, 227, 238, 239 Reisinger, O., 10, 86 Reiss-Husson, F., 6, 19, 85 Remsen, C. C., 2, 13, 88 Reporter, M., 14, 31, 88 Rest, M., van der, 18, 81. 90 Reyn, A,, 208, 242 Richards, W. R., 37, 43. 44, 46, 88, 8Y

Richmond, M . H., 137, 181 Riemersma, J . C., 174, 181 Rittenberg, S. C., 223, 241 Robichon-Szulmajster. De.

H.,

168.

181

Rogers, D. T., 97, 104, 106, 107, 114, 115, 117, 118, I20 Rohm, K., 152, 178 Rohova. L., 163, 178 Romano, A. H., 137, 161, 163, 169, 180, 182

Romijn, J . C., 19, 88 Rose, A . H., 123, 181 Roseman, S., 149, 161, 181 Rosenberg, Th., 165, 182 Rosenbusch, J . P., 110, 118 Rothman, L., 159, 181 Rothstein, A,, 160, 161, 166, 169, 178, 181, 182

Rowe, A, J., 205, 206. 242 Rowe, L. B., 97, 98, 11Y Ruzicka, F. J., 237, 242

AUTHOR INDEX

S Sackmann, E., 49. 88 Sammler, P., 165, 178 Sanadi, D. R., 220, 242 Sandermann. H., 35, 47. 88 Sandy, J. D.. 37, 38. 72, 73. 83, 87, 88 San Pietro, A., 24, 28, 34, 42. 58, 61, 66, 67, 68, 75. 86

Sanwal, 9. D., 200, 203, 204, 207, 225, 226, 242, 244

Satir, B., 10, 81 Satir. P., 10, 81 Sato. M., 150, 17Y Sauer, 22, 23 Sauer, K.. 22. 23. 24, 25, 88 Saunders, V. A..7. 37, 41, 66, 82, 89 Saville, D.. 107, 108, 109. 110. 112, 114, 119, 120 Scarborough, G. A.. 162, 163. 170, 171, 172, 181

Schachman, H . U., 4, 8Y Schaller, K., 117, I20 Schatzmann. H., 127, 128, 134. 135, 136. 140, 141, 145, 147, 148, 154, 155, 156, 157, 160, 181

Schekman, R., 107, 120 Scherrer. R., 162, I81 Schlegel, H. G., 137, 181. 223. 224, 239 Schmidt, K.. 40. 8Y Schmitz, R.. 26. 89 Schneider, E., 28. 89 Schneider, R. P.. 172. 181 Schoff, E., 152, 179 Scholes, P., 31, 89 Scholze, H., 203, 244 Schon, G., 10, 54, 71, 73, 8Y Schott, E., 152, 17Y Schroder, J.. 37, 38, 39.42. 44,47, 51.63. 77. XI, 87

Schuegraf, A., 153, 180 Schulte, Th. H., 163, 181 Schumacher, A., 34, 39,40,43,44, 57, 58, 59, 60, 61, 66, 77. 83, 8Y Schwartz, E. R., 228, 242 Schwartz, M., 108, I18 Schwulera, U . , 28, 89 Seaston, A,, 173, 181 Segen. 9. J., 4. 43, 84, 87 Self, C . H., 224, 225, 226, 242

255

Senior. 9. W . , 117, I20 Senior, P. J., 197, 205, 242 Serrano, R., 164, 170. 173. 174, I81 Shaw, M . A.. 43, 8Y Shen, L. C., 226. 228. 242 Sherman, D.. 108, 109, 110. l l Y Shifrin, S., 117. 121 Shiio. I., 193, 224, 242 Shinmyo, A,, 1 1 I , 120 Shioi. Y., 30. 8Y Shiokawa. K., 62, YO Shuman. H., 108. 118 Sidey, C., 205, 206. 244 Silhavy, T., 108, 118 Silverstein, S., 103. I21 Singer, S. J., 13, 48, 8Y Singh, M., 203, 242 Sistrom, W. R., 4, 5, 6, 18, 21, 31, 32, 40, 42, 53, 54, 55, 57, 58, 61, 66. 71. 72. 73. 74. 75. 81, 82, 84, 8Y. Y I

Skinner, A. J., 216, 242 Skipper, N., 108. 115. 117. I I Y . I21 Skipper, N . A., 110, I I I , I21 Slayman, C. L.. 170. 172, I82 Slayman, C. W.. 170. 172, 177, 182 Slooten, L.. 6, 19, 8Y Smith, A . E.. 106, I I Y Smith. L., 5, 7, 8Y Smith, R. A,, 221, 242 Smith, W. P., 44, 8Y Snozzi, M.. 32, 33, 44, 83. 91 Sojka, G. A,, 48, 53, YO Solomon, M., 216, 217, 242, 244 Sols. A.. 153, 155, 160, 17Y. 182 Somers, J . M., 95. 97. 101, 109, 110, 118, 119. 121

Sorge. J. R., 6, 83 Spector, L. B., 191, 243 Sperber, E., 127, I82 Speth. V., 10, 81 Spoerl. E.. 164, 166, 180, 182 Sponholtz, D. K., 29, 89 Srere, P. A,, 191. 192, 193, 194, 195, 203. 204, 231, 239, 241, 242, 243

Srinivasan, V. R.. 137, 173 Stachow, C. S., 225, 242 Staehelin. L. A,. 10. 12, 13. 14. 16, 26, 31, 35, 36. 62. 81, 8Y

Stanier, R. Y., I . 2, 4, 13, 21, 26. 35, 40. 53. 54, 55. 73. 14, 82, 84, 89,

256

AUTHOR INDEX

Starr. M . P., 54. YO Stebliak. A. G., 169, 182 Steere. R. L.. 10. 82 Steiner. L. A,, 18, 31, 8 7 Steiner, S., 48, 53, YO Steinlauf. R.. 117. I20 Stenback, W. A., 208, 230 Stern, J . R.. 191. 243 Stern, T. A.. 115, 117, I I Y Steveninck, J . van. 161, 163. 165. 166, 169. 170. 17Y. 182 Stickland. L. H., 124, 182 Stier, T. J., 134, 177 Stoeckenius. W., 2. 52. 81. 87, YO Straley, S. C.. 6, 18, YO Strasters, K . C., 137, 182 Strating, M., 230, 243 Strittmatter, C. F., 137, 182 Strouse, C. E., 19, 21, YO Stumm, C., 114. 115, 117. 118. 120. 121 Sturgeon, J. A., 95, 98, I18 Styles, C. A., 101, 102, 119 Sundaram, T. K., 230. 243

Tanaka, N., 193. 197, 200. 243 Tassi, F., 173, 177 Tate, A., 97. I21 Tauchert, H.. 193, 204, 205, 240 Tauschel. H.-D.. 10, 13, YO Taylor, B. F., 193, 195,200,201,205.207, 209. 21 I . 243 Taylor, I . J., 197, 211. 238 Tejwani, G . A,, 154. 182 Theuvenet, A. P. R., 162, 163, 167, 168, I 78 Thomas, P. D., 114, I20 Thompson, R. C . , 44. 8Y Thornber, J . P., 2, 19. 21. 22. 23, 24, 26. 40, 69, 82. YO Throm, E., 4. YO Tiger, H . A,, 220. 244 Tipper, D. J., 95, 97. 98, 104, 106, 107, 114, 118. l l Y Toh-e, A,, 102, I21 Tomasz, A,. 110, 118 Tong, E. K., 203, 243 Tonn, S. J.. 23. 24, YO Trentini, W. C., 54, YO

Suomalaken, K , 128, 156, 163. f7g. /&'I Trosper. ?'

Sutherland, P. A., 97, 98, 100. I20 Suzuki, T.. 237, 243 Suzuki, Y.. 26, YO Sweeney, T. K . , 97, I21 Swissa, M., 193, 209, 210, 215, 243 Switzer, R., 152, 179 Switzer, R. C., 117, I21 Sybesma, C., 7, 8, 9, 84. 90 Sykes. J., 26, YO Szulmajster, J., 137, 17Y

T Tabor. C. W . . 102, 119 Tai, P-C.. 44, 8Y Tait, G. H.. 38, 40, 83, 84, 87 Takacs, B. J., 14. 16, YO Takahashi. M., 62. YO Takamiya, K.. 6. 8, 30, 83. 89, YO Takamya, A,, 26, YO Takaoka, Y., 110, I I Y Takemoto, J . . 14, 40, 42. 43. 61, 66, 75, 77. 86, YO Talsky. G. 25. YU

L.. 14. 21, g6

Triiper. H . G., 2, 3. 13. 40. 88. YO, 137, I81 Tsao. M . S.. 44, 46, 88 Tuboi, S., 38. 72, 17, 85 Tzen. J . C.. 97. I21

U Ueki, T.. 14, 90 Ujigawa, K.. 193, 242 Umnov, A. M.. 169, 182 Unkeless. J., 193, 240 Unmova, N . S., 169. 182 Upper, C. D.. 221, 23Y

v Van Dongen. W., 230, 243 Vatter, A. E., 4, 90 Vaughan, M . , 1 1 1, I20 Veeger, C., 228, 229, 238, 239 Vermeglio, A., 35, 88, 90 Vernon, L. P., 7, 62, 84, YO

257

AUTHOR INDEX

Villoutreix, J., 10, 86 Visser, J., 228, 230, 238. 240, 243 Vodkin, M., 97, 104, 121 Voinovskaya, K . K . , 19, 86

W Wagner, E. K . , 95. 119 Wakim. B., 10. 52, Y0 van der Wal, H. N., 7, 9, YO Wall, J . D., 212, 238 Wallace, R. B., 44, 46. 88 Walton, G. M.. 158, 177 Warburg, O., 124, 182 Ward, B. A., 205, 244 Warner, R . C., 95, 119 Wassink, E. C . , 19, 61, 85. YO Waterbury, J. B., 2, 90 Waygood, E. R . , 220, 242 Weaver, P.. 53, 56, 8 7 Weber, M. M., 224, 225, 241 Weckesser, J., 2, 10, 44, 51, 83, 87, 90 Wedding, R. T., 220, 242 Wegener. W. S., 137, 182 Weibel,E. K., 141, 156, 157, 158, 159, 182 Weinstein, R. S., 10, 82 Weitzman, P. D. J., 191, 192, 193, 194, 195, 196, 197, 198,200.20 I , 202,203, 204,205,206,207,208,209,2 10,2 1 1, 212.213.214,215.216,217.219,220, 221,222,224,225,226,228,229,233, 234,235,237,239,240,241,242,243, 244 Welsh, J. D., 103, I21 Wenner, C. E., 154, 182 Werkman, C . H.. 208, 240 West, I . C., 170, 182 Whatley, F. R., 234, 240 White, J., 127, 182 Wickerham, L. J., 129, 182 Wickner, R. B.. 95,97. 100, 101, 102. 103, 106, 107, 11 I , I I Y , 120, 121 Wickner, W., 116, 121 Wider, E. A.. 220, 244 Wiegand, G., 203, 244 Wiken, T. O., 133, 182 Wilbrandt, W., 163, 165, 166, 182 Wiley, W. R . , 172, 181 Wilkins, P. 0.. 166. 178

Wilkinson, A . E., 230, 243 Williams. B., 137, 181 Williams, J. P., 164, 182 Willsky, G. R., 113, 121, 170, 182 Wilson, D. B., 165, 182 Wimpenny, J. W. T., 127, 137, 178, 182, 190, 23Y Winkler, K. C., 137. 182 Withers, N. W., 2, 90 Woese, C . R., 2, 90 Wolf, D., 152, 182 Wolfe, R. S., 4, YO Wolin, M. J., 223, 238 Wood, H. A,, 116, 121 Woods, D. R., 108, I 1 I , 121 Worden, P. B., 54, 57, Y l Wraight, C. A,. 5, 45, 47, 72, Y I Wright, 1. P., 230, 243 Wright, .I.A., 200. 203. 204, 207, 244 Wu, J. Y., 203, 244 Wyman, J., 204. 241

Y Yagiu, M., 99, 114, 115. 117, I21 Yamamoto, Y . , 109, I20 Yamazaki, A,, 228, 244 Yang, J. T., 203. 144 Yates, D. W., 41, 82 Yen, H. C., 39, 77, Y I Yike, N. J., 30, 85 Yip, A. T., 225, 239 Yoch, D. C., 29, 82, Y l Yoneyama, Y., 38, 91 Yotsuyanagi, Y . , 147, 183 Young, T. W., 99. 114, 115, 117,120, I21 Yubisui, T., 38, 91

Z Zahand, J. P., 164, 182 Zankel, K. L., 5, 91 Zannoni, D., 5, 7, 8, 81, 91 Zatman, L. J., 190, 193, 197, 209, 211, 223, 239 Zimmerli, A,, 137, 142, 183 Zorin, N . A., 30, 84 Zuber, H., 21, 23, 24, 32, 82, 83 Ziirrer. H., 32, 33, 44, 83, Y I

Subject Index

A Acetobacter aceti, isocitrate dehydrogenase. 223 Acetobacter calcoaceticus, citrate synthase, molecular size, 202 succinyl-coenzyme A inhibition 202 Acetobacter peroxydans, isocitrate dehydrogenase, 223 Acrtobacter suhoxydans, isocitrate dehydrogenase. 223 Acrtobacter xylinurn citrate synthase. NADH, inhibition and, 210 mutants, citrate synthase, 21 5 Achromobacter liyuefuciens, citrate synthase, taxonomy and, 208 Acinetobacter spp. citric acid cycle, multipoint control, 232 isocitrate dehydrogenase isoenzymes, 226 Acinetobacter anitratus, citrate synthase, NADH inhibition and, 21 1 Acinetobacter calcoaceticus citrate synthase. allosterism. 204. 205, 206 inhibition by, NADH, 196 molecular size, 203, 207 isocitrate dehydrogenase, molecular weight, 225 mutants, citrate synthase, 215, 216 succinate thiokinase, nucleotide. specificity, 222 Active transport, sugars, 169-1 75 Adenylate control, citric acid cycle, Acinetobacter, 233

Aerobacrer arrogenes, mutants, citrate synthase, 21 1 Aerobic yeasts active sugar transport, 170- 172 facultative, active sugar transport. 172-1 75 Aerobiosis, yeast cells, 136-141 Aeromonas jormicans, citrate synthase, taxonomy and, 208 Allosterism, citrate synthase, 204-207 6-Aminolaevulinate synthase inhibition, 72 regulation, 38 AMP, pyruvate dehydrogenase stimulation by, 228 Anaerobic conditions, Crabtree effect, 144-149 Anaerobiosis, yeast cells, 133-1 36 Aspartate transcarbamylase Citrobacter freundii, 2 18 Escherichia coli, 207 Asprrgillus sp., double-stranded ribonucleic acid plasmids. 99 ATP citrate synthase inhibition by, 192-195 isocitrate dehydrogenase inhibition by, 224 microbial production, I57 ATPase. intracytoplasmic membranes, 14 Azotobacter hrijerinckii, citrate synthase, allosterism. 205 Azotobaeter vinelandii citrate synthase, molecular size, 203 isocitrate dehydrogenase, molecular weight. 224 258

SUBJECT INDEX

B Bacillus sp., malate dehydrogenase, 230 BuciNuJ caldolyticus, pyruvate dehydrogenase, 230 Bacillus culdo tenax, pyruvate deh yd rogenase, 230 Bucillus muceruns, citrate synthase, aoxoglutarate inhibition, 200 Bacillus polymyxu citrate synthase, a-oxoglutarate inhibition by, 200 pyruvate dehydrogenase, 230 Bacillus strurorhermophilus isocitrate dehydrogenase, molecular weight, 225 pyruvate dehydrogenase, 229, 230 Bacillus subtilis malate dehydrogenase, 230 mutants, citrate synthase, 212 pyruvate dehydrogenase, 230 Bacteria, citric acid cycle enzymes, 185244 Bacteriochloroph yll genes, 77 phototrophic bacteria, biogenesis, 37 synthesis, regulation, 73 Blue-green bacteria see Cyanobacteria Bordrtellu bronchisepticu citric acid cycle, multipoint control, 234 isocitrate dehydrogenase isoenzymes, 226 Branhumella sp., citrate synthase, NADH inhibition and, 210 Brevibucteriurn leucinophugum citric acid cycle, multipoint control, 234 citrate synthase, taxonomy and, 208 isocitrate dehydrogenase isoenzymes, 226

C Cundidu sp., susceptibility to glucose, 141 Cundida ulbicuns, effect of killer toxins on, 117

259

Cundidu bevrrwijkii, active sugar transport, 171 Cundiduguilliermondii, active sugar transport, 171 Cundidu parapsilosis, active sugar transport, 171 Cundidu tropicalis see Succhuromyws lipolyticu Capsid, killer yeast, 98-99 Carbohydrates catabolism, 186 Carotenoids genes, 77 phototrophic bacteria, biogenesis, 40 Catabolite inactivation, glucose metabolism regulation, yeast cells, 150153 Catabolite repression model, 176 glucose metabolism regulation in yeast cells, 149-150 Cellulomonas rossica, citrate synthase, taxonomy and, 208 Cell wall receptors for killer toxin, 109111 Chlorobiaceae growth, 4 properties, 3 Chlorobium spp. cytoplasmic membrane, 79 light-harvesting antenna pigment complexes, 25-26 Chlorobium limicolu chromosomes, 12 cytoplasmic membrane, 13 intracytoplasmic membranes, 16 light-harvesting antenna pigment complexes, 26 membrane, differentiation, light and, 62 organization, 1 1 photoreduction of N A D + , 8 reaction centre preparations, 19 Chlorobium thiosulp ha top hy Ilurn, c y t oplasmic membrane, ATPase, 31 Chloroflexaceae metabolism, 4 properties, 3 Cloroflexus spp. cytoplasmic membrane, 79 light-harvesting bacteriochlorophyll complexes, 80

260

SUBJECT INDEX

C'loroJle.uus aurantiacus chromosomes, 12 intracytoplasmic membranes, 14 membrane, differentiation, light and, 62 organization, I 1 Chloropseudomonas ethilica, membrane differentiation, light and, 62 Chloroplasts, evolution, 235 Chlorosomes, 13 supramolecular structur, 35 Chromatiaceae growth, 2 properties, 3 Chromatiurn vinosum bacteriochlorophyll synthesis, 73 chomatophores, electron-transport chains, 29 light-harvesting antenna pigment complexes, 23, 25-26 membranes, ATPase preparations, 27 differentiation, light and, 6 1 4 2 temperature and, 68-70 hydrogenase, 30 reaction centres, 18 acceptor, 6 Chromohacterium violaceum citric acid cycle, multipoint control, 234 isocitrate dehydrogenase isoenzymes, 226 Citrate synthase, 189 allosterism, 204-207 biosynthetic control, 198-202 characteristics, bacterial taxonomy and, 207-209 in citric acid cycle, 191-218 molecular size patterns, 202-204 Citric acid cycle, 185-191 enzymes, bacterial, 185-244 multipoint control, 231-234 Citrobacter freundii, aspartate transcarbamylase, 2 I8 Clostridium acidi-urici, citrate synthase. a-oxoglutarate inhibition, 200 Competitive acceleration, sugar transport and, 166-167 Corynebaclerium nephridii, citrate synthase, taxonomy and, 208 Counter-transport, sugars, 166-167

Coupling-factor ATPase, subunits, 3 1 Coupling-factor HEPases, phototrophic bacteria membranes, 27-28 Crabtree effect, 142-153 anaerobic conditions, 144149 Crjpptococcus neojormans, effect of killer toxins on, I18 Culture techniques, yeast cell growth and, 126-127 Cyanobacteria citrate synthase, inhibition, 195, 201 photosynthesis, 1 Cytochromes, phototrophic bacteria, biogenesis, 39 Cytochromes h, in photochemical electron transport of phototrophic bacteria, 7 Cytochromes c, in photochemical electron transport of phototrophic bacteria, 6 Cytochrome cz, phototrophic bacteria, 33 Cytochrome c oxidase, Rhodopseudomonas palustris membranes, 29

u Deharyomyces vanrijii, killer toxin, 1 16 Dehydrogenases, phototrophic bacteria respiratory chain, 35 Differentiation cellular membrane system, 36-78 phototrophic bacteria membranes, 191 external factors, 53-71 regulation, 71-78 Diffusion see Facilitated diffusion; Physical diffusion Dihydrolipoamide dehydrogenase, inhibition by NADH, 228 Double-stranded ribonucleic acid killer yeast, 95-98 replication, 103 Drosophila melanogaster, specific heat generation, 157

SUBJECT INDEX

E Electron-transport systems phototrophic bacteria membranes, 4-9 constituents, 29-30 Embden-Meyerhof-Parnas pathway, regulation, I53 Enzymes (See also specific enzymes) citric acid cycle, bacterial, 185-244 glucose metabolism, yeast cells, regulation, 142-144 Escherichia coli aspartate transcarbamylase, 207 citrate synthase, allosterism, 204. 205 inhibition by ATP, 195 inhibition by NADH, 196 molecular size, 203 succinyl-coenzyme A inhibition, 202 taxonomy and, 208 isocitrate dehydrogenase isoenzymes, 226 malate dehydrogenase, 230 membrane-bound proteins, 45 membranes, ATPase preparations, 28 mutants, citrate synthase, 21 I , 2 13. 214, 216 pyruvate dehydrogenase complex, 229 specific heat generation, 157 succinate thiokinase. molecular size, 219 Evolution, citric acid cycle and, 234-237

F F, complex, phototrophic bacteria membranes, 27 FI complex, phototrophic bacteria membranes, 27 Facilitated diffusion sugars, into yeasts, 163-168 plasma membrane proteins and, 167-168 Fermenting yeast, glucose sensitivity, 131

261

Pasteur effect and, 156 Gemella haemolysans, citrate synthase, 191 Glucose conversion to citric acid, 187 metabolism, regulation in growing yeast cells, 123-183 Green sulphur bacteria, membrane differentiation, light and, 62 Growth patterns, yeast types, 131-132 yeast cells, glucose metabolism regulation and, 125-141 physiology, 1 32- 141

H Haernophilus vaginalis, citrate synthase. taxonomy and, 208 Halobacterium spp. citrate synthase, 220 NADH inhibition and, 210 light-driven processes, 2 photosynthetically active membranes, 52 Hydrogenase. phototrophic bacteria membranes, 30 Hydrogenomonas eutropha, isocitrate dehydrogenase, 223

I Immunity, killer toxin, 114 Inhibition, sugar transport, specificity, 165-166 Isocitrate dehydrogenase, I89 citric acid cycle and, 223-227 isoenzyme, 224 molecular weight, 224

K Galactose metabolism in Saccharomyes ('erevisiae, facilitated diffusion, 167

Killer factor, yeast, physiology, 93-121 Killer (mak) genes maintenance, 101-102 mutations, 102

262

SUBJECT INDEX

Killer toxin. 1 0 4 1 18 cell-wall receptor, 109-1 1 I effect on pathogenic yeasts, 117-1 18 immunity, 114 in yeast genera, 1 1 4 - 1 16 membrane damaging by, 1 1 1-1 13 physiology of action, 107-109 post-wall receptor, 110 properties, 104106 secretion, 106-108 structure, 104106 synthesis, 106-108 Ustilago, 1 1 6 - 1 17 Kingella sp., citrate synthase, NADH inhibition and, 210 Klebsiella aerogenas, isocitrate dehydrogenase isoenzymes, 227 Kluyveromyces sp., active sugar transport, 173 Kluyveromyces drosophilanum, killer toxin, 116 Kluyveromyces nickerhamii, killer toxin, 116

L Lactobacillus plantarum, citrate synthase, 191 Light, differentiation of photosynthetic apparatus in bacteria and, 54-62 Light harvesting antenna bacteriochlorophyll-carotenoid-protein complexes, Rhodospirillales, 22-23 Light-harvesting antenna Bchl I1 complexes, 34 phototrophic bacteria membranes, 1926 regulation, 75, 77 L molecules, double-stranded ribonucleic acid, killer yeast, 95

M Malate dehydrogenase, 190 citric acid cycle and, 23G231 Mass transfer in yeast cell growth, 127

Membranes cytoplasmic, supramolecular structure, 35 interaction with killer toxin, 1 I 1 photosynthetically active, formation, 47-53 phototrophic bacteria, differentiation, 1&12 differentiation external factors, 5371 fine structure, 9-16 functional subunits, 16-30 intracytoplasmic, 12-16 topography, 3G36 organisation and differentiation, 193 supramolecular organisation, 9-36 Membrane system, cellular differentiation, 36-78 Menaquinone, acceptor in reaction centres, 6 Mitochondria, Paracoccus denitrifcans and, 234 M molecules, double-stranded ribonucleic acid, killer yeast, 95 Moraxella sp., citrate synthase, NADH inhibition and, 210 Mutants, citrate synthase activity and, 21 1-218

N NAD citrate synthase inhibition, 194 photoreduction. phototrophic bacteria, 8 NAD reductase, phototrophic bacteria membranes, 30 NADH citrate synthase inhibition by, 196-198 citrate synthase not inhibited by, 209 a-oxoglutarate dehydrogenase inhibition by, 228 pyruvate dehydrogenase inhibition by, 228 Neisseria spp., citrate synthase, NADH inhibition and, 210 Neurospora crassa active transport of sugars, 169-170. 171 facilitated sugar diffusion, I63

263

SUBJECT INDEX

Nuclear genes killer toxin immunity, 114 killer toxin production, 107 mutants. plasmid maintenance and control, 101-1 03 Nucleotides citrate synthase inhibition by, 192-195 succinate thiokinase specificity. 220223 Nutrition in yeast cell growth, 127 phototrophic bacteria membrane differentiation and, 70-71

0 Organisation, phototrophic bacteria membranes, 1-9 1 a-Oxoglutarate. citrate synthase inhibition by, 199, 201-212 a-Oxoglutarate dehydrogenase, 190 citric acid cycle, 227-230 Oxygen demand, Saccharomjws crrcvisiue, 126 photochromic bacteria membrane differentiation and, 63-67

P Paracoccus denitrijxans, citric acid cycle, evolution and, 234 Pasteur effect, 153-156 Pathogenic yeasts, effect of killer toxins on, 117-118 Penicillium sp., double-stranded ribonucleic acid plasmids, 99 Penicillium stolonijerum, PsV-S virus, double-stranded ribonucleic acid in, replication, 103 Peterkofsky model see Catabolite repression model Phenidate, methyl-sugar transport inhibition by, 166 Phenotypes, killer yeast, 96 Photosynthetic apparatus in phototrophic bacteria. 79 assembly in v i v o , 4 2 4 7 assembly, reconstitution experiments, 41-44 biogenesis. 37, 40

membranes, 4, 5 Phototrophic bacteria, membranes, organisation and differentiation. I91 Physical diffusion, sugars, through plasma membranes, 162~163 Pichia kluprri, killer toxin, 1 15 Pichia vanrijii, killer toxin, 116 Pigments, orientation in chromatophores, 34 Plasma membrane proteins, facilitated sugar diffusion and, 167 Plasmids genotypes, killer yeast. 96 nuclear gene mutants, maintenance and control, 101-103 replication, physiology, 100-101 Post-wall receptor for killer toxin, i 10 Prochlorophyta, photosynthesis, 2 Prosthecochloris drstuarii, membranes, NAD+ reductase, 30 Proteinases, Saccharomjws crrrvisiar. glucose repression, 152 Pseudomonas acmiginosu citrate synthase, 218 allosterism, 204, 205 mutants,citratesynthase, 215,216,217 pyruvate dehydrogenase, 230 succinate thiokinase. nucleotide specificity, 222 P.seu~lomona.s~uorescc~ns, pyruvate dehydrogenase, 230 Psc.udomonus iodinum, citrate synthase, taxonomy and. 208 Pyruvate dehydrogenase, 190 citric acid cycle, 227-230

0 Quinones, phototrophic bacteria, biogenesis, 39 Quinone Z in phototrophic bacteria, electron transport and. 6

R Reaction centres phototrophic bacteria membranes. 5 , 18-19

264

SUBJECT INDEX

Reaction centres -cant. molecular weight, 31 polypeptides, regulation, 77 Redox effectol. phototrophic bacteria, 73 Regulation glucose metabolism. in growing yeast cells, 123-183 yeast cells, 142-144 mechanism, 149-153 phototrophic bacteria membrane differentiation, 71-78 yeast cells, molecular background, 142-159 Replication double-stranded ribonucleic acid, 103 plasmids. physiology, 100-101 Respirative yeast, glucose sensitivity, 132 Respiratory chain facultative phototrophic bacteria. 7 in phototrophic bacteria, 79 membranes, 4 Rhodospirillaceae photoheterotrophic growth, 2 properties, 3 Rhodospirillineae, intracytoplasmic membranes, 12 Rhodospirillum sp., light-harvesting antenna pigment complexes, 24, 80 Rhodospirillum rubrum chromatophores, succinate dehydrogenase. 29 transhydrogenase. 30 citrate synthase, NADH inhibition and. 21 1 coupling-factor ATPase, 31 differentiation, 79 growth, 47 intracytoplasmic membranes, 48, 5 I , 53 chromatophores, 54 topography. 32 light-harvesting antennae pigment complexes, 2 1-24 regulation, 76 membranes, 50 ATPase preparations, 27 differentiation. light and, 54-57 nutrition and, 71 oxygen and, 6 3 4 4

functional subunits, I7 hydrogenase, 30 photosynthetic apparatus, assembly in vivo, 42 quinones, biogenesis, 40 reaction centres, 33 acceptor, 6 succinate dehydrogenase, 35 Rhodospirillum fenur cytoplasmic membrane, 52, 79 intracytoplasmic membranes, 48 light-harvesting bacteria chlorphyll complexes, 5 1 membrane differentiation, 10 Rhodotorula glutinis active sugar transport, 170 Rhodopsrudomonas sp. light-harvesting antenna pigment complexes, 24, 80 light-harvesting bacteriochlorophyll I, 80 light-harvesting bacteriochlorophyll I I complex, regulation, 76 membrane differentiation, oxygen and, 65-67 Rliodopscudot~~o~~us cupsulu tu bacteriochlorophyll biogeneses in, 39 cytochrome 1.2, 33 electron transfer, 6, 7 intracytoplasmic membranes, 14, 47, 51. 53 chromatophores, 54 topography, 32 light-harvesting antenna pigment complexes, 22, 24 light-harvesting Bchl 11 complexes, 34 regulation, 75, 76, 78 membranes, ATPase preparations. 27 differentiation, light and, 57-61 oxygen and, 65 temperature and, 67-68 electron-transport pathways, 5 functional subunits, 17 mutants, citrate synthase, 212 photoreduction of N A D + , 8 photosynthetic apparatus, 41 assembly it1 vivo, 42, 44, 45 reaction centres, 18, 31 Rhodopseudomonas gelatinosa membrane differentiation. 10

265

SUBJECT INDEX

Rliodo~ps1.udomoiia.Fgelatinosa4on t . reaction centre preparations, 19 Rhodopseudomonas palustris cytoplasmic membranes, 15 light-harvesting antenna pigment complexes, 24 light-harvesting bacteriochlorophyll I I complex, regulation, 76 membranes. Bchl complexes, 34 cytochrome c oxidase. 29 differentiation, light and, 57-6 I oxygen and, 66 functional subunits, 17 photosynthetic apparatus, 15 ubiquinone, biogenesis, 39 Rhodopseudornonas ruhrum electron transfer, 6, 7 intracytoplasmic membranes, 14, I6 photoreduction of N A D + , 8 reaction centres, 18 Rhodopseudomonas spheroides chromatophores, 14 electron-transport chains, 29 cytochrome cz, 33 cytoplasmic-membrane proteins, 49 electron transfer, 6 intracytoplasmic membranes, 48, 5051. 53 chromatophores, 54 topography, 31. 32 isocitrate dehydrogenase, molecular weight, 225 light-harvesting antenna pigment complexes, 22, 24 light-harvesting bacteriochlorophyll I I complex, regulation, 76 membranes, ATPase preparations, 27 Bchl, complexes, 34 cyctochromes h, 7 differentiation, light and, 57-61 oxygen and, 66 functional subunits, 17 photosynthetic apparatus, 41 assembly in vivo, 42, 45, 46, 47 photosynthetically active membranes, 52 proteins, biogenesis, 40 reaction centres, 18, 31, 33 acceptor, 6 preparations, 19

succinate thiokinase, nucleotide specificity, 221 ubiquinone, biogenesis, 39 Rhodopseudomonas fenue, intracytoplasmic membranes, 14 Rhodopseudomonas viridis intracytoplasmic membranes, 14 membranes, Bchl complexes, 35 functional subunits, 17 acceptor. 6 preparations, 19

S Saccharomyces sp., killer yeasts, 99 Saccharomyces carlshergensis active sugar transport, 173 sensitivity to glucose, 141 Saccharomyces cerevisiae active transport of sugars, 169-1 70, 172 aerobic growth, 138, 139 anaerobic growth, 134, 136 ATP production, 157 citrate synthase, inhibition, 195 inhibition by ATP, 192 facilitated sugar diffusion, 163-168 glucose metabolism enzymes, regulation, 142- 144 killer toxin, 115 killer yeast and, 94 membrane interaction with killer toxin, 1 1 1 micrographs, 146-147 oxygen demand, 126 Pasteur effect, 153 proteinases, glucose repression, 152 sugar transport, 160-162 Saccharomyces fragilis, active transport of sugars, 169-170, 173 Saccharomyces lipolytica glucose metabolism enzymes, regulation, 142-144 mitochondria1 organisation, I57 Salmonella typhimurium isocitrate dehydrogenase, isoenzymes, 227 molecular weight, 225 Saturation, sugars, sugar transport and, 163

266

SUBJECT INDEX

Sc~hi~o.saccliuroni~c~c~s pornhe facilitated sugar diffusion, 168 growth. 128. 129 sensitivity to glucose, 141 Sorratia rnarc~~~sccns. isocitrate dehydrogenase isoenzymes, 227 Sols model, 153-156 Specific heat. generation by microbes, 157 Strep/ococcu.s hovis, isocitrate dehydrogenase. 223 Strc~~~toc.oc~c~ir.\' /trc~c~rrli.v.citrate synthasc. 191 Succinate dehydrogenase from Rhodospirillurn ruhruni chromatophores. 29 Succinate thiokinase, 189 citric acid cycle and. 218-223 molecular size. 2 19-220 nucleotide specificity. 220-223 Succinyl-CoA. citrate synthase inhibition by. 201-202 Sugars, facilitated diffusion. plasma membrane proteins and. 167-168 Sugar transport active, 169- 175 facultative aerobic yeasts. 172-1 75 obligatory aerobic yeasts. 170-1 72 specificity. 164- 165 systems, through plasma membranes. 162- 175 yeast cells, glucose metabolism regulation and, 159-1 77 Super killer (ski) genes. 102-103

Thiobacillus ncapoli/anu.s, citrate syn-

thase, NADH inhibition and, 210 Thiohacillus thioosidans, isocitrate dehy-

drogenase, 223 Thiocupsa jloridana,

in t rac yt oplasm ic

membranes, I6 Thiocapsu rosropc.rsic,inu. membranes.

hydrogenase, 30 Torulopsis candida, active sugar trans-

port, 171 Torulopsis gluhrata

effect of killer toxins on, 1 17- I 18 killer toxins, 115 Transhydrogenase. Rhodospirilluni ruhrum chromatophores, 30 Trichosporon cutancum. aerobic growth, I39

U Ubiquinone in phototrophic bacteria membrane reaction centres, 18 Ubiquinone acceptor in reaction centres, 6 Uranyl ions, sugar transport inhibition by. 166 Ustilago maydis killer strains. 99 killer toxins. I 16- 1 17

W Wickerham medium, composition, 129

X T Taxonomy, bacterial, citrate synthase characteristics and, 207-209 Temperature, phototrophic bacteria membrane differentiation and. 6770 Thermus ayuat ic'us citrate synthase, 220 NADH inhibition and. 210 Thiohacillus sp.. citrate synthase. inhibition, 195 Tliiohucillus dcnitr~jican.s, citrate synthase, NADH inhibition and. 210

Xanthornonas hyacinthi

citric acid cycle, multipoint control. 2 34 isocitrate dehydrogenase isoenzymes, 226 Xan/hornonns pruni, isocitrate dehydrogenase, 223

Y Yeast, killer factor, physiology, 93- 121 Yeast cells. growing glucose metabolism. regulation, 123-1 83 Yeast types, growth patterns, 131-132

Rose/Morris: Advances in Microbial Physiology, Volume 22

Erratum

In the Contents List on p. viii the title of P.D.J. Weitzman’s chapter should be read Unity and Diversity in Some Bacterial Citric Acid-Cycle Enzymes In the list that follows Section I should read I. A view of the citric acid cycle

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