Advances in Microbial Physiology Volume 14

Advances in

MICROBIAL PHYSIOLOGY

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Advances in

MICROBIAL PHYSIOLOGY edited (y

A. H. ROSE School o f Biological Sciences Bath University England

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

Volume 14

1976

ACADEMIC PRESS London New York San Francisco A Subsidiary o f Harcourt Brace Jovanovich, Publishers

ACADEMIC PRESS INC. (LONDON) LTD. 24/28 Oval Road London NW 1 United States Edition published b y ACADEMIC PRESS INC. 111 Fifth Avenue New York, New York 10003

Copyright @ 1 9 7 6 by ACADEMIC PRESS INC. (LONDON) LTD.

All Rights Reserved No part of this b o o k may be reproduced i n any form by photostat, microfilm, o r any other means, without written permission from the publishers

Library of Congress Catalog Card Number: 67-19850 ISBN: 0-12-027714-X

Printed in Great Britain b y William Clowes and Soits Limited L o n d o n , Colchester and Beccles

Contributors to Volume 14 CLINTON BALLOU, Department of Biochemistry, University o f California, Berkeley, California 94720, U.S.A. J. A. COLE, Department of Biochemistry, University of Birmingham, Birmingham, B 1 5 2TT, England

D. E. F. HARRISON, Woodstock Laboratories, Shell Research Ltd., Sittingbourne, Kent R. E. MARQUIS, Department of Microbiology, University o f Rochester, School of Medicine & Dentistry, Rochester, New York 14642, U.S.A. A. H. STOUTHAMER,Biologisch Laboratorium der Vrije Universiteit, de Boelelaen 1087, Amsterdam-Buitenveldert, The Netherlands

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Contents Microbial Gas Metabolism J. A. COLE I. Introduction . 11. The Nitrogen Gases . A. Gaseous Intermediates in the Nitrogen Cycle . B. Gas Production from Nitrite . . C. Nitrogen Fixation D. Evolution of Nitrogenase . E. Genetic Regulation of Nitrogenase . F. Regulation of Inorganic Nitrogen Metabolism 111. Oxygen Metabolism by Micro-organisms . . A. Diversity of Oxygen Metabolism . B. Bacterial Cytochrome Oxidases C. Biochemical Basis of Oxygen Toxicity D. Control of Enzyme Synthesis by Oxygen. IV. Gaseous Carbon Compounds . A. Formation of Gaseous Carbon Compounds B. Utilization of Gaseous Carbon Compounds . . V. Hydrogen Metabolism A. Hydrogen Production B. Hydrogen Formation by Obligate Anaerobes C. Formate Hydrogenlyase Activity of Facultative Anaerobes D. Desulfovibrio Hydrogen Metabolism . E. Structure of Hydrogenase . . F. Aerobic Hydrogen Metabolism VI. Summary and Conclusions. VII. Acknowledgements . References .

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9 14 19 22 25 29 29 30 34 48 55 55 56 67 67 68 70 71 73 76 81 84 84

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Structure and Biosynthesis of the Mannan Component of the Yeast Cell Envelope CLINTON BALLOU I. Introduction . A. Organization of Mannan in t h e Cell Envelope . B. General Physical and Chemical Properties of Yeast Mannans C. Carbohydrate Composition of Mannans . VI I

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93 94 96 96

CONTENTS

viit

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99 99 100 100 101 103 104 105 107 . 107 . 126 . 128 . 129 . 136 . 137 138 . 138 147 . 151 . 153

11. General Methods for Structural Analysis of Yeast hlannans A. Mannan Isolation . B. Selective Acid Hydrolysis C. Selective Alkaline Degradation D. Selective Acetolysis E. Enzymic Degradation F. Nuclear Magnetic Resonance Spectroscopy G. Immunochemical Methods . 111. Detailed Structures of Specific Yeast Mannans A. Saccharomyces cerevisiae . B. Other Sacckaromyces Species . C. Kluyveromyces Species . D. Hansenula Species . E. Candida Species F. Other Yeast Mannans . IV. Mannan Biosynthesis A. Mannan Biosynthesis in Sacckaromyces species. B. Mannan Biosynthesis in other Yeasts . C. A Model for Mannan Biosynthesis . References .

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High-pressure Microbial Physiology ROBERT E. MARQUIS

I. Introduction . A. Background . B. Basic Methodology . 11. Information from High-pressure Chemistry . 111. Life and Death under Pressure . A. Long-Term Survival and Growth . B. Short-Term Survival and Death . IV. Effects of Pressure o n Biopolymers . A. Nucleic Acids . B. Protein Denaturation . C. Effects of Pressure o n Polymeric Interactions . V. Effects of Pressure o n Some Specific Microbial Cell Functions A. Permeability and Transport Reactions . B. Catabolic Processes . C. Biopolymer Synthesis . . D. Cell Division and hlorphological Differentiation E. Regulatory Functions . . F. Motility G. Luminescence . VI. Acknowledgements . . References

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159 159 163 168 174 174 183 191 192 195 202 211 211 213 222 229 231 233 233 234 234

CONTENTS

ix

T h e R e g u l a t i o n of R e s p i r a t i o n R a t e in G r o w i n g B a c t e r i a DAVID

E. F. HARRISON

.

I. Introduction . . . 11. Response of Respiration Rate t o Environmental Changes . A. Response t o Dissolved Oxygen Tension . B. Response t o Temperature . . . C. Response t o p H Value . D. Growth Rate . 111. Substrate Control of Respiration. . . . . IV. Adenosine Phosphates as Regulators of Respiration A. Steady-State Contents of Adenosine Phosphates in Growing Cells . B. Concept of Energy Charge C. Transient-State Studies. . V. Role of NADH in the Regulation of Respiration . . . A. Measurement of Nicotinamide Nucleotides B. Response of NAD(P)H Content t o Perturbations of the Steady State. . C. Oscillations in NAD(P)H Fluorescence . . D. Regulatory Role of NADH Dehydrogenase VI. Cytochromes as Regulators of Respiration A. T h e Inducibility of Bacterial Cytochromes . . B. Branched Electron-Transport Systems . . VII. Energy Conservation. VIII. General Conclusions . . . References

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243 245 246 256 257 258 259 263 265 267 269 277 278 281 285 290 290 291 297 303 306 309

B i o c h e m i s t r y and Genetics of N i t r a t e Reductase in Bacteria A. H. STOUTHAMER

I. Introduction . . 11. Properties of Nitrate Reductase . . . A. Differentiation of Nitrate- and Chlorate-Reducing Enzymes . B. Purification and Properties of Nitrate Reductase A . . . C. Role of Molybdate in the Formation of Nitrate Reductase . D. Role of Metals in Nitrate Reductase Activity . . . 111. Regulation of the Formation and Activity of Nitrate Reductase A. Regulation of the Formation of Dissimilatory Nitrate Reductase . B. Influence of Oxygen o n the Activity of Dissimilatory Nitrate . Reductase C. Regulation of the Formation and Activity of Assimilatory Nitrate Reductase . .

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315 316 316 318 325 329 332 332 339 341

CONTENTS

X

IV. Electron-Transport Chain t o Nitrate a n d Energy Conservation During Nitrate Respiration . . A. Electron-Transport Chain t o Nitrate . . B. Energy Conservation During Nitrate Respiration . . V. Genetics of Nitrate Reductase Formation . . A. Methods for the Isolation of Mutants Blocked in Nitrate Respiration. B. Genetic Mapping of Mutations Affecting Nitrate Reductase . Formation. C. Physiological Properties of Chlorate-Resistant Mutants . . D. Protein Composition of Membranes of Chlorate-Resistant Mutants E. In vitro Complementation Between Chlorate-Resistant Mutants VI. Concluding Remarks and Future Prospects . . VII. Acknowledgements . . References

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343 343 350 356

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357 360 362 364 367 369 370

Microbial Gas Metabolism J. A. COLE Department of Biochemistry, University of Birmingham, Birmingham 5 15 2TT, England

I. Introduction . 11. The Nitrogen Gases . A. Gaseous Intermediates in the Nitrogen Cycle B. Gas Production from Nitrite . C. Nitrogen Fixation . D. Evolution of Nitrogen , E. Genetic Regulation of Nitrogenase . F. Regulation of Inorganic Nitrogen Metabolism . 111. Oxygen Metabolism by Micro-organisms . A. Diversity of Oxygen Metabolism B. Bacterial Cytochrome Oxidases . . C. Biochemical Basis of Oxygen Toxicity D. Control of Enzyme Synthesis b y Oxygen IV. Gaseous Carbon Compounds . A. Formation of Gaseous Carbon Compounds B. Utilization of Gaseous Carbon Compounds V. Hydrogen Metabolism . A. Hydrogen Production . B. Hydrogen Formation by Obligate Anaerobes . C. Formate Hydrogenlyase Activity of Facultative Anaerobes D. Desulfovibrio Hydrogen Metabolism . E. Structure of Hydrogenase . F. Aerobic Hydrogen Metabolism . VI. Summary and Conclusions. VII. Acknowledgements . References .

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1 7 7 9 14 19 22 25 29 29 30 34 48 55 55 56 67 67 68 70 71 73 76 81 84 84

I. Introduction Four gases provide the biosphere with reservoirs of potentiallyuseful carbon, nitrogen and free energy: these are carbon dioxide, nitrogen, oxygen and hydrogen. Other gases which are substrates or products of microbial enzymes are listed in Table 1. Gas metabolism 1

2

J. A . C O L E

continues t o interest the microbial physiologist partly because of the underlying similarity in the properties of enzymes which metabolize gases, but mainly because it encompasses some of the oldest unsolved problems in chemical microbiology. The least o f these is the problem of semantics: in this review, the terms autotroph, chemolithotroph and hydrogcnomonad will refer, respectively, to organisms that can use carbon dioxide as the major source of cell carbon, organisms which can sjmthesize XTP at the expense o f energy released from inorganic redox reactions, and bacteria which can catalyse the Knallgas reaction (see Rittenberg, 1969; Davis et al., 1970). Many of the enzymes which metabolize gases have been described in earlier reviews in this series, and elsewhere. Only a summary of their properties will be presented. The cited references therefore fall into three categories: (i) those that are particularly informative t o the non-specialist; (ii) others that report data which were not available t o earlier reviewers; and (iii) conclusions that are based on controversial assumptions which should not pass unchallenged, but nevertheless mav be correct! Carbon dioxide is, by definition, the source of organic carbon for autotrophic growth; apart from biomass, it is also the predominant product of microbial respiratory processes. In the former role, carbon dioxide is the substrate for enzymic reduction, and in the latter the product of an oxidation. But why has the biological importance of carbon dioxide been emphasized, while methane has, by comparison, been ignored? hlethane is the catabolic product of certain anaerobes and a growth-supporting substrate for other microbes. It is only from hlan’s egocentric viewpoint that carbon dioxide is of more fundamental importance to the biosphere than methane, and if one accepts that carbon dioxide plays an important role as a metabolic regulator (rt’impenny, 1969a) then one should anticipate the possibility that methane has similar regulatory importance amongst the more evolved anaerobes. Even Man produces methane, albeit indirectl?.: trials at a recent Farnborough Air Dispiay, near London, detected one major air pollutant, namely methane, which had been evolved by those unacclaimed ruminants, the spectators! hlolecular oxygen, nitrogen and hydrogen are also both the end-products and the substrates of microbial metabolism. Oxygen is produced by both plants and cyanobacteria during photo-

3

MICROBIAL GAS METABOLISM

synthesis, and is reduced during aerobic respiration. Nitrogen production and consumption both involve reduction: it is formed during anaerobic respiration of nitrate or nitrite by denitrifiers (Painter, 1971) and it is the least desirable nitrogen source for nitrogen-fixing bacteria and cyanobacteria. Whether molecular hydrogen is formed or consumed depends on the balance between electron supply and demand-indeed, to the non-specialist, one o f the most confusing aspects of the metabolism of gases is t o predict which cultures will form or consume any particular gas. By definition, one should expect oxygen, carbon dioxide, nitrogen and hydrogen t o be consumed by aerobes, autotrophs, nitrogen fixers and hydrogenomonads, respectively, but these expectations are only TABLE 1. Gaseous Substrates or Products of Microbial Metabolism Origin or product Nitrite

Gas

NO N 2 0 N2 N2 NH3 0 2

Origin or product organic - NH2 (-NH) H2 0 ;H2 0 2 ; 0 2 -

CH4

co2

CH4 ; cell carbon

co

co2

H2 O;H+ Sulphate or sulphite

?

H2 H2 s

cysteine

?

-Oxidation

?

Reduction

-

reasonable in specific environments. Thus a facultative autotroph will produce, not consume, carbon dioxide during heterotrophic growth; and if the nif plasmid (a genetic determinant conferring ability t o fix nitrogen) were ever t o be successfully transferred t o Pseudornonas aeruGqinosa,nitrogen could be then evolved by a nitrogen-fixer. An aid t o an accurate prediction in many circumstances is that carbon dioxide is the product of oxidative metabolism (which unfortunately includes fermentation), but methane, nitrogen and hydrogen are products of anaerobic, reductive processes. Schlick (197 1) demonstrated that Rhodosjbirillurn ru brzirn either consumed or evoIved

4

J. A. COLE

hydrogen and carbon dioxide, depending on what other nitrogenand carbon-containing compounds were available. Thus this organism is a splendid advertisement for “High Speed Gas” in that it can survive with a diet of carbon dioxide, nitrogen and hydrogen. Other metabolizable gases in Table 1, such as hydrogen sulphide, nitric oxide and nitrous oxide and even ammonia, are intermediates of biological redox processes. We can therefore make the first generalization about gas enzymes: almost all of them catalyse electrontransfer reactions. An exception is carbonic anhydrase which catalyses hydration of carbon dioxide t o the bicarbonate anion. A second interesting problem of microbial gas metabolism concerns the special properties of enzymes which catalyse the transformation of substrates which, by definition, are volatile. One of the simplest models of an enzyme-catalysed reaction is: k+l

k+Z

k- I

k-2

E + S F ES S EP

k+3

E+P

where the enzyme E forms a complex ES with the substrate S, which in turn is transformed to a product complex, EP. The product complex then dissociates to yield product, P, and free enzyme (Haldane, 1930). The velocity, v, or a reaction which follows this mechanism is given by the expression:

vs

v = --. S+K Thus the rate of product formation depends on the optimal effectiveness (not efficiency!) of the enzyme V, the concentration of substrate S, and another specific property of the enzyme K. It would be intellectually satisfying if K were an equilibrium constant (or binding constant) for the formation of ES from E + S: in fact it is a function of the various rate constants, k, such that:

V also depends on the concentration of enzyme

MICROBIAL GAS METABOLISM

5

Notice that this simplistic model ignores: (i) the reverse reaction of E -F P to form EP, (ii) the formation of metabolic intermediates in the reaction, (iii) the requirement for a second substrate, that is an electron donor o r electron acceptor, and (iv) the possibility of multiple enzyme subunits, allosteric sites, or side reactions. Gases are volatile not only because they are small, but also because they interact poorly with other commonly available molecules. The solubility of many gases, such as nitrogen, oxygen, hydrogen and methane, is low and this property is especially significant to microbial gas metabolism. Organisms which produce gases as catabolic end products have no waste disposal problems because the product, P, never accumulates at concentrations sufficient t o inhibit its production. Thus photosynthetic organisms dispose of their excess oxidant as molecular oxygen, and heterotrophs release it as carbon dioxide. Many anaerobes dispose of their excess reducing potential as hydrogen gas or methane. Accumulation of these gases in the biosphere has subsequently favoured evolution of hydrogen- and methane-oxidizing bacteria to occupy the new ecological niche. To do so, these organisms had t o be capable of re-assimilating the volatile gases which had been lost so readily by the organisms that had produced them. Because of the low solubility of many gaseous substrates in aqueous media, v can only be rapid if V is high or K is low. Gas utilization will therefore be rapid only when either the enzyme concentration eo is high, or when k+ 1 ,k, 2 and k+ 3 are high. The Theory of Absolute Reaction Rates tells us that k , , , for example, is high when the energy of activation for the chemical transformation is low, or when considerable quantities of energy have been expended to synthesize a complex enzyme which optimizes the probability that the substrate will reach the active site. In summary, one would predict that enzymes which metabolize the least soluble gases either constitute a si
6

J. A. COLE

which are extremely high. The two most abundant gases in the atmosphere, oxygen and nitrogen, can both co-exist for long periods with molecular hydrogen, despite the fact that their conversion t o water or ammonia is thermodynamically favourable. The stability of molecular nitrogen and oxygen is due in part t o their chemical structures, which can be written as multiple-bonded molecules in which the outermost electron shell is full. b'hy, then, can the rate of oxygen reduction be accelerated by a far wider range of catalysts than the rate of nitrogen reduction? The reason can be deduced from the distribution of the outermost electrons that are contributed to the available molecular orbitals by each atom (Fig. 1).Each nitrogen atom contributes two electrons t o the two bonding and antibonding 2s orbitals (one electron in each) xnd three electrons t o the bonding r P x r P yand u p z orbitals. In nitrogen, therefore, the three available bonding orbitals are completely full, and the activation energy required t o displace any of them is consequently large. The oxygen molecule has available the same molecular orbitals at

10 outer e l e c t r o n s

Atomic orb1 t 01s

12 o u t e r e l e c t r o n s

Molecular orbitals

Atomic orbitals

FIG. 1. Distribution of outer electrons in the nitrogen and oxygen molecules.

MICROBIAL GAS METABOLISM

7

similarly spaced energy levels, but two extra electrons must be accommodated in antibonding orbitals. Two of these orbitals occur at the same energy level, so one electron is associated with each orbital, their spins being parallel. Oxygen is therefore a paramagnetic diradical rather than a double-bonded molecule, and the presence of unpaired electrons in higher energy orbitals means not only that the energies of activation of reactions involving oxygen are lower than those involving nitrogen, but also that oxygen will readily accept electrons, either one or two at a time, t o pair with its unpaired electrons. Thus oxygen is reactive, but nitrogen is inert. Extending these principles t o other gases, one can predict that hydrogen and methane are unreactive, nitric oxide is extremely reactive, and that carbon dioxide and nitrous oxide would show intermediate stability. It is interesting, therefore, to correlate the chemical stability of each gas with the degree of complexity of enzymes that metabolize it. 11. The Nitrogen Gases A. GASEOUS INTERMEDIATES IN THE NITROGEN CYCLE

An outline of the nitrogen cycle is shown in Fig. 2. Nitrate is formed from ammonia in well-aerated soils and water by a group of chemolithotrophic bacteria and a few fungi (Peck, 1968; Painter, 197 1). Hydroxylamine and nitrite are chemical intermediates in this process. In both assimilatory and dissimilatory nitrate reduction, nitrate is first reduced t o nitrite. Assimilatory nitrate reduction is far slower than dissimilatory nitrate reduction, but the latter occurs only in oxygen-deficient environments. There is no compelling evidence that these two processes involve two different enzymes, but variations in the intracellular location of nitrate reductase, its stability and its source of electrons have been noted (see, for example, Van’t Riet et al., 1968). Denitrification occurs when nitrate or nitrite is reduced t o gaseous products; these include traces of nitric oxide and nitrous oxide and large quantities of molecular nitrogen. Although the terms “denitrification” and “nitrate dissimilation” are frequently used interchangeably, one should realize that some micro-organisms can use nitrite as a terminal electron acceptor during anaerobic ,@owth in media rich in ammonium salts or amino acids, but the product is ammonia rather than a gas. Because the nitrite-nitrogen is

8

J. A. C O L E

not assimilated, this is essentiallv a dissimilatory process (see, for example, Prakash and Sadana, 1972). The three other major processes in the nitrogen cycle are the reduction of molecular nitrogen t o ammonia (“nitrogen fixation”), the incorporation of ammonia into organic nitrogen compounds (“ammonia assimilation”), and the deamination of organic nitrogen compounds. Four gases are included in Fig. 2, namely nitric oxide, nitrous oxide, molecular nitrogen and ammonia. Ecological and biochemical aspects of nitrogen fixation were reviewed by DaIton (1974) and by

,

/

Dissimilation

NO

Nitrification

z

NHzOH

; \

N2 0 ;_---------

Assimilation or Dissimilation

,,’ ?Assimilation?

y g

NH3 Deamination

Fixation

Organic Nitrogen

FIG. 2. Outline o f the biological nitrogen cycle.

Dalton and Rlortensen (1972), respectively. The review by Benemann and Valentine (1972) summarized the metabolic pathways which are used t o pass electrons t o the nitrogenase complex, and Eady and Postgate (1974) have provided a concise statement of currently accepted views of how these electrons are transferred t o molecular nitrogen. The reader is referred t o these reviews for information which has been omitted from Sections 11-C to 11-E (pp. 14-29). One might anticipate that the inclusion of oxides of nitrogen in Fig. 2 is somewhat speculative, but the case for considering ammonia as a metabolizable gas is clearcut. One aim of this section will be t o establish that the reverse is true. Ammonia is extremely soluble in water, but the PI<, for NH4+ (9.25 at 24OC) is so high that the

M ICRO BIAL GAS METABOLISM

9

concentration of unionized ammonia in neutral, aqueous media is extremelv low. Even if one accepts the dogma that ammonia can diffuse across cell boundaries at a sufficientlv rapid rate to maintain equilibrium, one should still require rigorous proof that NH, rather than NH4+ is the substrate for oxidation or assimilation reactions. The nearest that one can get is well illustrated in a recent paper by Suzuki et al. (1974). These authors determined the rate at which ammonia” oxidation was catalysed by cells and extracts of Nitrosomonas europaea at various pH values, and from their data showed that although the apparent K, for NH,’ was pH-dependent, the apparent K, for NH3 was constant. Reference t o the Introduction (p. 5) will remind the reader of a few of the many assumptions that must be made before one can conclude that NH3 is therefore the true substrate for “ammonia oxidase”. It would also be difficult t o design an experiment t o show whether there is an active transport mechanism for NH,’, and, if so, t o determine the relative rates of “ammonia” uptake by active transport and free diffusion. Although ammonia was probably a major component of the prebiotic atmosphere, and was therefore a potential source of nitrogen for primitive micro-organisms, contemporary microbes predominantly encounter the ionized species, NH,’. There are good reasons, therefore, for omitting ammonia from a list of typical gaseous metabolites. Because an earlier review in this series was entirely concerned with inorganic nitrogen metabolism (Brown et al., 1974), ammonia metabolism will not be considered in detail. A brief discussion of the role of glutamine synthetase in regulating the synthesis o f nitrogenase will, however, be included in Section 11-E (p. 22). The metabolism of nitrous and nitric oxides also will be considered because Painter (197 1) stated that, although nitric oxide was a well-established intermediate in nitrate reduction by both assiniilatorv and dissimilatory organisms, evidence that nitrous oxide was a biochemical intermediate was scanty. These points will now be re-examined in the light of results which have been published subsequently. ‘1

B. GAS PRODUCTION FROM NITRITE

Although nitrite reduction has been investigated with a variety of micro-organisms, the majority of our detailed biochemical information has been derived from studies with seven bacterial species:

10

J. A. COLE

these are Pseudomonas denitrificans, P. stutzeri, P. aeruginosa, Micrococcus denitrificans, Achromobacter fischeri, Alcaligenes faecalis and E. coli. The reader is therefore warned that generalizations in the literature are inevitably based on a small and unrepresentative sample of microbial types. An extensive review by Payne (1973) described various properties, cofactor requirements and cellular locations of nitrate and nitrite reductases in fungi and algae. These enzymes essentially have an assimilatory function, and their syntheses are usually repressed by ammonia and by organic nitrogen compounds. Contrary t o the situation with many bacterial species, hydroxyIamine is well established as an intermediate in the fungal conversion of nitrite t o ammonia (Magill, 1972; Payne, 1973). Although an anaerobic, respiratory function has been suggested for nitrate reductases from Neurospora crassa and Hygrophorus conicus, neither of these fungi reduces nitrate t o gaseous products (Nicholas and Wilson, 1964; Mehta and Siehr, 1973). The only relevance to microbial gas metabolism of nitrate or nitrite reduction by eukaryotes is that hydrogen and hydrogenase can donate electrons via ferredoxin t o nitrite reductase of the alga Chlorella fusca (Stiller, 1966). An analogous process is believed t o occur in Clostridium pasteurianum (Mortenson et a!., 1962). Payne (1973) has also reviewed the early literature concerned with the evolution of nitrogen oxides by micro-organisms, as well as his own work with the marine pseudomonad P. perfectomarinus. Nitrous oxide was shown nearly 1 0 years ago t o be the end product of denitrification by Corynebacterium nephridii (Hart et al., 1965), but its routine analysis in gas mixtures became possible only with the more recent development of sensitive gas chromatographic techniques. It has subsequently been established that the bacterial species which catalyse reduction of nitrite to nitrous oxide or molecular nitrogen include both autotrophs and a filamentous budding bacterium Hyphomicrobium which denitrifies vigorously with methanol (Payne, 1973; Sperl and Hoare, 1971). Pseudomonads are non-fermentative bacteria, but some of them will grow anaerobically when nitrate or nitrite is available t o replace oxygen as a terminal electron acceptor: oxidative phosphorylation is coupled to the reduction of both compounds. A long series of investigations with P. denitrificans, in T. Rlori’s laboratory, has established that the evQlution of nitrogen from nitrite is catalysed by

MICROBIAL GAS METABOLISM

11

the sequential activity of three enzymes, nitrite reductase, nitric oxide reductase and nitrous oxide reductase. Nitrite reductase from this species is a soluble blue copper protein which, when pure and oxidized, had an absorbance optimum at 594 nm. Its catalytic activity was inhibited by cyanide and by diethyldithiocarbamate, but the cyanide-inhibited enzyme could be re-activated by cupric ions (Iwaski et al., 1963). Electrons for nitrite reduction could be donated by cytochromes c 5 5 2 or c s s 3 of Pseudomonas sp, derived from lactate or from ascorbate and tetramethylphenylenediamine. The reduction product was nitric oxide (hliyata and Mori, 1969). When the electron donors were ascorbate and tetramethylphenylenediamine, the enzyme catalysed the reduction of molecular oxygen and hydroxylamine as well as nitrite. These investigations supplemented earlier work by Radcliffe and Nicholas (1968) who also purified a soluble nitrite reductase from the same source: reduced benzyl viologen and flavin mononucleotide (FMN) were the most effective electron donors, and 2,4-dinitrophenol inhibited the formation of nitric oxide from nitrite. This was not due to its activity as an uncoupler of oxidative phosphorylation, however, but to its preferential reduction t o 2-amino-4-nitrophenol. Flavin COenzymes were required to transfer electrons from particulate dehydrogenases to nitrite reductase (Radcliffe and Nicholas, 1968). The optimum pH value for this enzyme was 6.0 rather than 6.8- 7.4, but this difference may reflect differences in the assay procedures in the two laboratories (especially with respect to the electron donor) rather than to intrinsic differences in the enzyme preparations. Nitric oxide reductase activity has been detected in cell-free extracts of P. denitrz'ficans (hliyata et al., 1969). This enzyme was bound to the cytoplasmic membrane, and it catalysed reduction of nitric oxides, by lactate or formate, to nitrous oxide. Succinate, oxaloacetate and NADH, were ineffective electron donors, and unlike nitrite reductase activity, neither FhlN nor flavin adenine dinucleotide (FAD) stimulated the enzyme. hlercurials, such as P-chloromercuribenzoate, inhibited the enzyme (85% at 1 mhI), but 2,4-dinitrophenol had little effect. Unbroken bacteria converted nitric oxide to molecular nitrogen but evolution of nitrogen gas was strongly inhibited by azide (hliyata e t al., 1969). It was subsequently established that nitric oxide and nitrate reductases compete for electrons from substrates such as lactate: it was suggested that

12

J. A. COLE

electrons flow from lactate dehvdrogenase to a 6-type cytochrome, and then either to nitrate reductase and nitrate, or to aparticulate cytochrome c 5 5 2 , nitric oxide reductase and nitric oxide (h4atsubara, 1971). Far less is known about the conversion of nitrous oxide to molecular nitrogen. Alatsubara and hlori (1968) detected nitrous oxide reductase activity in unbroken bacteria when lactate was the electron donor, but the enzyme was inactivated by sonication. The optimum pH value for nitrous oxide reduction was 7.0 and the K , for nitrous oxide was 50 pm. Iodoacetate, carbon monoxide and copper sulphate were potent inhibitors of the enzyme, and K 2 formation was also inhibited by 99% by 0.5 mW1 azide, by 95% by 50 pM cyanide and by 100% by 0.1 mM 2,4-dinitrophenol. When P. denitrificans was grown anaerobically with nitrous oxide as the only inorganic oxidant, the bacteria actively reduced nitrous oxide t o N 2 , but activities of nitrate, nitrite and nitric oxide reductases were far less than in bacteria which had been grown with nitrate (Rlatsubara, 197 1). This observation suggests that the enzymes for denitrification might be reLp1ated sequentially rather than co-ordinately, and that phosphorylation is coupled to the reduction of nitrous oxide. Furthermore, nitrite reductase was more active than nitric oxide reductase, and more than 90% of the product of nitrite reduction was nitric oxide: this provides independent evidence that nitric oxide reductase has an important physiological role in nitrate dissimilation. Pseudomonas stutzeri and P. aeruginosa also reduce nitrate or nitrite t o molecular nitrogen, but like A. faecalis and M. denitrificans, these bacteria synthesize the cytochrome cd-type of nitrite reductase. This “haem d” component is abnormal because it is readily released by extracting the protein with methyl ethyl ketone at pH 4.5 (Xewton, 1969). This type of nitrite reductase also has cytochrome oxidase activity, but the K, for oxygen is high (27 phl; Lam and Nicholas, 1969). These bacteria only synthesize cytochrome cd during anaerobic growth with nitrate or nitrite, and they form alternative cytochrome oxidases during aerobic growth (Kodama and Shidara, 1969; Lam and Nicholas, 1969). It is therefore unlikely that cytochrome cd functions in vivo as a cytochrome oxidase. The haem c of reduced cytochrome cd must be attached in an irregular manner because its reduced form gives two absorbance maxima, at 552 and 558 nm. Identity has been established between the soluble nitric oxide reductase activity and cytochrome cd from

13

MICROBIAL GAS METABOLISM

A . fueculis Matsubara and Iwasaki, 1972). This activity is very low compared with the nitrite reductase activity, and an active nitric oxide reductase has been detected in membranes from the same species. hlatsubara and Iwasaki (1971) have selectively inhibited nitrous oxide reductase with 50 pM cyanide, and confirmed that nitric oxide and nitrous oxide are intermediates in the dissimilation of nitrite by A. fueculis. Part of their summary of the properties of enzymes which convert nitrite into gases is shown in Table 2. TABLE 2. Some Properties of the Nitrite Reduction Systems i n Denitrifying Bacteria

---

-

Assay

Pseudomonas stutzeri strain van Niel

__

Pseudomonas Alcaligenes denitrificans faecalis

UNBROKEN BACTERIA Main product from lactate + NOzMinor product from lactate

+ NO2-

Inhibition of gas evolution by 1 0 - 4 M CN-

NZ

N2O + N2

N2

NZ0

-

N2 0

68%

20%

N O + Nz 0

N2 0

75% N O + N2O

Product from lactate + NO2N2 0 Cytochrome cd-type NO2- reductase PRESENT

NZ 0 ABSENT

N2 0 PRESENT

Cu-protein-type of NO*- reductase

PRESENT

ABSENT

Main products when CN- present

CELL-FREE EXTRACTS

ABSENT

Data compiled fromMatsubara and Iwasaki (1971) and Miyata and Mori (1969).

Nitrous oxide and nitric oxide are now both well-established intermediates in the denitrification process. Nitrite reductase activity is, however, derepressed during anaerobic growth of many bacteria which d o not evolve molecular nitrogen, nitrous oxide or nitric oxide, and the product of nitrite reduction b y these species is usuallv ammonia. The NADH, -dependent nitrite reductase activities from A . fischeri and E. coli are just two examples of this type of nitrite dissimilation (Prakash and Sadana, 1972; Zarowny and Sanwal, 1963; Cole, 1968). The enzyme from E. coli has been purified to homogeneity but, unlike the A. fischcri enzyme, it does not contain haem c (K. J. Coleman and J. A. Cole, unpublished results). Nitrite

14

J. A. COLE

reductases from Bacillus licheniformis and Desulfovibrio desulfuricans also reduce nitrite to ammonia, and cytochrome c3 from the latter organism will also catalyse this reaction. hlany of these enzymes will reduce hydroxylamine t o ammonia, but the rates of hydroxylamine reduction are slower than the overall rate of nitrite reduction, and the K, for hydroxylamine is far higher than that for nitrite: it is therefore unlikely that hydroxylamine is a normal substrate for these enzvmes, or a free chemical intermediate in nitrite reduction (see, for example, Prakash and Sadana, 1972). Although there is ample evidence that the function of the NADH,-nitrite oxidoreductase from E. coli or A . fischeri is t o reduce nitrite, other enzymes are known which reduce nitrite t o ammonia, but function in uiuo as sulphite reductases (Kemp et al., 1963; Prabhakararao and Nicholas, 1970). The relevance of these enzvmes t o microbial gas metabolism is this: once it is firmly established that an enzyme is capable of transferring six electrons t o a substrate (for example, t o convert sulphite t o sulphide or molecular nitrogen t o ammonia), then it is unnecessary t o hunt for gaseous intermediates such as nitric oxide or nitrous oxide from microbes which assimilate nitrite. Hypothetical intermediates in the reduction or nitrite t o ammonia can safely be forgotten (see, for example, Fry, 1955; Painter, 1971). One interesting question which remains unanswered is whether oxides of nitrogen are chemical intermediates in nitrate assimilation by denitrifying bacteria. hlutants of P. aerziginosa were recently isolated which neither evolved gas when grown anaerobically with nitrate and ammonia, nor grew with nitrite as sole nitrogen source (H. Swain, H. J. Somerville a n d J . A. Cole, unpublished results). It is therefore probable that assimilatory and dissimilatory nitrite reductases share some common genetic determinants, and it is possible that nitric oxide is an intermediate in nitrate assimilation by these bacteria. Meyer (1973) has also reported that nitric oxide is produced when nitrite interacts with the reduced form of some cytochromes. C. NITROGEN FIXATION

3licrobial nitrogen fixation has attracted more interest from reviewers than any other aspect of gas metabolism. Its appeal to academics stems from the contrast between the mild conditions

15

M ICRO BIAL GAS METABOLISM

requircd to convert molecular nitrogen to ammonia enzymically, and the extremes of temperature and pressure that are used in chemical processes. Hardy e t al. (1971) estimated that micro-organisms were responsible for more than 80% of the 1.1 x 10’ kg of nitrogen gas which was converted to ammonia in 1970. Agricultural crop yields can usually be increased by adding “fixed” nitrogen to farmland, so any increase in the rate of biological nitrogen reduction in situ should permit a corresponding decrease in expenditure of eneqgy and manpower for chemical processes. A wide range of bacteria and cyanobacteria from unrelated genera synthesize nitrogenase, but no eukaryotes fulfil the current criteria for a nitrogen-reducing species. I t is conspicuously absent from the pseudomonads, a genus in which plasmids abound (Dalton, 1974). Nitrogenases from Clostridium pasteurianurn and Klebsiella pneumoniae have been purified and characterized in impressive detail (Chen et al., 1973; Eady et al., 1972), but pure preparations have also been obtained from other sources (see, for example, Bums et al., 1970; Kelly, 1969; Klucas et al., 1968). Some of these preparations have been used for electron paramagnetic resonance studies (Smith et al., 1972, 1973; Orme-Johnson et al., 1972; Orme-Johnson and Beinert, 1969; Mortensen et al., 1973), circular dichroism (Chen et al., 1973), Mossbauer spectroscopy (Smith and Lang, 1974) and kinetic analysis with various inhibitors (Hwang et al., 1973). The nitrogenase complex is polymeric, and several different designations are used to specify its component proteins. In this review, the term nitrogenase refers to the catalytically-active complex: molybdoferredoxin refers t o the larger component protein, and this has also been called component I or the molybdenum-iron protein. Azoferredoxin refers to the smaller component which contains iron but no molybdenum; it is also known as component I1 or the iron protein. Use of the terms azoferredoxin and molybdoferredoxin should not be taken to imply homology between these Proteins and ferredoxins from plants, photosynthetic bacteria, or clostridia, and neither does it indicate their mode of action. Nitrogenase from Cl. pasteurianum probably contains one molecule of molybdoferredoxjn and two molecules of azoferredoxin (Dalton and Mortensen, 19 72). hlolybdoferredoxin is a tetramer with two subunits of mass 59,500 daltons and two of mass 50,700 daltons, but azoferredoxin is a dimer with identical subunits of mass

’

16

J. A. C O L E

27,500 daltons. hlolybdoferredoxin contains 18 iron and two molybdenum atoms as well as calcium and magnesium, but the only metal which is bound firmly to azoferredoxin is iron (Dalton et al., 1971; Mortensen et al., 1967). Table 3 compares the properties of nitrogenase proteins isolated from K. pneumoniae with those from Cl. TABLE 3 . Comparison of Components of the Nitrogenase System Present in Clostridium pasteurianum and Klebsiella pneumoniae Composition or property

Clostridium pasteu ria nu ma

Kle bsiella p n eu m o n iae

MOLY BDOFERREDOXIN Molecular weight Number and molecular weight of subunits Number of atoms of iron Number of atoms of molybdenum Acid-labile sulphide groups

220,000 2 x 59,500 plus 2 x 50,700 18

2 20,000

2 x 59,600 plus 2 x 51,300 17.5

2

1

16

17

NO YES NO

YES NO YES

Complements azoferredoxin from:

Klebsiella pneumoniae Clostridium pasteuriarium Ar o t o bac ter vinelandii AZOFERREDOXIN Molecular weight Number and molecular weight of subunits

55,000

62,000

2 x 27,500

2 x 34,600

Number of atoms of iron

4

4

Number of atoms of molybdenum

0

0

Acid-labile sulphide groups

11

11

Cold labile

YES

NO

a Data from Dalton and Mortensen (1972). Data from Eady et al. (1972).

pasteuriunum. Although nitrogenases from these and other species vary in detail, their superficial similarity is particularlv striking. All require ATP or an ATP-generating system, Mg2+ and a source of reductant for activity. Both component proteins are inactivated by oxygen, and it is essential t o purify azoferredoxin anaerobically if activity is to be retained. Various small molecules with C-C or C-N

MICROBIAL GAS METABOLISM

17

triple bonds are substrates for the enzvme, and the many inhibitors include gases such as oxygen, nitric oxide, hydrogen and carbon monoxide. The only physiological inhibitor is .4DP (hloustafa and >lortensen, 1967). Nitrogenase will catalyse a reductant-dependent hydrolysis of ATP, the ATP-dependent evolution of molecular hydrogen, and the reduction of acetylene to ethylene. Low concentrations of ethylene can be measured accurately by gas chrornatocgraphy, s o this last reaction provides a sensitive assay for “nitrogenase” activity. Current interest in nitrogenase stems from the above properties. It is a large protein which has yet to be sequenced, so the composition of the catalytic site is unknown. It contains six subunits of three different types (Eady c t al., 1972; Huang e t al., 1973; but not as in J e n g and Mortenson, 1969), s o numerous assumptions have t o be made to deduce its catalytic mechanism. Purified azoferredoxins show a range of cross-reactivity with moljibdoferredoxins from other species; and they d o not occur in eukaryotes despite the existence of environments in which a nitrogen-fixing plant would have a demonstrable advantage. Knowledge of the mechanism of nitrogen reduction has been derived largely from hlossbauer and electron paramagnetic resonance spectroscopy which provide independent probes of the density of d electrons around the iron atoms. Smith et al. (1972, 1973) have concluded that electrons are transferred from dithionite or the physiological electron donor t o azoferredoxin, and then to the substrate-binding site on molybdoferredoxin. Contrary to the conclusions of Mortensen et al. (1973), which were based on electron paramagnetic resonance spectroscopy alone, Smith deduced that electrons were transferred t o a reduced form of molybdoferredoxin to give a super-reduced form which was analogous t o reduced high-potential iron protein from Chromatiurn vinosum (Smith and Lang, 1974). The N,-binding site should therefore be on molybdoferredoxin but, because neither substrates nor the inhibitor, carbon monoxide, influenced the intensity of the hlossbauer signal from the super-reduced iron atom, it is unlikely that iron is directly involved in substrate binding. B r i n t h g e r (196613) has proposed a general mechanism for transferring six electrons t o molecular nitrogen without the formation of energetically unfavourable intermediates. This would involve doubleinsertion of a dinitrogen molecule into two metal-hydride bonds of a

18

J. A. C O L E

dimeric p-hydrido complex, as probably occurred in his nonenzymic reduction of molecular nitrogen (Brintzinger, 1966,). This mechanism has two merits. Firstly, because both molybdenum and iron can form hydrides, it eliminates the necessity for involving iron at the substrate binding-site by suggesting that the molybdenum atom is an equally plausible site. Secondly, it allows the ATPdependent hydrogenase activity in the absence of molecular nitrogen to be explained by the decomposition of the metal hydride when n o electron acceptor was present. The scheme is outlined in Fig. 3 .

P

M(gd

N;M(

II

red)

--- ----M (red 1

m

Ip I

tI + N p

PHX

I

Y

t

t 2 H - -2X-

1+2HX

r

Xp(oX)

XZM(OX)

t2NH3

m

YI

FIG. 3. Postulated mechanism for reduction o f molecular nitrogen to ammonia. hl(red) represents the metal in its reduced state, a n d M(ox) the metal in its oxidized state. HX represents an acidic reagent. From Brintzinger (1966b).

Attempts to distinguish between substrate and inhibitor bindingsites have been less successful. Hwang et al. (1973) measured nitrogenase activity with a variety o f substrates and inhibitors a t different concentrations, and applied Cleland’s (1967) methodology t o interpret the results. Five sites on nitrogenase were proposed-an N, -binding and H, -inhibition site, the CO-inhibition site, the Hz evolution site, the acetylene-reduction site, and a site for azide, cyanide and methylisocyanide reduction. Two attitudes have been adopted towards data such as this. Either one can accept it at its face

MICROBIAL GAS METABOLISM

19

value, in which case there are important implications for other aspects of nitrogenase research; or one can challenge the inevitable assumptions that are made about equilibria, chemical intermediates and the rate-determining step in order t o analyse kinetic data for an enzyme as complex as nitrogenase. What, for example, constitutes an independent site for substrates or inhibitors that are as small as molecular nitrogen, molecular hydrogen, hydrogen ion and carbon monoxide? If such doubts are unjustified, then the detection of independent sites for acetylene- and nitrogen-reduction raises the serious possibility that the acetylene reduction tests will not always give a reliable assay of “nitrogenase” activity. Hwang’s data established that the K, for nitrogen for the A . uinelundii enzyme was 0.1 atmosphere. If the K, could be taken as a measure of the affinity of the enzyme for dinitrogen, nitrogenase would be effective when the intracellular dinitrogen concentration was one-eighth that of air-saturated water. A permease or activetransport mechanism is of survival value to a cell only when the rate of eniry of the substrate is less than its rate of utilization. There is no evidence that molecular nitrogen enters bacteria other than by unfacilitated diffusion down a concentration gradient, so unless the permease remains undetected, it is probable that the rate-limiting step for nitrogen fixation is the electron-transfer process itself (see, for example, Hardy and Burns, 1968). This conclusion is consistent with the high activation energy for the reduction of nitrogen which, for A . uinelundii, is 14.6 Kcal/mole at temperatures above, and 39 Kcal/mole at temperatures below, 21°C (Burns, 1969). D. EVOLUTION O F NITROGENASE

From the preceding sections, it can be seen that hypotheses t o explain the origin and subsequent evolution of nitrogenase must be consistent with: (a) the gross similarities in the structure of nitrogenase from various sources; (b) the cross-reactivities of various azoferredoxins and molybdoferredoxins; (c) the selective advantage gained by the organism in which the enzyme developed; (d) the absence of nitrogenase from eukaryotes; (e) its oxygen-sensitivity; and (f) its genetic regulation. Nitrogenase used to be considered a primitive enzyme with a long evolutionary history because it occurs most frequently in anaerobes

20

J.

A. COLE

which, apart from being prokaryotes, had other traits which generallv have been considered primitive (De Ley, 1968). The earliest progenitors of nitrogen fixation were assumed to be photosynthetic bacteria (Burris, 1961) so, if mitochondria and chloroplasts originated from endosymbiotic prokaryotes, then nzf genes were either absent from the original endosymbiant, or have been lost during subsequent organelle evolution. If nitrogenase is indeed a primitive enzyme, it would have evolved when ammonia was freely available in the Earth’s atmosphere. It is therefore not immediately obvious how the ability to reduce molecular nitrogen to ammonia would give such an organism a selective advantage. T o answer this point, Silver and Postgate (1973) proposed that nitrogenase originally had an alternative function as a detoxification enzyme t o reduce, for example, nitriles, isonitriles or cyanogen which were also prevalent in the primitive atmosphere. The reduction of nitrogen to ammonia would then have been a side reaction which only provided an additional selective advantage much later when the local supply of fixed nitrogen had been exhausted. Postgate (19 74) has recently developed a third hypothesis for the origin of nitrogenase which suggests that the enzyme evolved recently in a prokaryote capable of transferring genetic determinants t o a variety of other genera. What are the relative merits of the three hypotheses? Are the properties of nitrogenase inconsistent with any of them? The gross similarities in structure and catalytic activity of the proteins from various sources would certainly be expected of an enzyme which evolved recently, or whose genes could be transmitted on a plasmid from species to species. If the latter were correct, however, why has this plasmid so conspicuously snubbed the pseudomonads? (Hill and Postgate, 1969.) One might also argue that very few juxtapositions of metal atoms, amino acids, cofactors and the electron donor are capable of catalysing the reduction of a molecule as stable as dinitrogen. If this is correct, nitrogenase should be an ultraconservative protein with little scope for el. olution. The considerable range of cross-reactivities of the component proteins is also compatible with all three theories, but the failure of azoferredoxin from Clostridium pasteurianium to complement molybdoferredoxin from Azotobacter species is an embarrassment t o the “recent origin” hypothesis. This lack of complete cross-reactivitv is sufficient reason for suggesting that azoferredoxin from Azotobacter species is more

MICROBIAL GAS METABOLISM

21

closely related to the Klebsiella protein than t o the Clostridium protein (Dalton, 1974). As soon as this point is conceded, the force of the argument for a recent origin is lost. Notice that all three h\rpotheses will still be tenable even if it is established that aminoacid sequences of the proteins vary little from species t o species, but g o s s differences in their sequences would suggest a long evolutionary history. It is possible that speculation about the evolution of nitrogenase mav soon be seen t o have been one of the most constructive of academic indulgences. The justification for this statement is as follows. For as long as it is accepted that nif genes evolved before the eukaryotic cell, it must also be accepted that sustained pressure of natural selection has failed t o establish nif genes in plants. Neither research councils nor industry will finance an attempt t o confirm a negative result, so there is little risk that the “scientific” dogma will be seriously challenged. According t o our most recent hypothesis, however, the late evolution of nif has not allowed natural selection sufficient time t o solve genetic or physiological restriction barriers to incorporate nif into the plant genome. In this case, there would be every reason t o hope for positive results from attempts t o accelerate a nz‘f epidemic in plants by laboratory manipulation, and the potential economic rewards would justify the necessary expenditure. Although similar genetic barriers t o success have been overcome with other experimental systems, the physiological barriers may b e simply too great. One such barrier is the universal inactivation of nitrogenase by oxygen. The correlation between oxygen sensitivity and the more frequent occurrence of nitrogenase in anaerobes has been considered to support thc view that nitrogenase is a primitive enzyme. Indeed, some cyanobacteria, legumes and Azotobacteriaceae have had sufficient opportunity t o evolve specialized mechanisms for protecting nitrogenase during aerobic growth (Stewart et al., 1969; Donze et al., 1971; Bergersen, 1962; Jones e t al., 1973). An alternative view is that oxygen inactivation is an inevitable consequence of the type of chemical reaction being catalysed. In order to reduce a molecule as stable as N, , nitrogenase inevitably passes electrons t o stronger oxidizing agents of comparable size or shape. An oxygen molecule might be expected t o cause g o s s changes in tertiary structure by accepting up to four electrons from super-reduced nitrogenase (Smith and Lang, 197-1). Such changes have been detected by

22

.J. A. C O L E

circular dichroism spectroscopy (Eady ct ui., 1972; Chen et ul., 1973). It is extremely doubtful, therefore, vchether the oxygen sensitivity of nitrogenase tells us anything about how the enzvme evolved. Because XTP is consumed when molecular nitrogen IS reduced t o ammonia, Silver and Postgate (1973) considered it unlikely that a primitive nitrogenase would be a useful terminal electron-transfer enzyme. I f nitrogenase originated in a phototroph, the growth rate of the organism rarely would have been limited by the available supply of ATP. The pertinent question is, therefore, whether a more favourable oxidant than molecular nitrogen would have been available t o accept unwanted electrons, and t o answer it we need t o know the chemical composition of th? primitive biosphere. Cyanogen, nitriles or isonitriles could have fulfilled the role of terminal electron acceptor but one cannot totally exclude the possibilitj- that molecular nitrogen has always been the physiological substrate. It would be interesting to know whether any of the alternative substrates are inducers, or their reduction products corepressors of nitrogenase synthesis. It is widely accepted that recplatory mechanisms are beneficial, and that they evolve more rapid1.l.~than structural genes, but how long does it take for a regulatory mechanism such as the ammonia repression of nitrogenase t o emerge? The fact that a reLp1atory gene can mutate spontaneously t o an ineffective allele is no reason t o assume that a sophisticated regulatory mechanism also developed overnight. This discussion could be protracted further, but the conclusions would be the same: our choice betiireen the three hypotheses for the evolution o f nitrogenase will be governed by the assumptions which offend u s least. For the future, it is possible that compxative amino-acid sequrnce data will lend strong support to the view that nitrogenase was a primitive enzyme, or that successful attempts t o introduce nif genes into plants will favour the opposite opinion. Until these formidable research programmes are complete, our speculation will continue t o be uninhibited by experimental data. E. G b S E I I C R E G C I A ” I 0 N

OF NITKOGENASE

Oxcr 20 years ago, mutants of azotobacters and clostridia were isolated \vhich were unable to fix nitrogen. Their usefulness was

MICRO BIAL GAS METABOLISM

23

1irn;ted by the lack of techniques both for separating and assa).ing nirrogeriase proteins in cell-free extracts, and for genetic transfer Lvitliin these genera. It vcas therefore impossible t o determine xvhich nitrogenase proteins were defective in these mutants, t o map the mutation sites, or even t o assign them 10 genetic complementation groups. Alutants of ilzotobacter vinelandii isolated by Fisher and Brill (1969) were probably similar t o earlier isolates, but these authors were able t o demonstrate differences between their two types of mutant. Extracts of neither class could reduce nitrogen, acet);lene, azide or cyanide but, while one was deficient in “component I” (molybduferredoxin), the other also lacked “component 11” (azoferredoxin). In order t o assay one component in vitro, one must add an excess of the other component. Mutants of the first type were therefore potentially useful as a source of uncontaminated azoferredoxin for assaying rnolybdoferredoxin at various stages during its purification. Azoferredoxin in crude extracts of this mutant was not only far easier t o prepare than purified azoferredoxin from the wild type, but it was also far more stable. Mutants of the second type could have arisen either from a deletion extending into two or more structural genes, or by alterations of a regulatory gene. Thus a single TINA base substitution in a regulatorv gene could result, amongst other things, in the synthesis of a super-repressor or a defective positive control element, either of which would prevent the expression of the structural genes. Despite the potential usefulness of these azntobacter mutants, they have not in practice stimulated any of the recent advances in nitrogenase research. The same cannot be said of mutants isolated by Xagatani e t nl. (1971), by Streicher et al. (1971) and by Dixon and Postgate (1971). In each of these cases mutants were generated in Kfrbsiella Pneumoiiiac so that biochemical genetics could be applied t o studies of nitrogen fixation. Klebsiellu pneunioniae is closely related t o E. c d i : it has a similar genetic linkage map, and many nitrogen-fixing strains have been isolated (Streicher et al., 1972). Unfortunately, nitrogenase was absent both from sexually fertile strains of KlebsieIla, and from K . aerogenes which is sensitive t o a Klebsiellaspecific transducing phage. Streicher et nl. (197 1) therefore isolated a series of Kif mutants in strain h14al which is sensitive t o the generalized transducing coliphage, P1. When transduced with P1

24

J. A. COLE

which had been propagated in the wild-type, transductants were obtained at a frequency of 1-4 nif' colonies/lO5 infective phage particles. These frequencies were similar to transduction frequencies for auxotrophic markers in E. coli. Recombination frequencies for a series of two-point crosses between different Nif mutants varied from 1x to 2.3 x 10-5/infective phage, and separate transductions with Nif- His- recipients established that the various nif mutations were 30-55% linked to the histidine operon. Taken together, these results indicate that several genes of nitrogen-fixation components are close to the histidine operon on the K . pneumoniue linkage map. Dixon and Postgate (1971) transferred the derepressed drug resistance factor R144drd3 from E. coli t o K . pneumoniue by conjugation. Klebsiellu pneumoniue StrS nif R144drd3 was then mated with strain M5alnif Str', and nif' Str' recombinants were isolated at a frequency of lo-'. Linkage of his and nif genes was again demonstrated and the frequency of double recombinants from conjugation experiments varied from 85% to 95%. Subsequently the nif genes were transferred with the derepressed R factor from K. pneumoniue to E. coli C-603 his urg S t r R , the recipient being chosen to avoid restriction and modification barriers which prevent recombinants arising from many intergeneric matings (Dixon and Postgate, 1972). His' StrR recombinants were selected at a frequency of /donor. In two experiments, ten out of twelve and two out of six purified his+ colonies were also able t o reduce acetylene and were therefore nif +.Most of these hybrids segregated tiny Nif- colonies when incubated anaerobically on ammonium-free agar with histidine and arginine, but hybrid R17 appeared t o be stable. When hybrid M7 was superinfected with the R factor "AST" (which belongs t o the same compatibility group as R144drd3), determinants characteristic of R144drd3 were lost, but the nz'ffhis+ phenotype was retained (Cannon et ul., 1974). The his and nifgenes of Klebsiellu were no longer associated with the R-factor in strain M7 but were probably integrated into the E. coli chromosome. Although intergeneric transfer of nitrogen-fixation genes was exciting in itself, it also provided important insight into how the expression of nif might be regulated. The low frequency of recombination between different n$ mutation sites and their linkage in transduction and recombination experiments provided presumptive evidence that several nitrogenase proteins are encoded in contiguous +

MICRO BIAL GAS METABOLISM

25

genes which might form an operon. Furthermore,'fi. hybrids of E. coli did not svnthesiLe nitrogenase when grown in media supplemented with ammonia (Dixon and Postgate 1972). Thus repilatory genes were transferred with the nif structural genes. Although there is still no formal proof that a complex of ammonia and the product of a regulator gene binds to an operator t o prevent transcription of a nifoperon (Jacob and Monod, 1961), Tubb and Postgate (1973) have recently shown that, in K. pneurnoniae, ammonia regulates nitrogenase synthesis by inhibiting the initiation of mRNA synthesis. Preformed nitrogenase proteins were essentially diluted out during growth with ammonia or other sources of fixed nitrogen after nitrogenase mRXA had decayed. The half-life o f this mRNA was 4.5 minutes at 30°C, under non-growing conditions, irrespective of whether ammonia was present or absent. These and earlier studies established that nitrogenase synthesis occurs in a vacuum, or under an atmosphere of argon and acetylene: molecular nitrogen is therefore not required to induce nitrogenase synthesis. The simplest model for .if regulation requires that nitrogenase structural genes are linked t o their promotor and operator or initiator genes, close t o the histidine operon. The regulator gene which codes for a repressor or activator protein could either map close to, and therefore be transferred with, the structural genes, or it could be unlinked to the .if operon. In the latter case, however, one must conclude that E. coli (2-603 has an equivalent unlinked allele which can effect ammonia repression in the nif' hybrids. In the last five years, extensive evidence has accumulated that enzymes of inorganic nitrogen metabolism are regulated interdependently. The possible identity of the nitrogenase repressor protein will therefore be considered in the wider context of the regulation of glutamate synthesis by micro-organisms. F. REGULATION OF INORGANIC NITROGEN METABOLISM

Nitrogenase is not the only enzyme whose synthesis is apparently repressed by excess ammonia (and/or ammonium ion). Other examples include nitrate assimilation by fungi (Pateman et al., 1967; Burn e t al., 1974), histidase and proline oxidase synthesis in Klebsiella aeroyenes (Prival and Magasanik, 197 l ) , and a permease that is required for tryptophan catabolism (Rlagasanik et al., 1974).

26

J. A . COLE

>Ian\. micro-organisms ieukaryotes as well as prokaryoies) use at least t x v o pathways f o r s)-nthesiz,ing glutamate from ammonia and 2-osoglutarate. In K . n t r o g c i t p s , for example, glutamine synthetase ar,d glutamate dehydrogenase activities varied inversely in different growth media. IShen t h r extracellular concentration of ammonia was lrss than 1 mhl, the glutamine synthetase-glutamate synthase pathway (GS:GOGA'I' pathway; Meers el al., 1970) was used: glutanline synthctase was induced but glutamate dehydrogenase ~ v a sapparently repressed. TVhen ammonia was in excess, glutamine synthetase was repressed, b u t glutamate dehydrogenase was derepressed. Glutamate dehydrogenase has a high Kwz for ammonia, so i t would appear that the GS-GOGAT pathway is preferred for ammonia assimilation whenever glutamate dehydrogenaze is ineffective. Not all microorganisms synthesize the enzymes for b o t h pathways: Erwinia species, Bacillus megateriiim and B. subtilis are deficient in glutamate dehydrogenase (Illeers c t al., 1970), arid some, b u t n o t all, eukaryotic micro-organisms lack the GS-COGAT pathway since glutamate synthase is absent (Brown et al., 1973). 'The importance of the CS-GOGAT pathway t o nitrogen-fixing ba,eteria was revealed by Nagatani et al. (1971) who isolated a series of Nif mutants of K . pnrumonicic. Some of these mutants failed t o ,grow with a variety of nitrogen sources which included molecular nitrogen and low concentrations of ammonia, b u t they grew well when the extracellular concentration of ammonia was high. Clutamate dehydrogenase activity was induced by high concentrations of ammonia b u t , in nitrogen-deficient media, nitrogenase was induced b u t GOGXT was absent. The defect in these Nif mutants was in the GS-GC>GAT pathway, which is seemingly essential fQr thr assimilation of molecular nitrogen by this organism. The reciprocal regulation of glutamate dehydrogenase and glutamine synthetase b y ammonia has heen investigated in Alagasanik's laboratory with cultures of K . aerogenes. In this organism, either histidine or proline can serve 2 s a nitrogen source for growth, b u t histidase and proline oxidase are both subject t o catabolite repression by glucose, except when the source of nitrogen is growth ratelimiting. Escape from catabolite repression could be correlated with glutamine s)-nthetase activity which was high in ammonia-limited cultures b u t low when ammonia was in excess. Llutations at two sites, g1nA and glrzR, gave strains which required glutaminc for yrowth, had

M ICRO BIAL GAS METABOLISM

27

undetectable activities of glutamine synthetase and could not utilize flistidine as a nitrogen source. In revertants of glnA the regulation of qlutamine synthetase was normal, but in revertants of glnB, enzyme activity was high even in bacteria grown with excess ammonia. The mutation responsible for the constitutive synthesis of glutamine S \ nthesis (gkzc), which suppressed the glnB mutation, was located close to gZnA. Mechanisms for reLq1ating glutamirie synthetase activity by adenylation-de-adenylation were normal in glnB glnC revertants, but histidase and proline oxidase were synthesized constitutively, and glutamate dehydrogenase was permanently repressed. The fact that a single mutation could have so many effects implied that a single protein was responsible for glutamine synthesis, for the relief of histidase from catabolite repression during nitrogen starvation and for the repression of glutaniate dehydrogenase. It was suggested that this protein was glutamine synthetase, the product of the qlnA gene. The glnB gene was assumed t o be the structural gene for a positive control element which is required for glnA transcription and glnC was the target for this activator. Ammonia and glutamine could act as corepressors of glutamine synthetase by binding to the glnB product, thus preventing it from activating glnA transcription, and mutations in glnC would make expression ofglnA independent of the glnB product (Prival et al., 1973). Transductional anal\Tsis has subsequently established that two glnA mutations, which result in loss of glutamine synthetase synthesis, map on either side of the putative “glnC” gene. It is apparent, therefore, that GlnC is a phenotype associated with the geneglnA, and the working model has been revised accordingly (Magasanik et al., 1974). It is now suggestcd that the glutamine synthetase protein represses the synthesis of its own messenger RNA, unless prevented from doing s o by the &B protein, and that the latter probably binds to the g h A gene when ammonia is absent. In mutants with the GlnC- phenotype, the target for glutamine synthetase “self-repression” has been mutated so that theglnB protein no longer plays an effective recgulatory role. Direct evidence that glutamine synthetase protein can activate t1-anscription of the histidase genes has recentlv been presented: the lmadenvlated hlg2+-insensitive form of this protein stimulated histidase mRXA synthesis, but the adenylated enzyme was ineffective (Tyler et al., 1974). The interesting question is therefore whether drlepression of nitrogenase is also mediated bv products of gliziZ and

28

J. A. COLE

glnB alleles in other organisms. If so, E . coli nif' hybrids which contain nitrogenase activity should also have high activities of glutamine synthetase. Results of experiments with nitrogen-limited continous cultures of E. coli OR 75 are entirely consistent with this hypothesis (Cole et al., 1974). Secondly, it should be possible to isolate a pleiotropic-negative mutant which is unable to fix nitrogen or ,grow with a variety of nitrogen sources because of a defect in a glutamine synthetase gene. One such strain has been isolated and partially characterized by R. S. Tubb (personal communication). In K . pneumoniue M5al-RT4, glutamine synthetase activity was neither fully repressed by ammonia and glutamine, nor was it fully derepressed when ammonia was absent. Nitrogenase proteins were not synthesized by this mutant in any grow-th medium, even when an F'nif'hi.~ plasmid was transferred to RT4. Unlike most nif mutations, the lesion in RT4 was either transdominant, or was unlinked to the his operon. It was tentatively concluded that a high concentration of glutamine synthetase and a low concentration of ammonia are prerequisites for nitrogenase synthesis, and that glutamine synthetase is probably a positive control element for nitrogenase Synthesis. This conclusion was strengthened by the observation that nitrogenase synthesis was partially constitutive when the F' nif'his plasmid was transferred t o two Gln C - strains, but was repressible by ammonia in the Cln C + parent. These conclusions may prove t o be premature, however, because attempts to isolate Nif' revertants of RT4 which were analogous to Gln C- strains of K. aerogenes were unsuccessful. Preliminary evidence has accumulated which suggests that nitrate reductase and nitrogenase of symbiotic nitrogen-fixing bacteria share one or more common genetic determinants. Cheniae and Evans (1960) noted that the nitrogen-fixing activity of soybean nodule preparations was positively correlated with the specific activity of nitrate reductase, and Nason et al. (1971) demonstrated that the molybdenum-containing subunit of nitrate reductase from Yeurospora crassa could replace the molybdoferredoxin of nitrogenase from soybean nodule bacteroids, hlore recently, cultures of Rhizobium meliloti were treated with a low concentration of nitroso'panidine to generate point mutants which \vere unable t o reduce nitrate (Kondorosi et ul., 1973). When sterile lucern plants (two days old) were infected with these mutants and then grown for +

+

MICROBIAL GAS METABOLISM

29

a further sixty days, nodulation occurred either at a normal or at an increased frequency. The dry weights of plants with the increased frequency of nodulation were n o
Oxygen is a product of photosynthesis by organisms which contain both photosystem I and photosystem 11: it is not, therefore, a product of bacterial photosynthesis. Some oxygen is evolved when catalase and peroxidase catalyse the decomposition of peroxides, but this is only part of the oxygen that is consumed when peroxides are formed. Enzymes that reduce oxygen include the di-oxygenases that catalyse insertion of both oxygen atoms into a substrate (usually with the concomitant cleavage of a C-C bond) ; the mixed-function oxidases or mono-oxygenases that insert one oxygen atom into a substrate while the other is reduced t o water by a second electron donor (usually NADH, or FADH,); flavoprotein oxidases such as the L - and D -amino-acid oxidases and glucose oxidase from fungi and manv bacteria; and a variety of cytochrome oxidases. Catalase, peroxidase and superoxide dismutase have all been implicated as enLvmes that protect various cell types against the toxic effects of ox vgen. Oxygen metabolism has already been reviewed in this series by Hughes and \Vimpennv (1969). These authors described in detail the t i pes of enL\-me t h a t reduce oxygen, the subcellular structures in

30

J. A C O L E

which they are located, the role of oxygen as a metabolic regulator, and w a \ s in which the oxygen concentration o f a microbial culture can be measured and controlled. 30 attempt will be made t o re-iterate or amplify those aspects of oxygen metabolism that are still adequately covered bv the earlier review. Furthermore, literature concerned with the development of fully-functional yeast mitochondria from promitochondria, in response to aeration, has been reviewed by Linnane et al. (1972). Irlitochondrial biogenesis will not therefore be considered here. The continuing evolution of ideas about how oxygen re*gulates the synthesis and activity of microbial components will be discussed, as will the implications of more recent research into the multiplicity of bacterial cytochrome oxidases. Greatest emphasis will be given t o the problem of oxygen toxicity, and t o how the enzyme superoxide dismutase might protect variou5 cell types against oxygen damage (see also Morris, 1975). B. BACTERIAL CYTOCHROME OXIDASES

In most obligate and facultative aerobes, oxygen is reduced b y electrons which flow from flavoprotein dehydrogenases through band c-type cytochromes t o a variety of cytochrome oxidases. Bartsch (1968) concluded his review by stating that the study of bacterial cytochromes was still largely in the collecting phase. The microbial biochemist was faced with an almost bewildering variety, but analysis of the properties of individual cytochromes had begun. I t was therefore inevitable that the dia,granis of microbial respiratory chains offered by Hughes and Wimpenny (1 969) were tinged with imagination as well as experimental fact: which chemo-organotrophs, for example, have the three alternative cytochrome oxidases, cytochromes a1 , d ( ( 1 2 ) and cis, but neither cytochrome a nor cytochrome o? During the last five years, however, there has been significant progress in establishing the metabolic function of some of these pigments. Convincing evidence has been presented that the major oxidase in Azotobactcr zli?zt.landii is cytochrome d (which used t o be referred to as cytochrome ciz). C. W. Jones and his colleagues have established that this bacterium has a branched electron-transfer chain, possibly as shown in Fig. 4 (see, for example, Jones and Redfearn, 1967; .4ckrell and ,Jcnes, 1971). Electron transport

31

M l C P O B l A L G A S METABOLISV

through tlvo segments o f the electron-transfer system ( b d +0 2 . and c4 0,) is not coupled t o oxidatile phosphorjlation, so the overall efficienc) of enerhy transductioii a t “site 111” is low. \fhen moleculai nitrog-en IS the sole source of nitrog-en for grotvth, it is essential that the nitrogenase complrx should be protected from exposure t o oxygen. Nitrogen-fixing cultiires their fore respond t o liii:re.iqed aeration b\- s\nthrsij.inq mole c-, tochrome d , and hv --f

--f

Succinate or M3late

/”

,/“

NXDH,

FpN

+

+ Q

c,.t , . ‘ ,* i ...

-’-+

<-

\

\\

ryt

o2

fll”

4

/* I

,/ fl

/

DCPIP ” TMPD

--_i

’_\

low [ i C S ]

‘~ \

, , 1

L

cyt

I

C 5 --

Cyt f l l Q

>-+0 2

FIG. 4. Branched electron-transfer chain of A z o t o b a c t c r rinelandii. The abbreviations used are as follows: F p ~ q , FpS and F ~ Mare fldvoprotein dehydrogenases for N.4DH2 , succinate and malate, respectively. DCPIP is 2,6-dichlorophenol indophenol; TMPD is N,N.N’,N’-tetramethyl-p-phenylenediamine; (2 is ubiquinone; HQNO is :!-n-a!kyl. l--hydros)i\iinolin.-S-oxide. Iahibitors a r e indir atcci b y dashed rlrrows.

increaqing theii respiratory capacitv. in this process o f “respiratory protection”, oxyqen is reduced as f,isl ‘is it enters the bacteria (Jones c’t nl., 1973). If respiiation arid cxidntive phosphnrvlation \\ere tiqhtly coupled at both site I and rite IT, howeler, oxygen ~ o u l r l accumulate . inside the cell a< soon as ail of t h r available ,%DP lvas phosphor\ lated to ATP. 1he third response ot nitrogen-fixing 1 zoto hnc ter t o incredsrd aeration is therefor? t o tincouple oxidative phosphor) lntion from electron floiz. through site I (the NXI)H2 to 1-Ibiquinnnr o r cvtochrome h seqml nt of the respiratory chain; lorips t’t nl., 1973). These e\plaii.itio~~-( * ) I h i 1:7(hecl eirction transfer

32

J. A. COLE

pathways in Arotobacter are extremelv attractive, but how convincing is the experimental evidence for their existence, and what is the relevance of these results to other bacteria? Azotobacter is highly specialized, and atypical even amongst the nitrogen-fixing bacteria because it aIone can reduce molecular nitrogen in well aerated environments. But why is cytochrome d in some bacteria apparently repressed by high aeration while the equivalent pigment in Azotobncter is derepressed? (see Kaufmann and Van Gelder, 1973). The evidence for branched electron-transfer pathways in Azotobacter was derived from a variety of kinetic experiments in which the rates of oxidation and reduction of individual electron carriers by physiological and artificial electron donors were determined. Carbon monoxide action spectroscopy implicated cytochrome d as the major terminal oxidase, and although reduction of this cytochrome was strongly inhibited by hydroxyquinoline N-oxide, it was insensitive to low concentrations of cyanide (Jurtshuk et al., 1967; Jones and Redfearn, 1967). Conversely, oxidation of cytochromes c 4 and c 5 by cytochromes al and CJ was extremely sensitive to low concentrations of cyanide. Kaufmann and Van Gelder (1973) have challenged this assi'gnment because the redox potentials of cytochromes a1 and CJ were unexpectedly low, cytochrome al apparently did not bind cyanide, and cyanide drastically altered the spectrum of oxidized cytochrome d. Part of the controversy was soon resolved by Jones (1973) who determined the effect of various concentrations of cyanide on the rates of oxidation of tetramethylphenylenediamine (TMPD), dichlorophenolindophenol (DCPIP), o r physiological substrates. Paterson ( 1972) had previously demonstrated that inhibition kinetics were bi- or polyphasic when respiring pseudomonads were incubated with various concentrations of cyanide. Apparent inhibition constants could be calculated from these data, and provided care was taken to identify the immediate electron donors, and to allow for changes in the rate of electron flow as their concentration decreased, one could assign the apparent inhibition constants to the various oxidases. Jones (1973) concluded that Azotobacter cytochrome d was indeed sensitive to cyanide, but that this sensitivity was far less than that of cytochromes a l and CJ.Because inhibition of ascorbate-T,ZTPD oxidation by cyanide was of the pure, uncompetitive type, but inhibition of ascorbate-DCPIP oxidation was pure, non-competitive, it was concluded, in qreement with earlier

MICROBIAL GAS METABOLISM

33

proposals, that cytochromes a1 and u were independent cytochrome oxidases on separate branches of the electron-transfer chain. Therefore, although Kauffman and Van Gelder (1973) summarized reasons for doubting the significance of cytochrome a 1 as a cyanide-sensitive oxidase, the roles of cytochromes d and u in Arotobacter appear to be beyond dispute. Further studies are needed to confirm the role of cvtochrome a l in this and other bacteria. During the last five years there have been several other significant advances in research into bacterial respiratory systems. The concept of branched electron-transfer pathways is now well established (see, for example, White and Sinclair, 1971). Although Lemberg and Barrett (1973) failed to unravel the multiplicity of bacterial pigments, their treatise “Cytochromes” is valuable not only for its wealth of factual information, but also because it clearly distinguishes between two different types of cytochrome d . According to their nomenclature, cvtochrome d refers to the membrane-bound cytochrome oxidases which occur in many aerobic bacteria, and cytochrome d, (which also occurs covalently linked to haem c ) is the soluble nitrite reductase from denitrifying bacteria. Kaufmann and Van Gelder ( 1973) have summarized the main distinguishing properties of the two types of haem d , but some of these differences were previously noted by Newton (1969). The interesting question now arises whether facultative anaerobes such as E. coli, which can also reduce nitrite, contain two different d-type cytochromes. Cytochrome d synthesis by this organism is derepressed during anaerobic growth with nitrate or nitrite (Cole and Wimpenny, 1968) but this membrane-bound pigment is also oxidized by molecular oxygen with a half-life of two milliseconds: it is therefore a potentially effective cvtochrome oxidase (B. A. Haddock, J. A. Downie and P. B. Garland, personal communication). Two experimental approaches have also made significant contributions to recent research. The first was the isolation of mutants deficient in specific cytochrome components (Ruiz-Herrera and De hloss, 1969). The second was the routine application of low-temperature spectroscopy to study the complexity of respiratory pigments. Shipp (1972a) has developed this technique still further by computing fourth differential derivatives of absorption spectra in order to resolve composite spectral bands into their individual components. In first differential derivative spectra, which can be obtained directly

34

.I.

&

{ ~ G tI

fl. d < i i 1 31 Y:,I .iilJton1cti.ls. oile 1.t.c.otds the r:lic o f changi. of absil1-halii C' p tinst wavelength. Points rjf intlexlon on dii-vcr s p c c t r d p q i h : , ;ipptdr as peaks or troughs in first differenLial spectra h u t , i n srcc,nd 01- fourth diiferential bpectra, the "stionldcrs" cnicrqr" :is d i s c x t c pcaks. To use these sophistic iIted. ) f e t icadily available, tci.hniques s u c c c ~ s f u l l yone must I)e sure thar : (a) samples ~ K - Cf r o z m in a repr<>duciblemaliner ( \ ~ ' i I s o n1967); ~ (1,) the sample tcniperature is constant and reproducihle; (c) sample concentrations are ac1,justcd t o w i t h i n the range in ivhic-h the signal is proportional to the cytochromc concen trarion; (d) the optical system of the sptctr:ipholci~netci- passes a minimum of scattered light; ( c ) electronic noise in the spc ctrophotometer and iecorder are minirnized; and ( f ) rhc slit width (which detrrmines the spcctral resolution) is the minimuin consistent Lvith jd) a n d (e) above. 1mprowniei:ts can frequently be achieved if a timgsrcn light sourcc is repliiccd b!, a high-intt.nsit)-halogen lamp. Three significant restilts have cmerged f i . ~ nr h~ r coinbint;:l gcncric and low-temperature studies. First, E. coii syiithesizes at Ieast three and probabl). four b-type q-tochromes, t w o of which are spectroscopically identical and donate electrons to nitrate retluctasc rather than t o cytochronie oxidase (Ruiz-Hererra arid Dc Aloss, 1969; Shipp, 1972a). Secondly, genes specifying many of the E . coli cytochromes are clustered in a 2 minute segnient of the linkage map, located close t o thegol operon (Shipp ct al., 1972). Finally, m d e r all growth conditions tested, a multip!icity of cJrtochromes is synthesized b y many bacteria: isith wholc tells, one cannot hope to study one t i , 0 01' c c . t q ~c).tochrome in isolation, despite large q u an ti t at i1.e changes 1 n :c: t o cl-Ir om e c o m pl ern en t in re s p o n se to different growth conditions (Shipp 1972a,b ) . Exciting adimices in our kno\\.Jcdge o f bacterial respiration can be anticipated in the next few years as these techniyilcs are coml~ined with careful kinetic st udi cs .

il rotc "Dcspit\x an ex:ensii t' (,)ften ret h r harinfiil effects o f o x ~ g e non the c(ilt1vation and metabolism of maiiv tiiicro-origanisnis. exceedinql) little is

l l o r i i s (1970) iecriitl\

iterative) literatuie

011

MI C R 0 B IA L G A S M ETAB O L ISh~i

35

~ e known t of the primary biochemical elents that lie at t h t root cf such expressions of ox) gen toxicity”. This literature reaches back for over 100 \ears, and from it hlorris summarized six hypotheses t o explain ox\geii toxicity. The first hypothesis, that oxygen itself is toxic, has received little consideration presumably because few biological molecules are known t o bind ox:;gen without reducing it (heamoglobin, for example). It is not inconceivable, howeber, that molecular ox)rgen could bind t o a transition metal t o displace an essential ligand from a metalloprotein, thereby inactivating that protein. The remaining five hypothmes are interrelated because they suggest that oxygen acts indirectly: (a) by conlertiiig cellular or medium components to toxic chemicals; (b) by generating an intracellular redox potential ( E h ) which is too high for normal metabolism t o continue; (c) by determining the concentration of a metabolic regulator which is directly or indirectly oxidized by oxygen; (d) b y inactivating the key enL\mes in which free -SH groups are oxidiied t o --S-Sbridges; and (e) by competing with biosvnthetic reactions for the cell’s reducing potential. Notice that if the nicotinnmide nucleotide redox potential were the Lritical r e p l a t o r y factor, (b), (c) and (e) become indistinguishable. Hypothesis ( a ) , however, includes the possibility that the toxic agent might be a product of oxygen reduction such as hydrogen peroxide, organic peroxides or the superoxide ion. hIorris concluded that, despite the temptation t o adopt a unitary hypothesis, it is unlikely that a single explanation M ould apply t o all organisms in all media-a view which has subsequently received experimental support. The problem, therefore, is t o identify the detrimental oxidation process in each specific case. Although different micro-organisnis show coiisiderable Inriatioris in their ox\ geii tolerance, anaerobes are usuall5- inhibited by lower extracellular concentrations thdn aerobes. The genus Vzhrzo contains both anaerobes and aerobes but the type species, Vzbrio c o m m a , grows best in abundant oxygen. Se\ errheless, partia! pressiires above 0.87 atmospheres were bactericidal for this organism, and oxygen pressures of 0.87, 1.87 and 2.87 atmospheres were progressively bacteriostatic f o r other xibrios, salmonellae and shigellae (Gottlieb and Pakman, 1968). Prolonged exposure to hJ-perbaric oxygen was required for full sensitivity t o develop, but neither pressure per se, nor oxidized media components were responsible for this inhibition.

36

J A COLE

Sensitivity decreased in richer growth media, and was partially dependent upon which carbohydrate supported growth. Because sulphisoxazole enhanced oxygen sensitivity, Gottlieb and Pakman (1968) proposed that oxygen inhibited growth by interfering with p-aminobenzoic acid metabolism. Gottlieb ( 1966) had previously demonstrated that Achromobacter P6 was sensitive to oxygen, but
'

'

37

MICROBIAL GAS METABOLISM

dbsent from extracts of aerated cultures (Cole and Rittenberg, 197 1; Cole, 1973). An alternative possible basis for oxygen toxicity is that hyperbaric oxygen “preferentially consumes the cell’s ‘reducing power’, leaving it insufficient to maintain normal biosyntheses” (Alorris 1970). This might either increase the steady-state redox potential of every couple in the redox chain by a mass action effect, or it might short circuit the normal respiratory chain by oxidizing nicotinamide nucleotidelinked enzymes directly. The first possibility is extremely unlikely because cytochrome oxidases from most sources are essentially saturated even at the lowest detectable concentration of dissolved oxygen (Sanadi and Fluharty, 1963). Furthermore, Chance et al. (1965) reported that the total respiratory activity of mitochondria was constant or declined slightly under hyperbaric conditions, indicating that the respiratory chain does not leak electrons to oxygen at a significant rate at any point except the terminal oxidase. Hyperbaric oxygen may nevertheless increase the nicotinamide nucleotide redox potential either by activating enzymes which oxidize NAD(P)H, , or by inactivating enzymes which reduce NAD(P). Chance ct al. (1965) measured 450 nm fluorescence changes which occurred when anaerobic pigeon-heart or rat-liver mitochondria, or yeast or ascites tumour cells were exposed to pure oxygen at one or eleven atmospheres. The fluorescence of baker’s yeast increased two minutes after the suspension was flushed with nitrogen: all of the nicotinamide nucleotides had been reduced (Fig. 5). The chamber ~ 7 a s

to reduced nicotinamide

nucleotides

Gas phase:-

1 a t m 0 2 +-Nz-d+l

atm 0 2 + l 1

atm 0 2

FIG. 5. Oxidation of nicotinamide nucleotides by hyperbaric oxygen. Based on Figure 4A of Chance et al. (1965).

38

J.

A. C0L.E

then flushed with pure oxygen and the fluorescence decreased t o a new plateau. \Yhen the oxygen pressure increased t o 11 atmospheres, the fluorescence again decreased, the response being quantitatively similar to that of anaerobic yeast t o pure o x ~en g at one atmosphere. These experiments demonstrated that up t o 50% of the cellular nicotinamide adenine dinucleotide remains reduced even when cytochronie oxidase is active and saturated with oxygen. How is this pool maintained, and why is it oxidized under hyperbaric oxygen? IVhen intact mitochondria were aerated and then incubated with succinate and A’TP, intramitochondrial KAD was completely reduced within two minutes. Reduction was severely inhibited when the mitochondria were exposed to 11 atmospheres of oxygen for 80-90 seconds before succinate and ATP were added (Chance et ul., 1965). Very little of this inhibition could be accounted for by an increased rate of NA4DH2oxidation. It was concluded that hyperbaric oxygen preferentially inhibits energy-dependent reversed electron transfer to S . 0 . Furthermore, when NAL)H2 oxidase activity was inhibited with antimycin ‘4 or sulphide, instead of NADI-I, being oxidized more slowly by hyperbaric oxygen, it was oxidized fourtimes more rapidly. The oxidation of NADH2 is therefore necessary for S A D t o be reduced! ‘The pool of reduced nicotinaniide nucleotides is maintained at the expense of ATP generated during normal cytochrome oxidase activity. Most of the above experiments were performed with mammalian mitochondria in an attempt t o resolve discrepancies between rates of inhibition of other cnzymes which have been postulated as the targets for oxygen toxicity, and the speed with which hyperbaric oxygen caused convu1si:)ns in the living animal. Excess oxygen produces cultural drop-outs rather than convulsions in microorganisms, but the sensitivity or reversed electron transfer to oxygen is likel), to he o f particular relevance t o chemolithotrophic bacteria which have no ( ~ t i i c i1% (,f generating rectuced iiicotinamide niucleotidcs (Peck, 1968). O’Bricn and hlorris ( 19 7 1 ) investigated \vhether molecular ox)-gen p c v st( \vas toxic to the obiigate anaerobe, C‘los tridiztm acetoh u t > ~ / i c u mo, r M-hcther growth was inhibited by the ele\Tated redox potential of the culture medium. The). Lvere surprised at this bacterium’s ability to adapt to increased aeration. The NADH2 oxidase activit?. and the extracellular redox potential increased with J

TARLF 4. Effect O F Aeration and the Extracellular Kedox Potential on the Growth Rate of Clostridium acc.toDutylicum. _ _ _ I I _

~.~___._~.___._____.I____.___._~.__

Cultural conditions

Dissolved oxygeit (PW __

Anaerobic Aerated Aerobic Anaerobic + potassium ferricyanidc Aerobic + potassium ferriryanitle Aerobic + dithiothreito!

nil 1

40 to 50 nil 40 49

__

~

~

E: valuc

(mv) ~

--370 to --40!: -50 to - 1 0 +loo to +150 +370 +370 -50

__

Mean generation time tor growtll (minutes) -

80 911 m

Sd

m

g 0

n

0

E!

9

r

0

2

<

rn -!

m

Data taken from 0’Hric.n and Morris ( 1 Y 7 1 j.

W

lD

40

J A. COLE

increasing rates of aeration and, although NADH, was being consumed for reductive detoxification of oxygen, the ,growth rate, molar growth yield and fermentation balance were little affected until oxygen was sufficienrlv in excess to be detected with an oxygen electrode. ,4t this point growth, DNA, RX,4 and protein synthesis stopped, and intracellular ,4TP was converted t o ADP. N o similar dramatic fall in ATP concentration occurred when growth was inhibited bv deprivation of required amino acids, treatment with diamide or with metromidar,ole. Each of these treatments inhibited the rate of glucose utilization. Thus three experimental cultural conditions could be distinguished-“anaerobic”, “aerated”, and “aerobic”. From Table 4 it can be seen that the extraceIluIar redox potential increased dramatically when “anaerobic” cultures were “aerated”, b u t only a small change occurred when “aerated” cultures became “aerobic”. If the gross physiological disruption of “aerobic” cultures was due t o an increase in Eh above a critical threshold value, this value must be between -10 and + l o 0 mV. When ferricyanide was added t o an anaerobic culture, the E h was poised a t +370 mV, but normal metabolism and growth were maintained. As soon as this culture was made “aerobic”, growth stopped. Conversely, an “aerobic” culture was poised at an E h of -50 mV by intermittently adding dithiothreitol. No growth occurred under these conditions, again indicating that the free oxygen in the medium (rather than the culture E h ) was the critical factor in causing growth inhibition. These resuIts are hardly surprising when it is realized that it is far more likely that intracellular redox couples will equilibrate with the 0, /H, 0 couple in organisms which respire than in those which d o not. The mechanism by which molecular oxygen inhibits clostridial growth is therefore probablv entirely different from the way it depresses activities of anaerobically induced enzymes in facultative anaerobes (Wimpenny and Cole, 1967).

2. E n z y In ic Pro te c tion Aguins t O x y g e n T o x i c i t y The experiments with Clostridium u c e t o b u t y l i c u m , described in the previous section, implicated NADH, oxidase as a protective enzvme which could be synthesized or activated in response t o changes in the supply of oxygen to the culture. Is this a universal protective enzyme against oxygen toxicity, or d o other en7ymes have

MICROBIAL G A S METABOLISM

41

a similar function in other micro-organisms? Until recently, catalase and peroxidase were assumed to be important in this respect. The); are usually absent from obligate anaerobes, but are drtectable in many fermentative bacteria which are aerotolerant. In the latter ,group are organisms such as Lactobacillus casei which were found t o ,grow with an optimal yield and specific growth rate under atmospheres containing from 1%to 46% oxygen (Erkama et al., 1968). In this organism, an IVADH2-dependent peroxidase activity was induced as the oxygen partial pressure increased. Unlike Clostridium acetob u t y l i c u m , NADH2 oxidase activity was similar in bacteria grown with or with ou t oxygen. Hydrogen peroxide is a reduction product o f oxygen which might be expected t o damage cellular components if it were allowed t o accumulate. Many aerobes synthesize an active catalase which can disproportionate hydrogen peroxide t o oxygen and water. Agromyces ramnosus is one of a group of catalase-negative bacteria which ,grow poorly when exposed t o air. Despite their widespread occurrence in soil, their sensitivity t o oxygen prevents them from forming discrete colonies on most selective media. Jones et a l . (1970) reported that A g r o m y c e s ramnosus grew well in mixed cultures with catalase-positive bacteria, and formed colonies if catalase was added t o solid media. Abundant g o w t h also occurred on media enriched with manganese dioxide which also catalyses the decomposition of hydrogen peroxide. Further evidence that catalase forms part of the mechanism for resistance to oxygen came from experiments with synchronous cultures of bacteria and fungi. Synchrony was induced in Bacillus szibtilis or Escherichiu coli by exposing cultures to 10 atmospheres of ox!.gen for 18 hours. Resistance to further exposure t o hyperbaric oxygen was then determined at various stages in. the cell cj,cle. hlaximuni resistance was observed immediately after cell division, co-inciding with the time of maximum catalase activity (Gifford, 1968). Exposure to 10 atmospheres of oxygen also prevented further cell division of exponentially growing Succharoinyces cereuisiue and C a ~ d i d uiitilis (Gifford and Pritchard, 1969). All cells died after several days, but Ionger survival was noted with ethanol as carbon source than with glucose. Stationarlz-phase cultures contained higher catalase activity and were more resistant t o molecular oxygen. In s!,nchronous cultures of' Sncch. cerciisiue, enz?.mes are synthesized at

42

J. A . C O L E

predetermined stages in tha growth cycle. Periods 0 1 ox)’geii resistance ;tnd catalase s)-nthesis co-incided at t w o points in the cell cycle, so once again it was concluded that catalase is a protective enzyme. Catalase is not a universal panacea against the toxic effects o f ox!-g:cn, however. Benbough (1969) reported that the bactericidal effect of oxygen or air t o airborne coliforms was greatest at lo\,vest relative humidities. Hydrogen peroxide did n o t enhance oxygen sensitivity, and catalase did not diminish it. I.Tiability\\?asprolonged if 0 2

1 2 outer electrons -~

0 2

02=(H202)

13 outer electrons

14 outer electrons

_____

PIG. 6. .\sslgnrnent of electrons t o niolrtu1,ir superoxide radical s n d hydrogen peroxide.

U”

orbitds

rbt

oxbgen,

the

water loss was decreased by coating bacteria with polyhydroxycompounds, b u t paramagnetic ions such as NO2:, hIn2+ o r I- \yere more effective. ‘These experiments implicated free radicals as mediators of oxygen toxicit!.. Oxygen itself is a paramagnetic diradical which can interact with the reduced form of redox enz)-mes which catalyse one-electron transfer reactions t o form another free radical, the superoxide ion (see Figs. 1 and 6). I t is significant, therefore, that all cells that can reduce oxygen contain an enzyme Ivhich dismutates the superoxide ion according to the equation:

M I C R O B I A L GAS METABOLISM

43

The physiological importance o f this enzyme has emerged from a brilliant series of papers from Fridovich's laboratory which began with a demonstration that erythrocuprein from human blood, or hemocuprein from bovine blood, had superoxide dismutase activity illcCord and Fridovich, 1969). Tetrazolium oxidase is also identical to superoxide dismutase (Lippitt and Fridovich, 1973). The purified enzyme from bovine erythrocytes contained two equivalents of copper per mole, and required this copper for activity. It was assayed lvith superoxide ion generated from molecular oxygen by electrolytic reduction in the aprotic solvent, dimethyl formamide. The superoxide ion was relatively stable in dimethylformamide, but it rapidly reduced ferricytochrome c. Superoxide dismutase can therefore be assayed by its ability t o inhibit cytochrome c reduction by competing for electrons from 0 2 ; (Foreman and Fridovich, 1973). Similar competition experiments demonstrated that the oxidation of epinephrine by milk xanthine oxidase is also mediated by the superoxide radical, as is the oxidation of xanthine by oxygen. IVhen tetranitromethane or ferricytochrome c was added to the last reaction, it was reduced by 02;t o nitroform or ferrocytochrome c, respectively. Reversal of the superoxide dismutase reaction was demonstrated by incubating the enzyme with hydrogen peroxide and molecular oxygen, trapping the 0 2 : produced with tetranitromethane, and measuring the rate o f accumulation of nitroform (Hodgson and Fridovich, 1973). A more direct, yet more sophisticated, assay for superoxide dismutase has been described by Bannister ~t al. (1973) who, by pulse radiolysis, generated 02: at lo-' Ll. This was sufficiently concentrated to be detected spectrophotometrically by its ultraviolet light absorbance at 245 nm. Half of these superoxide radicals were generated directly from molecular oxygen and hydrated electrons, and the remainder from secondary reactions between ethanol and H' or HO' radicals. Once again, human erythrocuprein was the source of enzyme. Double reciprocal plots of reaction velocity against the substrate concentration passed through the oriqin : obviously the rnzyme did not follow the r\lichaelis-Alenton mechanism. Subsequent experiments with 02:generated by pulse radiolysis showed that only half o f the active sites on superoxide dismutase were catalytically functional (Bra). et al., 1974; Fielden rt uf., 1974), and that when CU'+ was reduced b y hydrogen pcroxide t h e kinetics ! v e x complex

44

J. A. C O L E

and the reaction did not go t o completion. The half-life for enzyme reduction was 150 milliseconds, and on prolonged exposure t o hydrogen peroxide, the enzyme was inactivated. The second order rate constant for the dismutation reaction at 25°C was 2.37 x l o 9 If-' s - ' . When the viscosity of the reaction fluid was increased, the velocity decreased, showing that the reaction was almost diffusionlimited. Onll- one species of Cu2+ was distinguishable by electron paramagnetic resonance spectroscopy, but once again only half of the copper was involved in turnover. The following allosteric mechanism was proposed in which one Cu2+ was reduced t o R-Cu' as the other became transiently unreactive (R--Cu2+-+ K-Cu'+) :

K - Cu2+ I<+1 R - CLIZ+ k _ ,

~

7-

R - Cu+ k+Z & - CU2+ k-;

7

N R

Cu'

-~~ -

cu2+

k+3

R - Cu+ R - Cu'

7

k-,

In all of the previous experiments, the superoxide ion was generated artificially t o assay an enzyme extracted from mammalian blood. IVhat is the evidence that the superoxide radical is generated by intracellular redox reactions in z i u o , or that superoxide dismutase is physiologically important t o micro-organisms? Flavin nucleotides (FMN, FAD), and the vitamin K analogue menadione, all stimulate electron transfer from various enzymes t o cytochrome c (and presumably t o other electron acceptors as well!). The aerobic, but not the anaerobic, electron transfer from ferredoxin-NADP reductase, or xanthine oxidase, t o ferricytochrome c is inhibited in zu'tro up t o 70% by superoxide dismutase (h7cCord and Fridovich, 1970). This means that up to 70% of the electrons transferred t o ferricytochrome c are donated by the superoxide ion, and the experiment provides convincing evidence that 02: could be generated in uiuo when oxygen enters a cell. In addition, superoxide dismutase has been detected in every species of aerobic micro-organism that has been examined. The first bacterial superoxide dismutase t o be characterized was found in Escherichia coli strain B, and gross differences between this enzyme and erythrocuprein wrre immediatel!. apparent. The bacterial enq'me contained A h 2 ' instead of Cu2+ and Zn2+,arid its amino-acid composition was also dissimilar from the mammalian enzyme (Keele c,t al., 1970: \Veisiger and Fridovich, 1973). Similar enz!rmes were detected in a variet). of arrotolerant anaerobes which included streptococci and lactic acid bacteria, but it was abstnt from obligate anaerobes (McCord ct al., 197 1). Catalase

MICROBIAL GAS METABOLISM

45

activity was absent from both the obligate and the aerotolerant anaerobes so, although there was a good correlation between oxygen tolerance and superoxide dismutase, there was no similar correlation with catalase activity. Only one species, Lactobacillus plantarum, was both superoxide dismutase-negative and aerotolerant, but this bacterium was completely unable to reduce oxygen, so neither hydrogen peroxide nor the superoxide anion could be accumulated. A particularly interesting species was Bacillus popilliae, an obligate aerobe which is both catalase and peroxidase negative. This organism has an active superoxide dismutase, so Costilow and Keele (1972) concluded not only that superoxide dismutase provides sufficient protection against oxygen toxicity, but also that hydrogen peroxide generated bv superoxide dismutase activity is not toxic to Bucillus popilliae. I t seems a pity to spoil such a good story, but unfortunately one should realize that, if hydrogen peroxide rather than 02;is the toxic agent, superoxide dismutase could also “detoxify” hydrogen peroxide to give 02: which is not only relatively stable (Rabini and Njelsen, 1965), but also reacts harmlessly with cytochrome c! It is far more likely, however, that damage in vivo is caused by two decomposition products of the stable superoxide anion, namely singlet oxygen and hydroxyl free radicals (Paschen and M’eser, 1973; Gregory and Fridovich, 1974). If this is correct, Costilow and Keele’s (1 972) assessment is fully justified. A second superoxide disrnutase was later isolated from Escherichiu roli, but once again its amino-acid composition was unrelated to the mammalian enzyme (Table 5 ) . Its major difference to the first superoxide dismutase of E . coli was that Fe2+ replaced Mn2+as the prosthetic group (Yost and Fridovich, 1973). The iron enzyme could be released from unbroken bacteria by osmotic shock, so it appeared to be localized in the periplasrnic space. The hln2+ enzyme was retained in the cytoplasm and, unlike the Fez+enzyme, its synthesis was app:.rently induced by oxygen. Bacteria grown in iron-deficient media contained little of the periplasmic enzyme, so bacteria deficient in cither enzvme could be obtained by manipulating the culture conditions. Extracellular 02; was lethal to iron-deficient bacteria. but protection was afforded by bovine superoxide dismutase. The physiological role of the periplasmic superoxide dismutase therefore appears t o be to protect bacteria from extracellular 02:, while the Mn*+ cnt,yme protects it from Oz; which is generated

46

J. A. COLE

intracellularly (Gregory and Fridovich, 197 3). If these conclusions are correct, superoxide dismutase is the fourth microbial enzyme which can protect cells against detrimental effects of molecular oxygen or its reduction products. 3. Superoxide Dismutase and the Origin of Mitochondria The gross differences in metal content and amino-acid composition of superoxide dismutases isolated from bacteria and mammalian erythrocytes were surprising in view of their similar physiological functions. It seemed that no vestige of the prokaryote enzyme had been retained during eukaryote cell evolution. Weisiger and Fridovich (1973) realized, however, that the bacterial enzyme would have been inactivated by the chloroform and ethanol extraction which is routinely used to purify erythrocuprein. Furthermore, mammalian mitochondria were known t o contain avimanganin, a protein with high affinity for R4n3+, but n o measurable enzyme activity, and no known function (Scrutton, 1971). Fresh chicken liver was therefore used to purify two types of superoxide dismutase, one from the cytosol, and another which was localized in the mitochondria. The mitochondrial enzyme was a manganoprotein with a molecular weight of 80,000 daltons: it contained 2.3 atoms of manganese and four subunits of equal mass which were not covalently joined (Weisiger and Fridovich, 1973). Its enzyme activity was insensitive to cyanide, but was irreversibly lost on exposure to chloroform and ethanol. Comparable concentrations of avimanganin and superoxide dismutase were found in mitochondria, and the two proteins were not separated by precipitation with ammonium sulphate, at pH 7.2, or by DEAE-Sephadex chromatography. It was concluded that avimanganin is an inactive form of mitochondrial superoxide dismutase. The amino-acid compositions of the subunits of various superoxide dismutases are listed in Table 5 . It can be seen that, despite the differences between mitochondrial and cytosol enzymes from chicken liver, the mitochondrial enzyme is quite similar t o those from E. coli and S t r e p t o c o c c u s mutans. Not onlv is the distribution of amino acids similar in these three proteins, but also the total number of residues in each subunit is almost the same. It seems likely, therefore, that the mitochondrial enzyme has evolved directly from the bacterial enzyme.

TABLE 5 . Amino-acid Composition of Superoxide Dismutase Purified From Different Sources. All figures are t o the nearest intecrer number of residues uer sub unit.

Escheric hiu coli (Mn2+enzyme)a (Fez+ enzyme)b

15 6 5 21 9 11 18 8 13 24

Lysine IIis tidine Arginine Aspartate ‘Threonine Serine Glutamate Proline Glycine Alanine IIalf-Cystine Valine Methionine Isoleucine Leucine Tyrosine Phen ylalanine Tryptophan ~

_

Data Data Data Data Data

_

?

10 2 7 19

6 9 ? _

.

10 6 4 22 13 10 16 9 16 26 1 11 0 8 15 6 10 4

______

Streptococcus mutansC

Chickend mitochondria

10 6 3 21 10 4 22 7 12 29 12 2

12 7 5 19 10

10 7 4 16 9

10 19 8 16 13 11 3 8 17 8

7

12 19 8

7 ? ?

5 2 5

Chickend cytoplasm

12 6

25 11 14 2 7 8 1 4 7 0

Bovinee erythrocytes

11 8 5 18 13 10 12 7 25 11 14 0 8 10 1 5 ? 0

5

0

n

0 m

D

r

0 D vl

P rn

-i

D 0

(I:

1 v)

z

from Kecle et al. (1970). from Yost and Fridovich (1973). from Vance et al. (1972). from Weisiqer and Fridovich (1973). from Keele et al. (1971). P

4%

J. A. COLE

If amino-acid sequence data proves consistent with this conclusion, the properties of superoxide dismutase will add important additional evidence in favour of the hypothesis that mitochondria evolved from an endosymbiotic association of two primitive prokaryotes (see Stanier, 1970). D. CONTROL O F ENZYME SYNTHESIS BY OXYGEN

Facultative anaerobes are micro-organisms which respond to the presence or absence of oxygen by derepressing the synthesis of some enzymes and repressing the synthesis of others. .J. W. T. Wimpenny and his colleagues have documented most thoroughly the changes in enzyme complement which occur when anaerobic batch or continuous cultures of Escherichia coli K 1 2 are aerated (Gray et al., 1966a, b; Wimpenny and Cole, 1967; Cole and Wimpenny, 1968; Wimpenny, 196913; Wimpenny and Firth, 1972). Similar investigations have been made with this and otfier micro-organisms (Pichinoty, 1962; references 1-9 cited by Gray et at., 1966a; Harrison and Pirt, 1967; Sapshead and Wimpenny, 1972; Wimpenny and Warmsley, 1968; Moss, 1952, 1956). Wimpenny (1969a) focussed attention on the regulation of enzyme synthesis by asking whether oxygen is directly involved as a reLgulator molecule and, if not, whether the regulator molecule is a single metabolite which controls the appearance and disappearance of large ,groups of enzymes. Is the formation of groups of enzymes regulated co-ordinately or sequentially, and how does oxygen in particular regulate the formation of new structures? Are the r e p latory inechanisms the same as those reported for other repressible or inducible enzymes? Pichinoty (1962) reported that hydrogenase and formate hydrogenlyase activities were absent from aerobic cultures of Klebsiella (Aerobacter) aerogenes, Escherichia coli, Proteus vulgaris and Salmonella oranienburg. These catalytic deficiencies were not due t o irreversible inactivation by oxygen, to the presence of an inhibitor, or t o iron deficiency, so it was suggested that molecular oxygen was a corepressor which could combine with a repressor molecule to switch off hydrogenase and formate hydrogenlvase synthesis in the same way that the lac repressor prevents transcription of P-galactosidase and lactose permease mRNA Uacob and RiZonod, 1961). It was subsequently established that many of the

MICROBIAL GAS METABOLISM

49

enzymes which were “repressed” by oxygen were also repressed by alternative electron acceptors: it was therefore suggested that the synthesis of enzymes which are induced or repressed by oxygen was regulated not by oxygen p e r se but by the redox potential of the culture (Cole and Wimpenny, 1966; Wimpenny and Cole, 1967; Cole and Wimpenny, 1968; Wimpenny, 1969b). It was implicit in this suggestion that the extracellular redox potential would be determined by the supply of oxidants to the culture, and that both the microbes and the redox electrode were capable of detecting the redox potential of the culture. Nevertheless, subsequently it was necessary t o state these points explicitly (Harrison, 19 72). The importance of the correlation between the extracellular redox potential and the rate of intracellular enzyme synthesis is that it suggests that the regulator molecules interact not only with the genes which they control, b u t also approach equilibrium with extracellular redox couples, at least one of which can act as a mediator to equilibrate with the redox electrode. If it is assumed that these regulator molecules are proteins, then they must be either redox proteins in which the prosthetic group can be oxidized or reduced, or proteins which bind redox-sensitive corepressors. The many possible corepressors include the oxidized and reduced forms of ffavin and nicotinamide nucleotides, ubiquinone, vitamin K, haematin, folic acid, organic redox couples and even compounds which equilibrate with redox couples (ATP and ammonium ion, for example). The problem is, therefore, to identify the extracellular redox couples which equilibrate with a platinum electrode; t o discover the basis for the correlations between the redox state of these couples, the availability of oxidants t o the culture, and the activity of the intracellular regulatory proteins; and t o identify the regulatory proteins and their corepressors. The first part of the problem has been considered by Harrison (1972) who summarized the reasons for doubting that a platinum electrode is an ultrasensitive substitute for an oxygen electrode. Even in a solution as chemically complex as a microbial culture, it is unlikely that a redox electrode can equilibrate with oxygen per se. Most culture media contain more than 10 ph4 ferrous or ferric ion, however, and redox electrodes equilibrate with many transition metal ions at this concentration. Furthermore, the activity of cytochrome oxidase is far higher than the rate-limiting step in the

50

J. A . COLE

electron-transfer chain, SO the 0 2 / H 2 0 couple should equilibrate rapidly with the oxidized and reduced forms of cytochrome oxidase. Alternative electron acceptors such as nitrate, nitrite and hydrogen ion can also interact rapidly with nitrate reductase, nitrite reductase and hydrogenase, respectively. Iron has been implicated in the formation of each of these enzymes-for the haem groups of cytochromes a, d, and cr of the cytochrome oxidases, for cytochromes 6 5 5 5 of nitrate reductase, for c- and d-type cytochromes of nitrite reductases and for the non-haem iron of hydrogenase (Gel’man e t al., 1967). It is reasonable t o suppose, therefore, that extracellular oxidants regulate enzyme synthesis by oxidizing components of the various electron-transfer chains which are in equilibrium with the redox probe via electro-active transition metal ions. The response of aerated cultures of facultative anaerobes to a m e m biosis should therefore depend on the activity of the terminal reductases. Since the earlier review in this series (Hughes and Wimpenny, 1969), preliminary evidence has accumulated which suggests that this hypothesis is correct. Showe and De Moss (1968) investigated the kinetics of nitrate reductase derepression in E. coli. Nitrate reductase activity of bacteria g o w n anaerobically without nitrate was only 5% of those grown with nitrate, but this was far higher than the activity of bacterial grown aerobically. Two control mechanisms appeared t o operate: repression of nitrate reductase synthesis by oxygen was superimposed upon repression in the absence of the inducer, nitrate. When the air supply to an aerobic culture was stopped, an exponential burst of nitrate reductate synthesis was followed by a less rapid phase during which the specific activity of nitrate reductase either increased slowly, or remained constant (Fig. 7). When isopropy1-0-D-thiogalactoside was added simultaneously with the onset of nitrate reductase derepression, the kinetics of p-galactosidase synthesis were monophasic rather than biphasic. These observations were interpreted as indicating that nitrate reductase synthesis is repressed by a redox-sensitive repressor which is inactive when reduced but active when oxidized, and by a nitrate-sensitive repressor which binds to a nitrate-reductase operator gene when nitrate is absent. When aerated cultures become anaerobic, the intracellular redox potential would fall and the redox-sensitive repressor would be inactivated: nitrate reductase synthesis would proceed slowly or

51

MICROBIAL GAS METABOLISM

/

A : T o t a l activity n i t r a t e reductase,

a-1200

,’

-

:-i

\,/I

Z‘

= .5

/

O E

/

-?: 2g

/ /

W \

/

.r g 8 0 0 -

- 0.0

/

/

\

-..’

C O -c

/ 1 A:Specific activity

._.._> .--

- 6.0

rU -c 0 -

0

- 4.0

400-

+0-

- 2.0 0

40

80

120

160

200

0

pg Protein / m l

FIG. 7. The biphasic derepression of nitrate raductase in Esckerickiu coli. In experiment A , a broth culture was incubated with glucose and nitrate, and aeration was stopped at the point indicated by the arrow. In experiment B , the broth culture was incubated with pyruvate. Again aeration was stopped at the point indicated by the arrow, and isopropyl-0-D-thiogalactoside added simultaneously. From Showe and De Moss (1968).

rapidly depending on whether the nitrate-specific repressor was active or inactive. The second, slower phase of nitrate reductase synthesis would be due to re-oxidation and therefore re-activation of the redox-sensitive repressor by nitrate and nitrate reductase. Notice that it is not necessary for the redox-sensitive regulator t o be a repressor: it could equally well be a positive controI element which promotes transcription when it is reduced. Either hypothesis clearly implicates an intracellular redox couple as a re‘gulatory factor, and emphasizes the role of the terminal reductases for equilibrating this redox couple with extracellular oxidants. These conclusions are re-inforced rather than weakened by the experiments of O’Brien and Morris (1971) with Clostridium acetobutylicum (see p. 39). Their observation that growth was insensitive to the extracellular redox potential was entirely consistent with the suggested role of electron-

52

J. A. COLE

transfer components in regulating the metabolism of facultative anaerobes. Clostridia are haem-deficient : they therefore cannot synthesize cytochrome oxidase or other haemoproteins of the respiratory chain. Negative evidence cannot establish a hypothesis, however, and if the postulated regulatory protein exists, it should be possible to isolate derepressed mutants which have lost it. Alternatively, if positive control is operative, it should be possible to isolate pleiotropic-negative mutants which are unable to synthesize a variety of anaerobically-induced enzymes. Experiments to tests these predictions are long overdue, and even if they are unsuccessful, the results would have important implications. Any component of the electron-transfer chain could be the redoxsensitive component of the regulator complex, but one candidate which has received serious consideration is the NAD/NADH, couple. One can make the teleological objection to this suggestion that NAD and NADH, are essential t o so many aspects of metabolism that microbial survivaI depends upon maintaining these and similar couples within narrow limits. The concentration of these intermediary metabolites must be regulated: they are not regulators, except as rapid, fine controls responsible for phenomena such as the Pasteur effect. Despite this objection, a decrease in fluorescence due to NAD reduction was observed when an aerated culture of K . aerogenes was made anaerobic (Harrison and Chance, 1970). In one experiment, the reduced nicotinamide nucleotide concentration changed from 0.08 t o 0.8 nmole/ml when aeration was stopped. If it is assumed that the total concentration of reduced and oxidized nicotinamide nucleotides was 0.88 nmole/ml and constant, this corresponded t o a decrease in the nicotinamide nucleotide redox potential of 60 mV (from -290 mV t o -350 mV). If the total concentration was substantially greater than 0.88 nmol/ml, then the change in this potential was far less than 60 mV. Wimpenny (1969b), in similar experiments, has shown that the extracellular redox potential changed by approximately 400 mV, from +250 mV to -150 mV; obviously the nicotinamide nucleotide and extracellular redox potentials are not in equilibrium, and if they were, the bacteria would be dead! Wimpenny and Firth (1972) subsequently determined the concentrations of NAD and NADH, that could be extracted rapidly from various bacteria growing aerobically and anaerobically. They concluded that the intracellular concentration of NADHz was remark-

MICROBIAL GAS METABOLISM

53

ably constant under different growth conditions, but the nicotinamide nucleotide redox potential changed by at most 30 mV because of variations in the NAD concentration. If NAD is the oxidized active corepressor for anaerobic metabolism, it must interact in an extremely sophisticated way with the repressor. This would be necessary t o amplify a weak signal sufficiently t o account for the high repression ratios which are observed for an anaerobic reductase such as hydrogenase (see, for example, Cole and Wimpenny, 1966). Furthermore, Ruiz-Herrera et al. (1969) have isolated mutants of E. coli K12 which are totally unable to reduce nitrate because they lack formate dehydrogenase. Nitrate is therefore unable to oxidize NADHz in these mutants, so presumably the same is true of the wild type from which they were derived. The rate-limiting steps in many electron-transfer chains are the flavoprotein dehydrogenases, so the most likely candidates for regulatory molecules are those which transfer electrons from the dehydrogenases to the terminal electron acceptors. If this is correct, one should be able to perturb the regulatory signal in either direction by adding inhibitors of the terminal reductases (carbon monoxide, azide, cyanide) or of the dehydrogenases (rotenone, atabrine, amytal or thenoyltrifluoroacetone). The former should stimulate a partial switch from an “aerobic” to an “anaerobic” type of metabolism, and the latter should stimulate the opposite change. Similarly one can investigate whether the redox regulator is before or after cytochrome b by studying the effect of inhibitors of cytochrome b oxidation (hydroxyquinoline-N oxides, antimycin A) on reductase synthesis. Results from inhibitor studies could be confirmed if mutants were available which lacked specific components of the electron-transfer chain. Mutants with defects on the electron acceptor side of the regulator molecule should be constitutive for anaerobically-induced enzymes, but those with blocks earlier in the chain should show decreased derepression ratios. Before such a programme can be undertaken, it is essential to identify the components of the various aerobic and anaerobic electron transfer chains: this work is well advanced in many laboratories. Furthermore, Simoni and Shallenberger (1972) have isolated a mutant of E. coli that lacks a component of the electron-transfer chain between cytochrome b and oxygen: this mutant is indeed constitutive for nitrate reductase synthesis during aerobic growth!

54

J. A. COLE

Preliminary results from A. H. Stouthamer’s laboratory are also consistent with the preceding predictions, but they suggest a slightly modified mechanism for oxygen regulation (de Groot and Stouthamer, 1969, 1970a, b, c). Metabolic inhibitors, haem-deficient and chiorate-resistant mutants were used to investigate the regulation of reductase formation by Proteus mirabilis, a bacterium which can use thiosulphate and tetrathionate as well as nitrate and hydrogen ion as anaerobic electron acceptors. Nitrate repressed the synthesis of other anaerobic reductases, and oxygen also repressed nitrate reductase formation. Proteus mirabilis grew both aerobically and anaerobically with 1 mM azide, but azide inhibited both formate hydrogenlyase and nitrate reductase activities. Because neither formate dehydrogenase nor hydrogenase is sensitive t o azide, this inhibitor was thought to inactivate one of the unidentified formate hydrogenlyase electron carriers, XI or X2 (Peck and Gest, 1957). During anaerobic growth with azide (but without nitrate) formate hydrogenlyase formation was repressed, but nitrate reductase was derepressed. In a leaky mutant which formed little nitrate reductase, the other reductases were repressed during growth with nitrate, but derepressed when both azide and nitrate were present. Nitrate did not inactivate thiosulphate reductase if azide was also added, and when bacteria were grown anaerobically with azide and then aerated, neither thiosulphate nor tetrathionate reductases were inactivated. Two conclusions were drawn from these complex results. First, the repressor for nitrate reductase synthesis is likely to be the reduced form of one of the formate hydrogenlyase electron carriers, XI or X2 (de Groot and Stouthamer, 1970a). Secondly, inactivation of anaerobically-induced reductases and repression of their synthesis are linked phenomena: synthesis is repressed whenever electron transfer to the reductase cannot occur (de Groot and Stouthamer, 1970b). The authors realized that their first conclusion could only explain the regulation of nitrate reductase synthesis during anaerobic growth, but they thought it unlikely that there is a single redox sensitive repressor which regulates the formation of all of the reductases. A simple alternative explanation for oxygen regulation will be offered to conclude this section, namely that when electron transfer to a reductase cannot occur, the reductase is oxidized and converted to a catalytically inactive form which is a specific repressor for that

MICROBIAL GAS METABOLISM

55

operon. This does not exclude the possibility that electron-transfer chains with common or interacting components can be regulated by a common, oxidized repressor.

IV. Gaseous Carbon Compounds A. FORMATION O F GASEOUS CARBON COMPOUNDS

Volatile carbon compounds that are products of microbial metabolism include carbon dioxide, methane, primary, secondary and tertiary amines and organic sulphides. Carbon dioxide formation is catalysed by a variety of decarboxylases which include nicotinamide nucleotide-dependent dehydrogenases (isocitrate dehydrogenase), thiamin pyrophosphate and lipoic acid-dependent dehydrogenases such as 2-oxoglutarate and pyruvate dehydrogenases, the amino-acid decarboxylases for which pyridoxal 5-phosphate is the co-enzyme and bacterial lysine and lactate oxygenases. Carbon dioxide is the major product of aerobic microbial metabolism, but it is also formed during anaerobic respiratory and fermentation processes. I t is an extremely soluble gas which interacts chemically with water t o form a weak, dibasic acid: many of its metal salts are insoluble in water, so both the Earth’s crust and its atmosphere are reservoirs of carbon dioxide that originated in the biosphere. The partial pressure of carbon dioxide in the atmosphere is very low, however, so despite rapid rates of carbon dioxide formation due t o biological oxidation processes, the concentration of carbon dioxide (plus HCO3 - and Cog-) dissolved in natural waters is low (about 0.1 mM), and any extra carbon dioxide generated locally by biological activity is rapidly lost to the atmosphere. At the opposite extreme, methane is the ultimate reduction product of a group of obligate anaerobes. Methane formation has been reviewed twice in this series (Wolfe, 1971; Quayle, 1972), so repetition of data presented previously would be superfluous. Quayle (1972) commented about the scale of methanogenesis by ruminants, and students of D. D. Woods will recall his tale of the fate of the inebriate farmer who entered a barn with a naked light: other folklore has linked methane and Will 0’ the Wisp. Methane is probably formed from methanol by a sequence of reactions in which

56

J. A. COLE

the single carbon atom is complexed with cobalamin derivatives; methanol is not believed t o be a free intermediate in the methane fermentation, but to be formed from carbon dioxide as a transitory intermediate complexed t o folic acid coenzymes. Electrons for methane production are supplied by molecular hydrogen. Bacteria which can form methane from molecular hydrogen and carbon dioxide include Methanobacterium barkerii and Methanobacterium M.o.H., the latter being the methanogenic partner of the symbiotic association which used t o perform under the pseudonym “Methanobacterium omelianskii”,(Bryant et al., 1967; Quayle, 1972). Volatile primary, secondary and tertiary amines occur as minor products of decaying plant and animal tissue, and higher concentrations are encountered in the fishing industry. Little biochemical detail is available about the role of microbial enzymes in forming these degradation products. B. UTILIZATION OF GASEOUS CARBON COMPOUNDS

1. Carbon Dioxide Wimpenny (1969a) has summarized the detrimental effects that a carbon dioxide deficiency has on heterotrophic micro-organisms: if carbon dioxide generated by oxidative metabolism is removed from a culture as it is formed, the anaplerotic enzyme phosphoenolpyruvate carboxylase will be unable to catalyse formation of oxaloacetate t o replace tricarboxylic acid-cycle intermediates which are also substrates of biosynthetic enzymes (Kornberg, 1965). Carbon dioxide is therefore essential for heterotrophic growth in all but the most complex media. Many groups of photosynthetic and chemolithotrophic microorganisms are autotrophs. Within these groups, there are significant variations in the ability of individual species to supplement their diet with organic carbon compounds (see articles by Rittenberg, 1969, 1972). In the same way that the availability of products of dinitrogen reduction appear to regulate nitrogenase synthesis, organic carbon compounds appear t o repress the synthesis of enzymes which catalyse carbon dioxide assimilation. Genetic analysis of autotrophic organisms is still something of a pipe dream, so their regulatory mechanisms will remain hypothetical for some years yet. Indeed,

MICROBIAL GAS METABOLISM

57

when faced with the task of summarizing the role of carbon dioxide as a metabolic regulator, Wimpenny (1969a) concluded that there seemed to be very little to discuss! If discussion of the role of carbon dioxide as a metabolic regulator is inhibited by the lack of definitive experimental data, the same cannot be said of metabolic pathways for carbon dioxide assimilation. Despite extensive evidence for the existence of at least three, and probably more, such pathways, most reviewers choose to ignore some of the alternatives. An attempt will therefore be made to summarize the various postulated pathways, and t o draw attention to the major gaps in the experimental evidence on which they are based. Critical summaries of some of this evidence have been included in reviews by Quayle (1972) and by McFadden (1973). The first pathway for carbon dioxide assimilation to be discovered was the reductive pentose phosphate or Benson-Calvin cycle (Benson and Calvin, 1950). Autotrophs which use this pathway must synthesize two enzymes that are not normally found in heterotrophs. The first is ribulose 5-phosphate kinase that catalyses the synthesis of ribulose 1,5-diphosphate from ribulose 5-phosphate and ATP, and the second is ribulose 1,5-diphosphate carboxylase which catalyses the carbon dioxide-fixation step to generate two molecules of phosphoglyceric acid. Ribulose 5-phosphate is then regenerated in a series of reactions that are common t o most cell types. With the possible exception of a few tropical grasses, some dicotyledons, methanogenic bacteria and photosynthetic bacteria of the genus Chloro b i u m , these two enzymes are still considered t o catalyse carbon dioxide fixation in all autotrophic organisms. The pathway is also used b y Pseudomonas oxalaticus, “Bacterium formoxidans” and probably b y at least one strain of Rhodopseudomonas palustris, when these bacteria are grown with formate as carbon source. The second pathway is the reductive carboxylic acid cycle in which the low midpoint redox potential of ferredoxin is exploited to reverse the reactions of the citric acid cycle (Fig. 8). One molecule of acetate, acetyl-CoA, pyruvate, phosphoenolpyruvate or any intermediate of the citric acid cycle can be withdrawn for biosynthesis for each turn of the cycle: any of these compounds can be regarded as the product of the cycle (Gehring and Arnon, 1972; Quayle, 1972). Nett synthesis of one molecule of citrate would require six molecules of carbon dioxide, two molecules of ATP and 18 electrons of which

58

J. A. COLE

jcit,ate]\ Citrate<

6

cis-Aconitate

\

[Acetate]

/ 2:

Isocitrate k k

NADPHz

J

a-Oxoglutarate

Acetyl-CoA

k?

I

B-FReduced

)+m A\Lm

ferredoxin

Reduced ferredoxin

Succinyl-CoA 44

&

ATP CoA

Pyruvate

-4

Succinate

Reduced flavin and/or

Fumarate .A#

Phosphoenolpyruvate

\

Pi

NADHz

FIG. 8. The reductive carboxylic acid cycle of carbon dioxide fixation. Modified from Quayle (1972).

six must be donated by reduced ferredoxin, eight by reduced nicotinamide nucleotides, and four by reduced flavin nucleotides. Key enzymes of the complete cycle include citrate lyase, acetate kinase, pyruvate synthase, phosphoenolp yruvate c arboxylase, fumarate reductase and 2-oxoglutarate synthase. Once again, the cycle is regenerative. It is believed to be the major or even the sole route for carbon dioxide assimilation by photosynthetic green sulphur bacteria of the genus Chlorobium (Sirevag and Ormerod, 1970; Buchanan et al., 1972). Insufficient experimental data are available for its role in other photosynthetic or chemosynthetic anaerobes t o be assessed. The third pathway also involves carboxylation of phosphoenolpyruvate to oxaloacetate (Fig. 9): this is the Hatch-Slack-Kortschak

z

W

v,

~~~~P~

OXALOACETATE

%Fs

/

PY RUVATE

n W

r

r

ASPARTATE

v,

RIBULOSE 1,5 DIPHOSPHATE

3-PHOSPHOGLYCERIC ACID

\

BENSON-CALVIN CYCLE

1

8 3

r r

STARCH

FIG. 9. Modified Hatch and Slack pathway for the assimilation of carbon dioxide by some plants.

pathway which has been considered in some detail by Walker and Crofts (1970). Slack and Hatch (1967) proposed that oxaloacetate, or some other carboxylic acid, was the substrate for a transcarboxylation reaction in which a pentose phosphate, possibly ribulose 1,5-diphosphate, was the carboxyl group acceptor. This hypothetical transcarboxylase was postulated in order to explain how the three-carbon carbon dioxide-acceptor molecule might be regenerated. Subsequent studies have established, however, that leaves of tropical grasses, in which this pathway is believed to

60

J. A. COLE

operate, contain at least two cell types, mesophyll cells and bundle sheath cells. It now seems likely that chloroplasts of mesophyll cells contain an active phosphoenolpyruvate carboxylase which accounts for 85% or more of the total carbon dioxide fixed by the entire leaf. Aspartate or malate is the fixation product, and either or both products diffuse to the bundle-sheath cells where they are decarboxylated. The three-carbon decarboxylation product diffuses back to the mesophyll cells to be recarboxylated, while the carbon dioxide in the bundle-sheath cells combines with ribulose 1,5diphosphate, catalysed by ribulose 1,5-diphosphate carboxylase. Starch is then synthesized in the bundle-sheath cells of “Hatch and Slack” plants by the same reactions that are used in “Benson-Calvin” plants. The extensive and very recent evidence for the operation of this third pathway for carbon dioxide fixation has been reviewed by Black (1973), who has also summarized the variations of this scheme which probably occur in different types of higher plant. One is left with the impression that the criteria on which this pathway is based are far more rigorous than those of the earlier two pathways, but questions now arise about the evolutionary origin of the pathway. For example, is there sufficient cell differentiation within the cyanobacteria for such a pathway to operate in this group of micro-organisms, and have sufficient species been rigorously investigated to exclude the possibility? (see Jansz and MacLean, 1973; Wolk, 1973; Stanier, 1974). Alternatively, can one trace the origin o f the Hatch and Slack pathway to a particular group of eukaryotic algae, and if so, what biochemical advantage can be derived from a three-stage carboxylation process when most organisms seem to prefer a single carboxylation step? A plausible answer to the final question can be suggested by examining the experimental evidence for the other two pathways. The criteria on which the significance of a carbon-dioxide assimilation pathway is assessed should include: (1) the results of short-term C 0 2 -fixation experiments; (2) the demonstration that the necessary enzymes are present; ( 3 ) characterization of kinetic parameters to establish that the enzymes are sufficiently active; (4) identification of the products of CO, -fixation experiments, and the distribution of 4 C in the products; (5) use of inhibitors of enzymes of the postulated pathway; ( 6 ) the results of experiments in which carbon dioxide is replaced by postulated intermediates of the

’

’

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pathway; and (7) the demonstration that in facultative autotrophs, synthesis o f the appropriate enzymes is re
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from a variety of higher plants, and its structure and biosynthesis have been investigated. The polymer has a molecular weight of 550,000 daltons, and contains two different types of subunit o f molecular weight 55,000 and 15,300 daltons, respectively. Criddle et al. (1970) reported that chloramphenicol specifically inhibited the incorporation of l 4 C-leucine into -the large subunit of greening barley leaves, but cycloheximide preferentially inhibited the incorporation of label into the small subunit. It would appear, therefore, that only the large subunit was synthesized within the chloroplast. Elegant experiments with the purified enzyme and its subunits from the French Bean Phaseolus vulgaris indicated that the large subunit contained the active site (Gray and Kekwick, 1974a, b). Enzyme activity was inhibited by antisera against the complete enzyme or the large subunit, but antisera against the small subunit were ineffective. Furthermore, partial immunological identity of the bean and spinach enzymes was located in the large subunits. Subsequent experiments with these specific antisera demonstrated that 55% o f the soluble protein synthesized during the greening process was ribulose 1,5-diphosphate carboxylase, and that the small subunit constituted 30% of the protein which was synthesized in. vitro on bean-leaf cytoplasmic polysomes. It was concluded that the sites of transcription and translation of the two subunits of ribulose 1,5-diphosphate carboxylase are spatially separated in the cells o f higher plants, and that the small subunit probably functions as a template on which the catalytic large subunits are assembled (Gray and Kekwick, 1974a, b). By contrast, relatively little is known about ribulose 1,5-diphosphate carboxylase from micro-organisms. Exceptions are the enzymes from Hydrogenomones eutropha and H. facilis which were purified to homogeneity from soluble extracts of bacteria which had been cultured with fructose as the carbon source (Kuehn and McFadden, 1969a, b). Only a 26-fold increase in specific activity was obtainable, so this enzyme constituted approximately 4% of the soluble protein in H. eutropha. Similarities with respect t o metal-ion requirements, inhibition by phosphate and kinetic mechanism between enzymes from H. facilis and spinach did not extend to the enzyme from H. eutropha. The molecular weights of the enzymes from H. eutropha and H. facilis were 515,000 and 551,000 daltons, respectively. Each enzyme could be dissociated into a single type of subunit of mass 40,700 or 38,000 daltons, respectively. It can be

MICROBIAL GAS METABOLISM

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TABLE 6. Relative Amino-acid Compositions of Ribulose 1,5-Diphosphate Carboxylases From Two Hydrogenomonads and From Spinach. Data taken from Kuehn and McFadden (1969b) Amino acid

Hydrogenomonas facilis

Hydrogenomonas e u trop ha

Spinach

Phenylalanine Threonine Aspartate Serine Glutamate Proline Glycine Alanine Valine Isoleucine Leucine Tyrosine Lysine Histidine Arginine Cysteine Methionine Tryptophan

1.00 1.25 2.58 1.00 2.08 1.08 2.00 2.08 1.58 1.08 1.75 0.69 0.80 0.56 1.42 0.58 0.68 0.34

1.00 1.51 2.44 1.16 2.20 1.29 2.02 2.44 1.64 1.02 1.80 0.82 0.88 0.56 1.60 0.69 0.76 0.40

1.00 1.37 2.01 0.68 2.18 1.24 2.05 1.89 0.84 2.00 1.07 1.15 0.67 1.34 0.46 0.45 0.64

-

seen from Table 6 that there are superficial similarities in the amino-acid compositions of the enzymes from these two hydrogenomonads and from spinach. Other studies of ribulose 1,5-diphosphate carboxylases from micro-organisms have included the partial purification and kinetic characterization of the enzyme from Thiobacillus denitrificans (McFadden and Denend, 19721, a comparison of the enzyme from various Athiorhodaceae with that from plants (‘4kazawa et al., 1969), and the determination of the sedimentation velocity of the enzyme from various photosynthetic organisms (Anderson et al., 1968). The major conclusions to emerge from these studies are that the molecular weights vary from 68,000 daltons in Rhodospirillum rubrum (but see Anderson et al., 1968), through 350,000 daltons in T. denitrificans, Rhodopseudomonas spheroides and R. palustris, to 550,000 daltons in H. facilzs, various cyanobacteria, higher algae and plants; and that the K, for bicarbonate is high in every case, with values from 4 to 22 mM being reported. The excellent review by McFadden (1973) contains a more complete discussion of autotrophic carbon dioxide fixation, and o f the role of ribulose 1,5-diphosphate carboxylase in this process.

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One of the interesting questions which remains to be answered is whether there is amino-acid sequence homology between the bacterial and cyanobacterial enzymes, or between specific groups of cyanobacteria and higher algae. If so, the latter would provide experimental evidence for the theory that chloroplasts arose by endosymbiosis of specific photosynthetic prokaryotes, and it might also indicate whether different
2. Metabolism of Reduced Carbon Compounds Quayle (1972) has summarized the known and postulated pathways by which reduced single-carbon compounds are metabolized. These include the serine pathway, the ribose phosphate cycle o f formaldehyde fixation, pathways involving N-methylated amino acids for assimilating gaseous amines, and possibly a fourth pathway

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involving corrinoid and folate derivatives. The serine pathway has been implicated as the major route for carbon assimilation in methylamine-grown Diplococcus PAR (Leadbetter and Gottlieb, 1967), methane-grown Methanomonas methano-oxidans (Lawrence et al., 1970) and in other methylotrophs with the characteristic “Type II” membrane structure. This membrane consists of a series of bundles of disc-shaped vesicles distributed throughout the cell (Wittenbury et al., 1970; not as in Quayle, 1972). In the serine pathway, methane or methanol is converted to glyoxylate which is then transaminated by serine t o yield glycine and hydroxypyruvate. Two features of this cycle are the requirement for the enzyme hydroxypyruvate reductase, and the low rate of incorporation of * O into cell material (QuayIe, 1972). Methane-carbon is assimilated at the oxidation level of formaldehyde by methylotrophs with “Type I” membrane structure which consists of paired membranes running throughout the cell or concentrated around its periphery (Whittenbury et al., 1970): typical species include Pseudomonas methanica and Methy locystis capsulatus. The key enzyme of this pathway is hexose phosphate synthetase which incorporates formaldehyde-carbon (and its 80-oxygen atom, if present) into C1 of a hexose phosphate. N-Methylated amino acids, such as N-methylglutamate, are early products of pseudomonads growing with methylated amines as carbon source, but the hypothetical corrinoid and folate pathway may well be used by methanogenic bacteria which are essentially autotrophs. It is known that these bacteria generate methane from carbon dioxide via a series of coenzymebound intermediates, and that Clostridium thermoaceticum can synthesize acetate from carbon dioxide via similar if not identical intermediates. It is possible, therefore, that some aerobes might assimilate methane, or anaerobes carbon dioxide, by a similar pathway in which a methyl corrinoid would be the common intermediate. Quayle (1972) commented upon the difficuIty in assessing the si‘gnificance of early claims that some micro-organisms can grow with carbon monoxide as carbon source. Yagi (1959) has cited reports of organisms which could either oxidize o r reduce carbon monoxide, and he subsequently demonstrated that extracts of Desulfovibrio desulfuricans, which had been grown with lactate as carbon source, would catalyse oxidation of carbon monoxide to carbon dioxide

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with water as the electron donor. The enzyme responsible for this reaction was not identified, but it was more heat-stable than hydrogenase or formate dehydrogenase (Yagi, 1959). This is no longer a sufficient criterion, however, for concluding that these enzymes were not responsible for carbon monoxide oxidation. Hmch (1968) described the isolation of both aerobic and anaerobic micro-organisms which apparently were assimilating carbon monoxide, and he commented that continuous illumination aided growth even of the non-photosynthetic carbon monoxide-oxidizing bacteria. Presumably, carbon monoxide bound to cytochrome oxidase (and other redox enzymes) was photodissociated by illumination. Hubley et al. (1974) reported that carbon monoxide-saturated buffer stimulated the rate of oxygen reduction by suspensions of Methylomonas albus and Methylosinus trichosporium, and that 4 C 0 was quantitatively oxidized to I 4 C O 2 . Although yet again no attempt was made t o identify the enzyme responsible for carbon monoxide metabolism, relevant data have subsequently been presented by Ferenci (19 74). Both Ferenci (1974) and Ribbons and h4ichalover (1970) were interested in the mechanism of methane oxidation by cell-free extracts, and they had established an apparent 1:1: 1 stoicheiometry for NADH2 and methane oxidation, and O2 reduction. When these data are considered with the meticulous 8 O isotope data of Higgins and Quayle (1970), one is tempted to suggest that the first enzyme of the methane oxidation pathway is a mono-oxygenase. Carbon monoxide is an inhibitor of mixed-function oxidases which require cytochrome P450 for activity, so Ferenci (1974) attempted to study the effect of carbon monoxide on the methane hydroxylase activity of a particulate fraction from Pseudomonas methanica. He was surprised to find that carbon monoxide stimulated rather than inhibited the rates of oxygen reduction and NADH, oxidation. Reduced nicotinamide adenine dinucleotide and molecular oxygen were removed in a 1:1 molar ratio while carbon monoxide was being oxidized, suggesting that this reaction was also being catalysed by a mono-oxygenase. Carbon monoxide inhibited methane oxidation by cell suspensions, presumably because it competed with methane for the co-substrate (NADH2) but was unable to supply electrons to regenerate NADHz. Conversely, carbon monoxide stimulated the rate of oxygen reduction by bacteria incubated with formate: formate dehydrogenase in P. methanica is NAD-linked, so electrons

MICROBIAL GAS METABOLISM

67

released from formate could be passed either to “carbon monoxideoxidase”, or to an NADH, oxidase (which is probably inhibited to carbon monoxide). Control experiments established that carbon monoxide was indeed oxidized: it was not simply an activator of enzymes of the methane oxidation pathway. These interesting results are entirely consistent with the suggestion that methane hydroxylase might also have carbon monoxide oxidase activity, but it was realized that they may only share one component of two multi-component enzyme complexes (Ferenci, 1974). The lingering controversy about the mechanism of methane oxidation should ensure that methane hydroxylase will soon be purified. The identity of the carbon monoxide-oxidizing enzyme from methylotrophs might simultaneously be established.

V. Hydrogen Metabolism A. HYDROGEN PRODUCTION

While nitrogenase research had made rapid progress during the last ten years, only scattered items of biochemical detail have been added to our knowledge about hydrogenase. The review by Gray and Gest (1965) still provides an accurate basis for discussing hydrogen formation by micro-organisms, and had they also been asked in 1965 to review hydrogen utilization, these authors would probably have been able to predict most of the results of more recent experiments. Hydrogen metabolism is catalysed by two types of enzyme, hydrogenase and nitrogenase. In nitrogen-fixing prokaryotes there is a reciprocal inhibition of dinitrogen reduction by molecular hydrogen, and of hydrogen evolution by molecular nitrogen. This has already been discussed in the section on “nitrogenase” (p. 1 7 ) in the context of the ultimate acceptor for electrons donated to the nitrogenase complex. N o one has successfully demonstrated with inhibitors, or with mutants that have lost the ability to evolve molecular hydrogen, whether the hydrogen-evolution reaction gives nitrogen-fixing bacteria a specific selective advantage. One is therefore free t o speculate about the significance of this hydrogenevolution reaction: either it wastes ATP and reductant when these are in excess, or it is useful as a mechanism for disposing of unwanted electrons.

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Hydrogen formation is essentially an anoxic process, even in an obligate aerobe such as Azotobacter. Many of the photosynthetic prokaryotes can fix molecular nitrogen, and when growth is not limited by either the avaiIabiIity of reductant or the light intensity, they too evolve hydrogen. Gray and Gest (1965) did not attempt to assess the relative contributions of hydrogenase and nitrogenase to the rate of hydrogen formation in nitrogen-fixing organisms, and the writer can d o little .better ten years later. Techniques are now available to separate and assay each of these enzymes from cell-free extracts so this point should soon be settled. Apart from the photosynthetic micro-organisms, Gray and Gest (1965) considered that bacteria which evolve molecular hydrogen fall into three groups. These are: (i) obligate anaerobes which do not synthesize cytochromes (clostridia, for example); (ii) heterotropic facultative anaerobes; and (iii) the genus Desulfouibrio. Hydrogen is evolved by each of these groups t o eliminate excess electrons generated by fermentation reactions in which large quantities of an organic substrate are oxidized. Little ATP is generated by these reactions: growth is slow, and large quantities of fermentation products accumulate. It is debatable whether these anaerobic growth rates are limited by the rate of ATP synthesis from ADP, the rate of NADH2 oxidation to NAD, or by some other reaction such as the rate of synthesis of an amino acid. Hydrogenase activity is probably advantageous to these bacteria because electrons which are ultimately lost into the atmosphere as gaseous hydrogen could come either directly or indirectly from NADH2. In the former case, NAD would become available to oxidize more molecules of the growthsupporting substrate when hydrogenase is present than when it is absent. Alternatively, hydrogenase could “spare” the cell’s NAD by diverting electrons away from reactions in which NAD is reduced t o NADH2, for example, those catalysed by lactate or alcohol dehydrogenases. Many fermentation products, especially from bacteria in group (iii), are acids: hydrogenase activity therefore probably confers an additional advantage by removing protons which otherwise would adversely lower the pH value of the environment. B. HYDROGEN FORMATION BY OBLIGATE ANAEROBES

Obligate anaerobes such as the clostridia are essentially fermentative bacteria which accumulate hydrogen as one of many fermen-

MICROBIAL GAS METABOLISM

69

tation products. This hydrogen is released from pyruvate, and various clostridia, Veillonella alcalescens (which used t o be called Micrococcus lactilyticus) and Peptostreptococcus elsdenii catalyse a rapid exchange of carbonate into the carboxyl group of pyruvate. This enzyme has been purified from Clostridium acidiurici: it has a spectrum similar to ferredoxin, and it copurified with thiamin pyrophosphate (Raeburn and Rabinowitz, 197 1a, b). It will reversibly catalyse the decomposition of pyruvate to carbon dioxide and acetyl-CoA if an electron acceptor of low redox potential and thiamin pyrophosphate are present. Clostridial ferredoxin was an effective electron acceptor for electrons released from pyruvate, and this low redox potential carrier is also an effective electron donor for hydrogenase from other clostridia (Cl. acidiurici does not synthesize hydrogenase). Reduced ferredoxin can also reduce urate to xanthine (catalysed by xanthine dehydrogenase), or NAD to NADH, (catalysed by NAD: ferredoxin oxidoreductase Valentine et al., 1963). Clostridia can therefore in theory use molecular hydrogen as electron donor for generating reduced nicotinamide nucleotides (Fredricks and Stadtman, 1965). It is more likely, however, that these enzymes catalyse nicotinamide nucleotide oxidation when the nicotinamide nucleotide redox potential falls too low, especially when one realizes that, at pH 5.0, the reduction of H+ by NADHz is thermodynamically more favourable than at pH 7.0. It is not surprising, therefore, that hydrogenase is essentially a constitutive enzyme in clostridia which functions predominantly in the direction of hydrogen evolution: it is incapable of coupling with other electron carriers from clostridia t o reduce inorganic oxidants (Gray and Gest, 1965). Despite the fact that bacteria have usually been the source of hydrogenase for biochemical studies, one should remember that hydrogen is aIso metabolized by some eukaryotes. Trichomonads are symbiotic or parasitic flagellates that live in the digestive or genitourinary apparatus of the host, and the cattle parasite, Tritrichonzonas foetus obtains ener'gy from fermentation reactions. Approximately 0.75 mole of hydrogen, one mole of succinate and one mole of acetate are formed for each mole of glucose metabolized. Rlitochondria have not been detected in trichomonads, but subcellular organelles with a single membrane and a granular matrix have been described. These contain dehydrogenases, but neither NADH2 oxidase

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nor catalase: they are therefore not peroxysomes. The isolated organelles will catalyse the exchange of C-carbon from carbon dioxide to pyruvate, pyruvate synthesis from molecular hydrogen, carbon dioxide and acetyl CoA, and reduction of FMN, FAD, clostridial ferredoxin or viologen dyes by molecular hydrogen (Lindmark and Muller, 1973). It- is likely, therefore, that these organelles play a central role in anaerobic pyruvate metabolism by generating electrons which reduce hydrogen ions by reactions similar to those occurring in clostridia. The term “hydrogenosome” has been proposed for this granular organelle (Lindmark and Muller, 1973). C. FORMATE HYDROGENLYASE ACTIVITY OF FACULTATIVE ANAEROBES

In many facultative anaerobes, hydrogenase is coupled through two other electron carriers and formate dehydrogenase to convert formate to hydrogen and carbon dioxide. This formate hydrogenlyase activity therefore tends to stabilize the pH value of the environment by decomposing the strongest acid which is formed during the mixed acid fermentation. Escherichia, Citro bacter, Klebsiella, Aero bacter, Salmonella, Proteus and Hafnia species all use oxygen in preference t o other terminal electron acceptors, but alternatives include nitrate, nitrite, fumarate (and other organic acids), sulphate, sulphite, tetrathionate and H+. These alternative oxidants not only inhibit hydrogenase activity by competing with H + for electrons, but also inactivate it and repress its synthesis (Gray, 1964). Thus hydrogenase synthesis in E. coli is repressed by oxygen or nitrate, and its activity is severely inhibited by nitrite, nitrate and oxygen (Pichinoty, 1962; Gray et al., 1966a). This pattern of regukation is consistent with hydrogenase being an inducible enzyme which functions predominantly to reduce H + when no better oxidant is available. Hydrogenase can catalyse the reverse reaction, however: it has long been known that E. coli can couple fumarate reduction to hydrogen oxidation, and that less hydrogen is evolved when this bacterium is grown anaerobically with fumarate than without it (Krebs, 1937). Hydrogen and hydrogenase have also been used as a source of reductant to assay nitrate reductases of facultative anaerobes, but the preferred source of the hydrogenase activity is the Hildenborough strain of Desulfovibrio desulfuricans. Hydrogenase from this organism is neither contaminated with nitrate reductase

MICROBIAL GAS METABOLISM

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activity, nor is it inhibited by nitrate (Pichinoty and Piechaud, 1968). Formate hydrogenlyase activity is regulated by at least three factors other than genetic defects or the availability of terminal electron acceptors. These are the iron content of the growth medium, its amino-acid composition and its pH value. Formate hydrogenlyase activity was absent from iron-deficient cultures of E. coli but was induced when the depleted bacteria were incubated with glucose, formate and iron salts. Hydrogenase was also absent from the iron-depleted bacteria, and re-appeared concomitantly with formate hydrogenlyase activity (Fukuyama and Ordal, 1965). Iron is probably part of the active sites of at least two proteins in the formate hydrogenlysase complex. These are hydrogenase itself, and a cytochrome which has been implicated as electron carrier for transferring electrons from formate dehydrogenase t o hydrogenase (Gray and Wimpenny, 1963). Contrary t o earlier suggestions, this cytochrome is probably not cytochrome c 5 5 2 (Douglas et al., 1974). Synthesis or activation of formate hydrogenlyase is also pH-dependent. When incubated with formate, cultures of E. coli which had been grown in a rich medium at pH 6.2 and 7.0 evolved 93 and 4 p1 H,/h/mg protein, respectively. There was no corresponding difference in the hydrogenase activity of the two cell types, but the formate dehydrogenase which donates electrons t o benzyl viologen was far more active in bacteria grown at an acidic pH value (Gray et al., 1966a). Hydrogenase activity can therefore only stabilize the pH value of an acidic medium in which it is most required; in alkaline media, it is uncoupled from formate, its source of electrons. A third nutritional factor which affects formate hydrogenlyase synthesis or activity is the complexity of the growth medium. Minimal salts cultures of some strains of E. coli will not release molecular hydrogen from formate, have very low formate dehydrogenase activities, and a diminished hydrogenase activity. The basis for this type of regulation is unknown (Gray et al., 1966a). D. DESULFOVIBRIO HYDROGEN METABOLISM

Although hydrogen is not formed during normal growth of the sulphate-reducing bacteria, they contain high activities of hydrogenase and will decompose formate to hydrogen and carbon dioxide

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(Gray and Gest, 1965; Le Gall et al., 1971a). Hydrogenase from D. g p can be released into the suspending medium by osmotic shock; it is therefore located at the surface of the cell, in the periplasmic space (Peck, 1974; Heppel, 1967). Peck (1974) has suggested that this cellular location indicates that the physiological role for desulfovibrio hydrogenase is to trap s m a l a m o u n t s of hydrogen from the environment. This is no mean task, however, because the solubility of hydrogen in aqueous media is extremely low, and its fugacity is such that it is readily lost from the earth’s atmosphere; hydrogen will therefore only accumulate in a closed ecological niche. Despite these problems, there is ample evidence that desulfovibrio hydrogenases can catalyse hydrogen utilization as well as hydrogen formation. Peck (1966) demonstrated that extracts of D. gigas, which had been grown with lactate and sulphate, could catalyse the reduction of sulphate by hydrogen, and that oxidative phosphorylation was coupled to this reaction. Synthesis of ATP was uncoupled by gramicidin, 2,4-dinitrophenol or pentachlorophenol, but not by oligomycin. The P:H, ratios were usually 0.1-0.2, but were occasionally 0.4. The enzyme preparation contained a sufficient concentration of adenine nucleotides to maintain optimal rates of ATP synthesis, but exogenous ADP was required if the cell extract was dialysed before use. Soluble proteins were also required t o catalyse electron transfer to sulphite, but in subsequent experiments, a particulate fraction alone transferred electrons from molecular hydrogen to fumarate (Barton et al., 1970). This redox reaction is also sufficiently exergonic t o be coupled t o ATP synthesis (AG = -20.4 Kcal/g mole). Once again phosphorylation was insensitive to oligomycin, but was inhibited by gramicidin, dinitrophenol and pentachlorophenol. The P:H, ratios were usually 0.3-0.4, and at best were as high as 0.9. This ATP was not formed by substrate-level phosphorylation from side reactions because the glucose used for the ATP trap was not metabolized beyond hexose phosphate, and all of the fumarate that was consumed was converted to succinate. Malate could only substitute for fumarate as electron acceptor if soluble proteins were added as a source of fumarate hydratase activity (Barton et al., 1970). Taken together, these observations provide convincing evidence that sulphate-reducing bacteria can couple ATP synthesis to the reduction of two of their normal intermediary metabolites, but how are electrons transferred to these oxidants?

MICROBIAL GAS METABOLISM

73

Gray and Gest (1965) considered the desulfovibrios t o be a separate group from other obligate anaerobes which catalyse hydrogen formation because they synthesize haemoproteins. Long after the discovery of cytochrome c3 (Postgate, 1956), it was realized that the sulphate-reducing bacteria contain several c-type cytochromes which are species- and even strain-specific. These cytochromes vary in their cellular location, molecular weight and haem content, and at least three types can be distinguished by the last two criteria. These are desulfovibrio cytochromes c 5 5 3 which have a single haem group and a molecular weight below 10,000 daltons, cytochromes c3 with two haems and a molecular weight of 13,000 daltons, and cytochromes cc3 with four haems and a molecular weight of 26,000 daltons (Le Gall et al., 1971a). Although cytochrome c3 from D. vulgaris is soluble and basic, cytochrome c; from D. gigas is particulate and acidic (Le Gall et al., 1965). These various cytochromes mediate electron transfer from formate to molecular hydrogen, and from hydrogen to sulphite and fumarate (Yagi, 1970; Le Gall et ul., 1971a). Furthermore, fumarate reduction by molecular hydrogen was inhibited by 2-n-heptyl-4 hydroxyquino1ine-Noxide and antimycin A which are specific inhibitors of cytochrome b oxidation. Cytochrome b has been detected in D. gigas membranes by low-temperature spectroscopy, and confirmed by converting its protohaem group to the characteristic pyridine haemochrome which absorbs 556 nm light (Hatchikian et al., 1972). E. STRUCTURE OF HYDROGENASE

Hydrogenases have been purified from several heterotrophic anaerobes. The enzymes isolated from Clostridium pasteurianum by Nakos and Mortenson (1971a, b) and from Desulfouibrio vulgaris by Le Gall et al. (1971b) were probably better than 95% pure, but there are good reasons for doubting the homogeneity of some of the other samples. For example, Kleiner and Burris (1970) detected molybdenum as a structural component of their hydrogenase preparation from Cl. pastrurianum. On the basis of kinetic studies with molybdenum-supplemented enzyme, they suggested that their enzyme had an H,-site linked by an electron-transfer bridge to Mo(vi) at the electron donor site. Their data were consistent with two possible catalytic mechanisms in which Mo(vi) was reduced to hlo(v) and then

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re-oxidized by ferredoxin or methyl viologen. As a single electron was transferred from one hydrogen atom t o Mo(vi), a proton would be released and be free to exchange with D 2 0 in the solvent. The second hydrogen atom would form either a stable S-H bond or a metal hydride at the H,-site. Kleiner and Burris (1970) estimated that their least contaminated enzyme was 80% pure after preparative gel electrophoresis, but this sample was largely inactive and contained only 0- 0.2 atom Mo per molecule. Less purified samples gave multiple bands of activity on polyacrylamide gels, but the Rf values were variable and different from that of the single band obtained from the purest samples. Kidman et ul. (1968) had also detected isoenzymes” of Cl. pusteuriunum hydrogenase, but they believed b<.

them to be interconvertible forms of a single protein of mass 50,000 to 60,000 daltons. Hydrogenase isolated from the same source by Nakos and Mortenson (1971a, b) did not contain significant quantities o f molybdenum: it did not catalyse reduction of Mo(vi) to Mo(v) by moIecular hydrogen, and it was not activated by molybdenum. These authors considered that apparent “isoenzymes” were preparative artefacts. Physical and chemical properties of the purified enzyme are summarized in Table 7. It is a golden yellow protein of the ferredoxin type with two identical subunits. The subunits associate strongly, even in 8M urea. Hydrogenase purified from D. uulguris was very similar to the clostridial enzyme (Le Gall et ul., 1971b). Both enzymes were inactivated by oxygen, and both lost iron and catalytic activity during purification. Once again there were approximately four atoms of iron and two identical subunits of mass 30,000 daltons per molecule (Table 7). The desulfovibno subunits did not associate as strongly in dilute solution as the clostridial subunits, and a sharp g perpendicular electron-spin resonance signal at 1.86 was detected at 220°K-an abnormally high temperature for electron spin resonance spectroscopy. Once again, hydrogenase appeared t o be an iron-sulphur protein of the high molecular-weight ferredoxin type. The Desulfovibrio enzyme was not activated by Mo, neither did it catalyse reduction of Mo(vi) to Mo(v). Less pure samples of hydrogenase have been isolated from these and other bacteria. Yagi (1970) solubilized the membrane-bound hydrogenase from D. uulguris and subsequently obtained a protein which migrated electrophoretically as a single co-incident peak of

TABLE 7 . Comparison of Hydrogenases Purified from Clostridium pasteurianum and Desuljovibrio vulgaris Property

Clostridium pasteurianum W 5 a Desulfouibrio uuIgaris “HiIdenborough”b

2 0 m

I

Molecular weight Number of subunits Electron paramagnetic resonance absorption by reduced enzyme Fe/60,000 daltons Acid-labile sulphide/60,000 daltons Electron donor for evolution of hydrogen Wavelength maximum in absorption spectrum of oxidized enzyme a

Data from Nakos and Mortensen (197 l a , b). Data from LeGall e t al. (1971a, b).

60,000 2

60,000

2

1.86 3.5 3.2 Cytochrome

1.94 4 4 Ferredoxin

9

r c, 9

v)

z

c3

rn

-I

0

400 nm (approx) -

408 nm

r v)

3

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J. A. COLE

protein and catalytic activity. The molecular weight was approximately 60,000 daltons, and the metal content was 7.7 atoms of iron, 0.6 atoms of zinc and 0.4 atoms of copper per 60,000 daltons. Although the zinc and copper were probably due t o contamination, it is possible that Yagi’s enzyme lost less iron during purification than other hydrogenases, and that the enzyme in uiuo contains eight or even more atoms of iron per molecule. The K, for molecular hydrogen at 30°C was less than 8 mm Hg (<8 pM), and the enzyme was specific for cytochrome c j . The soluble hydrogenase from D. desulfiuicans was also purified, and its properties were compared with those of Cl. pasteurianum hydrogenase. Desulfouibrio desulfuricans hydrogenase donated electrons to cytochrome cg but not to ferredoxin, but the opposite specificity was observed for the clostridial hydrogenase (Yagi et al., 1971). Molecular weights of unpurified hydrogenases from a variety of bacteria were determined by Kidman et al. (1969). These estimates agreed well with those for purified enzymes, and established that hydrogenase from Cl. butylicum is approximately twice as large as that of other clostridia. It seems likely that a gene-doubling event has occurred during evolution of this species. Even larger hydrogenases were detected in extracts of Arotobacter vinelandii (molecular weight of 130,000 daltons), Proteus uulgaris (180,000 daltons) and E. coli (210,000 daltons). Hydrogenases from clostridia were predominantly soluble proteins, but those from facultative anaerobes and desulfovibrios were particulate (Kidman et al., 1969; see also Riklis and Rittenberg, 1961). Hydrogenase from the photosynthetic bacterium Chromatium was associated with the chromatophore membrane. It could transfer electrons t o NAD either through ferredoxin and ferredoxin-NAD oxidoreductase, or through benzyl viologen. Relative rates of H-D and D2 formation from H2 and 100% D 2 0 were consistent with heterolytic cleavage of molecular hydrogen t o form a proton and an enzyme-bound hydride (Feigenblum and Krasna, 1970).

F. AEROBIC HYDROGEN METABOLISM

In a previous section it was established that the sulphate-reducing bacteria have evolved a sophisticated electron-transfer chain which couples ATP synthesis to oxidation of molecular hydrogen. Oxygen

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is a more powerful oxidizing agent than either fumarate or sulphite, so it is not surprising that Micrococcus denitrificans, and a variety of bacteria belonging to the genera Hydrogenomonas and Pseudomonas, can synthesize ATP at the expense of energy released from the oxidation of hydrogen by oxygen. All of these hydrogenomonads are facultative autotrophs. The redox potential for the H+/H2 couple and the mid-point potential of the NAD/NADH, couple at pH 7.0 are -0.42 V and -0.32 V respectively, so it is thermodynamically possible for molecular hydrogen to reduce NAD directly. A major problem of aerobic hydrogen metabolism has been to decide whether this reaction occurs in uz’uo, and if so, whether it is obIigatory if ATP synthesis is to be coupled to oxidation of molecular hydrogen. The following criteria were used to settle the controversy. Most electrontransfer chains accept electrons from NADH, before the rotenonesensitive flavoprotein, from succinate at cytochrome b , and from ascorbate at cytochrome oxidase. Electron flow through each of these sites is sensitive to specific inhibitors; atabrine, amytal and rotenone inhibit electron flow from NADH, to cytochrome 6; antimycin A and hydroxyquinoline-N-oxides inhibit flow from cytochrome 6 t o cytochrome c ; and cyanide and azide inhibit some b u t not all cytochrome oxidases. If electrons from molecular hydrogen are transferred to NAD, oxidation of molecular hydrogen should be inhibited by rotenone as well as by cyanide and hydroxyquinolineN-oxides. If, in addition, ATP synthesis is coupled t o electron flow across any of these sites, it should be inhibited by uncoupling agents as well as by inhibitors of electron-transfer reactions. It is now apparent that hydrogenomonads vary considerably in their requirement for NAD as an intermediate for electron flow from hydrogen to oxygen. Thus although NAD is an obligate intermediate in the oxidation of hydrogen by Hydrogenomonas eutropha, it is not involved at all in Hydrogenomonas H20 (Ishaque and Aleem, 1970; Bongers, 1967). Micrococcus denitrificans appears to lie between these two extremes (Knobloch et al., 1971). When extracts were prepared from AVl. dcnitrificans which had been ,grown with hydrogen, carbon dioxide and oxygen, phosphorylation coupled to NADH2 oxidation was completely inhibited by rotenone, but phosphorylation coupled t o the oxidation o f molecular hydrogen was only inhibited by 30-40%, and the rate of

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hydrogen oxidation was hardly inhibited at all. Because rotenone inhibits electron transfer from NADH, to cytochrome b, M . denitrificans must have an NAD-independent pathway for oxidizing molecular hydrogen which feeds electrons into the respiratory chain at or beyond cytochrome 6 . When hydrogen, NADH2, succinate and ascorbate were used as alternative substrates t o support oxidative phosphorylation, P:O ratios were 0.6- 1.6, 0.4- 1.2, 0.7- 1.0 and 0.3- 0.5, respectively. It was therefore concluded that all three energy-coupling sites were functional during autotrophic growth (Knobloch e t al., 1971). Hydrogen was not oxidized by bacteria which had been grown with succinate, s o hydrogenase appears to be an inducible enzyme which is repressed during heterotrophic growth. The H, -supported ATP synthesis was completely inhibited by low concentrations of various uncoupling agents or by cyanide, azide, hydroxyquinoline-N-oxide and antimycin A. Similar experiments with extracts of H. eutropha once again established that all energycoupling sites were functional, but NAD was considered to be an obligate intermediate for oxidation of molecular hydrogen by this bacterium (Ishaque and Aleem, 1970). At the other extreme, oxidative phosphorylation by extracts of autotrophically-grown Pseudomonas saccharophila was insensitive to rotenone, atabrine and amytal when molecular hydrogen was the electron donor. The P:O ratios for hydrogen and succinate oxidation were 0.45-0.73 and 0.15, respectively: because more ATP is formed from hydrogen than from succinate, there must be an additional coupling site between molecular hydrogen and cytochrome b. Evidence has also been presented for energy-coupling between hydrogen and cytochrome b in Hydrogenomonas H20 (Bongers, 1967). One can conclude that various hydrogenomonads metabolize molecular hydrogen by one or both of the pathways shown in Fig. 10. It should therefore be possible t o detect two types of hydrogenase activity in these bacteria, an NAD-dependent hydrogen dehydrogenase, and a hydrogen: cytochrome b oxidoreductase: both types have been characterized. Autotrophically
Rotenone Amytal Atabrine

/ \

\' i

Hydrogen deh ydrogenase

/

,I

TTFA

Fpn

',

'*+ FP,

\ i

CN-; N3I

HQNO; antimycin A

I

i

I

1

I V

5 0

n

D

r

-

FIG. 10. AIternative pathways for oxidation of molecular hydrogen h y hydrogenomonads. The pathways of electron flow are indicated by the solid arrows, and the sites of inhibition by the dashed arrows. TTFA is thenoyltrifluoroacetone, and HQNO is 2-n-alkyl-4-hydroxyquinoline-N-oxide.

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electron-transfer chain for ATP synthesis (Ishaque et al., 19 7 1) while the NAD-specific enzyme maintains the intracellular supply of reduced nicotinamide nucleotides. Hydrogenomonads are therefore the only chemolithotrophs which can reduce nicotinamide nucleotides without having to use ATP t o reverse electron flow from succinate t o NAD (Peck, 1966). Hydrogen dehydrogenase has been purified 300-fold from extracts of 11. rulzlandii (Bone et a[., 1963). In crude extracts, the K, for molecular hydrogen appeared t o be 480 pM, but the K, for the pure enzyme was 7.6 pM. This estimate agrees well with the K, for molecular hydrogen for the purified hydrogenase from Cl. pasteurianum (Yagi et al., 1971). The enzyme from H. ruhlundii was stabilized by Mn2+ during its purification, but n o cofactors were required for activity other than the electron acceptor, NAD (K, 6.6 pM). Hydrogen dehydrogenase of Hydrogenomonas H16 has also been isolated free from flavins and nicotinamide nucleotides (Pfitzner e t al., 1970). Reducing agents inhibited this enzyme but, unlike hydrogenases from anaerobes, it was not inactivated by oxygen. The K, value for hydrogen was estimated t o be 190 pM, and the activation energy was 10.4 Kcal/mole (Pfitzner et al., 1970). Biologists have for years focussed attention upon the carbon, nitrogen and sulphur cycles of the biosphere, but in this brief synopsis of microbial hydrogen metabolism one can detect the skeleton of another cycle, the hydrogen cycle. Photosynthetic and fermentative microbes contain soluble, constitutive hydrogenases which catalyse hydrogen evolution in highly reducing environments; these hydrogenases are inactivated by oxygen. The facultative anaerobes have inducible hydrogenases which are apparently repressed in oxidizing environments, but they too evolve molecular hydrogen in reducing environments. Sulphate-reducing bacteria can produce hydrogen, but their hydrogenases have evolved primarily t o scavenge for traces of gaseous hydrogen which can be used as an electron donor for anaerobic respiratory chains. Finally, the hydrogenomonads are an extremely sophisticated group which not only use molecular hydrogen as an electron donor t o support ATP synthesis, but have also evolved independent enzymes (which are presumably subject t o independent regulatory mechanisms) to transfer electrons t o nicotinamide nucleotides. The latter are in tremendous demand during autotrophic growth. Hydrogen is indeed an important compound in interspecies electron transfer! (Bryant, 1969; Hungate, 1967).

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VI. Summary and Conclusions 1. Errors and Omissions

Any review of a subject as far-reaching as microbial gas metabolism must be selective. In this review, the metabolism of volatile, reduced nitrogen compounds has largely been i
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might regulate enzyme synthesis in facultative anaerobes. Further experimentation might well establish that a reduced component of an electron-transfer chain, rather than a later, oxidized c,omponent,is the repressor for enzymes o f that pathway. If so, this will be an error of detail rather than of fundamental principle. My attention has been drawn to a review by Morris (1975) which is currently “in the press”, and in which problems associated with Eh measurements are again discussed. One should be aware that this excellent review discusses hypotheses t o explain oxygen toxicity and the physiology o f obligate anaerobiosis, and therefore amplifies Sections 111-A and 111-C of this review. Redox control of gene expression was invoked by Wimpenny and his colleagues only because there was a positive correlation between the extracellular redox potential of a complex culture medium and the intracellular concentrations of specific gene products. No significance would have been attached to a negative correlation, and hypotheses which were discussed in Section 111-D (p. 48 et seq.) do not conflict with those of Morris (1975).

2. Evolution of Gas Enzymes It was suggested in the Introduction that gaseous metabolites could be arranged in order of stability on the basis of their electronic structure; that they all participate in redox reactions; and that the low solubility o f the most stable gases would require highly specialized enzymes to catalyse reactions in which they are substrates. The structure of an enzyme should be such that its prosthetic group is not only effective as a catalyst, but is also usually available to organisms in their natural habitats. Redox enzymes have essentially two types of prosthetic group, organic redox couples synthesized intracellularly, and transition metal ions. The former group includes nicotinamide and flavin nucleotides, quinones, sulphydryl, pteridine and all other enzymes in which vitamins are structural components. Synthesis of enzymes of this type is relatively unaffected by fluctuations in the supply of trace metals, but equally these enzymes are unsuitable catalysts for stable gases, or for many inorganic redox reactions. Transition metals, however, provide a wide range o f electronic orbitals at various energy levels which offer more scope for overlap with atomic orbitals of stable substrates. The disadvantage nf these metalloproteins is that their substrate specificity decreases as a

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consequence of their high catalytic activity, and enzymes which interact with nitrogen, hydrogen and methane also interact with more reactive gases such as oxygen, carbon monoxide, nitric oxide and acetylene. It is inevitable, therefore, that this review has failed to discuss the metabolism of one gas in isolation without reference to other gases, and that the instability of molecular oxygen has influenced many other areas of gas metabolism. Although two different types of prosthetic group for redox enzymes can be recognized, there are many examples of both types occurring in a single enzyme complex. Many of the enzymes which metabolize stable or insoluble gases are of this type, and they therefore justify the initial predictions. The simplest examples include non-haem iron proteins of the ferredoxin type, and manganese-dependent superoxide dismutases. More complex examples include nitrogenase and cytochrome oxidase 11113. Although various comments have been made previously about the evolution of gas enzymes, the temptation t o suggest that an enzyme with a very complex structure is highly “evolved” has been resisted. Such a criterion for evaluating the age of an enzyme would not appear to be inferior to many of these discussed previously however (see the various references in “Evolution in the Microbial [World”: 24th Symposium of the Society for General Microbiology, 1974). Although it is debatable whether the complexity of the secondary, tertiary or quarternary structure of an enzyme reflects its evolutionary status, its cellular location is likely to be pertinent. If it is assumed that primitive enzymes are also unspecialized and relatively ineffective, one can trace from this review the evolutionary path taken by gas metabolism. When the concentration of a growthsupporting substrate is low, the most effective way to trap it is to locate an enzyme with a low K, as a monomolecular layer at the cell surface, that is, in the cytoplasmic membrane. The incorporation of a soluble enzyme into a membrane is but a small step in the evolution of specialized organelles or differentiated cells. It is therefore surprising that attempts to detect enzyme evolution by laboratory manipulation have focussed attention on increased rates of enzyme synthesis, or synthesis of an enzyme with a higher V,,, value o r a lower K, value, but have ignored possible changes in cellular location. By comparing the metabolism of hydrogen, nitrogen and oxygen in clostridia, with hydrogenomonads or azotobacters, one can

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see that, as enzymes evolved to fulfil a more specialized role, they also became associated with cellular membranes. Thus hydrogenases of clostridia are at least partially soluble, and they transfer electrons between molecular hydrogen and a soluble redox protein, ferredoxin. In some hydrogenomonads however, a particulate hydrogenase donates electrons to an electron-transfer chain to which oxidative phosphorylation is coupled. The branched electron-transfer chain and respiratory and conformational protection of nitrogenase in Azotobacter vinelandii are further examples of structural complexity in a highly evolved organism. Further information about the function of the intracellular membranes in methylotrophs is eagerly awaited. VII. Acknowledgements

I am grateful to Dr. C . M. Brown and Dr. R. S. Tubb for allowing me to see manuscripts in advance of publication, and to numerous collea
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Le Gall, J., Bruschi-Heriand, M. and DerVartanian, D. V. (1971a). Biochimica et Biophysica Acta 234, 499. Le Gall, J., DerVartanian, D. V., Spilker, E., Lee, J. P. and Peck, H. D. (1971b). Biochimica et Biophysica Acta 234, 525. Le Gall, J., Mazza, G. and Dragoni, N. (1965). Biochimica et Biophysica Acta 99, 385. Le Gall, J. a n d Postgate, J. R. ( 1 9 7 3 ) .Advances in Microbial Physiology 10, 81. Lemberg, R. and Barrett, J. (1973). In: “Cytochromes”, Academic Press, London and New York. Lindmark, D. G. and Muller, M. (1973). Journal of Biological Chemistry 248, 7724. Linnane, A. W., Haslam, J. M., Lukins, H. B. and Nagley, P. (1972). Annual Review of Microbiology 26, 163. Lippitt, B. a n d Fridovich, I. (1973). Archives of Biochemistry and Biophysics 159, 738. Magasanik, B., Prival, M. J., Brenchley, J . E., Tyler, B. M., Deleo, A. B., Streicher, S. L., Bender, R. A. and Paris, C. G. (1974). Current Topics in Cellular Regulation 8, 119. Magill, C. (1972). Genetics, Princeton 7 1 , 536. Matsubara, T. (1971).Journal ofBiochemistry, T o k y o 6 9 , 9 9 1 . Matsubara, T. and Iwasaki, H. (1971).Journal o f Biochemistry, T o k y o 6 9 , 859. Matsubara, T. and Iwasaki, H. (1972). Journal of Biochemistry, T o k y o 72, 57. Matsubara, T. and Mori, T. (1968).Journal of Biochemistry, T o k y o 64, 863. McCord, J. M. a n d Fridovich, I. (1969). Journal of Biological Chemistry 244, 6049. McCord, J. M., Keele, B. B., and Fridovich, I. (1971). Proceedings o f the Natzonal Academy o f Sciences of the United States of America 6 8 , 1024. McFadden, B. A. (1973). Bacteriological Reviews 37, 289. McFadden, B. A. and Denend, A. R. (1972). Journal of Bacteriology 110, 633. Meers, J. L., Tempest, D. W. and Brown, C. M. (1970). Journal of General Micro biology 64, 187. Mehta, J. M. a n d Siehr, D. J. (1973). Archiu fiir Mikrobiologie 88, 163. Meyer, D. J. (1973). Nature New Biology 245, 276. Miyata, M., Matsubara, T. and Mori, T. (1969). Journal of Biochemistry, T o k y o 66, 759. Miyata, M. and Mori, T. (1969). Journal o f Biochemistry, T o k y o 6 6 , 463. Morris, J. G. (1970). Journal of General Microbiology 60, iii. Morris, J. G. (1975). Advances in Microbial Physiology 12, 169. Mortenson, L. E., Morris, J. A. and Jeng, D. Y. (1967). Biochimica et Biophysica Acta 141, 516. Mortenson, L. E., Valentine, R. C. a n d Carnahan, J. E. (1962). Biochimica et Biophysica Acta 9 , 448. Mortenson, L. E., Zumft, W. G. and Palmer, G. (1973). Biochimica et Biophysica Acta 292, 422. Moss, F. (1952). Australian Journal of Experimental Biology and Medical Science 30, 531. Moss, F. ( 1 9 5 6 ) . Australian Journal of Experimental Biology and Medical Science 34, 395. Moustafa, E. and Mortenson, L. E. (1967). Nature, London 216, 1241.

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Structure and Biosynthesis of the Mannan Component of the Yeast Cell Envelope CLINTON BALLOU Department o f Biochemistry, University of California, Berkeley, California 94720, U.S.A.

I. Introduction A. Organization o f Mannan in the Cell Envelope B. General Physical and Chemical Properties of Yeast Mannans. C. Carbohydrate Composition of Mannans . 11. General Methods for Structural Analysis o f Yeast Mannans A. Mannan Isolation . B. Selective Acid Hydrolysis C. Selective Alkaline Degradation . D. Selective Acetolysis E. Enzymic Degradation . F. Nuclear Magnetic Resonance Spectroscopy . G. Immunochemical Methods 111. Detailed Structures of Specific Yeast Mannans . A. Saccharomyces cerevisiae . B. Other Saccharomyces Species . C. Kluyveromyces Species . D. Hansenula Species . E. Candida Species . F. Other Yeast Mannans . IV. Mannan Biosynthesis . A. Mannan Biosynthesis in Saccharomyces species . B. Mannan Biosynthesis in other Yeasts . C. A Model for Mannan Biosynthesis . . References

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I. Introduction Mannan is a major polysaccharide component of t h e cell envelope of many, although not all, yeasts (Phaff, 1971). It is easily extracted 93

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from the cell-wall matrix of glucan and chitin by autoclaving the cells in a neutral citrate buffer (Peat et al., 1961); in this way, it is obtained as an amorphous, water-soluble mixture o f macromolecules. Mannan isolated in this manner and then fractionated by selective precipitation (Haworth et al., 1937), ion-exchange chromatography (Thieme and Ballou, 1971), or sizing on gel columns (Thieme and Ballou, 1972), contains covalently-linked protein or polypeptide material. Consequently, most or all of the mannan is present in the cell wall as a mixture of glycoproteins or, if you prefer, proteoglycans. Recent studies revealing the polymorphic structures of yeast mannans (Ballou and Raschke, 1974) suggest that these polysaccharides may contribute species-specific information of importance for the organization of the cell wall and for cell-cell recognition. In the past, most of the work done with yeast mannan has dealt with heterogeneous mixtures; and only rarely, as with the studies on Saccharomyces FH4C invertase (Gasc6n e t a / . , 1968) or Hansenula wingei 5-agglutinin (Yen and Ballou, 1973, 1974b), have reasonably homogeneous molecular species been analysed. When important, distinction will be made between such studies and those that involve the bulk cell-wall mannan. Several reviews have appeared in recent years that deal with various features of the yeast cell envelope. Among these are discussions of the structure and biosynthesis of the yeast cell envelope (Phaff, 1971), the budding of yeast cells (Beran, 1968), regeneration of the cell wall by yeast protoplasts (Netas, 1971), yeast cytology (Matile et al., 1969), Saccharomyces cerevisiae cell cycle (Hartwell, 1974), and external enzymes of yeasts (Lampen, 1968). My aim will be to review new developments concerning yeast mannan structure and biosynthesis as they relate t o the organization of the cell wall and the roles played by these glycoproteins. The purpose is not to provide a comprehensive citing of the literature, but rather to attempt an integration of those studies that have led to new insights concerning the title subject. A. ORGANIZATION O F MANNAN IN THE CELL ENVELOPE

Although the plasma membrane and the cell wall are clearly distinguished in electron micrographs of thin sections of yeast cells (Matile et al., 1969), there is no strong evidence that the mannan and

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glucan are present in separate layers. Electron micrographs of thin sections o f yeast cells stained with the periodic acid-Shiff's reagent show an outer stained layer, attributed t o mannan, that is about one-fifth t o one-seventh the thickness of an inner unstained layer attributed to glucan (Mundkur, 1960). Although this is often cited to support the view that the glucan and mannan occur as distinct layers, that interpretation is not convincing. On the other hand, the evidence is good that most or all of the chitin in Sacch. cerevisiae is located in the bud scars (Beran, 1968). The surface location of some of the mannan is supported by immunochemical studies, for mannanspecific antibodies agglutinate whole cells that possess appropriate mannan antigenic determinants (Hasenclever and Mitchell, 1964; Ballou, 1970). Yet, much of the mannan also appears t o be located in the space between the plasma membrane and the cell wall, because essentially all of the mannan-invertase is assayable in whole cells and is released in soluble form when the cells are broken (Arnold, 1972, 1973). Whether the surface mannan completely covers the glucan is unknown, because a glucan-specific immunochemical probe does not appear to be formed by injection into rabbits of isolated glucan or cell-wall fragments. Antigenic determinants that react with mannanspecific antibodies are detectable on the surface of Sacch. cerevisiae protoplasts (C. E. Ballou, unpublished), although it has been reported that protoplasts from Candida utilis lack antigens characteristic of the cell surface and possess those characteristic of the inner side o f the cell wall (Mendoza et al., 1968). These were postulated to be fragments of the wall that remained attached to the protoplast membrane. Purely as a working hypothesis, it seems most fruitful t o consider that the cell-wall mannan is differentiated in location, with a structural component being interspersed with the glucan and covering the surface o f the cell, whereas mannan-enzymes such as invertase and phosphatase may be located mainly in the periplasmic space. Other nonglycosylated enzymes (glucanases) appear to be bound tightly within the insoluble wall matrix, and presumably function in wall expansion (Fleet and Phaff, 1974). The sexual agglutination factors of H. winLqeiare bound tightly on the cell surface, but soluble forms can be isolated from broken cells and may be intracellular precursors of the external mannan-protein (Crandall and Brock, 1968).

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B . GENERAL PHYSICAL AND CHEMICAL PROPERTIES O F YEAST MANNANS

Yeast mannan preparations are heterogeneous, with molecular species ranging from 25,000 to at least 1 million daltons in mass being present in bulk rnannan extracts. The external invertase activity of Sacch. cerevisiae X2180 can be fractionated by size (Smith and Ballou, 197413) as well as by charge (Smith and Ballou, 1974a), SO even a mannan-enzyme can be composed of different molecular types. The size heterogeneity most likely reflects differences in the carbohydrate content, although some strains do possess multiple invertase genes and could make invertase proteins with different properties. Different ratios of carbohydrate to protein are readily detected by sedimentation-equilibrium measurements on a caesium chloride gradient (J. E. Varner, unpublished). The charge differences that allow fractionation on DEAE-Sephadex columns probably reflect variable amounts of phosphate esterified to the polysaccharide component (Thieme and Ballou, 1971). Most mannan preparations are readily decreased in size by treatment with dilute alkali (0.1 M NaOH; Nakajima and Ballou, 1974a), with dithiothreitol (Smith and Ballou, 197413) and by pronase digestion (Thieme and Ballou, 1972). All three changes probably reflect degradation of the protein component that holds the polymannose chains together. The alkaline treatment also releases an assortment of small manno-oligosaccharides from most crude mannan preparations owing to 0 elimination of these fragments from linkage to serine and threonine units in the protein (Sentandreu and Northcote, 1968), but this alone should not alter the size si'gnificantly. The majority of the carbohydrate in most such crude mannan preparations is attached to asparagine units in the protein, probably by way of a di-N-acetylchitobiose unit (Tarantino et ad., 1974) as in many other glycoproteins (Hughes, 1973). As discussed below, some homogeneous mannan-proteins have only one o r the other of these two kinds of carbohydrate-protein linkage. In no instance has it been established that both kinds of linkage co-exist in the same mannan molecule, although this seems likely because such examples have been found for other glycoproteins (Carlsen et al., 1973). C. CARBOHYDRATE COMPOSITION OF MANNANS

As the name implies, the principal carbohydrate in mannans is D-mannose. However, it is by no means the only sugar (Gorin and

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Spencer, 1968). All preparations in which the polymannose chains are attached by alkali-stable linkage contain some N-acetvl-D -glucosamine that serves as the bridge between the polysaccharide and protein chains. This same hexosamine is also found as a terminal sugar in the side chains of the mannan of several Kluyveromyces species (Raschke and Ballou, 1972). Other monosaccharides found in yeast mannans include D -galactose, D -glucose, D -xylose, L -arabinose, D-glucuronic acid, L-fucose and L-rhamnose (Spencer and Gorin, 1973).

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FIG. 1. Representative structures illustrating the nature of the carbohydrate components in: (a) Saccharomyces cereuisiae mannan, (b) a mannan with some 1-6 linkages in the side chains, and (c) a mannan with some 12' linkages in the backbone. The mannan in Sacch. cereuisiae has short oligosaccharides attached to residues of serine and threonine in the protein, whereas large polysaccharide units are linked to asparagine residues. The structures (b) and (c) are analogous to the polysaccharide chains of Sacch. cereuisiae rnannan.

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Much of the thinking about mannan structure has been prejudiced by the concentration of past investigations on baker’s yeast, a strain or mixture of strains of Sacchuromyces cereuiszue. That may have been fortunate, for the mannan of this yeast happens t o have one of the more easily defined polysaccharide structures. Characteristically, baker’s yeast mannan is composed o f long manno nose chains in a1-6 linkage, with short side chains in al+2 and al+3 linkage (Fig. la). These macromolecules are attached to asparagine in the protein.

08 0

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FIG. 2. A schematic model of Saccharomyces cerevisiae mannan which emphasizes the highly branched nature of the molecule. The triangles represent mannose units attached to hydroxyamino acids (A), those that form the inner core (A)and those in the outer chain (a).The squares represent N-acetylglucosamine units that link the polysaccharide chains to the protein, while the circles indicate amino acids.

In addition, this mannan has short oligomannoside units with 011’2 and (~1-3 linkages that are linked t o serine and threonine in the protein. A schematic model for such a molecule is shown in Fig. 2. There are many subtleties t o be discussed later in this review, but this gives the general picture. By contrast, I can present a much less detailed structure for the mannans from other yeasts, in part, because they d o not appear to possess the uniform 1’6-linked backbone, but rather are ramified molecules like those shown in Figs. l b and lc.

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11. General Methods for Structural Analysis of Yeast Mannans A. MANNAN ISOLATION

The state of denaturation of the rnannan-protein complex isolated from the cell depends, naturally, on the conditions used for the extraction. The classical method of the carbohydrate chemist utilized hot alkali that dissolved the mannan and left most of the glucan and the chitin as insoluble residues (Haworth et al., 1937). At the same time, the alkali-labile bonds in the mannan were extensively disrupted. This would include the glycosyl-serine and glycosyl-threonine linkages, phosphodiester bonds, some peptide and disulphide bonds, and acyl ester linkages. Isolation by the milder extraction with hot water or hot, neutral citrate buffer (Peat et al., 1961) gives material with intact phosphodiester bonds and, presumably, intact peptide bonds, but in which the protein is denatured. Simple cell breakage gives the most intact mannan-protein preparations, yielding active external invertase and other mannan-enzymes that can be purified to maximum specific enzymic activity (Neuman and Lampen, 1967). The 5-agglutinin on the surface of H. wingei 5-cells is released by subtilisin digestion of the intact cells (Taylor and Orton, 1967), and it seems probable that this occurs by cleavage of a polypeptide chain that anchors the mannan-protein in the matrix of the wall. Such a mannan can be purified by affinity adsorption t o cells of the opposite mating type. Finally, the release of any mannan fixed in the glucan layer should be facilitated by the action of glucanases (Phaff, 1971), and Glusulase (Endo Laboratories) or Zymolyase (Kirin Brewery Co.) are convenient for this purpose. To understand the functional complexity of the yeast cell wall it will be important to determine how many different kinds of proteins are held in the wall as rnannan complexes or in a nonglycosylated form. The development of gel electrophoresis in sodium dodecyl sulphate has made it possible to answer such questions about cell membranes (Ames, 1974). However, mannan-proteins d o not resolve well on electrophoresis with or without the presence of detergent. One might hope to improve the resolution by enzymic removal of all of the carbohydrate from the mannan-protein preparation (Smith and Ballou, 1974a). However, even this does not reveal the presence of distinct protein components (W. L. Smith and C. E. Ballou, unpublished data).

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The phosphodiester bonds appear to be the most acid-labile linkages in mannans (Phaff, 1971). The (Y-D-mannopyranosyl phosphodiester linkage is broken by 0.01 N HC1 at 100°C for 30 minutes with quantitative release of free mannose (Rosenfeld and Ballou, 1974a). Recovery of the mannan and rehydrolysis releases very little additional mannose, thereby demonstrating the specifity of the reaction, although 0.1 N acid under the same conditions does hydrolyse other glycosidic bonds. Exocellular phosphomannans are extensively degraded in this reaction and yield oligosaccharide phosphates as a result of depolymerization of these teichoic acid-like molecules (Slodki, 1963).

C. SELECTIVE ALKALINE DEGRADATION

Treatment of mannan with 0.05 N NaOH at 50°C for 18 hours releases mannose and manno-oligosaccharides from their linkage t o serine and threonine (Sentandreu and Northcote, 1968; Phaff, 19 7 1; Nakajima and Ballou, 1974a). This reaction can be carried out in the presence of M sodium borohydride t o reduce the oligosaccharides and minimize their further degradation, a process that is particularly facile if the monosaccharide at the reducing end is substituted at position-3 (Ballou, 1954; Lloyd et al., 1966). By gel filtration, the mixture of oligosaccharides can be separated and each component then analysed, thus allowing characterization of all the carbohydrate units linked to hydroxy amino acids in the protein. The sensitivity of this procedure is enhanced by reduction with tritiated sodium borohydride (Yen and Ballou, 1974a). The mildly alkaline conditions needed to catalyse 0-elimination also dramatically decrease the size of the mannan-protein complex, suggestive of saponification of acyl ester linkages or alkaline oxidative hydroIysis of disulphide bonds. The large polymannose chains attached to asparagine can be released by a much more strenuous treatment with N NaOH at 100°C for 5 hours (Nakajima and Ballou, 1974a) owing to hydrolysis of the N-glycosylamide linkage between N-acetylglucosamine and the amino acid (Lee and Scocca, 1972).

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D. SELECTIVE ACETOLYSIS

Probably no reaction has played a more important role in the determination of yeast mannan structure than selective acetolysis (Gorin and Perlin, 1956; Kocourek and Ballou, 1969). This procedure has a relatively high specificity for splitting 1+6 linkages in preference t o others, and it converts many yeast mannans to a mixture of small oligosaccharides with the more stable 1+2 and 1+3 linkages (Lee and Ballou, 1965). Because the backbone of the mannan from Succh. cerevisiae is formed exclusively from 1+6linked mannose units, the acetolysis-stable fragments represent the side chains of the polysaccharide in which the mannose unit at the reducing end is derived from the backbone of the polymer (Stewart and Ballou, 1968; Stewart et al., 1968). The mannan side chains are the principal immunochemical determinants on the yeast cell, and the acetolysis reaction provides a particularly convenient method for preparation of haptenic groups with which t o analyse the structural specificity of the immunochemical reactions of yeasts (Ballou, 1970). The acetolysis reaction was first applied to a yeast mannan by Gorin and Perlin (1956), and the mechanism has been studied in detail (Rosenfeld and Ballou, 1974b; Guthrie and McCarthy, 1967; Govorchenko et al., 1973; Lindberg, 1949; Matsuda et al., 1961). The reaction usually employs a mixture of acetic anhydride and concentrated sulphuric acid, and the attacking species is the acetylium cation [ CH3 CO]'. As shown in Scheme I, this reagent can lead both to acetolysis and t o anomerization although, in Sacch. cereuisiue mannan, the latter reaction appears insignificant because the mannose units are already in the stable (Y configuration. The selectivity for 1+6 linkages is thought t o reflect the higher electron density at the glycosidic bond in such compounds relative to that in 1+2-, 1+3- and 1+4-linked units, and is a measure of the number of electron-withdrawing oxygen atoms on the neighbouring carbons. This electronic effect also rationalizes the difference in acetolysis rates between CY-D -mannopyrdnosyl-(1 + 6 ) - -mannose ~ and a - D mannopyranosyl-( 1 - + 6 ) --mannitol, ~ the latter being considerably more stable. &though the acetolysis reaction seems to behave in a straightforward manner with the a-mannans, steric factors play a dominant

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OR 5

OAc

T

(c) -AczO

,OAc

3

OAc

i

(d) -AcOR

OO ~ O A C

AcO

4

OAc

Scheme I

role with other sugars. For 0-linked glucosides, anchimeric assistance from the trans acetyl group at position-2 appears to have a controlling influence on the rate of acetolysis, whereas the high rates for galactosides may reflect participation by the axial acetyl group at position-4 (Rosenfeld and Ballou, 19 74b). The latter observation should be kept in mind when applying the acetolysis reaction to galactomannans. Selective acetolysis of yeast mannan is usually done in a 10: 10: 1 (v/v/v) mixture o f acetic anhydride-acetic acid-concentrated sulphuric acid, at 40°C, for 2- 1 2 hours depending on the extent of degradation desired. Although the mannan may be treated directly with this reagent, better control is possible by first acetylating the polysaccharide in acetic anhydride and pyridine s o that it becomes soluble in the acetolysis mixture. If this is done, careful removal of

STRUCTURE A N D BIOSYNTHESIS

OF YEAST M A N N A N

103

the pyridine is important so that neutralization of the acid catalyst does not occur. Higher temperatures increase the rate of acetolysis, but they also enhance the formation of sulpho-acetic acid (Jeffery and Satchell, 1962), a side reaction that decreases the concentration of the acid catalyst. Other acids, such as perchloric acid, catalyse acetolysis, but much of the specificity is lost. Under appropriate conditions, oligosaccharide fragments can be obtained from Succh. cereuisiue that retain one or two intact 1+6 linkages (Stewart et al., 1968), and this procedure has been used in a “nearest neighbour” analysis of the side chains of the mannan (Rosenfeld, 1974; Rosenfeld and Ballou, 1975) which suggests that the side chains may not be randomly distributed in the molecule (See Section 111-A-7,p.

123). E.

ENZYMIC DEGRADATION

This section deals only with those enzymes that have been applied t o structural analysis of mannans. Jack-bean a-D-mannosidase (Li, 1967) and the a- and 0-D-mannosidases from bormelain (Li and Lee, 1972) have had limited use in the degradation of yeast mannans (Nakajima and Ballou, 1974b). Somewhat more useful has been a bacterial exo-a-mannanase (Jones and Ballou, 1968) that was obtained from the cultural filtrate of a soil bacterium selected for the ability t o grow on baker’s yeast mannan as the carbon source. The partially purified enzyme hydrolyses manno-oligosaccharides with c~1+2,a1*3 and al+6 linkages, although the rate is slower with the latter type (Jones and Ballou, 1969a). Consequently, most of the side chains can be removed from Succh. cerevisiue mannan by the action of this enzyme, leaving the backbone essentially intact (Jones and Ballou, 1969b). Side chains substituted by phosphate (Thieme and Ballou, 1971) or other sugars such as N-acetyl-D -glucosamine (Raschke and Ballou, 1972) are not attacked by the exo-a-mannanase. However, the carbohydrate can be removed completely by this enzyme from a mannan-protein complex isolated from a mutant of Sacch. cereuisiue that lacks phosphate in its mannan (Smith and Ballou, 1974a). The combined use of the exoa-mannanase and the acetolysis reaction provides a powerful tool for structural analysis of mannans (Ballou, 1974). The nature of the acetolysis products before and

104

C. EALLOU

after exo-a-mannanase digestion of the mannan reveals directly the presence of mannanase-resistant fra
Lee and Ballou (1965) demonstrated the usefulness o f proton magnetic resonance spectroscopy for determination of the anomeric

S T R U C T U R E A N D BIOSYNTHESIS OF YEAST MANNAN

105

configurations of oligosaccharides obtained from mannan by acetolysis. This is facilitated by the unique chemical shifts of the protons on the anomeric carbon, and by the fact that the chemical shifts are also sensitive to the linkage between the sugars (Van der Veen, 1963). Gorin and Spencer (1970) made an extensive investigation of intact mannans by proton magnetic resonance spectroscopy and have concluded that the patterns have utility for the taxonomy of yeasts. All of the a-linked mannose units in mannan give anomeric proton resonance sicgnals that appear downfield from the water peak in deuterium oxide solution, and these can be assicgned according to whether the sugars are linked 1+2, 1+3 or 1+6. The method is particularly useful for distin
Rabbit antisera obtained by injection of whole heat-killed yeast cells contains antibodies directed against various features of the mannan molecule (Suzuki et al., 1968; Ballou, 1970). The most common determinant is the terminal 1+3-linked CY-D-mannopyranosyl unit and, for some reason, this structure seems more immunogenic than the 1-Q-linked unit. The (Y-D -mannopyranosylphosphate group in the mannan of some Saccharomyces strains is another important immunogenic group (Raschke and Ballou, 1971), but the al+3-linked mannobiosylphosphate unit in Sacch. cerevisiue S288C mannan does not seem to be differentiated immunochemically from any other terminal &l-+3-linked mannobiose unit

106

C.B A L L O U

(Rosenfeld, 1974). Antiserum against Kluyv. Luctis possesses antibody specificities directed against the side chains terminated by a 1 - + 3 - -mannosy1 ~ units and also against the side chains substituted by a1-+2-linked N-acetyl-D -glucosamine units (Raschke and Ballou, 1972). Antiserum obtained by injection of Sacch. ccreuisiue that lacks terminal a1’3-linked mannose in the mannan is specific for the terminal ~ ~ 1 + 2 - l i n k e mannose, d whereas that prepared against a mutant that does not add side chains t o the mannan backbone is specific for the unsubstituted al’6-linked mannose chain (Raschke et uL., 1973; Table 1). TABLE 1. Agents Employed for Detection of Different Mannan Structures Mannan structural unit

Detection agent

I

@Man(1+3)aMan( 1+2)aMan( 1’2)Man

1

I

aMan( l+P)aMan( 1’2)Man

I

I &Man(1+6)aMan( 1’6)Man I I aMan( l+Z)aMan( 1’2)Man I I

a( 1+3)Mannosyl-specific antibody

a(1’2)Mannosyl-specific antibody

a(1+6)Mannosyl-specific antibody

dvlannosylphosphate-specific antibody

aMan-+P

I

I

aMan+P or aMan( 1+3)aMan+P

Alcian-blue dye binding

From these results, it is clear that antisera of various specificities can be prepared with relative ease, and that the specificity of any one serum can be enhanced by adsorption with celIs of other strains to remove cross-reacting antibodies. Such antisera can be used t o identify new or known determinants on the mannan of new yeast strains and even to detect cross-reacting determinants on animal cells (Sarkar, 1974). In many examples, the precipitin reaction between isolated mannan and the antiserum made against the intact yeast cell can be inhibited completely by isolated mannan fragments, thus allowing identification of all of the immunochemical determinants in the mannan. However, some mannans possess an acid-labile determinant that is not reco‘gnizable in any of the products from the selectively degraded polymer (Yen and Ballou, 1974a).

STRUCTURE AND BIOSYNTHESIS OF YEAST MANNAN

107

One of the most valuable applications of immunochemistry is in the isolation of yeast mutants with altered mannan structure (Raschke et a[., 1973). Using a specific antiserum directed against the wild-type mannan, one can agglutinate and remove the unchanged cells in a mutagenized culture and enrich it for cells in which the surface determinants have been altered in such a way that they no longer react with the antiserum. From such a mixture of mutagenized cells, individual clones can be isolated and then analysed to determine the nature of the structural change in the mannan. By this approach, it is potentially feasible to obtain a series of mutants related to each other by single alterations of mannan structure owing to the stepwise loss of biosynthetic enzyme activities (Ballou et al., 1973). Such mutants are useful for studies of mannan biosynthesis, but they can also reveal new features of mannan structure as outer layers of the polysaccharide are stripped away (Nakajima and Ballou, 1 974b). 111. Detailed Structures of Specific Yeast Mannans A. SACCHAROMYCES CEREVISIAE

1. General Structures of the Outer Chain Methylation analysis of baker’s-yeast mannan established the highly branched nature of this polysaccharide and the presence and ratios of 1+2, 1+3 and 1+6 linkages, as well as the linkages in the branchpoints (Haworth et al., 1941). The composition of the acetolysis fragments revealed that the molecule was composed of D -mannose, a-D-mannosyl-(1-+2)-D -mannose, a-D -mannosyl-(1+2)a - D -mannosyl-(1+2)-D -mannose, and a - D -mannosyl-(1+3)-0(-D mannosyl-( 1+2)-a-D -mannosyl-(1+2)-D -mannose, all somehow interconnected by 1+6 linkages (Lee and Ballou, 1965). The way in which this occurred and the nature of the backbone structure of this mannan were established by exo-a-mannanase digestion that removed the side chains and left the backbone intact, analysis o f which (Jones and Ballou, 1968). revealed it to be exclusively 1’6-linked The proton magnetic resonance spectra, the optical rotation and the susceptibility to digestion by exo-a-mannanase support the assignment of the a configuration to most or all of the mannose units.

108

C.BALLOU

Baker’s-yeast mannan and mannan from Succh. cereviskze strain S288C can be separated by column chromatography on DEAESephadex into fractions of low and high phosphate content (Thieme and Ballou, 1971). By titration, the phosphate was shown to be diesterified and, on mild acid hydrolysis, it is converted t o a mono-ester form concomitant with the release of an equimolar amount of mannose and mannobiose in variable ratios (Thieme and Ballou, 1971; Colonna and Lampen, 1974a). The mannobiose has the structure a-D-mannopyranosyl-( 1 - q - D -mannose (Rosenfeld and Ballou, 1974a). These results indicate that one ligand to the diester phosphate group is mannose or mannobiose and, from investigation of the resultant mono-ester phosphomannan, the other position of attachment has been determined (Rosenfeld and Ballou, 1974a). Partial acetolysis of the mannan yields, in addition t o the neutral oligosaccharides, mainly one phosphorylated fragment, a mannotetraose phosphate. By a series of degradative reactions, the phosphate was shown t o be attached to the mannose unit next to the one at the reducing end of the fragment. The exact position of attachment on that mannose unit was established by a selective degradation, namely periodate oxidation followed by base-catalysed elimination (Cawley et al., 1972; Rosenfeld, 1974) that caused release of most of the phosphate as inorganic phosphate. This would occur only if the phosphate had been attached to position-6 of the mannose unit. Other investigators had previously demonstrated the production of mannose 6-phosphate by total acid hydrolysis of baker’s-yeast mannan (Mill, 1966), although the possibility of phosphate migration tempers the significance of this observation. The attachment of phosphate to position-6 of mannose in Kloeckera brevis mannan has been established by other means (Thieme and Ballou, 1971). From these studies, one may conclude that baker’s-yeast mannan, as well as that in Sacch. cereuisiae strains S288C and its derivative X2180, contain some mannotetraose side chains with mannobiosylphosphate groups esterified to the mannose unit linked to the backbone (Fig. 3b). The mannans of some other strains, such as Sacch. cerevisiae A364A, differ in that mild acid hydrolysis yields only mannose, not mannobiose, and that acetolysis of the mannan yields no fragment larger than the trisaccharide (Antalis et al., 1973). Chemical and immunochemical analysis of this mannan revealed that

(b) Saccharomyces cerevisiae X2180

(a) Kloeckera brevis 6 6

1

6

-aM-aM--+@M P-aM TI

aM

t: t: aM

1

6

t:

6

1 6 1 --+&I-

6

P-CZM

OLM

1

6

1

6

1

t: i: t: i: aMt: ti t! t: t:

aM

aM

aM

aM aM

aM

aM

aM

6

1

6

1

aM

aM

t:

t; t;

aM

@M

aM

(c) Saccharomyces italicus 6

1

tl

aM

-aM-aM-aM+aM

6

i: t: t; 1: aM t: t; 1; aM

t:

7

1

6

1

-~M+(YM+cYM*~M-CYM-~M--~

7:

6

1

f:

aM

ffl

?I

C

0

CYM

CrM

-I

C

3

rn

B

CYM

z 0

6

1

6

1

-aM-aM-

i: 1: aM t:

&I

6

E

1

t: t:

(d) Kluyveromyces lactis --+

6

1 6 1 a M --+ &I

t:

CYM

&I 1 2

aGNAc-aM

1:

1:

CYM

6

aM

at t: aM

CYM

1

6

--j

-

1 6 1 a M --+ (YM

t:

aM

aM

1:

CYM

FIG. 3. Representative structures illustrating the outer chain portions of four yeast mannans with linear &1+6 backbones. All are assumed to have the same general overall structure established for the mannan from Saccharomyces cerevisiae X2180 (Fig. 2). Saccharomyces cerevisiae A364A mannan has a structure very similar t o the polymer in Kloeckera brevis.

0,

<

2 -I

I rn

2 ffl

%

<

rn

E;-i H B

2

2

B 2

-1

0 (D

110

C. BALLOU

it lacked terminal 1-+3-linked mannose units (Fig. 3a), and genetic analysis demonstrated that these two structural changes were under the controI of a single gene designated the mnn 1 locus. Strain A364A and several brewing strains from the Guinness Laboratory at Park Royal, Dublin, Eire (Cawley and Ballou, 1972) have the mannan chemotype of the mnnl mutant of baker’s yeast and presumably lack the enzyme activity for making the 1+3 linkage.

2. Fragments Released b y Mild Alkaline Degradation The release of mannose and small oligosaccharides on treatment of Sacch. cerevisiae bulk mannan with 0.1 N-NaOH at 20-40°C for 24 hours has been reported by Sentandreu and Northcote (1968) who found mannose and a mixture of disaccharides, by Thieme and Ballou (1971) who found a mixture of di-, tri- and tetrasaccharides, and by Cawley et al. (1972) who isolated mono-, di-, tri- and possibly tetrasaccharide fragments. The last result has been confirmed (Colonna and Lampen, 1974b), and the fraction of the mannan released in this form was about 10% of the total mannose. That the oligosaccharides were derived from 0-glycosidic linkage to serine and threonine in the mannan-protein complex was indicated by an increase in absorbance of the mannan solution at 240 nm, owing to the formation of dehydrohydroxyamino acids, and by the decrease in serine and threonine contents in the recovered mannan-protein residue as revealed by amino-acid analysis. The exact structures of the oligosaccharides from Sacch. cerevisiae X2 180 mannan have been established (Nakdjima and Ballou, 1974a) and are identical with those of the di-, tri- and tetrasaccharide fragments produced by acetolysis of the mannan outer chain (see Section 111-A-1, p. 109). Although some of the side chains of the mannan outer chain are phosphorylated in Sacch. cerevisiae mannan, it has not yet been demonstrated that the serine- and threonine-linked oligosaccharides carry phosphate substituents. Some o f the inconsistency in the literature concerning the nature of the fragments obtained from Sacch. cerevisiae mannan by alkaline elimination or by partial acetolysis is now readily explainable by the fact that different strains have been analysed and that there exists polymorphism of structure from one strain to another (Ballou and Raschke, 1974). The most obvious structural variation is that dealing with the terminal 1-+3-linkedmannose unit, a structure found in

STRUCTURE A N D BIOSYNTHESIS OF YEAST M A N N A N

111

baker’s yeast and related mannans but absent from other strains including one studied by Cawley e t al. (1972). The enzymic activity for adding this unit is required for synthesis of the tetrasaccharide side chain and the mannobiosyl phosphate units. Apparently this activity is missing in many wild-type strains of Sacch. cereuisiae. As will be discussed later in this review, there are also several Saccharom y c e s species, all interfertile with Sacch. cereuisiae, that possess a side chain one mannose unit longer than those found in the latter species. Thus, a five-peak acetolysis pattern is obtained from Saccharo m y c e s cheualieri and related mannans (Ballou et al., 1974; Fig. 3c). 3. Isolation and Characterization of Mannan M u t a n t s of Saccharomyces cerevisiae The study o f Salmonella mutants with altered lipopolysaccharide has played an important role in elucidating the structure and mode of biosynthesis of this complex cell-surface macromolecule (Wright and Kanegasaki, 1971). Similar approaches can be used for such investigations on yeast mannans, and this section summarizes some attempts in that direction. Following ethyl methane sulphonate mutagenesis of haploid cultures of Sacch. cereuisiae X2180 (a or CY mating type), four nonallelic mutants with altered mannan structure (Raschke e t al., 1973; Ballou et al., 1973) have been isolated which show the phenotypic properties indicated in Table 2. These differences are rationalized by the structures of the mannans shown in Fig. 4, which were established by chemical methods. The mnnl mutant lacks the terminal al+3-linked mannose units, and makes a mannan similar to that o f Sacch. cereuisiae A364A, suggesting that it and strain A364A lack the a1-+3-mannosyltransferase activity needed t o add this sugar unit to the mannan side chains. A second mutant, TABLE 2. Phenotypic Expressions of Mannan Mutants of Saccharomyces cereuisiae. From Raschke e t ul. (1973) and Ballou et al. (1973). Structural probe

a(1’3)Mannosyl

antiserum l+2)Mannosyl antiserum a(lP6)Mannosyl antiserum &Mannosy1phosphate antiserum Alcian blue dye binding (u(

Wild-type X2180-1A

+ +

mnnl -

+

-

-

+

+

+

Mutants mnn2 mnn3 -

+ -

-

+

mnn4

+ -

-

-

-

-

-

C. BALLOU

112

-M-+M-M-+M+M-M~~

T2 t: 1: t: t: t: t: t: I: t: t: t: 6

1

P-M

M

M

M

M

M

M

M

tl

M

M

M M M Saccharomyces cerevisiae X2180 'wild type'

mnnl [a1+3-mannosyltransferase-]

P-M Tl M

6

M

r:

t:

M

mnnl, mnnl [Q- 1+3-mannosyltransferaseand mannosyl phosphate transferase-]

M

M

M

t:

M

M

mnn2 [Q- 1+2-mannosy1transferase I-]

-+

-111

M-M-

mnn4 [mannosyl phosphate transferase-] --+M---+PyI-M-M-+M-

mnn3 [~-1+2-mannosyltransferase 11-1 -M-Mt 2 I1

M

t: t; T i t:

M t2

I1

M

M

t3 I1

M

M t 2

M

I1

M

73 I1

M

FIG. 4. Illustrations of the outer-chain mannan structures for the various Saccharomyces cerevisiae X2 180 mannan mutants. The assumed enzymic defects that lead to the structural changes are given in brackets, although the lesions have not yet been shown to result from structure-gene mutations. All backbone structures have the a1+6 linkage.

mnn2, makes a mannan that lacks all side chains in the outer chain, and it is presumed t o have a defective 01 l+2-mannosyltransferase-I activity. The mannan from this mutant is predominately a linear al'6-linked polymer; but, as described in the next section, it does still possess a branched inner-core structure (Nakajima and Ballou, 197413).

S T R U C T U R E A N D BIOSYNTHESIS OF YEAST MANNAN

113

A second 1+2 linkage is found in the side chains of Sacch. cereuisiae mannan, and the mnn3 locus regulates the activity of the transferase that adds this mannose unit. This is designated the al-+2-mannosyltransferase-II. Finally, a fourth locus (mnn4) has been defined by the isolation o f a mutant which lacks the mannosyl phosphate transferase activity that is involved in adding this unit to the mannan side chains. This strain was first isolated by mutagenesis of the mnnl mutants, and thus was a double mutant. By crossing this double mutant with the wild-type strain X2180 and dissecting the sporulated diploid, the mnn4 single mutant was obtained as a recombinant. As illustrated in the structures in Fig. 4 and supported by the properties listed in Table 2, the mnn2 and mnn3 mutants do not possess the mannosyl phosphate unit in their mannans because they fail to make the acceptor side chain to which this group is attached. Two of these mannan mutants are centromere-linked and have been mapped on two different chromosomes. The mnnl gene is on chromosome V between the ura3 locus and the centromere (Antalis et al., 1973), whereas the mnn2 gene is on chromosome I1 between the gal locus and the centromere (D. L. Ballou, unpublished results). As yet, it has not been established that these loci are the structural genes for the indicated transferase activities. In fact, the pleiotropic nature of the mnnl mutation suggests that it might have a regulatory function. The evidence has already been cited that this mutation alters the mannobiosyl phosphate unit (Rosenfeld and Ballou, 1974a) as well as the tetrasaccharide side chains in the mannan outer chain (Raschke e t al., 1973). Interestingly, the mutation also alters the tetrasaccharide chains attached to serine and threonine (Nakajima and Ballou, 1974a) as well as the tetrasaccharide units in the inner core (T. Nakajima and C. E. Ballou, unpublished results; see p. 117). The first three of these effects are illustrated in Fig. 5 which compares the products of mild-acid hydrolysis, alkaline 0-elimination, and partial acetolysis for the mannans from Sacch. cereuisiae X2180 wild-type and the corresponding mnnl mutant. In each, the absence of the al+3-mannosyItransferase activity is apparent in the changed gel-filtration pattern. The terminal al-+3-linked mannose unit gives a characteristic signal in the proton magnetic resonance spectrum (Lee and Ballou, 1965), and Spencer et al. (1971) used this property in the first

C. BALLOU

114

MZ

Acetolysis fingerprints

M i l d acid hydrolysis products

1

M

I\

:\

n

;:

1

, , I

I I I

I

;

I

(

I

'

I

I

I

I I

(

t

'

' I,

I

I

( I

#I

I I

M2

I 1

PS

4 80

120 Fraction number

160

200

STRUCTURE AND BIOSYNTHESIS OF YEAST MANNAN

115

demonstration that the presence or absence of this structural unit in Sacch. cerevisiae mannan was under the control of a single gene. The genetic control of alcian blue dye-binding, a property also regulated by the mnn4 locus, has been studied by Friis and Ottolenghi (1970) with Sacch. cerevisiae A1640B and Sacch. diastaticus S. Sp. IA. The dye-binding property was regulated by a gene linked to the ural locus, which would place it on chromosome XI of the Sacch. cerevisiae genome. Recently the mnn4 locus has been mapped on this same chromosome (D. L. Ballou, unpublished results), but it was shown t o segregate independently from the Sacch. diastaticus dyebinding locus. Thus, this chromosome carries two genes that are somehow involved in the addition of mannosyl phosphate groups t o the mannan. The assignment of the four different mannan mutations to different genes is based on the fact that they segregate independently, and all, except the mnn4 mutant which is dominant, complement each other so that the diploid cell produces a wild-type mannan (Raschke et al., 1973). The diploids from a cross of the m n n l , m n n 2 and mnn3 mutants back t o the wild-type have the wild-type phenotype, indicating that the mutations are recessive. But, on dissection of the sporulated diploids, the mutant and wild-type phenotype are found t o segregate 2+:2-in the haploidspore clones. The diploid obtained by a cross of the mnnl mutant with the natural isolate of strain A364A has the mutant phenotype, which suggests that the same locus is involved in both strains. 4. Phenotypes of Mannan Mutants The availability of mannan mutants allows one t o ask whether these alterations have any effect on the physiology of the cell. Among the properties that might be affected are: (1) the activity, stability, o r release of cell-wall mannan-containing enzymes; (2) altered cell viability, osmotic fragility, or sensitivity t o glucanases; FIG. 5. Comparison by gel filtration on Bio-Gel P2 columns of the partid degradation products of the wild-type Saccharomyces cerevisiae X2180 mannan (solid lines) and the mnnl mutant mannan made by a strain that lacks the ~1+3-mannosyItransferaseactivity (dashed lines). M, M z , M 3 and M4 stand for mannose, mannobiose, mannotriose and mannotetraose, respectively; M3P and M,P for the corresponding phosphate esters, and PS for the polysaccharide residue that resists degradation to small products. In each experiment, the results confirm that the fragments lack the terminal 1+3-linked mannose unit.

116

C. BALLOU

(3) defective cell division or sporulation, or altered morphology; and (4) altered mating efficiency, sexual agglutination, or response to a-factor. In the first category, we have no evidence that these mutations alter the activity or stability of external invertase (Smith and Ballou, 1974a). In fact, we have demonstrated that all of the mannan can be removed from the mannan-invertase without altering the activity or stability of the enzyme when in the presence of bovine serum albumin. There is still the question of whether the mannan has a non-specific stabilizing action that is replaced by adding serum albumin t o the solution (Arnold, 1969) but, at physiological temperatures and in the presence of other cellular constituents, the mannan does not appear to have an important role in stabilizing the protein moiety of the complex. Others have reported similar findings (Tarentino et al., 1974). On the other hand, it was found that the invertase of the mannan mutants is more easily released from the wall on treatment of the cells with dithiothreitol (Smith and Ballou, 1974b). Surprisingly the mnnl mutant shows a greater change than the mnn2 mutant, even though the latter has a more dramatic alteration in its mannan structure. Possibly the unbranched chains of this mannan are longer or pack more tightly because of their uniformity, and in such ways compensate for the loss of the side chains so that the mannan-invertase is still reasonably well immobilized in the matrix of the wall. With respect t o the second group of changes, n o decrease in cell viability or increase in osmotic fragility was observed, but the cells clearly are altered in sensitivity to glucanases such as Glusulase; P. N. Lipke, unpublished results). Again the result is not quite what one might expect, with the mnnl and mnn4 mutants showing enhanced sensitivity whereas the mnn2 mutant is similar t o the wild-type cells. All of the mutations except mnn4 are recessive and, in the haploid cells, they have no obvious effect on morphology (Hawkins, 1973) or on mitosis. When the homozygous diploids were tested for the ability to sporulate, only the mnnl/mnnl strain showed a decreased tendency to form spores (D. L. Ballou, unpublished results). The haploid mutants all respond normally to a-factor (P. N. Lipke, unpublished results) and show the usual sexual agglutination reaction (D. N. Radin, unpublished results) that occurs on shaking together a mixed culture of u and (X cells (Sena et al., 1973).

STRUCTURE A N D BIOSYNTHESIS OF YEAST M A N N A N

117

The general conclusion from studies of these four mannan mutations is that they have no dramatic effect on the cell, with the possible exception of the alteration at the mnnl locus. In retrospect, this is not too surprising because these mutations lead t o gross alterations of structure only in the outer chain and on one of the oligosaccharides linked to serine and threonine. Only the mnnl mutation appears to affect the structure of the inner core, and this may explain the diminished tendency of the homozygous diploid to sporulate. Apparently, the outer chains of the mannan-protein complexes have been adapted in yeasts mainly to anchor them in the cell wall. However, the inner core and some of the serine- and threonine-linked units may have more fundamental roles in regulating the biosynthesis and translocation of the mannan-proteins. Once mutants of the inner core are obtained it will be possible to test such hypotheses. 5 . Characterization of the Mannan Inner Core of

Saccharomyces cerevisiae The discovery of the inner-core structure of yeast mannans occurred by chance during an investigation of the mnn2 mutant mannan. The evidence that this mannan has an unsubstituted polysaccharide chain was based in part on the acetolysis pattern that revealed mainly mannose. However, it was noted that small amounts of di-, tri- and tetrasaccharides were stilI present in the acetolysis product, and this was attributed to a postulated “leakiness” of the mutation (Raschke et al., 1973). It is now recognized that these fragments came from a portion of the mannan molecule that retained a branched structure even though the majority of the molecule was an unsubstituted al-+6-linked chain. This became clear when the mannan prepared from the mnn2 mutant was used for isolation of a soil organism by enrichment culture, the aim being to find an al+6-mannanase for use in other structural studies. A bacterial strain was obtained that produced two mannan-degrading enzymes, namely an endo-al+6-mannanase and an endo-P-N-acetylglucosaminidase (Nakajima and Ballou, 197413). The products that accumulated in the growth medium from the action of these enzymes on the mannan are shown in the gel-filtration pattern in Fig. 6 , and the mechanism by which this presumably resulted is outlined in Fig. 7.

C.BALLOU

50

I00

150

200

Fraction number

FIG. 6. Fractionation by gel filtration on a Bio-Gel P4 column of the products from digestion of the mannan from Saccharomyces cereuisiae mnn2 mutant with The FIII a bacterial endo~l-+6-mannanaseand endo-fi-N-acetylglucosaminidase. component is a mixture of homologous al+6-manno-oligosaccharides with degree of polymerization 1 to 6; the FII fraction is a branched oligosaccharide with about 12 mannose units and one N-acetylglucosamine unit representing the inner core fragment; and FI is an endo-0-N-acetylglucosaminidase-resistant glycopeptide fragment containing FII-like units still linked to the peptide. The elution position of mannose (Man) and the void volume (Vo) are indicated.

With the discovery and characterization of the inner-core structure, a more detailed description of Sacch. cereuisiae mannan can now be presented (Fig. 8). The carbohydrate component of the molecule is differentiated into three parts, the outer chain, the inner core and the base-labile oligosaccharides. Some heterogeneity was observed in the inner-core structure, possibly because the work was done on the bulk cell-wall mannan. Analysis of a pure mannanprotein, such as invertase, may reveal a homogeneous moleculespecific inner-core structure. What is the significance of the mannan inner core? The most striking feature is the structural homology to glycopeptides that have

[M -+ M -+ M -+ M +M],+M

t i t t

M

M

M

+M

M

M

M

-+

M -+ GNAc -+ GNAc

-+

M TM v

M+P

M

+M

T T t t

t t t t t t t

M

+M

M

M

Ak

I

Saccharomy ces cerevisiae X2 180-1A mannan

Mutagenesis with ethylmethane sulphonate

M

M

Y

n

M

C

I

V

0 -I

I

[M + M + M + M +M],+M

-+

M -+ M + M -+ M + GNAc+ GNAc + Asn

t r t t

I

t Mt

M

M - + M - + M - + M + M-+GNAc

Saccharomyces cerevisiae mannan-protein linkage fragment (FII)

< rn

R

-I

H

FIG. 7. Reaction diagram illustrating the process by which the inner-core linkage fragment, isolated as described in Figure 6, was produced. The abbreviations stand for mannose (M), N-acetylglucosamine (GNAc), phosphate (P) and asparagine (Asn).

2

1

6

1

[M-M-M-M-

t:

M

6

1

6

1

6

t: t: t: t: t: t: t: ti t:

M

M

M-P

M

M

M

M

M

M

tl

M Outer chain

t: ti t: T:

6 1 6 1 -+ M ---+M

1

M -In-

M

6

M

M

1

6

1

M-

6

1

M

M

M

M

M

4

GNAc

1 4

GNAc

I

Asn

t; t: t; M

Inner core

? W

D

Ser (Thr) 1

2

1

2

1

2

1

2

M-M-M1

3

M-M-M-M-

Base-labile oligosaccharides

FIG. 8. Detailed structure of the mannan from Saccharomyces cereuisiae showing the base-labile oligosaccharides attached to hydroxyamino acids, the inner core and the outer chain. All anomeric linkages have the a-configuration except for the trisaccharide unit: pMan( 1+4)pGNAc(l-%I)flGNAc linked to asparagine. The configuration of the mannose attached directly to m i n e and threonine has not been determined.

r

6 C

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121

been obtained from mammalian glycoproteins (Hickman et al., 1972), and this suggests that similar roles may be served in both kinds of molecule. A prevalent hypothesis on the role of the carbohydrate portion of mammalian glycoproteins is that they function in the translocation, secretion and “homing” of the molecule (Eyiar, 1965; Ashwell and Morreli, 1974). Present evidence indicates that mannan-proteins are made in the cell and are translocated through the plasma membrane into the extracellular space (KoSinovi et al., 1974). Whether the mannan molecule undergoes modification during its movement across this membrane is not known, but it would be reasonable for the elaboration of the outer chain to occur at that time. It has been demonstrated by a study of mutants altered in the outer chain that translocation of the mannanprotein is not seriously affected. Thus, the inner core may play the more important role during the earlier steps in mannan-protein synthesis, thereby having a function analogous to that proposed for the carbohydrate chains of mammalian glycoproteins. In fact, one might speculate that the yeast mannan-protein chain is an evolutionary variation on an earlier glycoprotein molecule in which the carbohydrate portion that functions during synthesis and translocation has been further elaborated to serve the second function of immobilizing the external glycoproteins in the cell waII of this unicellular organism. 6. Macromolecular Structure of the Mannan of Saccharomyces cerevisiae One of the few careful studies on the physical properties of an undenatured mannan-protein has been carried out by Lampen and his coworkers (Neumann and Lampen, 1967; Gasc6n and Lampen, 1968; Gasc6n et al., 1968) on the invertases from Saccharomyces FH4C. Reportedly, this strain came from a hybrid of Saccharomyces chevalieri, Saccharomyces italicus, and Saccharomyces carlsbergensis (Colonna and Lampen, 1974a), so that the mannan is not strictly analogous to that of Sacch. cerevisiue X2180 (Ballou e t al., 1974). However, the acetolysis pattern of the bulk mannan of strain FH4C is very similar to that of Succh. cerevisiue X2180 mannan. The external invertase was obtained as a fairly homogeneous mannanprotein with 50% carbohydrate and a molecular weight of 270,000. The carbohydrate is essentially all in the form of polymannose chains

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C. BALLOU

attached through di-N-acetylchitobiose t o asparagine, and these can be removed by the action of an endo-0-N-acetylglucosaminidase without altering the activity of the invertase (Tarentino et al., 1974). About 20 oligosaccharide chains of two size classes, one with 54 and the other with 26 mannose units per mole of glucosamine, were released. Thus, the protein part, which would be composed of about 1300 amino-acid residues probably in more than one polypeptide chain held together by disulphide bonds, carried only about 20 carbohydrate chains, or one for every 65 amino-acid residues. Although it is possible that all of these polysaccharide chains are concentrated in a short portion of the protein, this kind of mannanprotein is quite different from other forms that possess serine- and threonine-linked oligosaccharides (Greiling et al., 1969; Yen and Ballou, 1974b). The bulk mannan, isolated from Sacch. cerevisiae X2180 by extraction with hot neutral citrate buffer, precipitation with Cetavlon (Lloyd, 1970), and fractionation by DEAE-Sephadex column chromatography, is a fairly homogeneous preparation composed of 90% carbohydrate and 10% protein, with an average molecular weight of about 133,000 (Nakajima and Ballou, 1974a). Treatment of this mannan with 0.1 N-NaOH for 24 hours releases about 15% of the carbohydrate as manno-oligosaccharides with an average size of a disaccharide. From these results one can calculate that there are about 40 oligosaccharide fragments linked to serine and threonine in the protein part of about 130 amino-acid residues. If the long polysaccharide chains have an average of 100 mannose residues (Jones and Ballou, 1968), there would be about six such chains. Thus, a total of 46 carbohydrate chains would have t o be attached t o a rather short polypeptide chain, and would require a protein with at least 30% serine and threonine, a not uncommon observation among yeast mannan-proteins (Thieme and Ballou, 1972). This kind of bulk mannan may represent mainly a structural component of the wall in which the other mannan-proteins are embedded. The mannan of Succh. cerevisiae X2180 is dramatically decreased in size by treatment with dilute alkali (0.1 N-NaOH) and by Pronase digestion, both giving material with a mass of about 40,000 daltons (Nakajima and Ballou, 1974a). The large amount of serine- and threonine-linked oligosaccharides must impair the action of Pronase,

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123

so that only a few positions in the chain are susceptible to attack. The size decrease following alkali treatment cannot be due solely to elimination o f the oligosaccharides because they constitute but 15% of the carbohydrate in the molecule. Therefore, some other crosslinking structure must be broken. The role of disulphide bonds in cross-linking yeast mannanproteins is not well understood although much has been written on the subject (see Beran, 1968). Neumann and Lampen (1967) found that perhaps two of the five half-cystine residues in Saccharomyces FH4C external invertase may exist as a disulphide bridge. Partially purified external invertase from Sacch. cerevisiae mnnl, mnn4 double mutant can be freed of carbohydrate by digestion with an exo-cx-mannanase, a step that does not appreciably change the position of elution from a Sephadex G-200 column. However, subsequent treatment with dithiothreitol produced an invertase activity that was included on the column to a greater extent, indicative of a decrease in size of the enzyme protein (Smith and Ballou, 1974b). This suggests that the protein part of the invertase is made of subunits that are cross-linked by disulphide bonds. Dithiothreitol treatment had a slight effect on the gel-filtration property of the intact enzyme, but the invertase activity was precipitated by antimannan serum before or after treatment, indicating that the dithiothreitol did not release from the complex a carbohydrate-free protein with invertase activity. Thus, if the external invertase is a multi-subunit mannan-protein with catalytic and regulatory components, as suggested by some of the studies of Lampen already referred to, the catalytic subunit of this external invertase must also be a mannan-protein even though the internal invertase of Saccharomyces FH4C lacks carbohydrate.

7. Do Mannan Side Chains Have a Sequence? Yeast mannan acetolysis patterns are strain-specific with respect both to the number of fragments and their ratios (Thieme and Ballou, 1970; Ballou, 1974), which suggests that a precise regulation is exerted over biosynthesis of the polysaccharide. This could occur in various ways. The side chains could be preformed on a carrier and then polymerized in a specific order with formation of the backbone linkages. Alternatively, the mannose units could be added one at a

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C. BALLOU

time, but the particular transfereases could recognize a portion of the chain preceding the point of new synthesis and make the appropriate addition. A third possibility is that the side chains may be ordered randomly, but that the activity of each transferase is closely regulated so that only a certain amount of each side chain is made. To select from these possibilities, it would be helpful to know whether the mannan side chains occur randomly o r in some ordered sequence. Two approaches to sequencing a mannan seem feasible. One is to release the polysaccharide at the glucosamine linkage to the protein, label the end of the molecule by reduction with tritiated sodium borohydride, then carry out a partial acetolysis and isolate the fragments of different sizes with intact 1+6 linkages. By subjecting each such labelled intermediate t o further acetolysis and then characterizing the resulting fragments, it might be possible to fit together longer and longer segments, thus establishing an overlapping sequence. This approach loses its attraction with the discovery of an inner-core structure of Succh. cereuisiue mannan and the demonstration of its heterogeneity (Nakajima and Ballou, 19 74b), although the idea would be worth pursuing i f one could obtain a sufficient amount of a homogeneous mannan-protein. An alternative procedure for obtaining evidence for a sequence might be called a “nearest neighbour” analysis (Rosenfeld, 1974; Rosenfeld and Ballou, 1975). For this study, a mannan from Succh. cereuisiue with a relatively simple structure was selected, namely one that gave only mannose, mannobiose and mannotriose in ratios near 1 : Z : l on acetolysis for 1 2 hours at 40°C in the 1 O : l O : l mixture of acetic anhydride-acetic acid-concentrated sulphuric acid (Fig. 9). The same mannan, when acetolysed for only one hour, gave the pattern shown in Figure 9. Assuming that the mannan had a simple order and sequence of one mannose unit, two mannobiose units, and one mannotriose unit, there are three ways these could be arranged, namely: (a) -M+M2+M2+M3-, (b) -M+M2+M3+M2and (c) -M-+M3-+M2+M2-. Assuming also a random cleavage of linkages during acetolysis, it is clear that each sequence would give a different mixture of fragments with one intact 1+6 linkage. For example, sequence (b) would not give a tetrasaccharide fragment but it would give pentasaccharides of two types. Table 3 lists the possibilities for the different kinds of tetra- and pentasaccharides, the expected ratios for a random order, and the observed values. Two results are worthy of comment. FirstIy, the amount of pentasaccharide is much larger

STRUCTURE AND BIOSYNJHESIS OF YEAST MANNAN

125

M3

-

i 1

Fraction number

FIG. 9. Comparison by gel filtration on a Bio-Gel PZ column of the products froma 1-hour (-) and 12-hour (---) acetolysis of the mannan from Saccharomyces cerevisiae 4484-24D which has a structure similar to that in Fig. 3a. The oligosaccharides larger than mannotriose (M3) have one intact 1+6 linkage.

than the amount of tetrasaccharide; and, secondly, the two kinds of pentasaccharides differ significantly in amount. Although the results favour sequence (b), they are not completely consistent with it. Of course, it is possible that the acetolysis of such a mannan is not a random process and that some linkages are protected more than others by st eric hinder ance . TABLE 3. Predicted and Observed Molar Ratios of Tetra- and Pentasaccharide Fragments with One 61’ linkage Obtained by Acetolysis of the Mannan from Saccharomyces cerevisiae Sequence fiedicted SequenceA Sequence B SequenceC Random Observed

MpM2

M+M3

1 0 1

0 0 1

4

1 0.11

0.17

M3+M

1 0 0 1 0.03

Total Mz-+M3 M3-+Mz M4 2 0 2 6 0.31

1 1 0 2 1

0 1 1

2 0.22

Total M5

1 2 1 4

1.22

126

C. BALLOU

Although this study leaves many questions unresolved, it clearly suggests that the order of side chains in Sacch. cerevisiae mannan is not random. Other polysaccharides, such as the 0-antigen chain of Salmonella lipopolysaccharides and the capsular polysaccharide of Aero bacter aerogenes, have repeating sequences of three or four sugars, and it is easy to understand.the formation of such repeating sequences in terms of the mechanism of biosynthesis from a lipid-linked oligosaccharide intermediate (Nikaido and Hassid, 1971). Synthesis of a repeating sequence for mannan poses a slightly different problem because the units extend to form side chains rather than extend the backbone of the polysaccharide. There are several ways in which the specificity of glycosyltransferases might be channelled to yield such repeating sequences, and the answer should be attainable from a study of the relevant enzymic reactions in cell-free systems. A polymer with sequence (b) could have interesting properties, for the side chains would project to form a symmetrical and repetitive wave along the backbone. A consequence of this shape could be that two strands might fit together in a complementary way. Hydrogen bonding could stabilize such interchain associations and facilitate immobilization of mannan-enzymes in the structural mannan-protein matrix. In fact, symmetry is not an important feature of this model; rather it is the regular complementarity of the two strands that would determine the stability of their interaction. Because many different complementarity patterns could occur together in the sarne cell-wall mannan, the ability to detect unique patterns might depend on the analysis of homogeneous mannan-protein preparations. Although this idea is highly speculative, it does provide a role for the well established polymorphism of yeast mannan structures (Bdlou and Raschke, 1974). B. OTHER SACCHAROMYCES SPECIES

No other yeast mannan has been subjected to the detailed analysis described above for baker’s-yeast and the closely related Sacch. cerevisiae S288C, X2180 and its mutants. Therefore this section will deal only with a comparison of the structures of the mannan outer chains and with their immunochemical properties.

STRUCTURE A N D BIOSYNTHESIS OF YEAST MANNAN

127

1. Comparative Structural Features Gorin and Perlin (1956) reported the first acetolysis of a yeast mannan when they showed that Saccharomyces rouxii gave three products, namely D -mannose, a-D-mannosyl-(1+2)-D -mannose and a - D -mannosyl-( 1+2)-(X-D -mannosyl-(1+z)-D -mannose. From the methylation data it was clear that these products arose by acetolysis of 1+6 linkages, and characteristically missing was any fragment with a 1+3 linkage. It is now known that the outer chains of this mannan are analogous to those of the Sacch. cerevisiae X2180 mnnl mutant, although the presence of mannosyl phosphate substituents has not been established. A typical acetolysis-gel filtration pattern has been published (Sandula and VojtkovL-LepSikovi, 1974), and the same pattern is given by some brewing strains (Cawley and Ballou, 1972) and Sacch. cerevisiae A364A (Ballou and Raschke, 1974), the latter strains possessing the mannosyl phosphate group on some o f the side chains. The mannans of the above group of yeasts give acetolysis-stable fragments no longer than the trisaccharide. A second group, including Saccharomyces carlsbergensis and Saccharomyces italicus, give acetolysis patterns with side chains up to the pentasaccharide in size (Ballou e t al., 1974). The di-, tri- and tetrasaccharide fragments have the same structures as those fragments from Sacch. cerevisiae S288C, whereas the pentasaccharide is a derivative of the mannotetraose with one additional al+3-linked mannose unit (Fig. 3c, p. 109). These strains are all interfertile with Sacch. cerevisiae (van der Walt, 1970) and we assume that they possess an additional al+3-mannosyltransferase activity that is specific for adding the second 1+3linked mannose unit. From a cross of the homothallic Sacch. chevalieri with the heterothallic Sacch. cerevisiae X2180, a heterothallic recombinant haploid clone with the mannan chemotype of these species has been recovered (D. L. Ballou, unpublished observations). With this strain, it should be possible to study the interaction of this putative c~1+3-mannosyltransferase-II with the transferase activity regulated by the mnnl locus (Raschke et al., 1973). A still more complex acetolysis pattern has been reported for Saccharomyces rosei (Sandula and VojtkovA-Lepgikovi, 1974) which

128

C. BALLOU

shows a t least seven oligosaccharide peaks that are reminiscent of the patterns obtained with several Candida species (Kocourek and Ballou, 1969).

2. Zmmunochemical Characteristics From the above comparison of acetolysis patterns, it is clear that all of these mannans from Saccharomyces species have closely related structures and, in those instances in which the mannan backbone has been investigated, the al-+6-Iinked structure characteristic of Sacch. cerevisiae strains was found. Thus, one would expect considerable immunochemical cross-reactivity, as is observed. Heterologous precipitin reactions of Sacch. cerevisiae mannan with Sacch. chevalieri antiserum, or of Sacch. chevalieri mannan with Sacch. cereuisiae antiserum, occur to about the same extent and both are inhibited t o the same extent by the mannotetraose and mannopentaose acetolysis fragments (Ballou et al., 1974; iandula and VojtkovP-LepSikovi, 1974). Thus, there does not seem t o be an antibody population that discriminates between the mannotetraose and mannopentaose side chains even though they must have rather different shapes. On the other hand, the cross-reaction of Sacch. cerevisiae antiserum with Sacch. rouxii mannan is weak, and may be related t o terminal al+Z-linked mannose units or t o antibodies directed against a mannosyl phosphate determinant, although this was not investigated (Sandula and Vojtkovi-LepSikovi, 1974). C. KLUYVEROMYCES SPECIES

Ktuyveromyces lactis (syn. Saccharomyces lactis, Fabospora lactis) is the most carefully studied species of this genus (Raschke and Ballou, 19 72), although there is evidence that Kluyveromyces dobzhanskii and Kluyveromyces marxianis have similar structures (P. Hsiao and C. E. Ballou, unpublished data). The mannan from Kluyu. lactis has the outer chain structure shown in Fig. 3d (p. 109). It is closely analogous to that of Sacch. cereuisiae S288C except that it lacks phosphorylated side chains and instead possesses N-acetylglucosamine substituents on some of the mannotetraose units. The acetolysis patterns of Kluyu. dobzhanskii and Kluyv. marxianis are similar t o those of Kluyv. Zactis, and the largest fragment of these mannans also contains N-acetylglucosamine. The a-linked terminal N-acetylglucosamine is unusual among glycoproteins, and it is also

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found on the fragments released by mild alkaline degradation of Kluyu. lactis mannan. Rabbit antiserum prepared against Kluyu. lactis possesses two main antibody specificities, one directed against the terminal a1+3linked mannose of the mannotetraose chains, and one against the atl+2-linked N-acetylglucosamine unit (Raschke and Ballou, 1972). Both determinants appear to be about equally immunogenic, because the precipitin curve with intact mannan gives only about twice the amount of precipitate as the exo-at-mannanase-digested mannan from which all of the side chains are removed except those substituted by N-ace t ylglucosamine. Mannan mutants of Kluyu. lactis have been obtained (Smith et al., 1975) that are analogous to those described in Sacch. cereuisiae. One class, designated mnnl, makes a mannan that lacks both the mannotetraose units and the N-acetylglucosamine substituents. Genetic analysis has shown that this alteration is under the control of a single locus, and we conclude that it affects the al+3-mannosyltransferase that adds the terminal mannose unit to the side chains. Apparently, the absence of mannotetraose side chains prevents the N-acetylglucosamine transferase from acting. This assumption was confirmed by demonstrating the presence of a wild-type N-acetylglucosamine transferase activity in this mutant. A second class of mutants was obtained that lacked only the N-acetylglucosamine substituent in the outer chain. This mutation was designated as being at the mnn2 locus, and has been shown to involve an alteration in the at1+2-N-acetylglucosamine transferase activity. This mnn2 locus is not to be confused with the Sacch. cereuisiae mutation that affects the atl+2-mannosyltransferase-I activity. The accepted convention is to number the genetic loci in each organism in the order in which they are obtained (Mortimer and Hawthorne, 1969). D. HANSENULA SPECIES

Much of the work done on mannan from these yeasts has dealt with the exocellular phosphomannan (Slodki, 1963), and it is not clear what relation this mannan has to the cell-wall polysaccharides. A characteristic feature of this class of phosphomannans is that they are rapidly depolymerized by heating in dilute acid concomitant with hydrolysis of the phosphodiester bonds. Thus, they are teichoic-acid like polymers (Archibald et al., 1970).

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C . BALLOU

1. Ex0 cellular Ph osp ho m a n nuns Slodki (1963) concluded that the major product from acid degradation of H. capsulata phosphomannan was an al+Z-mannobiose phosphate of the structure in Fig. lOa, and that the intact polysaccharide was formed by phosphodiester bridges involving the CH2OH

H203POCH2

H203M cs'/ 0

OH

FIG. 10. Structures proposed for the mannobiose phosphates obtained by acid-catalysed depolymerization of Hansenula hotstii phosphomannan and for the intact mannan. Structures (a) and (c) are according to Slodki (1963) and (b) and (d) according to Gorin (1973).

reducing end of each unit and the phosphate unit on position-6 of the monomer unit (Fig. 1Oc). More recent investigations by Gorin (19 73) employed carbon-13 magnetic resonance spectroscopy to show that, in fact, the phosphate was located on the mannose unit at the reducing end of the disaccharide fragment. Thus, the mannan would consist of a mannosylphosphate diester polymer, with a

STRUCTURE AND BIOSYNTHESIS OF YEAST MANNAN

131

mannose unit (or a mannobiose unit) substituted on position-2 of the mannose phosphate group (Fig. 10d). A somewhat more complex phosphomannan is found in the culture medium of Hansenula holstii (Jeanes and Watson, 1962), and the major component appears t o be a polymer of: P-6-cxMan(l+3)aMan( 1+3)cxMan( l+3)cxMan( l+2)Man in which the phosphodiester bridge links the reducing-end mannose glycosidically to the phosphate on the end of the next pentasaccharide unit (Bretthauer et al., 1973). About 10% of the mannan, however, was resistant t o acid-catalysed depolymerization, and methylation analysis showed that it possessed some 1 6’ linkages. This was proposed t o represent a core mannan, similar to that of Sacch. cerevisiae, to which the pentasaccharide phosphate diester polymer chains were attached by phosphodiester linkages. Slodki et al. (1970) have reported that the phosphomannans of H. capsulata and H. holstii are replaced by neutral mannans with increased 0-acetyl content when the organisms are cultured in a medium lacking inorganic phosphate. Blas and Cunningham (1974a, b) have reached similar conclusions, although they found a mannan with high phosphomonoester content as well as the usual neutral and phosphodiester mannans. This clear demonstration of metabolite regulation of mannan structure deserves further study aimed at defining the mechanisms by which, in response to the environment, the yeast cell alters the nature of the mannan synthesized.

2. Hansenula wingei Cell-Wall Mannans Hansenula wingei Y-2340 has played an important role in studies on the biochemical nature of cell-cell recognition and agglutination in yeasts (Crandall and Brock, 1968). The haploid forms of this yeast, called 5- and 21-cells, show a strong constitutive agglutination reaction when intermixed. A heat-stable factor released from the surface of 5-cells by subtilisin digestion will agglutinate 21-cells, and a heat-labile factor released from the surface of 21-cells by trypsin digestion will inhibit that agglutination reaction although it will not agglutinate 5-cells. Thus, the 5-factor is multivalent and the 21-factor is monovalent. Both factors have the properties of mannan-proteins, although the 21-factor has never been purified sufficiently to make this characterization definitive.

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C . BALLOU

a. Structure of the Bulk Cell-Wall Mannan. Mannan, isolated from 5-cells by extraction with hot citrate buffer and precipitation with ethanol, shows a broad elution pattern when fractionated on a Bio-Gel A-5m column (Yen and Ballou, 1974a), a matrix with a fractionation range of 10,000 to 5 million daltons. The mannan contains 90% carbohydrate (mostly mannose), 2-5% protein and variable amounts of phosphate. Acetolysis of the mannan yielded a mixture of mono- t o pentasaccharides. The mannobiose and mannotriose components were a1+2- and al+3-linked in all possible combinations, whereas the mannotetraose had the structure: aMan(l+S)aMan( 1+2)0Man( 1+2)Man and the pentasaccharide was: &Ian( 1+3)&Ian( l+Z)&Ian( l+Z)c&Ian( 1+2)Man. Exo-a-mannanase digestion of the mannan released about one-fourth of the mannose, and the resistant residue gave the same acetolysis pattern as the intact mannan. Thus, most of the side chains are protected from the action of this enzyme. In part, this is due t o the presence of phosphate, but the methylation data suggest that some of the 1+2 linkages are in the backbone, so this mannan has some of the features of the structure in Fig. lc. Some of the phosphate is present as a-D-ghcosylphosphate units, as is that of the mannan from HansenuLa polymorpha (formerly Hansenula angusta; Lipke e t al., 1974). About 10% of the carbohydrate in the 5-mannan was released under the conditions of alkaline P-elimination, and the released material could be fractionated by gel filtration into a series of oligosaccharides ranging in size from mono- to about a decasaccharide. The intact mannan contained about 30% serine and threonine, and the polymer recovered following the alkaline treatment contained 14% of those two amino acids. Antiserum prepared in rabbits against 5-cells reacts well with the isolated 5-mannan, but only about 30% of the precipitin reaction can be inhibited by the isolated acetolysis oligosaccharides and a-D-glucosylphosphate. The chemical basis of the remaining 70% of the reaction was not identified, but it was destroyed by heating the mannan in 0.01-N HC1 at 100°C for 30 minutes, a treatment that released a small amount of glucose and a trace of mannose. 6 . Structure o f t h e 5-Agglutinin. The 5-agglutinin is released in soluble form from 5-cells by subtilisin digestion (Taylor and Orton, 1967), and can be purified by affinity chromatography on a column of 21-cells linked covalentIy to cellulose (Taylor and Orton, 1968).

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The agglutinin binds to the column at pH 4, and is eluted by a shift to pH 1.8. Further purification is obtained by gel filtration on Bio-Gel A-5m, during which the active material is eluted near the void volume of the column. Activity is assayed by measuring the ability of serially-diluted samples to agglutinate suspensions of 21-cells. About 50 mg of pure 5-agglutinin can be obtained from 50 g of wet cells by the above procedure (Yen and Ballou, 1974b). The material has a molecular weight of about 960,000, and is composed of 85% carbohydrate (mostly mannose), 10% protein and 5% phosphate. The protein component has an unusually high content of hydroxyamino acids, 55% serine and 6-8% threonine, and 85% of these two amino acids is destroyed on treatment of 5-agglutinin with 0.1 N-NaOH at 23°C for 24 hours. Simultaneously, 90% of the carbohydrate is released as a mixture of manno-oligosaccharides that range in size from one to 1 5 sugar units, the principal fragment being the octasaccharide. Thus, the 5-agglutinin is a novel glycoprotein in which, on the average, every other amino acid is substituted by carbohydrate. This gives a structure very different from that of the bulk cell-wall mannan in which most of the carbohydrate is present as long branched polymannose chains attached t o the protein by an alkali-stable linkage (Yen and Ballou, 1974a). Models for these two kinds of mannan are shown in Fig. 11. In spite of this difference, the 5-agglutinin and the bulk cell-wall mannan give very similar acetolysis patterns and methylation results, and they have similar immunochemical properties. Thus, the structures of the carbohydrate components, which these analyses reflect, have many features in common. The agglutination activity of 5-agglutinin is stable t o heating at 100"C, but it can be destroyed by digestion with Pronase and by the action of an exo-a-mannanase (Yen and Ballou, 1974b), which indicates that both the protein and carbohydrate components are important for activity. Dithiothreitol also inactivates 5-agglutinin (Taylor, 1964) and causes the release of a small fragment of about 12,000 daltons which binds weakly t o 21-cells (Taylor and Orton, 1968). This binding fragment is thought to represent the active site of the agglutinin, and there appear to be five or six per molecule of agglutinin. Its composition is quite different from that of the intact agglutinin and the central core remaining after dithiothreitol treat-

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C. BALLOU

(a)

(b)

FIG. 11. Models for the structure o f Hunsenulu wingei bulk cell-wall mannan (a) and the 5-agglutinin mannan (b). The solid line is the polypeptide and the open circles are mannose units.

ment (Table 4), being enriched in threonine and showing a decreased content of serine (Yen and Ballou, 1974b). A model has been proposed for 5-agglutinin (Taylor and Orton, 1971) that consists of a central core (molecular weight about 900,000) to which are attached six active binding sites in disulphide linkage. The known composition of the central core and the binding fragments allows a further elaboration on this model as shown in Fig. 12. If the central core contained a single protein chain, it would be 900 amino acids long. However, digestion of 5-agglutinin with the exo-a-mannanase appears t o bring about its dissociation into fragments the size of which suggests that there may be about six protein chains (Yen and Ballou, 1974b). The 5-agglutinin obtained by subtilisin digestion may be onIy a part of the whole mannan-protein complex. In fact, Taylor (1965) isolated a form of the agglutinin with a particle mass greater than lo8 daltons by digestion of cell walls with Glusulase. The smaller form of 5-agglutinin was released from this material by subtilisin digestion. Material with 5-agglutinin activity can be obtained directly in soluble form by breaking H. wingei cells, and the several active components can be separated by gel filtration on Bio-Gel A-5m, the largest being about 100,000 daltons.

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TABLE 4. Composition of 5-Agglutinin and the Dithiothreitol-Reduced Products from the Mannan of Hansenula wingei Amino acids (residues/ 100 residues)

Intact 5-agglutinin

Carboxymethylcysteine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-Cystine Valine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Mannosed

0.0 2.8 9.2 55.3 7.0 2.0 1.6 5.1 0.3 6.3 3.5 3.5 0.9 0.4 0.2 0.2 1.2

After reductiona Central core Binding fragmentb 0.1c 1.9 7.1 66.5 5.2 1.5 1.2 3.5 0.5

4.8 2.8 2.4 0.6 0.3 0.1 0.2 0.9

3.5 (1) 10.1 (3) 26.8 (7) 17.3 (5) 7.1 (2) 2.2 (1) 2.7 (1) 7.0 (2) 0.3 8.3 (2) 3.7 (1) 5.9 (2) 0.8 0.1 0.1 0.1 3.8 (1) (60)

a The reduced product was fractionated on a Bio-Gel A-5m column to separate the large central core and the small fragment with binding activity. Values in parentheses represent the proposed molar composition of the active binding fragment, assuming one mole of cysteine per subunit. The low yield of carboxymethylcysteine indicates that cysteine is unreactive in the large central core. Calculated from the ratio of polypeptide to carbohydrate in the fragment.

The nature of the interaction between 5-agglutinin and 21-factor has not been defined, although presumably it is analogous t o an antigen-antibody reaction. The 21-factor is readily inactivated by heating which suggests that its activity is associated' with an easily denaturable protein component (Crandall and Brock, 1968). Although the solubilized 21-factor is monovalent, there must be many such molecules on the cell surface, thus making the cell itself multivalent. The binding of the multivalent 5-agglutinin to the multivalent 21-cells shows a very high standard-free energy of association (-14.5 kcal/mole of 5-agglutinin) at pH 4, whereas the monovalent binding fragment obtained by dithiothreitol reduction of 5-agglutinin has a standard-free energy of association that is less than

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F 2. 12. lodels for the 5-agglutinin from Hansenula wingeii showing the central core and the six binding fragments attached by disulphide bonds. The heavy line indicates protein and the lighter lines manno-oligosaccharide chains attached to hydroxyamino acids.

-7 kcal/mole (Taylor and Orton, 1970). These values are reasonable for a co-operative model of the antigen-antibody type (Karush, 1962; Hornick and Karusch, 1972). E. CANDIDA SPECIES

Several members of this genus (Candida albicans, Candida stellatoidea, Candida parapsilosis) are human pathogens (Gentles and La Touche, 1969). Special attention has been given to C. albicans, it being the most pathogenic member of the genus for humans. The mannan fraction of C. albicans is an important immunogen as evidenced by the observation that the isolated polysaccharide gives a positive skin test in human individuals with deep-seated candidiasis (Akiba e t al., 1957). Summers e t al. (1964) studied the cross-reactivity o f mannan preparations from different Candida species, and demonstrated the

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important point that two serotypes of C. albicans can be distinguished in this way. The acetolysis patterns of Candida mannans are more complex than those of most Sacch. cereuisiae strains and give as many as seven or eight oligosaccharide peaks on gel filtration (Kocourek and Ballou, 1969). Suzuki and Sunayama (1968, 1969) partially characterized these fragments, finding that the di-, tri- and heptasaccharides were a1+2-linked, whereas the tetra-, penta- and hexasaccharide contained a single a1+3 linkage in addition t o the 1+2 linkages. The most potent inhibitor of the homologous precipitin reaction was the hexasaccharide. In later studies, a comparison of C. albicans serotype A (NIH A-207) and serotype B (NIH B-792) showed that both strains gave oligomannosides that were almost completely al+Z-linked, although serotype A gave longer acetolysis fragments than serotype B (Sunayama, 1970; Sunayama and Suzuki, 1970). Both of these mannans give methylation results that support a 1+6-linked backbone structure. It was proposed that the difference in side-chain length could account for the immunochemical differences observed by Summers e t al. (1964). The small amounts of al+3 linkage might also be important in view of the dominant role of this structure in Sacch. cerevisiae mannans. Candida atmospherica and Candida diddensii give acetolysis patterns that are quite different from those of the above species (Kocourek and Ballou, 1969), and this pair of yeasts is placed together in a different taxonomic group. F. OTHER YEAST MANNANS

Many other yeast mannans have been described with only tentatively defined structures (Gorin and Spencer, 1968, 1970). Among those worthy of mention are galactomannans and mannans with both a- and p-linked mannosyl units. Several of the galactomannans have al+6-linked mannose backbone structures with the galactose attached as short side chains, whereas the mannans with P-linked mannose also seem t o have these units in the side chains. Thus, there appears t o be a considerable variety in side-chain structure with a more conservative regulation of the backbone structure.

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IV. Mannan Biosynthesis Our understanding of the mechanism of mannan biosynthesis is still in a rudimentary state, partly because of the insufficient knowledge concerning mannan structure and partly because of the inherent difficulties in working with such a complex macromolecule. When we consider the slow advances made in unravelling the secrets of biosynthesis of glycoproteins with much less complicated carbohydrate structures (Hughes, 1973), we can appreciate the problems faced by those working with yeast mannans. However, as indicated in the previous sections of this review, a fairly well detailed picture of Saccharomyces cerevisiae mannan can now be sketched, and this picture should facilitate the planning of experiments on, and the interpretation of results from, biosynthetic studies. Moreover, the availability of mannan mutants with known polysaccharide structural alterations should prove advantageous. Perhaps this is a good place to second the recommendation made informally by several yeast geneticists that “yeastologists” adopt a common strain for their studies (analogous t o Escherichia coli?), so that an integrated pool of knowledge can be assembled concerning one organism. The strain now most widely employed is Sacchuromyces cerevisiue X2180, selected and promoted by R. K. Mortimer, and a strain whose genetic map is well developed (Mortimer and Hawthorne, 1969). We have adopted this strain for the selection of mannan mutants, and these mutants are available to anyone who is able to use them. A proposal to concentrate efforts on a single strain should not be taken t o negate the importance of comparative studies on different yeast strains. In fact, discovering the evolutionary and biochemical significance of the polymorphism of yeast mannan structure (Ballou and Raschke, 1974) is one of the most interesting and challenging problems ahead. A. M A N N A N BIOSYNTHESIS IN SACCHAROMYCES SPECIES

Studies of mannan biosynthesis have taken three general tacks. One has been directed toward an elucidation of the overall process of mannan formation and translocation within the cell using “pulsechase” techniques and autoradiography. A second has dealt with the formation and secretion of intact mannan molecules by yeast

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protoplasts, and the inhibition of this process by compounds that prevent protein or polysaccharide synthesis. The third approach has concerned investigations of the enzymic processes involved in mannan synthesis, using cell-free extracts and following incorporation of radioactive mannose into endogenous and exogenous acceptors. Studies of the first type can be helpful in deciding such questions as whether the mannan is processed in vesicles or while free in the cytoplasm, but “pulse-chase” methodology has an inherently low resolving power and caution is needed in interpreting the results. Studies of the second type have thus far established only that protein synthesis is coupled to mannan synthesis but not to glucan synthesis. Should inhibitors be found that lead to accumulation or secretion of incomplete mannan molecules, further work in this direction could be very rewarding. The interpretation o f studies of the third type has been difficult because of the complicated structure of the carbohydrate portion of yeast mannans. In the endogenous system, mannose may be incorporated onto serine and threonine units and these may then be extended; mannose may be incorporated into the inner core or the outer chain o f the polysaccharide units attached to asparagine; and, lastly, the mannose may be attached to the outer chains as rnannosyl phosphate units that also can undergo extension to mannobiosyl phosphate units. As a conservative estimate, there must be at least ten mannosyltransferase activities involved in adding mannose to mannan in Sacch. cerevisiae X2 180. Although this sounds like a complicated biosynthetic problem, it is possible to dissect the larger problem into several smaller ones by selective product analysis and by use of mannan mutants. Thus, the endogenous mannan product may be isolated and hydrolysed in dilute acid t o release those mannose units attached in phosphodiester linkage. This hydrolysed product may then be subjected to p-elimination in dilute alkali to release the units attached to serine and threonine. Finally, the resistant polymer may be recovered and degraded by acetolysis to give the side-chain fragments from the backbone. Because all of the oligosaccharide fragments obtained in these selective degradation reactions are short (I to 4 mannose units in Succh. cerevisiue mannan), it is relatively easy to determine by chemical analysis into which position or positions of the oligosaccharide the radioactive mannose was incorporated. This whole

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study can be simplified by using mutants that make mannan lacking one or another of the various kinds of mannose units. For example, the mnnl mutant has none of the terminal 1+3-linked mannose, the mnn2 mutant lacks the outer chain side chains, and the mnn4 mutant mannan is devoid of mannosyl phosphate groups. 1. In corpora tio n Studies with Guanosine Dip h osp h a t e[ l C ] -Mannose Behrens and Cabib (1968) published one of the first definitive studies on incorporation of mannose from the sugar nucleotide into endogenous mannan in a particulate membrane system. Structural analysis of the product was performed on material isolated according to Cifonelli and Smith (1955), which invoIves extraction with alkali and precipitation with ethanol, so it appears that they were following incorporation only into the alkali-stable polymannose chains. Their results demonstrated that mannose was added in a Mn2+-dependent reaction into all positions of the acetolysis fragments, which means that both the a1+6- and al+2-mannosyltransferase activities must have been expressed. For some reason, very little mannose was added in a1+3 linkage, even though this structure was present in the bulk mannan of this yeast (Succharomyces carisbergensis). They estimated that the rate of incorporation in the particulate system was only about 1% of that of mannan synthesis in the intact cell. The next logical step in defining the process was t o utilize exogenous acceptors, and to attempt to solubilize and separate the individual enzymes, but here the progress has been slow. Lehle and Tanner (1974) have demonstrated a membrane-bound enzyme in Sacch. cerevisiae that catalyses hexose transfer from GDP-mannose to mannose, mannobiose and mannotriose to yield the next higher homologues, but this activity may reflect enzymes that are responsible for synthesis of the oligosaccharide chains found on serine and threonine. The transfer activity is Mn2+-dependent, and the same fraction transfers hexose from GDP-mannose to exogenous acidtreated mannan-protein giving a labelled product from which most of the radioactive mannose is released by mild alkaline treatment. The acid treatment presumably exposes more serine and threonine acceptor sites. FarkaS et al. (1974) have reported similar but less detailed findings. These resuIts suggest that the enzymic activities for synthesis of

STRUCTURE AND BIOSYNTHESIS OF YEAST MANNAN

141

the serine- and threonine-linked oligomannosides are much more active in the particulate preparations than are those involved in synthesis of the polysaccharide chains attached to asparagine, in spite of the fact that 80-90% of the mannose in yeast mannans is present in the latter form. The a1+6 linkage is characteristic of these polymannose chains, whereas Lehle and Tanner (1974) found that the mannobiose formed in their study was almost exclusively al+Z-linked. Thus, the mannan backbone-forming enzyme activity apparently is not very active in this system. The mnn2 mutant of S Q C C ~cereuisiue . that makes an unbranched backbone has been exploited to facilitate study of the a1+6-mannosyltransferase activity (T. Nakajima and C. E. Ballou, unpublished results). Protoplast particles from this mutant efficiently catalyse transfer of mannose from GDP-mannose to al+6-linked mannooligosaccharides, free mannose having no acceptor activity. Acetolysis of the product gives about equal amounts of mannose and mannobiose. Thus, about half of the mannose was attached by 1+6 linkage and half by an acetolysis-stable linkage, indicating the involvement of two enzymes. Transferase activity was solubilized by treating the particles with Triton X-100 containing urea (Garewell and Wasserman, 1974), and the soluble enzyme has no lipid requirement but is activated by both Mn2+ and Mg2+. This kind of study illustrates the utility of the mannan mutants. Because the mutant used in this example lacks the transferase activity for adding mannose in a1+2 linkage to the backbone, the acetolysis-stable product probably represents activity for one of the enzymes involved in making the inner-core portion of the mannan. It is unlikely that this activity represents an enzyme for adding mannose to serine and threonine because the acceptor employed had ~ 1 - 6 linkages, and these are not found in the oligosaccharides on the hydroxyamino acids. It may be instructive to re-emphasize the value that can be derived from the isolation of more mannan mutants. With the few Succh. cereuisiue mutants already obtained, it is possible to fit them into a plausible pathway for biosynthesis of the mannan outer chain (Ballou, 1974). The structures a-d in Fig. 1 3 represent the incomplete polysaccharide chains made by the various mutants, the phenotypes of which suggest that they lack one or another mannosyltransferase activity. Although the nature of the lesion, whether in

C.BALLOU

142

(a)

6

1 6

1 6

-M-M-M-M-M-M-

1 6

1 6

1 6

1

I

(u(I+Z)mannosyltransferase I

(b)

6

1 6

1 6

1 6

-M-M-M-M-M-M-

1 6

r:

t: t: t:.

M

M

M

1

1 6

t;

M

M

I

a( 1+2)mannosyltransferase 11

(c)

6

1

6

1

6

1

-M-M-M-M-M-M-

6

1

6

1

1

6

t: t: t: ti t: t: t; t:

M

M

M

M

I (d)

6

M

M

M

mannosyl phosphate transferase

1 6

1 6 1 6

1 6

-M-M-M-M-M-M6

P-M t1 M

M

t: Mt: Mt: M7: t: t: Mt: M M

t:

M

I

(u( 1+3)mannosyltransferase

(el

6

1

6

1

6

1

-M-M-M-M-M-M-

6

1

1 6

1

6

1

6

I 1

t; t: ti t: t: ti t:

tl

M

M

M

M

M

M

M

M

FIG. 13. Partial structures of the outer-chain portion of yeast mannan illustrating the postulated steps in biosynthesis of the side-chain structure inferred from the structures of the various Saccharomyces cerevisiae mannan mutants. All linkages have the &-configuration.

STRUCTURE AND BIOSYNTHESIS OF YEAST MANNAN

143

a structural or regulatory gene, has not been established for each, the practical consequence is that synthesis of a particular linkage has been prevented so that the mannan molecule is made in a simplified form. By a judicious selection of mutant, the incorporation of labelled precursors into selected parts of the mannan molecule can be controlled so that specific processes can be analysed even with particulate enzyme systems.

2. The Role of Lipid-Bound Intermediates In their investigations, Behrens and Cabib (1968) sought but did not detect participation of a mannosyl-lipid intermediate in mannan synthesis. The first indication that there might be such an intermediate was published by Tanner (1969) who showed that a substance with the properties of a mannosyl-P-lipid was formed in yeast membrane particles, and provided kinetic evidence that it was a mannan precursor. The formation of the substance occurred in the presence of Mg2+ or Mn2+, whereas the further transfer of the incorporated [’ ‘C] -mannose into mannan required Mn2+. Labelled mannose in the lipid was exchanged on incubation with GDP and GDP-mannose in presence of the particulate enzyme according to the reactions: Man-P-Lipid + GDP + GDP-Man + P-Lipid P-Lipid + GDP-Man + Man-P-Lipid + GDP Sentandreu and Lampen (1971) confirmed these observations and showed that a radioactive mannosyl-lipid could be isolated by chloroform-methanol extraction of yeast membranes that had been incubated with GDP- [ C] -mannose. The probable nature of the lipid was suggested by Tanner and Jung (1971) who found that dolichol phosphate isolated from yeast could serve as the acceptor of mannose from GDP-mannose. The stability of the product to acid hydrolysis (85% degradation in 1 5 min at 20°C in N-HCl) was typical of the phosphodiester linkage of such mannosyl-lipid derivatives (Warren and Jeanloz, 1975). A similar product was isolated by Sentandreu and Lampen (F972), but they concluded that “the kinetics of mannose incorporation into this crude lipid fraction are not those expected if a single compound were being formed or for a lipid(s) acting as a true intermediate in mannan synthesis”. This

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observation takes on significance in view of a subsequent observation of Babczinski and Tanner (1973) and of Sharma et al. (1974) discussed below. Jung and Tanner (1973) have isolated and characterized the lipid part of the mannosyl-lipid as a mixture of five dolichols with 14 to 18 isoprene units, of which 10-20% is found in the yeast cell as the monophosphate ester. One role of the mannosyl dolichol phosphates in yeast mannan biosynthesis has been clarified by studies (Babczinski and Tanner, 1973; Sharma et al., 1974) which indicate that, in the system employed, this intermediate may be involved principally in addition of the first mannose to the serine and threonine units of the protein. That is, most of the incorporated [’4C] -mannose was released by 0-elimination conditions. Subsequent additions appear to involve GDP-mannose. If this interpretation is correct, then the failure of Behrens and Cabib (1968) to observe a lipid requirement in mannan synthesis would be understandable if they were following incorporation of mannose into the asparagine-linked polymannose outer chains or on to mannose units already attached to serine or ‘threonine. Moreover, the kinetic pattern observed by Sentandreu and Lampen (1972) may, in part, reflect this restricted role of the mannosyl dolichol phosphates in mannan synthesis. The ability t o “chase” labelled mannose out of the intermediate could be limited by the availability of serine and threonine acceptor sites in the endogenous system. A part of the labelled mannose incorporated in the experiments of Tanner was not released by alkali, and this may reflect reactions of the inner core (Nakajima and Ballou, 1974b), a part of the mannan-protein that could be made in a manner similar to that of the core region of animal glycoproteins (Hsu et al., 1974). Sharma et al. (1974) have demonstrated in several ways that transfer of mannose from mannosyl dolichol phosphate and from GDP-mannose can be uncoupled. Ageing the membrane particles leads to a loss in the transfer activity from GDP-mannose to dolichol phosphate, and consequently to a decrease in the transfer of mannose to serine and threonine. With Mg2+ alone, the mannosyllipid is formed and the mannose transferred to hydroxyamino acids, but Mn2+ is required for the subsequent mannosyl transfer reactions. Sharma et al. (1974) have proposed the following scheme to rationalize these results:

STRUCTURE A N D BIOSYNTHESIS OF YEAST M A N N A N

GDP-Man + Dolichol-P

Mg2+ or Mn2+

Dolichol-P-Man + Protein-(Ser/Thr)

145

Dolichol-P-Man + GDP

Mg2+ or Mn2+ >

Protein-( Ser/Thr)-Man + Dolichol-P Protein-(Ser/Thr)-Man + GDP-Man

Mn2+

Protein-(Ser/Thr)-Man-Man + GDP Additional transfers from GDP-mannose would lead t o formation of serine- or threonine-linked mannotriose and mannotetraose units. This proposal seems reasonably well substantiated for synthesis of the oligosaccharide units that are released by 0-elimination. However, until one can follow independently the activities of the several other mannosyltransferase reactions, it will not be possible to delimit further the role of lipid-bound mannosyl derivatives in yeast mannan biosynthesis.

3. Secretion of Mannan b y Yeast Protoplasts Under appropriate conditions, protoplasts prepared from Sacch. cereuisiae by enzymic removal of the cell wall are able t o regenerate a new cell wall (NeEas, 1971). This regeneration requires synthesis and secretion of both glucan and the mannan-protein. Cycloheximide selectively prevents synthesis of the latter as demonstrated by its inhibition of incorporation of [' C] -glucose into mannan relative to glucan, and by inhibition of the incorporation of [' C] -threonine into the cell wall (Elorza and Sentandreu, 1969). Parallel studies have been carried out by FarkaS et al. (1969, 1970) who found that 2-deoxyglucose, which had no effect on protein synthesis in Sacch. cereuisiae, inhibited secretion of both carbohydrate and protein by yeast protoplasts. Although they confirmed that cycloheximide inhibited mannan secretion, it did not affect the mannan synthetase system of Behrens and Cabib (1968). Subsequent studies have demonstrated that 2-deoxyglucose is converted to various phosphorylated intermediates, and is even incorporated into mannan and glucan (Biely e t al., 1972, 1974). The mechanisms of inhibition of cell-wall regeneration by such analogues appear very complex (Biely et al., 1973; Bauer et al., 1974).

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C. BALLOU

Whereas these studies all deal with secretion of bulk mannan, Lampen (1968) investigated factors that affect secretion of mannaninvertase, a glycoprotein whose synthesis is regulated by glucose repression. Using a derepressed mutant, it was demonstrated that secretion was inhibited by cycloheximide. The attractive idea that a small, internal, carbohydrate-free . invertase is a precursor of the mannan-invertase that is secreted is yet t o be established (Gascbn e t al., 1968). Cytochalasin A has been shown to inhibit invertase secretion, not because of a direct affect on its synthesis, but rather owing to a general inhibition of the energy-dependent accumulation of its precursors and their conversion t o macromolecules (Kuo and Lampen, 19 74). 4. Intracellular Location of Mannan Synthetases The location of the synthetic apparatus for mannan formation has been investigated by Cortat e t al. (1973). Using the assay procedure of Behrens and Cabib (1968), they followed incorporation from GDP- [’ C] -mannose into ethanol-insoluble material, apparently avoiding alkaline treatment during product isolation. The synthetase activity was much higher in exponentially growing cells than in stationary-phase cells, and the greatest activity was localized in the light membrane fraction composed of fragments of the endoplasmic reticulum. Smaller amounts of activity were associated with the glucanase vesicles and fragments of the plasmalemma, and it was postulated that all three membrane fractions may be involved in mannan synthesis and secretion. Using a “pulse-chase” technique with tritiated mannose, KoSinovi et al. (1974) have shown by autoradiography that the initial sites of mannose incorporation occur in the cytoplasm, probably on membranes of the endoplasmic reticulum, and that the incorporated tritium is eventually translocated to the periphery of the cell. To enhance incorporation of labelled mannose into mannan, these workers grew the yeast on a galactose-containing medium and kept the mannose concentration low to minimize its conversion to glucose. These tricks led t o a very efficient incorporation of mannose into mannan. Employing the Saccharomyces FH4C strain of Lampen (1966), Beteta and Gasc6n (1971) have isolated the “vacuole” fraction from protoplasts by the procedure of Matile and Wiemken (1967). They

STRUCTURE A N D BIOSYNTHESIS OF YEAST M A N N A N

147

found most of the intracellular invertase in the vacuoles, and they showed it t o be a mixture of enzyme activities that were separable on a Sephadex G-200 column. The supposition is that these were carbohydrate-free and mannan-protein forms of invertase, and that the vacuoles represent secretory organelles destined for exocytosis by fusion with the plasma membrane. An alternative interpretation has been offered (Holley and Kidby, 1973). B. MANNAN BIOSYNTHESIS IN OTHER YEASTS

1. Kluyveromyces lactis The mannan of Kluyv. lactis is very similar in structure to that of Sacch. cerevisiae, the major difference being the replacement of mannosylphosphate groups in the outer chain with N-aCety1-D glucosamine units (Fig. 3d, p. 109). The presence of this sugar provides an opportunity to investigate the biosynthesis of what appears t o be a terminal step in maturation of the mannan polysaccharide chain. Particles from lysed protoplasts prepared from Kluyu. lactis are quite active in incorporation of N - a ~ e t y l - [ ' ~ C- ] glucosamine from UDP-N-acetyl-[' C] -glucosamine into endogenous mannan side chains to give a product that yields: orMan( 1+3)aLMan(1+2)arMan( 1+2)Man,

I

[I4C] crGNAc( 1+2)

designated Man4-['4C] -GNAc, on acetolysis (Smith et al., 1975). The reaction requires Mn2+ and is inhibited by UDP. The same particles utilize exogenous mannotetraose and produce Man4 - [ C] GNAc directly. Other oligosaccharides (Table 5 ) are also active, the most important requirement being the terminal ali3-mannobiose unit, although two sequential a1+3 linkages destroy the acceptor activity. Each of the acceptors yields a product with the gel filtration and chromatographic properties expected for addition of a single N-acetylglucosamine residue. In the intact mannan, this hexosamine is linked al+2 to the penultimate mannose residue of the mannotetraose side chains. Evidence that the particulate enzyme has the same specificity came from analysis of the product formed with d+3-mannobiose as the acceptor. Reduction of this product with

'

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C. BALLOU

TABLE 5. N-Acetylglucosamine Acceptor Activity of Various Oligosaccharides with the Transferase System from Kluyueromyces lactis. From Smith e t ol. (1975) Acceptor

Relative rate

&Man(1’2)Man &Man( 1+2)&Man(l+Z)Man &Man(l+Z)aMan( 1+2)aMan( 1’2)Man &Man(1’3)Man &Man(1-+3)&Man(1’2)Man &Man(1+3)aMan( 1+2)&Man( 1’2)Man &Man(1+3)&Man( 1+3)aMan( 1-+2)aMan(1’2)Man &Man(1+6)&Man(1+6)PMan( 1’4)GNAc

16 4 1 350 220 220 15

0

I

&Man(1’3) The incubation mixture contained protoplast particles (1 mg protein), 1 nmole UDPN-acetyl-D-[ 1-14C]-glucosamine, 175 nmoles of acceptor, 5 pmoles MgC12 in 0.05 M imidazole-HC1 buffer (pH 6.5) in a final volume of 0.5 ml. After incubation at 37°C for 30 minutes, the reaction was passed through a small column of Dowex 1 and the radioactivity in the neutral effluent was determined in a scintillation counter, Under these conditions, the disaccharide aMan( le3)Man accepted 0.35 pmoles of N-acetyl-D-[ 1-l4C]glucosamine per minute per mg of protoplast protein.

tritiated sodium borohydride followed by partial acid hydrolysis gave [’ C] -GNAc-+Mannitol-[ H] . Thus, the hexosamine had been added to the mannose at the reducing end of the disaccharide, which corresponds to the penultimate mannose of the tetrasaccharide acceptor: &an( 1+3)Man

+ UDP-GNAc

Mn2+

orMan(1+3)Man + UDP

I

GNAc From studies of the properties of mannan mutants of this yeast, some idea has been obtained as to the possible complexity of the regulation of mannan biosynthesis and modification (Smith et d., 1975). Mutants that lack N-acetylglucosamine in ttie mannan side chains were selected by their failure t o agglutinate with rabbit antiserum directed against this determinant. One class simultaneously lost the mannotetraose side chains, which suggests that the N-acetylglucosamine was missing because the acceptor was not made to which this hexosamine could be transferred. This supposition was confirmed by the demonstration that protoplast particles still possessed full wild-type N-acetylglucosamine transferase activity with exogenous acceptors.

STRUCTURE A N D BIOSYNTHESIS OF YEAST MANNAN

149

The other class of mutants retained the mannotetraose side chains, so it appeared that they had a defective N-acetylglucosamine transferase. This was established for one subclass by assay of the protoplast particles, but another mutant of this class surprisingly showed the wild-type N-acetylglucosamine transferase activity with exogenous acceptors. Thus, some defect in this mutant prevented expression of the transferase activity in the intact cell. Several explanations for this observation have been tested. For example, the mutant transferase could have a high K, value for one of the substrates. However, it was found that the affinities for mannotetraose, UDP-N-acetylglucosamine and Mn2+ were similar to those for the wild-type enzyme. The mutant could overproduce an inhibitor that was normally required to regulate the transferase activity in the intact cell, but no evidence for such an inhibitor was detected in mixing experiments. Finally, the enzyme could be made as an inactive pro-enzyme that was activated by a specific protease, and in the mutant this protease could be defective but be replaced by nonspecific proteases that were released when the protoplast particles were prepared for assay. However, we were unable to detect such an inactive pro-enzyme in the mutant by preparing the membrane particles in the cold or in the presence of protease inhibitors. Although the nature of the lesion in this mutant has not been defined, its properties reveal the important fact that the mannanprotein can be processed in a cell containing an active N-acetylglucosamine transferase without this enzyme activity being expressed. Either the enzyme is inhibited in the intact cell or perhaps it is not inserted in the appropriate place in the assembly line where it normally acts. Either explanation implies a subtle form of regulation of mannan biosynthesis.

2. Hansenula Species Mannan biosynthesis in this yeast is complicated by the fact that two very different kinds of mannan are made, one being a phosphomannan with a teichoic acid-like structure (Slodki, 1963) and the other a neutral mannan similar to that of Sacch. cerevisiae (Kozak and Bretthauer, 1970). Bretthauer e t ul. (1969) demonstrated transfer of mannosylphosphate units from GDP-mannose to endogenous acceptors with a particulate enzyme preparation from Hamenula

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C . BALLOU

holstii. The [' 'C] -mannose was released from the product by mild acid hydrolysis and the 3 2 P was recovered as mannose 6-phosphate following strong acid hydrolysis of the residue. Thus, the reaction was postulated as involving direct transfer of the mannosylphosphate group: GDP-Man + Mannan

-I MnZ+

Mannan + GMP P+Man

Subsequently, Kozak and Bretthauer (1970) showed that this reaction required Mn2+. The product was separated into a neutral mannan fraction that yielded mannose, mannobiose, mannotriose and mannotetraose on acetolysis as well as a phosphomannan fraction that gave mannose and mannobiose on mild acid hydrolysis. All of these products are characteristic of Succh. cerevisiae mannan, and the fact that mannobiose was released by mild acid hydrolysis suggests that transfer of mannose t o mannosylphosphate groups in the mannan had also occurred. Somewhat different results have been reported by Mayer (1971) with Hansenulu cupsuluta. He obtained mannose, mannose 6-phosphate and mannobiose phosphate by mild acid hydrolysis, whereas mannose, mannobiose and mannotriose were formed by acetolysis of the synthesized mannan. The products of acid hydrolysis and acetolysis were those expected for the neutral and phosphomannan structures proposed by Slodki (1963). He concluded that the radioactive mannosyl dolichol phosphates are also formed in H. holstii. There is kinetic evidence that they have a role in mannan biosynthesis in this yeast (Bretthauer e t al., 1973). 3. Cryptococcus laurentii In contrast to the difficulty in demonstrating mannan-related glycosyl-transferase activities with exogenous acceptors in Sacch. cerevisiae particulate-enzyme systems, considerable success has rewarded such studies with a heteroglycan in Cryptococcus laurentii, an encapsulated yeast-like organism (Schutzbach and Ankel, 1969). This organism contains in the cell wall heteropolysaccharides that are clearly different in structure from the usual yeast mannans, but some aspects of their structures appear related to mannan. For example,

STRUCTURE AND BIOSYNTHESIS OF YEAST MANNAN

151

partial acetolysis of Cr. laurentii polysaccharide yields al+3-mannotriose. Particulate enzyme activities have been demonstrated that transfer mannose from GDP-mannose to exogenous acceptors to form d + 2 , a1+3 and a l + 6 mannosyl linkages (Schutzbach and Ankel, 1971). The organism also catalyses transfer of D-xylose (Schutzbach et a/., 1974) and of D -galactose (Raizada e t al., 1974) to mannose acceptors. These systems may be good models for similar reactions that must be involved in biosynthesis of yeast mannans that contain D -xylose and D -galactose. C. A MODEL FOR MANNAN BIOSYNTHESIS

For a glycoprotein as complex as yeast mannan, one might suppose that, as the molecule is elaborated, its construction occurs in a sequence of steps ordered either by a progressive change in substrate specificity or by a compartmentation of the various enzymes. The proposal outlined in Fig. 14 assumes that the protein portion is synthesized first, presumably on ribosomes, and that different parts of the mannan molecule are then built up sequentially. The inner core di-N-acetylchitobiose-mannosyl unit might well be made on a lipid carrier in the form:

dolichol-PP-GNAc-GNAc-M-M-M-M

H I I I M M I

M M M

M

and then be transferred to the polypeptide chain (Hsu e t al., 1974). Similarly, the first mannose attached t o serine and threonine apparently is derived from a dolichol-P-mannose intermediate, whereas lengthening of these chains involves GDP-mannose as the donor (Sharma et al., 19 74). Glycosyltransferase reactions which involve participation of lipid intermediates generally occur in close association with membranous organelles such as the endoplasmic reticulum or the plasmalemma. However, such lipid-bound sugar derivatives are still subject to further glycosylation by sugar nucleotides, such as GDP-mannose, an indication that they are potentially accessible to the cytoplasm of the cell.

\

f

\Asn-GNAc-GNAcM-M-M-M

Asn-GNAc-GNAcM-M-M-M

I l l I I M M I

pE++]

M M M

Asn-GNAc-GN

Ac-M-M-M-M-[-M-M-M-M]

I l l M M M I 1 M M I

I I

M M

-

1

I l l I I M M I

M M M

Asn-GN Ac-GN AcM-M-M-M

I

l

I l l I 1 M M I

l

M M M

M M M M M

I

M

M

I I

M M

FIG. 14. A postulated sequence for glycosylation of yeast mannans, showing addition of N-acetylglucosamine (GNAc) and mannose (M) residues t o asparagine (Asn) and serine (Ser) residues in the protein. Details are discussed in the text.

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The outer-chain portion of the mannan polysaccharide might be built up on the inner core, as illustrated in Fig. 14, and preliminary evidence indicates that the donor is GDP-mannose because a lipid requirement is not apparent with the al+6-mannosyltransferase (Behrens and Cabib, 1968; T. Nakajima and C. E. Ballou, unpublished observations). Side-chain modification in Succh. cerevisiae consists of the transfer of mannosylphosphate groups from GDPmannose to certain portions of the outer chain. This reaction has been investigated in detail only in Hunsenulu species (Kozak and Bretthauer, 1970; Mayer, 1971), and GDP-mannose appears to be the donor. Side-chain modification in Ktuyverom yces lactis involves addition of N-acetylglucosamine, from UDP-N-acetylglucosamine, in a process that is inhibited by UDP (Smith et al., 1975). However, there does not appear to be a lipid requirement in the solubilized enzyme system with exogenous oligosaccharide acceptors. These examples suggest that all side-chain modifications may involve the sugar nucleotides as donors rather than lipid intermediates. Mannan biosynthesis is a directional process in the sense that the molecules eventually are secreted from the cell. Studies with mutants altered in the outer chain demonstrate that secretion is not dependent on the maintenance of the wild-type structure in that part of the molecule (Ballou, 19 74). In addition, the terminal al+3-linked mannose units in the outer chain, the inner core, and on serine and threonine, can be eliminated by mutation without affecting the ability of the cell to make and secrete mannan. However, attempts to isolate Succh. cerevisiue mutants that lack mannan completely have been uniformly unsuccessful, so it seems probable that some portion of the molecule will be found to serve a critical function in the secretory process. REFERENCES Akiba, T., Iwata, K. and Inoue, S. (1957). Japanese Journal of Microbiology 1 , 11. Arnes, G . (1974). Journal of Biological Chemistry 249, 634. Antalis, C., Fogel, S. and Ballou, C. E. (1973). Journal of Biological Chemistry 248,4655. Archibald, A. R., Baddiley, J. and Blumsom, N. L. (1970). Advances in Enzymology 30,223. Arnold, W. N. (1969). Biochimica e t Biophysica Actn 178, 347. Arnold, W. N. (1972). Journal of Bacteriology 112, 1346. Arnold, W. N. (1973). Physiological Chemistry and Physics 5 , 11 7.

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s. 5.

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Fleet, G. H. and Phaff, H. J. (1974). Journal of Biological Chemistry 249, 1717. Friis, J. and Ottolenghi, P. (1970). Comptes Rendus des Travaux Laboratoire Carlsberg 37, 327. Garewal, H. S. and Wasserman, A. R. (1974). Biochemistry, New York 13, 4063. Gascon, S. and Lampen, J. 0. (1968). Journal of Biological Chemistry 243, 1567. Gascbn, S., Neumann, N. P. and Lampen, J. 0. (1968). Journal of Biological Chemistry 243, 1573. Gentles, J. C. and La Touche, C. J. (1969). In “The Yeasts”, (A. H. Rose and J. S. Harrison, eds.), vol. 1, pp. 107-182. Academic Press, New York. Gorin, P. A. J. (1973). Canadian Journal of Chemistry 51,2105. Gorin, P. A. J. and Perlin, A. S. (1956). Canadian Journal of Chemistry 34, 1796. Gorin, P. A. J. and Spencer, J. F. T. (1968). Advances in Carbohydrate Chemistry 23, 367. Gorin, P. A. J. and Spencer, J. F. T. (1970). Advances in Applied Microbiology 13, 25. Gorin, P. A. J., Spencer, J. F. T. and Bhattacharjee, S. S. (1968). Canadian Journal of Chemistry 47, 1499. Govorchenko, V. I., Gorbatch, V. I. and Ovodov, Yu. S. (1973). Carbohydrate Research 29,421. Greiling, H., Vogele, P., Kisters, R. and Ohlenbusch, H. D. (1969). Zeitschrift fur Physiologische Chemie 350, 517. Guthrie, R. D. and McCarthy, J. F. (1967). Aduances in Carbohydrate Chemistry 22,ll. Hartwell, L. H. (1974). Science, New York 183,46. Hasenclever, H. F. and Mitchell, W. 0. (1964).Journal of Immunology 93, 763. Hawkins, E. R. (1973). Journal of Biological Chemistry 248, 4671. Haworth, W. N., Hirst, E. L. and Isherwood, F. A. (1937). Journal of the Chemical Society 784. Haworth, W. N., Heath, R. L. and Peat, S. J. (1941). Journal of the Chemical Society 833. Hickman, S., Komfeld, R., Osterland, C. K. and Kornfeld, S. (1972).Journal of Biological Chemistry 247, 2156. Holley, R. A. and Kidby, D. K. (1973). Canadian Journal of Microbiology 19, 113. Homick, C. I. and Karush, F. (1972). Immunochemistry 9, 325. HSU,A.-F., Baynes, J. W. and Heath, E. C. (1974). Proceedings of the National Academy of Sciences of the United States of America 71, 2391. Hughes, R. C. (1973). Progress in Biophysics and Molecular Biology 26, 189. Jeanes, A. and Watson, P. R. (1962). Canadian Journal of Chemistry 40, 1318. Jeffery, E. A. and Satchell, D. P. N. (1962). Journal of the Chemical Society 1913. Jones, G. H. and Ballou, C. E. (1968). Journal of Biological Chemistry 243, 2442. Jones, G. H. and Ballou, C. E. (1969a). Journal of BioZogical Chemistry 244, 1043. Jones, G. H. and Ballou, C. E. (1969b). Journal of Biological Chemistry 244, 1052. Jung, P. and Tanner, W. (1973). European Journal of Biochemistry 37, 1.

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Karush, F. (1962). Advances in Immunology 2, 1. Kocourek, J. and Ballou, C. E. (1969).Journal of Bacteriology 100, 1175. Koide, N. and Muramatsu, T. (1974). Journal of Biological Chemistry 249, 4897. Koiinovi, A., FarkaS, V., Machala, S. and Bauer, 5 . (1974). Archiv fti’r Microbiologie 99, 255. Kozak, L. P. and Bretthauer, R. K. (1970). Biochemistry, N e w York 9, 1115. Kuo, S.-C. and Lampen, J. 0. (1974). Annals of the New York Academy of Sciences 235, 137. Lampen, J. 0. (1968). Antonie van Leeuwenhoek 34, 1. Lee, Y. C. and Ballou, C. E. (1965). Biochemistry, N e w York 4, 257. Lee, Y. C. and Scocca, J. R. (1972). Journal of Biological Chemistry 247, 5753. Lehle, L. and Tanner, W. (1974). Biochimica e t Biophysica Acta 350, 225. Li, Y. T. (1967). Journal of Biological Chemistry 242, 5474. Li, Y. T. and Lee, Y. C. (1972). Journal of Biological Chemistry 247, 3677. Lindberg, B. (1949). Acta Chemica Scandinavica 3, 1153. Lipke, P. N., Raschke, W. C. and Ballou, C. E. (1974). Carbohydrate Research 37, 23. Lloyd, K. 0. (1970). Biochemistry, N ew York 9, 3446. Lloyd, K. O., Kabat, E. A., Layng, E. J. and Gruezo, F. (1966). Biochemistry, New York 5, 1489. Matile, Ph., Moor, H. and Robinow, C. F. (1969). In “The Yeasts”, (A. H. Rose and J. S. Harrison, ed.), vol. 1, pp. 219-302. Academic Press, London. Matile, Ph. and Wiemken, A. (1967). Archiv fti’rhfikrobiologie 56, 148. Matsuda, K., Watanabe, H., Fujimoto, K. and Aso, K. (1961). Nature, London 191, 278. Mayer, R. M. (1971). Biochimica e t Biophysica Acta 252, 39. Mendoza, C. G., Lopez, M. D. G., Uruburu, F. and Vallanueva, J. R. (1968). Journal of Bacteriology 95, 2393. Mill, P. J. (1966). Journal of General Microbiology 44, 329. Mortimer, R. K. and Hawthorne, D. C. (1969). In “The Yeasts”, (A. H. Rose and J. S. Harrison, eds.), voI. 1, pp. 385-460. Academic Press, London. Mundkur, B. (1960). Experimental Cell Research 20, 28. Nakajima, T. and Ballou, C. E. (1974a). Journal o f Biological Chemistry 249, 7679. Nakajima, T. and Ballou, C. E. (1974b). Journal of Biological Chemistry 249, 7685. NeEas, 0. (1971). Bacteriological Reviews 35, 149. Neumann, N. P. and Lampen, J. 0. (1967). Biochemistry, New York 6,468. Nikaido, H. and Hassid, W. Z. (1971). Advances in Carbohydrate Chemistry 26, 351. Peat, S., Whelm, W. J. and Edwards, T. E. (1961). Journal of the Chemical Society 29. Phaff, H. J. (1971). In “The Yeasts”, (A. H. Rose and J. S. Harrison, eds), vol. 2, pp. 135-210. Academic Press, London. Raizada, M. K., Kloepfer, H. G., Schutzbach, J. S. and Ankel, H. (1974). Journal of Biological Chemistry 249, 6080. Raschke, W. C. and Ballou, C. E. (1971).Biochemistry, New York 10, 4130. Raschke, W. C. and Ballou, C. E. (1972). Biochemistry, N e w York 11, 3807.

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Raschke, W. C., Kern, K. A., Antalis, C. and Ballou, C. E. (1973). Journal of Biological Chemistry 248, 4660. Rosenfeld. L. (1974). Ph.D. thesis: Universitv of California. Berkelev. Rosenfeld; L. ‘and Ballou, C. E. (1974a). Jburnal of Biological Chemistry 249, 2319. Rosenfeld, L. and Ballou, C. E. (1974b). Carbohydrate Research 32, 287. Rosenfeld, L. and Ballou, C. E. (1975). Biochemical and Biophysical Research Communications 63, 571. Sandula, J. and Vojtkovi-LepHikovi, A. (1974). Folio Microbiologiya 19, 94. Sarkar, S. (1974).Journal of Reproductive Medicine 13, 93. Schutzbach, J. S. and Ankel, H. (1969). Federation of European Microbiological Societies Letters 5, 145. Schutzbach, J. S. and Ankel, H. (1971). Journal of Biological Chemistry 246, 2187. Schutzbach, J . S., Raizada, M. K. and Ankel, H. (1974). Journal of Biological Chemistry 249, 2953. Sena, E . P., Radin, D. N. and FogeI, S. (1973). Proceedings of the National Academy of Sciences of the United States of America 70, 1373. Sentandreu, R. and Lampen, J. 0. (1971). Federation of European Biochemical Societies Letters 14, 109. Sentandreu, R. and Lampen, J. 0. (1972). Federation of European Biochemical Societies Letters 27, 331. Sentandreu, R. and Northcote, D. H. (1968). Biochemical Journal 190,419. Sharma, C. B., Babczinski, P., Lehle, L. and Tanner, W. (1974). European Journal o f Biochemistry 46,35. Slodki, M. E . (1963). Biochimica e t Biophysica Acta 69, 96. Slodki, M. E., Safranski, M. J., Hensley, D. E. and Babcock, G. E. (1970). Applied Microbiology 19, 1019. Smith, W. L. and Ballou, C. E. (1974a). Biochemistry, New York 13, 355. Smith, W. L. and Ballou, C. E. (1974b). Biochemical and Biophysical Research Communications 59, 314. Smith, W . L., Nakajima, T. and Ballou, C. E. (1975). Journal of Biological Chemistry 250, 3426. Spencer, J . F . T. and Gorin, P. A. J. (1973). Biotechnology and Bioengineering 15, 1. Spencer, J . F . T., Gorin, P. A. J. and Rank, G. H. (1971). Canadian Journal of Microbiology 17, 1451. Stewart, T. S. and Ballou, C. E. (1968). Biochemistry, New York 7, 1855. Stewart, T. S., Mendershausen, P. B. and Ballou, C. E. (1968). Biochemistry, N e w York 7, 1843. Summers, D. F., Grollman, A. P. and Hasenclever, H. F. (1964). Journal of Immunology 92, 491. Sunayama, H. (1970). Japanese Journal of Microbiology 14, 27. Sunayama, H. and Suzuki, S. (1970).JapaneseJournal of Microbiology 14, 371. Suzuki, S. and Sunayama, H. (1968).JapaneseJournal of Microbiology 12, 413. Suzuki, S. and Sunayama, H. (1969). Japanese Journal of Microbiology 13, 95. Suzuki, S., Sunayama, H. and Saito, T. (1968). Japanese Journal of Microbiology 12, 19. Tanner, W . (1969). Biochemical and Biophysical Research Communications 35, 144.

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Tanner, W. and Jung, P. (1971). Federation of European Biochemical Societies Letters 16, 245. Tarentino, A. L. and Maley, F. (1974). Journal of Biological Chemistry 249, 811. Tarentino, A. L., Plummer, T. H. and Maley, F. (1974). Journal of Biological Chemistry 249, 818. Taylor, N. W. (1964). Journal of Bacteriology 88, 929. Taylor, N. W. (1965). Archives of Biochemistry and Biophysics 111, 181. Taylor, N. W. and Orton, W. L. (1967). Archives of Biochemistry and Biophysics 120, 602. Taylor, N. W. and Orton, W. L. (1968). Archives of Biochemistry and Biophysics 126,912. Taylor, N. W. and Orton, W. L. (1970). Biochemistry, New York 9, 2931. Taylor, N. W. and Orton, W. L. (1971). Biochemistry, New York 10, 2043. Thieme, T. R. and Ballou, C. E. (1970). Biochemical and Biophysical Research Communications 39, 621. Thieme, T. R. and Ballou, C. E. (1971). Biochemistry, New York 10,4121. Thieme, T. R. and Ballou, C. E. (1972). Biochemistry, New York 11, 1115. Van der Veen, J. M. (1963). Journal of Organic Chemistry 28, 564. van der Walt, J. P. (1970). In “The Yeasts. A taxonomic study”, 0.Lodder, ed.). pp. 555- 718. North-Holland Publishing Co., Amsterdam and London. Warren, C. D. and Jeanloz, R. W. (1975). Biochemistry, New York 14,412. Wright, A. and Kanegasaki, S. (1971).Physiological Reviews 51, 748. Yen, P. H. and Ballou, C. E. (1973). Journal of Biological Chemistry 248, 8316. Yen, P. H. and Ballou, C. E. (1974a). Biochemistry, New York 13, 2420. Yen, P. H. and Ballou, C. E. (1974b). Biochemistry, New York 13, 2428. NOTE ADDED IN PROOF

Several publications related to this review have appeared since the manuscript was sent to the printer. Lehle and Tanner (1975) have described the formation in yeast membrane particles of a substance with the composition Man2 GNAc2 Pz- dolichol, a potential intermediate for the synthesis of the polysaccharide to asparagine linkage of manno-protein. This provides support for the biosynthetic scheme proposed herein. A more detailed structural analysis of the inner core linkage fragments from mannan has been reported by Nakajima and Ballou (1975a). Nakajima and Ballou (1975b) have also described procedures for the solubilization and selective assay of four mannosyltransferases that are involved in mannan biosynthesis in Saccharomyces. Finally, Ballou (1975) has published results concerning the genetic mapping of the mnn2 and mnn4 mutants of Sacch. cerevisiae x2180. ADDITIONAL REFERENCES

Ballou, D. L. (1975).Journal ofBacteriology 123, 616. Lehle, L. and Tanner, W. (1975). Biochimica et Biophysica Acta 399, 364. Nakajima, T. and Ballou, C. E. (1975a). Biochemical and Biophysical Research Communications 66, 870. Nakajima, T. and Ballou, C. E. (197513).Proceedings o f t h e National Academy of the United States oj.America 72, 3912.

High-pressure Microbial Physiology ROBERT E. MARQUIS Department of Microbiology, University of Rochester School of Medicine & Dentistry, Rochester, New York 14642 U.S.A.

.

I. Introduction A. Background B. Basic Methodology 11. Information from High-pressure Chemistry 111. Life and Death under Pressure A. Long-Term Survival and Growth B. Short-Term Survival and Death . IV. Effects of Pressure on Biopolymers A. Nucleic Acids B. Protein Denaturation . C. Effects of Pressure on Polymeric Interactions V. Effects of Pressure on Some Specific Microbial Cell Functions A. Permeability and Transport Reactions B. Catabolic Processes C. Biopolymer Synthesis . D. Cell Division and Morphological Differentiation E. Regulatory Mechanisms F. Motility G. Luminescence . VI. Acknowledgements . References .

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159 159 163 168 174 174 185 191 192 195 202 211 211 213 222

2 B 231 233 233 234 234

I. Introduction A. BACKGROUND

Although this article will focus primarily on physiological aspects of barobiology, it seems appropriate at the onset t o point out that the subject had its origins in marine ecology. As a result of the 159

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weight of the water column in the ocean, or in other bodies of water, hydrostatic pressure increases by about one atmosphere for every ten metres depth. The average pressure on the ocean floor is approximately 380 atmospheres, and the greatest pressure, at the bottom of the Challenger Deep in the Pacific Ocean, is some 1160 atmospheres (Kinne, 1972). In many ways, the.ocean depths can be considered inhospitable places-cold, dark and with a high ambient pressure. In fact, the adjective “hadal” that is applied to parts of the ocean deeper than about 6500 metres has a forbidding sound. Many old legends suggest that the deep was the home of abominable benthos. However, the view that developed as a result of early Nineteenth Century exploratory samplings was that the deep was devoid of life. For example, in his cataloguing of the life in the Aegean Sea, the Nineteenth Century oceanographer Edward Forbes proposed that the region below 300 fathoms was an azoic region. Since sunlight cannot penetrate so far, it could also be concluded that any plants that might exist below this depth would have t o be non-photosynthetic. The results of the voyages of oceanographic vessels in the latter part of the Nineteenth Century showed clearly that Forbes was incorrect in his proposal. During the voyage of the French Talisman in 1882-1883, living organisms, including animals, were recovered from dredgings at 6000 metres. The finding of life in the deep regions stimulated two French scientists, P. Regnard and A. Certes, to assess independently the effects of deep-sea pressures on a variety of physiological processes in a variety of organisms, including microorganisms. There is no need here to review the details of the early work in barophysiology. Summaries of various parts of it, with references, appear elsewhere. Useful background information with an historical perspective is presented in the book by Johnson, Eyring and Polissar (1954) and in the review by Cattell (1936). Also, Fenn (1970) has written an interesting review of Regnard’s book (1891), “Recherches expirimentales sur les conditions physiques de la vie dans les eaux”. In addition, a number of books have been published recently on the general subject of barobiology, including one edited by Zimmerman (1970), one by Brauer (1972) and one by Sleigh and Macdonald (1972). This particular article will focus on the barophysiology of micro-organisms, primarily bacteria, with the aim of developing some feeling for the molecular aspects of the pressure responses. Unfortunately, molecular barophysiology is still very

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much in its infancy, and most of our knowledge is still at the level of observing and recording phenomena. However, interest in the details of the effects of hydrostatic pressure on biopolymers has risen sharply in the last few years, and the time seems appropriate to synthesize a more molecularly oriented picture of the action of high pressure on physiological processes. Throughout this article, units of pressure will be expressed as atmospheres since this is still the most familiar unit. One atmosphere is equal to 14.696 Ib/in2, 1.033 kg/cm2 or 1 . 0 1 3 ~ lo5 Newtons/m*. High pressures can occur also in the biosphere in oil and sulphur wells, where the pressure may be as high as 400 atmospheres (ZoBell, 1970). The deep-sea environment is characterized by low temperatures, close to O°C, and high pressures; deep wells are characterized by high temperatures in the range of 60 to 105OC and high pressures. Samplings from both types of compressed environments have yielded viable bacteria, although there is currently some debate as to whether these bacteria actually grow in situ or are in a state of suspended animation. However, animals have been recovered from all depths of the ocean and have been photographed with the aid of remote cameras. Even though most of them are relatively lower forms of life, they still have a microbial flora associated with them. In their historic descent in the bathyscaph Trieste, Piccard and Walsh reported seeing what looked like fish and a red shrimp at the bottom of the Challenger Deep. In all probability, they saw some o f the giant amphipods that live as scavengers in abyssal waters. These animals are voracious, and the photos taken by Hessler et al. (1972) show them rapidly devouring fish used as lures to attract them. The gut bacteria of these amphipods must be adapted to function at abyssal pressures, and attempts are currently underway to grow them in the laboratory. There are a few known bacteria that can grow under laboratory conditions at pressures extant in the deep sea. For example, Pseudomonas bathycetes can grow at 1000 atmospheres. The greatest pressures encountered in fresh-water regions are considerably lower than those of the deep sea. The pressure at the nadir of the world’s deepest lake (Lake Baykal in the Soviet Union) is only about 160 atmospheres. However, even lower pressures can have very definite effects on microbial cells. For example, pressures of only about 100 atmospheres can completely inhibit flagella

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formation by Escherichia coli that is capable of growth at pressures as high as 550 atmospheres (Meganathan and Marquis, 1973). Walsby (1971, 1972) found that the gas vacuoles of Halobacterium strains can be collapsed by pressures of only about two atmospheres. The vacuoles of blue-green algae proved to be somewhat more resistant to collapse, but still one can envisage a situation in which these vacuoles would be collapsed by pressure in relatively shallow waters, and that this collapse would decrease the tendency of the organisms to rise to the surface where the light for photosynthesis is most intense. Obviously, the extreme pressure sensitivities here are due to the highly compressible gas phases within the vacuoles. Much higher pressures are needed to bring about any major changes in the volumes of the condensed liquid and solid phases that make up the substance of most microbial cells. Cells that lack gas vacuoles undergo almost no change in volume when compressed to 1000 atmospheres. The data of Pollard and Weller (1966) indicate a change of only 1.1% in relation to the initial average volume of E. coli cells after compression to 890 atmospheres. In fact, the point will be developed later that pressure affects the microbial cell primarily by inhibiting condensed phase biochemical reactions that result in an increase in volume, and by stimulating those reactions that result in a decrease in volume. In a compressed microbial culture, or suspension, pressure is rapidly distributed throughout the system immediately after it is applied. Therefore, there is no point in the system at which the pressure is greater than at any other point. The compressibilities of various components may differ, but still the pressure is uniform throughout the system. This uniformity is in contrast to the situation that occurs when bacteria are suspended in hypotonic media. Here water moves across the water-permeable plasma membrane to equalize the water activity on both sides of the membrane. The cell swells as a result of this water movement but, as it does, the elastic surface structures become more and more tense and compress the interior of the cell. This compression results in an increase in the hydrostatic pressure within the cell and an increase in the intracellular water activity. In the final turgid state of the cell, the surface structures are tensed, the water activity within the cell is equal to that outside, and there is a difference in pressure between the interior and the exterior of the cell. This difference is at most only about 30 atmospheres, and

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there is no knowledge concerning whether or not these low pressures have any significant physiological effects on cells that do not have gas vacuoles. Again, as mentioned, the application of hydrostatic pressure to cell suspensions generally does not result in an uneven distribution of pressure or change in turgor, unless of course pressure induces some loss of intracellular solutes. Compression causes transient increases in temperature, which have been measured with a bead-type thermistor by ZoBell (1959). Rapid compression to deep-sea pressures resulted in temperature increases ranging from 2 to 5°C. Decompression resulted in cooling of the same magnitude. These changes in temperature ordinarily do not create major problems, although they could cause damage at extreme temperatures. There are devices that employ pressure for breaking intact cells or fragmenting polymers. However, the damage is not due simply to compression and decompression. Thus, in the French pressure cell, the compressed material is forced out through a small opening in a needle valve, and in the Pan apparatus high-pressure nitrogen is used to supersaturate the material, which is then rapidly decompressed to produce cavitation. Simple compression and decompression do not seem to harm micro-organisms, and multiple cycles of pressurization do not result in decreases in viable counts of bacterial suspensions. As a final background note, it seems worthwhile to point out that the importance of hydrostatic pressure in microbiology is not confined t o aquatic ecology. Pressure has great potential, in both basic and applied microbiology, as a perturbing force that may be used to control many microbial activities, for deciphering the molecular details of physiological processes, for selecting conditional mutants, for preserving labile materials and for many other uses. B. BASIC METHODOLOGY

Morita (1970) has written a chapter describing in some detail procedures for applying high pressures to microbial cultures or suspensions. One of the basic set-ups used in the author’s laboratory is shown in Fig. 1. Here an hydraulic pump, with an attached Bourdon tube gauge for measuring pressure, is connected to a combined inlet-bleeder valve. There is a reservoir from which hydraulic fluid is fed into the pump. The fluid may be oil, or water,

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FIG. 1. Typical apparatus, described in the text, for subjecting micro-organisms to high pressure.

or more commonly in biological studies, an equal mixture of glycerin and water. In the mixture, the glycerin serves to diminish corrosion and retards fouling microbial growth. One of the exit ports of the inlet-bleeder valve is connected to a pressure vessel by means of high-pressure steel tubing. ZoBell has suggested that the term barokarn be used to refer to a pressure vessel. The barokam shown in Fig. 1 is the type most commonly used in microbiological work. It is of the general type described by Johnson and Lewin (1946), with the modifications of Oppenheimer and ZoBell (1952), which include an O-ring seal and a threaded male fitting on the cap that will take a high-pressure needle valve with a female fitting. Basically, the barokam is a hollowed out cylinder of 300-series steel with a threaded top that is closed with an overlapping threaded cap. As mentioned, it is usually convenient to have a male fitting on the top of the cap so that a standard high-pressure valve can be attached. Then the tubing from the pump can be connected to the valve by means of a high-pressure gland-nut fitting. The system can be

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pressurized after this connection is made, the needle valve closed to seal in the contents of the barokam, and the system up to thevalve decompressed by opening the bleeder valve. The pressurized barokam, and its attached valve, can then be disconnected and incubated under the desired experimental conditions. There are many modifications of the basic barokam, and there are numerous manufacturers of high-pressure apparatus, many of them with their own designs. A list of suppliers of high-pressure equipment appears in the book “Barobiology and the Experimental Biology of the Deep Sea” edited by Brauer (1972). Barokams can be fitted with electrical leads so that measuring devices such as conductivity cells, pH electrodes or other specific ion electrodes can be placed in the chambers, and changes in conductivity, pH value or ion activity under pressure can be recorded from outside the chamber. It is possible to purchase barokams with optical windows made of quartz or sapphire, such as those manufactured by the American Instrument Co. (Silver Springs, Maryland, U.S.A.). For pressures below about 50 atmospheres, transparent Lucite chambers have been used (Fenn and Marquis, 1968) but, for higher pressures, steel vessels are required. Many investigators prefer to have custom-made optical barokams, for example, according to the designs of Mohankumar and Berger

(1972). There is often a need to mix various ingredients under pressure, and there are relatively simple ways of doing this. Landau and Thibodeau (1962) described a two-compartment chamber with a glass cover-slip barrier that can be broken under pressure by means of a steel ball that is rolled from one compartment through the glass to the other. An even simpler method, which we have used, is to make up a solution or suspension of one of the ingredients to be mixed under pressure in a concentrated solution of Carbowax 4000 (polyethylene glycol from Union Carbide Co., New York). The highly polymerized Carbowax molecules are too large to pa% through the pores in the walls of bacteria and so do not harm the cells. However, Carbowax increases the viscosity and density of the solution, or suspension, so that a very stable interface can be formed. We generally include a glass bead in the Carbowax phase and then layer a less dense phase on top. The interface is stable during a series of pressurization and depressurization cycles. Then, when the solutions or suspensions are to be mixed, it is only necessary to invert the

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barokam a number of times and allow the glass bead to roll back and forth. For more complicated mixing under pressure, custom-built apparatus is necessary. Another recurrent problem in barophysiological experiments is that of supplying terminal electron acceptors for respiration of cells in enclosed pressurized environments. Obviously, one way around the problem is to use bacteria that do not require oxygen, but this approach is often more of a retreat than a solution. For many facultative or aerobic bacteria, non-gaseous electron acceptors such as nitrate can be used. Unfortunately, because of its toxicity, the level of potassium nitrate that can be used with cultures of bacteria, such as E. coli, is limited to about 0.1% (w/v) or about 9.9 mM. One millilitre of a 9.9 mM solution of potassium nitrate is equivalent, in electron-accepting capacity, to a solution containing from 158 to 396 pg O2 per millilitre, depending on the final state of reduction of the added nitrogen. Water, in equilibrium with air at 25"C, contains about 8 pg O2 per millilitre. However, a culture with a Y o (yield of cells in grams dry weight per gram-atom of oxygen consumed) of 10, and a culture yield of 1 gram dry weight of cells per litre, would require 0.1 gram-atom of oxygen per litre, or 1.6 mg O2 per millilitre, or 1600 pg O 2 per millilitre. Thus, even when nitrate is added to a rich culture medium containing a bacterium such as E. coli, it is used up before growth is complete, and the final stages of growth are anaerobic. Another method for supplying oxygen under pressure is that of Berger and Tam (1970). They used gas-permeable silicone membranes to make heat-sealable sacs for dialysis cultures. The fluid outside the sacs could then serve as a reservoir for oxygen for the cultures within the sacs. The solution we have found most effective is the use of fluorocarbon liquids manufactured by the Minnesota Mining and Manufacturing Company (St. Paul, Minnesota, U.S.A.). The compounds are biologically inert and have high capacities as solvents for oxygen and other gases. They have been used dramatically for liquid breathing in small animals, and it has been found that mice can respire and remain aIive when totally submerged in oxygenated fluorocarbons (Clark and Gollan, 1966; Kylstra e t al., 1967). The fluorocarbon liquid we have used most extensively is designated

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FC-80. It is composed largely of perfluorobutyltetrahydrofuran and its isomers. After FC-80 is equilibrated with air at 25"C, it contains 0.093 gram (1.90 mmole) oxygen per kilogram. This value compares with about 0.008 gram (0.25 mmole) oxygen per kilogram water. Thus, one kilogram of FC-80 is equivalent in oxygen-dissolving capacity to 11.6 kilograms of water. The fluorocarbon liquid FC-80 has a high density of 1.76 gram per millilitre and thus, as an oxygen reservoir, one millilitre of FC-80 is equivalent to 20.4 millilitres of water. However, it would still take between 9 and 1 0 millilitres of FC-80 per millilitre of culture to satisfy the oxygen demand of the culture described above, and so we often use a combination of nitrate and FC-80. When oxygenated FC-80, which is immiscible with water, is added to an enclosed culture, there is initially an equilibration leading to equal oxygen activities in the aqueous and fluorocarbon phases. Then, as the bacteria in the culture use oxygen, the gas moves from the fluorocarbon phase to the aqueous phase to maintain equality of activity until essentially all of the oxygen, or at least all of it that is detectable by the azide modification of the standard iodometric method, is used up. The rate of movement of oxygen is, of course, limited by the surface area between the culture and the FC-80. Fortunately, when mixtures are $haken, the FC-80 breaks into small beads, thus increasing the surface area and the rate of oxygen transfer. Literature on the properties of fluorocarbon liquids is available from the Minnesota Mining and Manufacturing Company. It should be pointed out that not only do fluorocarbon liquids act to supply oxygen to cultures, but they also serve as sinks for carbon dioxide and other gases. This removal of carbon dioxide from the cultures may or may not be desirable. With many bacteria, it is possible to introduce some oxygen into the pressure vessels used, and to expose the cells to mildly hyperbaric oxygenation without harming them. We have found that at least some strains of E. coli can tolerate a few atmospheres of oxygen at pressures up to 400 atmospheres. However, there is always the danger of toxic oxygen effects, and extra caution is needed since pressure potentiates the damaging action of hyperbaric oxygen (ZoBell and Hittle, 1967).

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11. Information from High-Pressure Chemistry

The most basic tenet for interpreting pressure effects is that pressure enhances processes that result in volume decreases and diminishes those that result in volume increases. Therefore, one can obtain an estimate of just how pressure will affect any process from a knowledge of the net change in volume that accompanies the process. Conversely, if one knows how pressure affects the process, we can estimate the volume change. There are two types of volume change that are considered in pressure chemistry, the volume of reaction (AV) and the apparent volume of activation (AVZ). They are defined by the following basic equations:

(y)

= -AV/RT

where K is the equilibrium constant, k is the reaction rate constant, R is the gas constant (82.06 ml-atm/mole/°K), T is the absolute temperature in degrees Kelvin, and P is the pressure. For derivations of these equations from standard thermodynamic relationships, books on high-pressure chemistry, such as that by Weale (1967), shouId be consulted. Also, a recent review article by Johnson and Eyring (1970) is helpful in that it considers the kinetic bases for biological pressure effects. Equations 1 and 2 indicate that a plot of In K versus pressure should yield a straight line with slope -AV/RT and a plot of In k versus pressure should yield a straight line with slope -AVZ /RT. Thus these basic equations imply that AV and AVZ are independent of pressure, which has been found to be more the exception than the rule for biological processes, or, for that matter, for most chemical processes. In part, deviations arise from differences in the compressibilities of reactants and products, but other factors also may be involved. However, it is still clear that, despite any such deviations, AV and AVZ are proper measures for quantitative comparisons of the pressure sensitivities of various processes. In barobiology, it is common to use the integrated forms of the equations and to

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calculate AV or AVt: from data obtained at one atmosphere pressure and at some particular higher pressure. Then:

Av=

2.30 RT p - 1 log K 1 IKP

2.30 RT AVS = p log k , /kP

(3)

(4)

where K1 and K, are equilibrium constants at one atmosphere and under pressure, and k , and k, are reaction rate constants. Thus, a positive AV value indicates that pressure shifts the equilibrium of the reaction towards reactants, and a negative AV value indicates a shift towards products. A positive AV* value indicates that the reaction rate is slowed by pressure, and a negative AVS value indicates that it is accelerated. The value of AV can be estimated in two essentially independent ways: (i) from a knowledge of the change in K with change in pressure, and (ii) from more direct measurements of the change in volume of the reaction mixture in a dilatometer vessel. The value of AVS can be estimated in only one way, that is from the effects of pressure on reaction rate. van’t Hoff (1901) was one of the first to consider the effect of pressure on reaction rates in terms of a volume change, although he does refer to earlier work by Planck. van’t Hoff’s equations were developed by use of arguments that are similar to those used to develop the Arrhenius equation. They contain an exponential “Av” term, which was subsequently considered in transition-state theory to be the volume of activation o r AV$ . It is common to refer to AV$ as the apparent volume of activation, largely because it has two, somewhat different, meanings. In the most general sense, it is simply a modulus that relates changes in reaction rate to changes in pressure. In a more specific sense, related to transition-state theory, it is the change in volume associated with formation of the activated complex. Equations (2) and (4)can be applied to any sort of process for which rate data can be obtained, including complex ones such as microbial growth or bioluminescence. However, it often requires a fair bit of dissection of any complex sequence of reactions to find out which steps in the sequence are primarily responsible for barosensitivity. What are the bases for the changes in volume that accompany

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chemical reactions? In general terms, it can be said that in most chemical reactions the sum of the partial molar volumes of products differs from that for reactants, and that this difference is the primary basis for the volume change. For a biological example, we could choose to consider glycolysis by homofermentative lactic-acid bacteria in which glucose is broken down to lactic acid. The partial molar volume of glucose is about 111.8 ml/mole, and that for lactic acid is 67.7 ml/ mole. Therefore, the volume change for glycolysis should be ( 2 x 67.7) - (1 x 111.8), or 23.6 millilitres per mole of glucose used, or 11.8 millilitres per mole of lactic acid produced. Values very close t o the predicted values were measured (Marquis and Fenn, 1969) with simple dilatometers, each of which consisted of a large glass reaction vessel connected to a calibrated capillary tube. Volume changes of the reacting system were monitored by following the movement of a meniscus in the capillary tube. Of course, i t is also possible to assess volume changes by monitoring changes in the density of the reacting system. In glycolysis, the volume change can be related primarily to that accompanying breakage of a carbon-carbon bond, which is about 1 2 ml/mole (Whalley, 1964), and volume increases can be expected in any reactions involving the breaking of covalent bonds. Another component of the volume change here is that involving changes in the interactions of reactants or products with solventwater in biological systems. A major component of the volume change in glycolysis is due to ionization of the lactic acid. If glycolysis takes place at, say, pH 7, well above the pK, of lactic acid (about 3.9), the acid will dissociate t o yield a proton and lactate anion. The dissociation is accompanied by a volume change of -11.7 ml/mole, largely due to electrostriction of water in the vicinities of the ions. Thus, the AV value of dissociation is opposite to that of carbon-carbon bond breakage, and sufficiently large nearly t o eliminate the dilatation of the system due to bond breakage. Why then is there a volume increase during glycolysis? Generally cells are buffered, and when lactic acid dissociates, the proton does not remain free but associates with a buffer ion. If this association decreases the number of ions in the system, there will generally be an increase in volume. The amount of the increase depends on the particular buffer present. For example, it is unusually large (approximately +24 ml/mole) for phosphate buffer in the physiological pH range, and is some +11 mlfmole for carboxyl buffers. For some

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buffers, there is no change in the number of ions as the result of protonation, and moreover, there can even be a decrease in volume associated with the buffer reaction. For example, protonation of imidazole buffer results in a volume decrease of -1.1 ml/mole, and protonation of the amino group of diglycine results in a volume decrease o f -4.4 ml/mole. Distiche (1972) has written an excellent review of pressure effects and volume changes for acid-base reactions. Other useful information on AV values for buffer reactions can be found in articles by Neuman et al. (1973) and Kauzmann et al. (1962), and in the book by Johnson et al. (1954). Therefore, it is necessary when studying volume changes of biochemical reactions to take into account all processes that occur, including buffer reactions; and in the study of glycolysis cited, it was possible t o alter considerably the measured volume change per mole of lactate by changing the buffer. However, the net volume change was always equal to that predicted from a knowledge of the partial molar volumes of all of the reactants and all of the products. With intact cells, the process of glycolysis does not result in a net build-up of ATP, and so phosphorylation reactions need not be considered. Also, although biosynthetic reactions do involve changes in volume, the extent of the synthetic reactions is small compared with the extent of catabolic reactions, and so nearly all of the volume change in growing or in non-growing cell suspensions is due to catabolism. In other words, it matters little whether the cells are growing or not when one determines the AV value for glycolysis. A similar situation occurs with microcalorimetric determinations (Forrest and Walker, 1971) in which the heat output of growing cultures is due mainly to catabolism, with an insignificant contribution from biosynthetic reactions. Indeed, dilatometry is in many ways similar to calorimetry. It yields information on the overall change in a basic thermodynamic parameter (that is, volume) in any reacting system. It is then possible to calculate the energy directed into pressurevolume work in the system simply by multiplying the measured AV value by the pressure. The amount of pressure-volume work in most biological processes is small at one atmosphere, and even under pressure is not large. For example, the change in volume accompanying glycolysis in Streptococcus faecalis in growth medium at 410 atmospheres was found by Marquis e t al. (1971) to be 15.7 millilitres per mole lactate. Therefore, the pressure-volume work is: 15.7

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cm3/mole x 410 atmospheres x 1033.3 g/cm2/atmosphere, or 6.65 x 1O6 g-cm/mole. The energy derived from glycolysis is approximately 29,000 calories per mole lactate, or 12.37 x lo8 g-cm/mole. Therefore, the work of dilatation or volume increase is only (6.65 x lo6 x 100)/(12.37 x l o 8 ) , or 0.54% of the energy obtained from forming one mole of lactic acid. If -glycolysis were carried out at the bottom of the Challenger Deep in the Pacific Ocean, where the ambient pressure is about 1170 atmospheres, the work of dilatation would still consume only about 1.5% of the total energy available from glycolysis. Thus, pressure-volume work does not represent much of an energetic drain, even at the highest pressure in the biosphere. In general, reactions that result in an increase in the number of ionized groups in aqueous systems are accompanied by a decrease in volume, which is attributable mainly to electrostriction of water in the vicinity of the ions. As a consequence, pressure tends to dissociate electrostatic interactions so that more ions are exposed to water. One of the most studied examples is the dissociation of hydronium and hydroxyl ions from water itself. The reaction: H2 0 H+ + OH- is accompanied by a volume decrease of 21.3 ml per mole at 25°C (Bodanszky and Kauzmann, 1962). Pressure then favours dissociation of water, and the pH value of pure water changes from 7.00 at one atmosphere, and 25”C, to 6.27 at 1000 atmospheres (Owen and BrinMey, 1941). Pressure tends also to dissociate hydrophobic interactions, and again, much of the volume change in dissociation is thought t o be due to changes in water solvent. As Kauzmann (1959) pointed out, the formation of water clathrates or “icebergs” around polar groups results in a decrease in volume-in contrast to the dilatation that occurs during ordinary ice formation. Hydrogen bonding results in a decrease in volume and so is enhanced by pressure. Since the structure of water is very much dependent on hydrogen-bonding between molecules, pressure has significant effects on the properties of water, even in the biospheric pressure range below 1200 atmospheres. A review of the effects of pressure on the properties of water has been written by Drost-Hansen (1972). Table 1 presents a summary of the effects of pressure on biologically important chemical bonds with specific examples. --f

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Knowledge o f AV values for biological reactions or processes, especially those involving polymeric interactions, has been very useful in barobiology since, in many cases, the observed pressure effect is due to the shifting of an equilibrium; for example, the equilibrium between the native and denatured states of a protein, or between multimeric and monomeric forms of an enzyme. However, very often, the pertinent volume change is not the AV value of reaction but is the AVZ value, the apparent volume of activation, which can be calculated only from knowledge of the effect of pressure on reaction rate. In the living cell, biochemical reactions are multiply coupled t o one another, and we usually introduce a somewhat erronTABLE 1. Volume changes associated with chemical bond breakage at 25°C Bond type Covalent

Example

AV (ml/mole)

c-c

+12 -21

Inhibits bond breakage

-23

Disrupts hydrophobic interactions

+4

Enhances hydrogen bonding

Ionic

HzO -+ H+ + OH-

Hydrophobic

CH4 in hexane + CH4 in water

Hydrogen

-OH.

I I . . 0 + -OH + 0 I I

Effect of pressure

Disrupts electrostatic interactions

eous abstraction when we consider one, or only a few, reactions in the huge interlaced network of reactions. The overall equilibrium for a coupled series of reactions such as glycolysis in a cell lies very far in the direction of products. The equilibrium constant for glycolysis with lactic acid as an end product is between 10' and 10' 5 , depending on pH value (Neilands and Stumpf, 1965). Thus, even if pressure were to change the glycolysis equilibrium constant by a few orders of magnitude, it would have almost no measurable effect on the amount of glucose converted to lactic acid. Pressure does significantly affect the rate of glycolysis and commonly, in biological experiments, we are concerned with rate-how rapidly glucose is broken down to lactic acid and how rapidly ATP is supplied for biosynthetic reactions. Therefore, the pertinent volume change is the AVz value.

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111. Life and Death Under Pressure A. LONG-TERM SURVIVAL AND GROWTH

Before considering specific biochemical and physiological processes, it seems worthwhile to consider the most vital issue in barobiology-whether organisms live or die under pressure. Survival under pressure can be considered in both the short-term and the long-term. Micro-organisms, including bacterial endospores, may be killed rapidly by pressure, particularly by pressures in the kilobar range, or they may die slowly at lower pressures simply because pressure induces physiological imbalances that make survival, for more than one or two generations, impossible. On reviewing the literature on the maximum pressures at which various micro-organisms can grow and survive, one is struck by the wide range of barotolerance among various types, and by the lack of any readily discernible pattern with respect to taxonomic groups. Extensive tables of barotolerance of terrestrial and marine bacteria were prepared by ZoBell and Johnson (1949), and by Oppenheimer and ZoBell (1952). Extreme examples from these catalogues include bacteria such as Achromobacter thalassius, Micrococcus euyhalis and Pseudomonas marinopersica which were killed by only 200 atmospheres pressure, when incubated in sea-water broth at 27OC for eight days. At the baroduric end of the spectrum, bacteria such as Bacillus borborkoites, Micrococcus aquiuiuus and Pseudomonas perfectomarinus were able to grow under these same conditions at pressures as high as 600 atmospheres. Moreover, there are a few still more baroduric bacteria, such as Pseudomonas bathycetes, that can grow at pressures as high as 1000 atmospheres. The highest pressure at which any organism has been reported to grow is 1400 atmospheres (ZoBell's work cited by Vallentyne, 1963). ZoBell (1958) has also cultivated bacteria (that were believed to be thermophilic DesuIfouibrio organisms) under the very extreme conditions of 1000 atmospheres pressure and 104OC, but it was not possible to subculture them. Although it does appear that at least some bacteria can grow under the highest pressures in the biosphere, there is still an important, and presently unanswered, question regarding whether any bacteria can become specifically adapted to growth at high pressures. Highly

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barotolerant bacteria have been isolated from deep ocean samples, but this does not necessarily imply that they are specifically adapted to this environment, although there may have been a selective survival of barotolerant forms. Moreover, there are reports, such as those of Kriss (1962), that highly barotolerant bacteria can be isolated from garden soil, where, presumably, there is no selective pressure favouring barotolerance. In fact, the bacterial population in the garden soil used by Kriss grew better under pressure than did those from deep-sea mud samples, and a highly barotolerant bacterium that could grow at 850 atmospheres was isolated from the soil. A subsequent paper (Kriss and Zaichkin, 1971) dealt with the distribution of barotolerant forms in various types of soil, but barotolerance was assessed only at pressures up to 600 atmospheres. It was pointed out that actinomycetes and fungi in the soil were all completely inhibited by pressures higher than 300 atmospheres, and the authors suggested that at least part of the basis for the sparsity of these forms in the oceanic depths is their barosensitivity. However, we have been able to cultivate a streptomycete in the laboratory at 600 atmospheres and room temperature, and so there do seem to be members of Actinomycetales that are relatively barotolerant, at least at higher temperatures. Attempts to isolate highly barotolerant mutants from stock cultures, with or without mutagen treatment, have been uniformly unsuccessful, although pressure-sensitive mutants, and mutants with slightly enhanced barotolerance, can be obtained. There are claims that pressure itself is a mutagen (Palmer, 1961) as indicated by the finding that incubation under pressure resulted in greater numbers of strep t o my cin-resistan t mutants in Serra t ia marinoru bra cultures. Attempts to repeat this work have been unsuccessful, although a recent report by Pope and Ogrinc (1975) indicates that a streptomycin-resistant mutant of E. coli exhibits increased barotolerance. McElroy and de la Haba (1949) found that pressure increased the numbers of biochemical mutants obtained after nitrogen-mustard treatment of Neurospora crassa, but decreased the numbers of morphological mutants recovered. It seems on review that pressure is probably not acting as a mutagen in these experiments, but that it may favour some mutant types. One interesting change caused by pressure is that reported by Vacquier and Belser (1965). They found that a pressure of 500 atmospheres, for one or two hours, caused a

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sex change from male t o female in eggs of the marine copepod Tigriop us califo rnicus. One might object to the whole notion of barotolerant mutants, and microbial adaptation to growth under high pressures, on the grounds that barotolerance depends on very many biochemical reactions and physiological processes..Therefore, any such adaptation involving changes in large numbers of characters would be highly unlikely. However, the same objection could be raised for the adaptation of micro-organisms t o temperature, and yet there are some bacteria that can more readily adapt reversibly or irreversibly, to growth at higher or lower temperatures than others. One of the most surprising examples of temperature adaptation is that described by Dowben and Weidenmuller (1968). They were able to train strains of Bacillus subtilis, with maximum growth temperatures of about 50°C, to grow at temperatures as high as 72°C by increasing the growth temperature in steps of one or two degrees. This training was not the result of selection of thermotolerant mutants since all of the population became trained, and the ability to grow at high temperature was lost following a few generations of growth at the low temperature. Adaptation of thermophilic bacteria to growth at low temperatures also has been reported (Allen, 1953), and there has been some speculation (Crabb et al., 1975) that changes in the thermostabilities of enzymes in facultative thermophiles may be related to changes in intracellular ion content. In all, although it seems that relatively rapid adaptation to changes in physical factors, such as temperature and pressure, might be prohibited because these factors affect so many cell functions, there is substantial evidence that short-term adaptations t o growth at abnormally high or low temperatures do in fact occur. However, as mentioned, there has been no demonstration of similar, rapid, genetic or physiological adaptation to growth at high pressure. Repeated subculturing of organisms grown under pressure has not to date produced barotolerant variants. Another fundamental question that remains unsatisfactorily answered is whether there exist obligately barophilic bacteria, that is, bacteria that grow only under pressure. During the Galathea expedition, ZoBell and Morita (1957) sampled sediments collected from depths as great as 10,210 metres and determined most probable numbers of bacteria by the tube dilution technique. They found that

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the apparent numbers of aerobes or anaerobes were greater when the samples were incubated under 1000 atmospheres pressure, than when they were cultured at one atmosphere, at 3" to 5°C. Their findings are the primary evidence for the existence of obligately barophilic bacteria. In fact, it appears from their data that these bacteria have become irreversibly adapted to life in the ocean depths. However, barophilic bacteria do not seem to grow well on subculture, and so they have not been subjected to laboratory studies t o find out just how they differ from the more usual barophobic bacteria. Morita and ZoBell (1956) indicated that, at best, their barophiles grew very slowly, and it appears from studies such as those of Jannasch e t al. (1971) that all types of microbial activity may be extremely slow in the deep-sea environment, partly as a result of the high ambient pressures. Pressure can affect all phases of microbial growth. It can prolong the lag phase, especially in cultures inoculated with cells that have to undergo adaptation to a new medium before growth is possible. The most commonly determined effect of pressure on growth is a slowing of the exponential growth rate. ZoBell (1970) has presented summary data, taken from the paper by ZoBell and Budge (1965), to show that pressure also caused a decrease in the yield of biomass with cultures of 30 species of marine, facultatively anaerobic, bacteria grown at 25°C and various pressures up to 1000 atmospheres. As shown in Fig. 2, which was prepared from ZoBell's data, yields declined with increasing pressure for both barophobic and barophilic types. Actually, it appears that, if yield had been used as the growth criterion, the latter type would have been called baroduric or barotolerant instead of barophilic. Marquis and ZoBell (1971) found that Streptococcus faecalis behaved as a typical barophobic species, in terms of decrease of growth rate and yield at higher pressures, with a maximum growth pressure of about 550 atmospheres at 25°C. In fact, the curve for growth rate versus pressure was very similar to that of the yield value. Other studies (Marquis et al., 1971; Matsumura et al., 1974; Matsumura, 1975) revealed that, for Strep. faecalis, the decrease in biomass produced under pressure had at least two causes. First, growth under pressure was inefficient in terms of Y A T P (that is, the yield of cells in grams dry weight, per mole of ATP produced by catabolism). For example, the Y A T P value for exponential-phase cultures in a complex

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Pressure (atmospheres)

FIG. 2. Biomass production at various pressures by cultures of 30 species of marine, facultatively anaerobic bacteria at 25'C. Data are taken from the paper of ZoBell and Budge (1965).

medium, with glucose as the main catabolite, declined from about 15 at one atmosphere to about 10 at 408 atmospheres. Pressure did not appear to affect the pathway for glucose degradation in this bacterium, and essentially the sole product of glycolysis was lactic acid at both one atmosphere and at 408 atmospheres. Chumak and Blokhina (1964) had reported earlier that pressure does alter the mode of glucose degradation by Pseudomonas desmolyticum organisms; more acid and less carbon dioxide was produced under pressure. The inefficiency of exponential growth of Strep. faecalis under pressure appeared to be in large part due to stimulation of membrane ATPase, and yields could be improved by adding to the

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cultures dicyclohexylcarbodiimide, which is an ATPase inhibitor (Matsumura, 1975). Biomass production by Strep. faecalis was also decreased under pressure owing to the growth inhibitory action of low pH values that was potentiated by pressure. Growth of this homo fermentative lactic-acid bacterium, in the medium used, ceased due to the build-up of lactic acid, and neutralization of the acid resulted in renewed growth. The range of pH values over which growth can be initiated in complex medium is about 4.4 t o 9.5, at one atmosphere, but this range was found by Matsumura et al. (1974) to be narrowed t o about 5.3 to 7.5 at 408 atmospheres. Growth in cultures at 408 atmospheres stopped at a considerably higher pH value than did growth in cultures maintained at one atmosphere. Therefore, the biomass produced under pressure was much less than that produced at one atmosphere. Moreover, since glycolysis was less sensitive t o low pH values than was growth, yields per mole of ATP produced declined under pressure. It should be mentioned that inefficient growth under pressure, by exponentially-growing cultures, is not related t o acid sensitivity. In other words, there are two effects here, both of which lead to low Y A T p values. It was possible to increase biomass production by Strep. faecalis cultures under pressure by buffering the growth medium, or by intermittent neutralization of the culture, although YA T p values under pressure were still decreased. Narrowing of the range of pH values for growth, by pressure, appeared to be nearly a universal phenomenon for a whole range of micro-organisms. Decreased yields under pressure, for a number of species including E. coli and S. marinorubra, appeared to be due at least in part to enhanced acid sensitivities, since yields could be increased by neutralization of metabolic acids that accumulated in the closed culture vessels which were used. In all, it appeared that bacterial growth in general is inefficient under pressure, and that low yields can be related in at least some cases to enhanced acid sensitivity and t o disruption of normal ATP coupling between catabolic and biosynthetic processes. ZoBell (1970) found also that four of his isolates, which he referred to as preferential barophiles, grew better, in terms o f yield, at 100 or 200 atmospheres than at one atmosphere. However, this enhancement of growth by low pressures may not be unique to barophiles. Marquis and Keller (1975) found that the rate, and

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extent, o f growth of E. coli B organisms, in a defined medium at 3OoC with 0.1% potassium nitrate, was greater at 200 atmospheres than at one atmosphere, or at higher pressures. This stimulatory effect of pressure is apparent also in certain other growth data to be found in the literature. For example, plots of growth rate of E. coli versus the reciprocal of the Kelvin temperature, presented in the paper by Johnson (1957), show that the rate of growth at 68 atmospheres and about 4OoC is greater than the rate at one atmosphere, at any temperature, and that this relatively low pressure results in a slight upward shift in the optimum growth temperature. Kriss and Mitskevich (1967) found that growth yields (weight of organisms/ml) of a marine pseudomonad were greater at 350 atmospheres than at one atmosphere or 500 atmospheres, at 28” to 29°C in a number of media, and they refer to the barophilic character of the bacterium. Similarly, we have found (Fig. 3) the optimal growth pressure for E. coli is not one atmosphere, or at least not under all conditions of cultivation. The yield values indicated in Fig. 3 for cultures grown at 100 or 200 atmospheres, at relatively high temperatures, are higher than the yield values, at any temperature, at one atmosphere pressure. It has been claimed that bacteria originated at some depth in the ocean (Sagan, 1973) where they were protected from solar radiation. Possibly, their physiology was originally adjusted to function best under moderate pressure. The barotolerance of any particular micro-organisms is very much dependent on growth conditions. Interactions among the different factors that affect growth and barotolerance commonly involve both antagonisms and synergisms. Therefore, it is extremely difficult experimentally to construct an “envelope of life”, as Scheie (1970) proposed, in an n-dimensional space with axes for various factors such as pressure, temperature, pH and Eh values. Moreover, the literature on the subject is highly confusing and contradictory, partly because it is difficult to control some factors while varying others. For example, pressure affects ionization processes, and compression changes the pH value of a buffered solution or suspension. Therefore, it is difficult to change pressure without also changing the pH value. The interactions that have been most extensively studied in microbial barobiology are those between pressure and temperature. The studies of ZoBell and Johnson (1949) clearly showed that mesophilic bacteria are generally more sensitive to pressure at 20°C

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200 atmospheres

0

d

I

1

I

I

I

I

I

15

20

25

30

35

40

45

Temperature

1

("C)

FIG. 3. Growth yields of Escherichia coli cultures, grown at one atmosphere ( O ) , 100 atmospheres (A) and 200 atmospheres pressure (O), in relation to the growth temperature. The medium used was trypticase-soy broth containing 0.1% (w/v) potassium nitrate.

than at 30°C, and more sensitive at 30°C than at 40°C. The need to specify temperature when considering pressure effects is obvious. The results of ZoBell and Johnson (1949) indicate an antagonistic relationship between temperature and pressure, as one would expect from the ideal gas law. However, microbial growth occurs in a condensed aqueous system in which major deviations from ideality occur. In fact, growth involves changes in a multiphasic system with most of the polymeric constituents of the cell in a series of solid phases. Figure 4 presents data obtained in this laboratory, by Dr. R. Meganathan, on the barotolerance of Strep. fueculis at temperatures

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:

6

-

o

0 5 t ---7 35°C

'b

Id0

2 h 360 4A0 Pressure ((1tmospheres)

'\

\ __ 560

6 0

FIG. 4. Rates of growth of Streptococcus faecalis at 2 5 ° C ( O ) , 35°C (A) and 45'C (m) in relation to growth pressure. The growth medium was tryptoneglucose-yeast extract broth.

below, close to and above the optimum. It can be seen that barotolerance is greatest near the optimum temperature. Pressure is known also t o cause a slight upward shift in optimum growth temperature. Very similar results were obtained for pressuretemperature effects on growth of a whole series of bacteria, including a psychrophile, Vibrio marinus (fischeri), and a thermophile, Bacillus stearothermophilus. In all, it appears that increased temperature and increased pressure are antagonistic in their effects on microbial growth at temperatures below the optimum, but that they act synergistically to inhibit growth at temperatures more than a few degrees above the optimum. In effect, expectations based on the ideal gas law are not fulfilled. Moreover, pressure responses appear t o be essentially independent of any adaptations to low or high temperatures. It seems then that those adaptations which permit psychrophilic bacteria and psychrophilic enzymes t o operate at low temperatatures do not also permit them to operate at high pressures. In short, temperature adaptation does not appear to result in correlated changes in barotolerance. How can this seemingly contradictory behaviour be interpreted in terms of the constituents of living cells?

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In their illuminating review article on the effects of pressure on biopolymers, Suzuki and Taniguchi (1972) present a summary table, that is reproduced here in somewhat modified form (Table 2), of the effects of pressure and temperature on the types of bonds that are most important in maintaining biopolymers in their native states. It can be seen that the volume change for hydrogen bond formation is negative, and so hydrogen bonding is enhanced at increased pressures. The enthalpy change (AH) for hydrogen bonding is also negative, which indicates that increased temperature disrupts hydrogen bonding. Therefore, pressure and temperature act as antagonists with respect to hydrogen bonding. TABLE 2. Volume changes and enthalpy changes accompanying bond formation' Type of bond formed

Value for AV

AH -

Hydrogen Hydrophobic Ionic

+c

+ or ~~

'This Table is adapted from one presented by Suzuki and Taniguchi (1972). Generally the sign of AV changes at pressures above 1000-3000 atmospheres. Generally the sign of AH changes at temperatures above about 6OoC.

In contrast, hydrophobic interactions result in volume increases and are disrupted by increased pressures. The AH value for hydrophobic bonding is positive so that increased temperature generally favours hydrophobic bonding and antagonizes the inhibitory effects of increased pressure. However, at pressures above about 1000 to 3000 atmospheres, AV values f o r hydrophobic interactions generally become negative so that now pressure and temperature act in concert to stabilize these interactions. The transition pressure may be even lower than 1000 atmospheres in certain systems. Moreover, above about 60°C, AH values for hydrophobic interactions change from positive to negative (Scheraga, 1963) so that, above 60°C and at low pressures, increased pressure and increased temperature can act in concert t o disrupt hydrophobic interactions. The volume changes for ionic bond formation in aqueous systems are positive, and pressure is inhibitory. However, AH values may be either positive or negative, and pressure and temperature may act in concert or antagonistically to affect electrostatic interactions.

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It is clear then that pressure effects on biopolymers are not simply the inverse or mirror image of temperature effects, as one might expect on the basis of applying the ideal gas law. For example, one would expect that pressure denaturation of a protein would be a uniquely different process from cold denaturation or heat denaturation, and that the series of bond breakages or formations leading to pressure denaturation would be different from the series leading to cold denaturation or heat denaturation. Although there have been some interesting demonstrations of extreme behaviour, such as the demonstration that malate dehydrogenase of B. stearothermophilus will function at a temperature as high as 105OC, but only when the pressure is raised to about 1700 atmospheres (Morita and Mathemeier, 1964; Morita, 1972), it still must be concluded that the inhibitory effects of high pressure cannot be overcome, in general, simply by increasing temperature. In fact, increased temperatures may potentiate inhibitory effects of high pressure. It also is clear that temperature adaptations can occur independently of pressure adaptations and involve different sets of changes in biopolymers. The effects of other growth parameters on barotolerance have been less fully described. Previous mention was made of the finding that the range of pH values for bacterial growth is markedly narrowed by increased growth pressures (Matsumura et al., 1974). In addition, ZoBell and Hittle (1967) found that pressure significantly enhanced the toxic effects of oxygen for bacteria. It is not known if this effect is specific for oxygen, or if it could also be demonstrated with other agents that raise the culture Eh value. It is a fairly general finding that bacterial growth in complex media can occur at higher pressures than can growth in defined media. For example, the maximum growth pressure for E. coli at 37OC is about 550 atmospheres in a complex medium such as trypticase-soy broth. In defined, minimal, glucose-salts medium, the maximum growth pressure is decreased to about 450 atmospheres. In contrast, Kriss and Mitskevich (1967) found that growth of a marine pseudomonad was better under pressure in a minimal, glucose-salts, medium than in a complex peptone-glucose-salts medium. However, growth in another minimal medium, with less magnesium, was poorer than in the complex medium. Also, addition of 0.01 or 0.1% (w/v) case in hydrolysate to the former defined medium allowed for even better growth under pressure. Growth in the minimal media was better at 350 atmospheres than at one atmosphere, but this barophilic be-

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haviour was not apparent in cultures grown in the complex medium. Growth in all of the media was essentially completely stopped by 600 atmospheres pressure at 28 to 29°C. Palmer and Albright (1970) also have reported that the maximum growth pressure for Vibrio marinus, at 4OC, was only 280 atmospheres in a complex medium, versus 327 atmospheres in minimal medium. The ionic composition of growth media has significant effects on barotolerance, and there seem to be specific ion effects. Palmer and Albright (1970) found that V. marinus grew best under pressure in the presence of 35 parts per thousand sodium chloride. Other ions could be substituted for Na+ or C1-, but they tended t o be less effective; monovalent cations were more effective than divalent cations. A subsequent study (Albright and Henigman, 1971) indicated that this requirement for sodium chloride for optimal growth under pressure is common among marine bacteria, but not among terrestrial species. In fact, in a defined medium, high concentrations of salt decreased the barotolerance of E . coli and Micrococcus luteus, but n o t of the marine bacteria. A considerably different set of responses was described (Marquis and ZoBell, 1971) for Strep. faecalis and two marine cocci. For these organisms, the divalent cations Mg2+ and Ca2+ markedly enhanced barotolerance, while monovalent cations were inhibitory. Anions appeared t o have little effect. The observed enhancement of growth rates and yields under pressure in cultures supplemented with 50 mM magnesium chloride or calcium chloride was specifically due to Mg2+o r Ca2+, and other divalent cations were ineffective or inhibitory. The enhancement of yield was subsequently found (Matsumura et al., 1974) to be a relatively common response, demonstrable even with cultures of the marine bacterium Serratia marinorubra and due t o increased resistance t o acid conditions under pressure in the presence of magnesium or calcium ions a t concentrations of about 100 mM. It is of interest, from a comparative point of view, that Ponat (1967) found that Ca2+ enhanced barotolerance of gill tissue of a marine mussel, and Schlieper et al. (1967) found that Ca2+ increased the resistance of the hermit crab t o the lethal effects of high pressure. B. SHORT-TERM SURVIVAL AND DEATH

Clearly, for long-term survival under pressure, bacteria must be able t o grow in compressed environments. However, pressure is

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known to have adverse effects on short-term survival, and relatively early in the development of microbial barobiology there was a great deal of interest in the use of pressure as a sterilizing agent. For the rapid killing of micro-organisms, it is generally necessary to use kilobar pressures. Even before the turn of the century, Regnard and, independently, Certes found that they could greatly retard spoilage of natural materials such as meat, milk, eggs and urine by application of pressures of 350 t o 700 atmospheres. Subsequent work by other investigators who used apparatus capable of producing much higher pressures showed that micro-organisms could be killed rapidly by pressures greater than those normally found in the biosphere. A relatively complete summary table of early results is presented in the book by Johnson et al. (1954) (pages 292 and 295). This past work, and more recent work, has yielded at least an outline of the hierarchy of relative sensitivities of various microbial types, although much work remains t o be done before a full series can be constructed. The current relatively sketchy hierarchy of pressure sensitivities bears some resemblance t o the more thoroughly worked out series for heat sensitivities. Simple acellular viruses are generally more pressure resistant than are more complicated cellular types. For example, the data of Rutberg (1964a, b) indicate that the exponential death rate constant for E. coli at about 2000 atmospheres and 37°C is about 0.125 min-' , compared with values of about 0.045 and 0.033 min-' for the bacteriophages T, and T,. Pressure also affects the growth cycles of viruses in susceptible cells. Foster and Johnson (1950), and later Rutberg (1964a), found that pressure prolonged the latent period, slowed virus release and decreased the burst size for phageinfected E. coli cells. Rutberg (1964c) found also that pressures of about 200 to 1000 atmospheres had an inducing effect on E. coli organisms that were lysogenic for lambda phage, with an optimum near 890 atmospheres at 37°C. Contrarily, some viruses do appear t o be very sensitive t o pressure, and Rautenshtein and Muradov (1966) report that pressures as low as 500 to 700 atmospheres, at 28"C, can kill certain phages of actinomycetes. There is one major case of disparity between heat resistance and pressure resistance. Bacterial endospores are highly heat resistant, usually by some orders of magnitude relative to vegetative forms, and they may also exhibit resistance t o very high pressures. For example, some spores are not killed by pressures in excess of 12,000 atmos-

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pheres (Larson et al., 1918; Basset and Macheboeuf, 1932) that rapidly kill all vegetative forms. In fact, it has been found by Johnson and ZoBell (1949) that pressure can even prevent heat inactivation of spores. However, lower pressures of 600 t o 8000 atmospheres may be rapidly fatal, especially at elevated temperatures. This apparently paradoxical behaviour has been shown by Clouston and Wills (1969), and by Sale et al. (1970), t o be due to triggering of germination by low pressure, as reflected by changes in microscopic appearance and the other common indicators of germination. The sequence of events in low-pressure killing seems to involve first germination and then death of the germinated forms. Higher pressures do not induce germination, and the spores which remain in the dormant state are pressure resistant. Among the various bacteria tested, there was a variable sensitivity with Bacillus pumilus at the sensitive end of the spectrum. Spores of this bacterium could be made to germinate by pressures as low at 545 atmospheres at 25"C, and could be killed by pressures as low as 610 atmospheres (Clouston and Wills, 1969). Based on change in rate, the germination process exhibited an apparent volume change of some 150 ml/mole in the range from 800 t o 1010 atmospheres (Clouston and Wills, 1970). The effects of pressure could be enhanced with phosphate buffer. In contrast, Sale et al. (1970) found that either sodium chloride o r calcium chloride had a protective effect for Bacillus coagulans. They found also that inactivation was greater at higher temperatures, at least partly because of the heat sensitivities of the germinated forms. The optimum pH value for inactivation was about 8.5; killing was somewhat decreased at higher pH values, and markedly decreased at low pH values. Furthermore, Could and Sale (1970, 1972) found that pressures as low as 200 atmospheres, at 30"C, enhanced the effectiveness of chemical germinants for heat activated Bacillus cereus spores, and also increased the range of compounds that could act as germinants. For example, D -alanine is not a germinant at one atmosphere pressure, and, in fact, it antagonizes the germinating activity of L -alanine. However, at 200 atmospheres, D -alanine acts as a germinant, possibly due to pressurestimulated racemase activity. Moreover, other compounds such as cysteine and guanosine could be made to act as germinants by increasing the pressure. It is possible that pressure-induced germination may depend on enhancement of electrolyte dissociation.

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Germination is associated with a massive conversion of spore electrolytes from immobile forms to mobile forms that should interact with water to cause electrostriction and a large decrease in volume. However, the pressure dependence of germination, with stimulation by low pressures and inhibition by higher ones, is difficult to interpret along these lines. As mentioned, the volume change for hydrophobic bond formation undergoes a change in sign at high pressure. Low pressures disrupt hydrophobic interactions, while high pressures stabilize them. This same pattern is apparent for spore germination. The complete sterilization of materials by means of pressure is often difficult because of “persisters”. For example, Timson and Short (1965) attempted t o sterilize milk with pressures as high as 10,300 atmospheres for 30 minutes at -25 to +25”C, but found that 0.05% of the initial population in the milk persisted, despite the initial rapid rate of killing. They found that the survivors of pressure treatments included cocci, both Micrococcus and Streptococcus organisms, but were mainly spore-forming bacilli. It is of interest in this connection that spore-formers make up a large fraction o f the bacteria that are recovered from deep-sea bottom samples, even though they are not present in large numbers in the water column (ZoBell, 1970). Timson and Short (1965) found that glucose, potassium citrate, sodium chloride or dipotassium hydrogen phosphate were protective for the milk flora, while ethylenediaminetetraacetate and Dowex 50 resin potentiated the lethal action of pressure. They found also that this lethal action of pressures, above about 2000 atmospheres, could not be counteracted by high temperatures of up to 100°C. They concluded that high pressure and high temperature inactivate bacteria by different mechanisms. Just how pressure kills micro-organisms is not clear: In fact, there is amazingly little information on the subject. Pressure disinfection is similar t o disinfection by heat, or with chemical agents, in that intensity and time both are factors in determining the extent of disinfection. At high pressures, above about 2000 atmospheres, killing can be achieved in only a few minutes, and it may well involve denaturation of proteins since kilobar pressures do denature isolated proteins. Lower pressures generally kill more slowly, and their action may sometimes simply be an acceleration of the death that normally occurs in non-growing cells. It is of interest that Holyoke and

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Johnson (1951) found that pressures of 272 and 544 atmospheres, at 35" C, accelerated lysis of intact Micrococcus lysodeikticus cells by lysozyme over a wide range of pH values. We have found (Matsumura et al., 1974) that pressure induced lysis of Strep. faecalis and Serratia marinorubra at high pH values, but the reaction occurs slowly and is probably only secondary t o other types of damage. In general, pressure-killed cells do not appear microscopically t o be greatly damaged, and so presumably, some more subtle damage causes death. Killing by low pressure is affected by temperature, but more often than not it differs from chemical disinfection in being more rapid at lower temperatures than at higher ones, within the range of temperatures over which heat inactivation or cold inactivation does not occur. In fact, pressure can, t o a degree, antagonize the lethal action of high temperatures (ZoBell, 1970). As mentioned earlier, salts appear t o have a protective effect against pressure killing of bacteria. Morita and Becker (1970) indicate that pressure-tolerant bacteria isolated from deep-sea samples could be protected by placing them in a medium used for growth of halophilic bacteria, even though this medium was toxic for the deep-sea bacteria at one atmosphere pressure. Results of some of our recent experiments indicate that salts are protective for both marine and terrestrial bacteria, but the effect does not seem to be very specific, and depends as much on ionic strength as on the specific ions used. We have been particularly interested in the pressure responses of Pseudomonas bathycetes. This bacterium is considered t o be indigenous t o the deep ocean, and it was first isolated during the Dodo Expedition (by R. Y . Morita, Jean ZoBell and Claude ZoBell) from samples collected at depths greater than 9000 metres in the Philippine Trench and in the Challenger Deep. Despite its barotolerance, Ps. bathycetes may have had difficulty surviving in the ocean depths because its optimum growth temperature is near 37°C. It will grow slowly at deep-sea temperatures, but it is very barosensitive at these low temperatures. Moreover, it is rapidly killed by pressure in sea water at 4°C. We found that the viable count of a suspension of Ps. bathycetes ATCC 23597 in sea water declined from 8 x 10' to 6 x l o 4 bacteria per miIliIitre after 24 hours incubation at 4°C and 1000 atmospheres. There was no decline in the viable count of a parallel, uncompressed suspension. For this experiment, the bacteria were grown in the medium

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described by Quigley and Colwell (1968), and the same medium solidified with agar was used for plating samples. Killing was found to be less extensive at 30°C than at 4"C, and in growth medium as opposed to sea water. The protective effect of inorganic ions was reflected in the finding that cell suspensions in 250 mM magnesium sulphate solution at 1000 atmospheres, and 4"C, showed only 50% killing in 24 hours, and only slightly more than a 90% decrease in count by 48 hours. Protective effects could also be demonstrated for cells in supplemented sea water or growth medium. In an independent study, Schwarz and Colwell (1974) found that killing of the bacterium at 1000 atmospheres was less extensive at 25°C than at 15°C. It is possible that this bacterium has changed since its initial isolation and has now become adapted t o growth at higher temperatures. However, Schwarz and Colwell (1975) reported that they were able to cultivate Ps. bathycetes at 1000 atmospheres and 3"C, but the cultures had lag phases of some four months, cell generation times of about 33 days and final viable counts of only one one-hundredth those of control cultures at one atmosphere. Certainly, it is apparent from the literature that there is need for more basic information on mechanisms by which pressure exerts its lethal effect. There are many possible ways that high-ionic-strength environments could be protective, since both pressure and salts affect the tertiary and quaternary structures of biopolymers. Since charged states are favoured by pressure, electrostatic interactions of both the attractive and repulsive types should be enhanced by compression. These electrostatic interactions are muted in high ionic-strength environments, and this muting could well counteract pressure effects. However, a great deal more experimentation is needed before any definite conclusions can be reached regarding the precise mechanisms involved. More detailed knowledge of pressure killing could have many practical applications. It is surprising that pressures of, say, 1000 atmospheres do not denature isolated proteins and yet are lethal for many bacteria. Thus, pressure may be useful for decreasing the number of viable bacteria in many labile materials. Moreover, there is some interest in information concerning which non-indigenous organisms deposited in the ocean might survive the slow descent t o the bottom, there to remain in a state of suspended animation. Along this line, we have recently assessed the effect of pressure on the

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longevity of Arthrobacter crystallopoietes in sea water at 4°C. At one atmosphere pressure, the bacterium in either the rod o r the sphere form has the longevity for which it is famous, and remains viable for weeks. However, at 1000 atmospheres, it is killed rapidly in a matter of hours. Somehow or other, compression upsets the mechanisms by which this organism preserves its viability for such long periods in the soil.

IV. Effects of Pressure on Biopolymers In a previous section, it was pointed out that simple chemical reactions such as single-bond breakage or single-group ionization are accompanied by volume changes that generally amount t o less than 30 ml/mole. Even the ionization of phosphate buffer at pH 7 has a AV value of onIy about 24 mlfmole, which is unusudly large for a simple ionization reaction. However, many polymeric reactions exhibit much larger volume changes, of some hundreds of ml/mole, and these large changes are due to co-operative intramolecular or intermolecular interactions. In other words, the large volume changes for these polymeric reactions are the sums of the small volume changes for monomeric ionizations and changes in bonding. The measured volume changes are net changes, and some component reactions may have positive changes while others have negative ones. In fact, some complex polymeric reactions do not exhibit large volume changes simply because of compensatory monomer volume increases and decreases. It seems important here t o stress the co-operative aspect of polymeric reactions since this co-operativity is the basis for the extremely high sensitivities of many polymeric reactions t o pressure. An excellent example of co-operativity is seen in pressure denaturation of metmyoglobin shown in Fig. 5. Here denaturation, estimated from changes in absorbance at 536 nm, occurs over a relatively narrow pressure range between 4000 and 5000 kg/cm2. Pressure denaturation is very much Iike acid or heat denaturation in that there is initially no change in the polymer as more and more pressure is applied; then at some critical pressure, the initiation of denaturation results in a co-operative transition in response to a relatively small additional increase in pressure. Apparent volume changes for physiological processes, such as growth, protein synthesis, movement, differentiation o r survival, all

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I

I

I

2000

4000

6000 2

Pressure (kg/cm )

FIG. 5. Pressure denaturation of metmyoglobin at 60°C indicated by changes in absorbance of light of 536 nm wavelength. The phenomenon is reversible as or indicated by the results obtained when the native protein was compressed (0) when the denatured protein was decompressed (a).From Zipp and Kauzmann

(1973).

tend to be Iarge and to increase markedly with increasing pressure. The large volume changes for these vital processes suggest that co-operative polymeric reactions are involved in pressure responses. Moreover, there are generally pressure thresholds for inhibitory effects, that is, low pressures have little or n o effect, but somewhat higher pressures are strongly inhibitory. In all, it seems appropriate here to review the major effects of pressure o n biopolymers since many biological responses to pressure probably are the result of changes in co-operative interactions among these polymers. Moreover, recent studies of the effects of pressure on protein denaturation have led to a realization that we may have to revise our views on the forces that determine the tertiary structures of proteins in aqueous media. A. NUCLEIC ACIDS

The most surprising finding from studies of the effects of pressure on nucleic acids is that these biopolymers, unlike proteins, are actually stabilized in their native states b y pressure. At least part of

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the surprise comes from knowledge that, in a caesium chloride density-gradient centrifugation, the denatured or single stranded form of DNA behaves as if it were more dense than the native double-stranded helix. This behaviour suggests that denaturation should have a negative volume change and so should be enhanced by pressure. HedPn et al. (1964a, b) found that exposure of transforming DNA from Bacillus subtilis to 10,000 atmospheres for 30 minutes at room temperature was without detectable effect on its transforming activity. Moreover, they found that pressure raised the melting or denaturing temperature. For example, the melting temperature of B. subtilis DNA, a t 2700 atmospheres, was some 6°C higher than that at one atmosphere pressure, as indicated by changes in optical density at 260 nm. Transforming activity was also spared from heat destruction by pressure. In addition, pressure increased the optimum temperature for renaturation of previously separated DNA strands. Subsequently, Weida and Gill (1966) pointed out that this stabilizing effect is curious in that one would expect DNA denaturation to be accompanied by a volume change of -3 ml/mole, on the basis of a difference in specific volumes of native and denatured DNA of about 0.005 ml/g. They, like Hedkn et al. (1964a, b), found that pressure raised the melting temperature for DNA and they applied the Claussius-Clapeyron equation t o their experimental data to obtain an apparent volume change of +4.5 ml/mole of base pairs for denaturation. Here the form of the equation is: dP - AH dT, T,AV where T, is the melting temperature, AH is the enthalpy of melting, P is the pressure and AV is the volume change. More recent work by Gunter and Gunter (1972) has yielded a AV value of +2.7 ml/mole of base pairs. The Gunters concluded that, “As it presently stands, helix-coil transition theory is incapable of explaining these results”. Suzuki et al. (1971, 1972) have investigated helix stabilization by pressure in some detail. They found that salmon sperm and calf thymus DNA were not denatured, even after one hour at 10,000 atmospheres in the temperature range from 25 t o 40°C. They considered the possibility that DNA might become denatured under pressure but then rapidly renature when pressure was released.

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However, no such renaturation was possible in their initial experiments in which formaldehyde was included in reaction mixtures to stop renaturation. In later experiments, they used an optical pressure cell with sapphire windows to measure, in situ, changes in light absorbance at 260 nm. Thus, they found that pressure retarded the rate of heat denaturation of DNA, but that, unexplainably, the slowing effect increased up to a pressure of 4000 atmospheres and then remained constant when the pressure was further increased. From rate constants, they were able to calculate a AVt value for denaturation of i l l - 1 4 ml/mole. One of their interesting findings was that, although the 260 nm absorption coefficient of native DNA changed little with pressure, that of single stranded DNA was reversibly decreased by pressure. They interpreted the latter change in terms of transformation of coiled chains t o chains with stacked bases hydrogen bonded together. In an effort to reconcile the apparent disparity between the AV value estimated from pressure studies and that estimated from buoyant density measurements, Chapman and Sturtevant (1969) have measured dilatometrically the volume change that accompanies denaturation of DNA by heat. They found that denaturation of calf thymus DNA in dilute buffer of ionic strength 0.02 was accompanied by essentially a zero volume change. It is well known that the interaction of DNA with salt solutions is complex and involves both hydration effects and Donnan effects (Eisenberg, 1969; Bauer and Vinograd, 1969). Chapman and Sturtevant (1969) attributed their finding of a zero volume change initially t o decreased hydration of DNA in the denatured state. Bound water was reasonably assumed to have a higher density than bulk water, as does so-called clathrate water. However, extrapolation of density determinations, at various water activities in salt solutions t o zero water activity, indicated no difference in density between native and denatured DNA, even in the dry state. Therefore, it seemed that, in denaturation, there had t o be some compensatory volume change to offset the positive volume due to dehydration of DNA. The major compensatory process that was proposed was a decrease in counterion binding by the single-stranded coil form. Such a decrease would result in greater ionization and electrostriction of water. In all, it seems that it is really not so surprising that the AV value calculated from pressure sensitivities of denaturation, in relatively low ionic-strength media, should not be

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equal to that calculated from the difference in densities of native and denatured DNA, in relatively high ionic-strength media such as concentrated calcium chloride solutions. Moreover, DNA is somewhat compressible, especially in the denatured form, and the high pressures generated in the ultracentrifuge would have effects on the apparent densities of DNA molecules which should be taken into account in calculations of absolute density (Bauer et al., 1971; Chun et al., 1973). B. PROTEIN DENATURATION

Hydrostatic pressure in the kilobar range is a denaturing agent for proteins, as demonstrated by numerous studies starting with those of Bridgman (1914) on egg albumin. Denaturing pressures are generally greater than the maximum biospheric pressure (1170 atmospheres), and so protein denaturation is often not thought to be of major importance in the pressure responses of organisms in natural environments. However, since the denaturing action of pressure is very much dependent on other factors such as temperature, pH value, ionic strength and the concentrations of specific ions, biospheric pressures may play a role in limiting microbial growth through their effects on cellular proteins. Recent work on pressure denaturation has been aimed at understanding mechanisms in terms of the component processes that lead to denaturation. We can again consider the example presented in Fig. 5, which shows a plot of absorbance of light a t 536 nm against pressure, for a solution of metmyoglobin in 0.05 M acetate buffer, maintained at 6OoC. When the protein is denatured, the local environment around the haem moiety is altered so that the absorption coefficient at 536 nm is increased. As the figure shows, no denaturation results from increased pressurization until a value of about 3800 kg/cma is attained. (One atmosphere is equal to 1.033 kg/cm2 .) Then, over a range of about 1000 kg/cm2, denaturation proceeds co-operatively to completion in a fully reversible manner. Obviously, the process of protein denaturation is composed o f many component reactions, involving the breaking of many bonds and the unfolding of the molecule. The intrinsic viscosity of myoglobin increases from about 3.1 to about 20.9 cc/g as a result of this unfolding from the globular to the random coil form. However, as

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the co-operative nature of the process indicates, we can consider the volume change for denaturation as the sum of the volume changes for each of the component reactions. Indeed, volume changes for polymer reactions may be very large because they are composed of a whole series of coupled "monomeric" reactions, so that the pertinent volume change for the process is the total of all of the component volume changes. The volume change associated with any particular denaturation process can be determined in two ways, dilatometrically or from a knowledge of (a lnK/aP)T. Unfortunately, both types of measurements are difficult to perform, and it was not until recently that reliable values have been obtained. However, there are now carefully determined values for denaturation of ribonuclease, metmyoglobin and chymotrypsinogen, all of which undergo major unfolding from the globular form to the random coil form during denaturation. In their detailed study of ribonuclease denaturation under a variety of combinations of pressure, temperature and pH values, Brandts et al. (1970) recorded a range of AV values from -45 ml/mole at 25°C and pH 2 to -5 ml/mole at 50°C and pH 4. They assumed that denaturation was a two-state process, N + D, where N represents the native form and D the denatured form. The denaturation process was fully reversible under the experimental conditions and so: AF" = -RTInK = -RT In

(D)/(N)

The relationship, AV = (aAF"/aP), = -RT(a InK/aP)= could then be used to estimate AV. Some recent evidence for the two-state hypothesis for denaturation has come from the pressure studies of Hawley (1973). He took advantage of the slow renaturation reaction of chymotrypsinogen under pressure and was able to separate two distinct species of the protein by means of electrophoresis of pressure-denatured mixtures. In the study by Zipp and Kauzmann (1973) mentioned previously, it was found that the AV value for the denaturation of metmyoglobin, estimated from determinations of (a lnK/aP)T, varied from -51 to -114 ml/mole of protein. Independently, Katz et al. (1973) obtained a value of -98 ml/mole of protein for acid denaturation at 30°C. Their value was determined dilatometrically and included corrections for the AV values of protonation reactions. The values

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obtained by the two methods, dilatometry and pressure sensitivity determinations, are in excellent agreement. The volume change for denaturation of chymotrypsinogen determined by Hawley (1971) from values for (alnK/aP)T was only about -14.3 ml/mole of protein at pH 2.07. Brandts et al. (1970) calculated a higher value of -48.6 ml/mole of ribonuclease at pH 2. Thus, although chymotrypsinogen is much larger than ribonuclease, its reversible denaturation is accompanied by a smaller volume change. In effect, it seems that it must involve a different set of component reactions. Zipp and Kauzmann (1973) found that plots of In Kversus pressure for metmyoglobin denaturation yielded essentially straight lines, indicating that the value for A V was essentially independent of pressure. However, both Hawley (1971) and Brandts et al. (1970) found that the denatured forms of the proteins they studied appeared to be more compressible than were the native forms, and at higher pressures this difference in compressibility contributed significantly to the observed volume change. For most biological reactions (aAV/aP) is not zero, and it is possible that at least part of this variation in the value of A V with pressure is due to differences in compressibilities of products and reactants. Presumably, changes in the compressibilities of proteins on denaturation result mainly from changes in solvent-solute interactions. In complex systems, such as living cells, it is much more difficult to define the basis for changes in the value of A V with changes in pressure. In the protein denaturation studies cited, the three main variables were pressure, temperature and pH value, and if some particular value of the N D equilibrium is considered, say, that for which AF" = 0 and (N) = (D), then it is possible to construct a three dimensional graph with axes corresponding to the variables, pressure, temperature and pH value. In Fig. 6, which is taken from the paper by Zipp and Kauzmann (1973), a slice of such a graph for metmyoglobin denaturation, cut in the pH-temperature plane, is shown. The inside of each of the closed figures indicates conditions in which the native state is favoured, while areas outside of the figures indicate conditions in which the denatured form predominates. It can be seen that the range of pH values over which the native form of the protein is favoured at any one temperature becomes narrower and narrower as pressure increases. A parallel

*

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'.____-

Temperature ("C)

FIG. 6. Constant-pressure contours in the pH-temperature plane at which the free energy for metmyoglobin denaturation is zero. Within each contour, the native state predominates, while outside the contour, the denatured state predominates. From Zipp and Kauzmann (1973).

effect has been observed (Matsumura et al., 1974) in regard to microbial growth under pressure-the range of pH values for growth becomes progressively narrower as the growth pressure is increased. It can also be seen that, for any one pH value, the temperature range over which the native state is more stable decreases with increasing pressure. Hawley (1971) prepared similar constant-AFo plots, on a temperature versus pressure plane, for denaturation o f chymotrypsinogen. The plots turned out to be elliptical in shape. Therefore, it appears that, at any particular pressure, there is a limited range of temperature over which the protein was in the native state. In other words, there seem to be two critical temperatures for denaturation corresponding to a unique cold denaturation temperature and a unique heat denaturation temperature. Both types of denaturation are clearIy defined from studies of many proteins. However, the finding that the curves for AFo = 0 were closed ellipses also indicates that there were two critical pressures, and that the native protein was stable only over a limited range of pressure at any given temperature. If we attempt to apply these findings for protein denaturation (or

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preservation of the native, functional states of proteins) to studies of processes such as microbial growth, we can readily identify unique maximum and minimum growth temperatures, which of course vary with changes in pH value, growth medium, and other parameters, but are unique for any one set of conditions. It seems that it should also be possible to identify maximum and minimum growth pressures. Much attention in barobiology has been paid to the former, but IittIe to the latter. In fact, it seems that the minimum growth pressure at temperatures below, or near to, the growth optimum may be negative. Although negative pressures are commonplace in plant physiology (so-called suction pressures), they are difficult t o maintain in the laboratory, and to date, at least as far as I know, there are no studies of microbial growth at negative pressures. At growth temperatures above the optimum, the optimum pressure for growth is generally greater than one atmosphere, and this topic was reviewed in Section I1 (p. 179). The results of pressure studies of protein denaturation have proved to be both a frustration and a stimulus to protein chemists. A central dogma of protein chemistry is that native conformations are stabilized in aqueous environments mainly by hydrophobic interactions, and that denaturation involves a breaking of hydrophobic bonds with exposure of polar groups to water. As indicated in the preceding section, transfer of polar groups from a hydrophobic medium, such as the interior of a globular protein, to an aqueous medium is accompanied by a volume decrease of some 1 0 to 20 ml/mole. With a protein such as ribonuclease, denaturation should be accompanied by a change in volume (AV) of -200 to -500 ml/mole of protein due to disruption of hydrophobic interactions (Tanford, 1968). The AV value for denaturation of metmyoglobin or chymotrypsinogen should be even larger, and yet, measured AV values are only a fraction of those expected (e.g. the AV value for denaturation of chymotrypsinogen is only -14.3 ml/mole of protein at pH 7.02). In fact, the AV values for protein denaturation tend to be only somewhat larger than those of monomer reactions. One’s first thought in relation to this apparent dilemma is that there must be some component process in denaturation that results in a volume increase which acts to offset the volume decrease due to disruption of hydrophobic interactions. Brandts et al. (1970), and subsequently others, have considered other processes which could

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accompany protein denaturation, and that could result in a compensatory volume increase. There is some evidence (Schellman, 1955) that the rupture of hydrogen bonds in peptides, and the subsequent formation of hydrogen bonds with water molecules, are accompanied by a volume decrease of about -2 ml/mole. However, Suzuki et al. (1970) found that- dissolution of diketopiperazine crystals in water, which seems to be a reasonable model process here, was accompanied by a volume increase of 4.5 to 5.9 ml/mole. Regardless of the sign of the volume change, it seems highly unlikely that breakage of hydrogen bonds would make much of a contribution to the volume change of protein denaturation. Some time ago, Noguchi and Yang (1963) found that the transition from helix t o coil in the poly-L-glutamate molecule was accompanied by a AV value of -1 ml/mole of peptide. However, it is difficult to interpret this often cited volume change since, presumably, a number of processes accompany the transition. Moreover, the measured change is a negative one, and so helix-to-coil transitions should not result in dilatation during protein denaturation. Yet another source of volume change is the change in charged groups that occurs during denaturation. Clearly, if protons are taken up or released from the protein itself, or from buffers, it is necessary to take account of any volume changes accompanying proton transfer. Kauzmann et al. (1962) found that carboxyl groups of proteins behave very much like carboxyl groups in monomeric compounds in that measured AV values for ionization were about -11 ml/mole. However, they found that protein amino groups differed from those of model, monomeric compounds. For example, the volume change for the reaction R-NH3+ + OH- -+ R-NH2 + H,O is commonly about 23 to 25 ml/mole but, for protein amino groups, the dilatation was only about 16 to 18 ml/mole. Protein imidazole groups behaved similarly with AV values for protonation that were some 5 to 9 ml/mole less than those for model compounds. Thus, it appears that amino and imidazole groups within a protein occupy larger volumes, by some 5 to 9 ml/mole, than do similar groups in aqueous solutions of model monomeric compounds. Therefore, denaturation should be accompanied by a large negative volume change (e.g. about -245 mg/mole for metmyoglobin) due to neutralization of basic groups. This contraction would then be in addition to the contraction due to disruption of hydrophobic interactions.

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Thus, a detailed consideration of component processes that might be involved in protein denaturation only makes matters worse and leads to the conclusion that the contraction accompanying protein denaturation should be even greater than that due to changes in hydrophobic interactions. Fortunately, there have recently been some indications that the AV value for disruption o f hydrophobic interactions could be positive. For example, B$je and Hvidt (1972) pointed out that the nonpolar groups in proteins are in a sense in concentration solutions rather than in dilute ones since they are bonded together in the polymer and so are never very far from each other. They concluded from studies of poly(N-methyl acrylamide) and poly(ethy1ene oxide) that the volume change for interaction of a nonpolar group with water is positive, rather than negative, at high concentrations. Also, Weber et al. (1974) have recently concluded from measurements of fluorescence under pressure that formation of a complex between aromatic rings in adenine and the isoalloxazine of flavin mononucleotide is accompanied by a decrease in volume rather than an increase. There do, then, appear to be possible solutions to the dilemma posed by the small volume changes associated with protein denaturation. However, there are still other problems that clearly indicate a need for obtaining more basic information on just how pressure affects polymers. When a protein is denatured, its heat capacity increases markedly, as one would expect, because the molecule has many more modes for heat absorption in the unfolded state, as compared with the folded state. For example, the change in heat capacity for denaturation of chymotrypsinogen at one atmosphere, pH 2.07, is 3800 cal/mole/"K (Hawley, 1971). If protein denaturation were predominantly a disruption of hydrophabic interactions, one would expect that the heat capacity change should become less positive at higher pressures and have a zero value in the region of, say, 5000 kg/cm2. Moreover, the AV value should become less negative at increased pressures, and change sign in the region of 1500 to 2000 kg/cm2. No such changes have been found. Clearly, one possibility here is that the model compounds, and model monomeric reactions, that have been studied are the wrong ones and are not good models for the component processes that occur during protein denaturation. It also is possible that our views of the forces that stabilize the native configurations of proteins are wrong. Zipp and Kauzmann (1973) suggest, in fact, that the view that the native

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conformations of proteins in aqueous solutions are stabilized chiefly by hydrophobic interactions may not be entirely correct. Certainly, this is an exciting area of biochemistry in which high-pressure studies are leading to a questioning of cherished, basic notions. Hopefully, the answering of questions regarding the nature of pressure denaturation of proteins will lead to a much fuller understanding of the forces that keep proteins in living cells in a functional state and the ways in which pressure alters these forces. C. EFFECTS O F PRESSURE ON POLYMERIC INTERACTIONS

1. Protein Associations To be functional in a living cell, many proteins must not only be in the native state but must also associate with other polymers to form water-soluble or insoluble aggregates. In fact, it seems that most of the proteins in cells are associated with other proteins, and most of them are not in the same chemical phase as the aqueous continuum of the cell and its environment. In effect, cells are multiphasic with most of the cell poIymers in solid aggregates. Moreover, even the proteins that are in solution commonly occur as multimeric forms that are functional only as multimers. Pressure can have major effects on polymeric interactions, at least in the test tube, and it is probable that many of the most dramatic effects of pressure on living cells can be traced to alterations in polymeric associations. As indicated in Table 3, these associations may be accompanied by large volume changes of some hundreds of ml/mole, and so are exquisitely sensitive to pressure changes. Simple, relatively non-specific aggregation of proteins can be caused by compression and it often accompanies pressure denaturation, as early studies showed (Bridgman, 1914;Johnson et al., 1954; Joly, 1965). Low pressures, below 1000 atmospheres, tend to decrease protein aggregation or precipitation, while higher pressures enhance them. Recently, Jaenicke (1971) has shown, by use of a quartz spring balance and dilatometers, that aggregation of serum albumin caused by isoelectric heating is accompanied by a positive volume change of 80 ml/mole of protein. This AV value is approximately equal to one determined previously by means of simple dilatometry for ovalbumin (Heymann, 1936). The volume change in Jaenicke’s experiments can be reasonably considered as the change

TABLE 3. Measured volume changes for some processes involving polymeric interactions

-

Process

AV Value

Temperature

Effect of Pressure

Reference

I t,

$

n

rn

(ml/mole)

("C)

Ribosomes -+ ribosomal subunits

-500

5

Causes dissociation

-52 Infante and Baierlein (1971) 5

Myosin filament + myosin monomers

-400

5

Causes filament breakdown

Josephs and Harrington (1968)

Native metmyoglobin + denatured metmyoglobin

-60 to -100

20

Causes denaturation

Zipp and Kauzmann (1973)

Flagellin

+157

22-23

Gerber and Noguchi ( 1 967)

+203

39

Inhibits filament formation Inhibits aggregation

v)

rn 7

2

0

9

0 w

P-0

--f

flagellar filaments

Aggregation of poly-L-valylribonuclease

-

?2 Kettman e t al. (1965)

E<

t,

N

0

W

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for aggregation alone since the albumin did not become denatured. Previously, Cassel and Christensen (1967) had found that phase conversion of tropocollagen, in dilute solution, t o the native collagen aggregate, was accompanied by a volume increase of 0.8 x mg/g collagen, as determined dilatometrically. They also found (Christensen and Cassel, 1967) that collagen denaturation was accompanied by an increase in volume of from 5 to 1 0 x ml/g protein, and that these values were not substantially different for denaturation in ethylene glycol. Therefore, hydration of the amorphous denatured polymer apparently did not contribute substantially to the volume change of denaturation. The formation of specific functional aggregates of bacterial polymers also appears to be accompanied by an increase in volume, and so is inhibited by pressure. Gerber and Noguchi (1967) measured, by use of dilatometers, the volume increase associated with the formation (at neutral pH values) of flagellar filaments from flagellin. They obtained a value of +157 ml/mole of flagellin. Therefore, pressure should inhibit the formation of flagellar filaments, and also cause disaggregation of previously formed elements. However, Meganathan and Marquis (1973) were unable to detect loss o f flagella by living bacteria at pressures up to 612 atmospheres, although formation of new flagella was extremely sensitive t o pressure, and could b e inhibited in E. coli by pressures as low as 100 atmospheres. In effect, there appeared to be some element that stabilized the flagellar filaments formed in vivo. There are other examples of positive volume changes associated with formation of protein aggregates that may be related to the known effects of pressure on higher organisms. For example, Josephs and Harrington (1968) studied the effect of pressure and other environmental factors on the equilibrium between myosin monomers and long filamentous myosin aggregates. They estimated a AV value for specific aggregation of some +380 ml/mole of monomer. In their studies, they used the ultracentrifuge to obtain pressures of up t o 40 atmospheres. Indeed, when one uses the ultracentrifuge, there is n o way to avoid subjecting the experimental materials to pressurization, and, in recent years, it has become apparent that the interpretations of the behaviour of macromolecules in the ultracentrifuge must take into account pressure effects. Kegeles and Johnson (1970) were able t o use the data obtained by Josephs and Harrington (1968) to

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predict quantitatively the shapes of Schlieren patterns obtained when myosin was centrifuged, i f allowance was made for pressure effects. Ikkai et al. (1966) estimated, from dilatometric measurements, that the formation of fibrous, double-stranded, F-actin helices from globular, monomeric G-actin, in the presence of magnesium and ATP, results in a volume increase of 391 ml/mole of G-actin, after making allowance for the AV value of +22 ml/mole for Mg2+binding to ATP, and the AV value of -18.6 ml/mole for phosphate cleavage from ATP. Jaenicke and Lauffer (1969) estimated a volume change of 1-257 ml/mole of monomer for polymerization of tobacco mosaic virus protein. However, because of a lack of knowledge regarding the pressure sensitivities of the other processes involved in virus maturation and assembly, their results cannot readily be applied to studies of inhibition of virus production by pressure. Similarly, it has been found (Collen et al., 1970) that the conversion of fibrinogen t o fibrin is accompanied by a large volume increase, but it is not known if pressurization interferes with blood coagulation in uivo. But of course, the pressure studies are revealing in regard to the molecular mechanisms of association even if they cannot be used to predict fully the responses to pressure in vivo. However, pressure-induced alterations in aggregation of certain proteins are considered to be the causes of certain observable pressure effects on living cells. One of the best examples is the inhibition by pressure of the formation of microtubules from the monomeric protein tubulin. These protein aggregates are important as structural elements for many eukaryotic organelles such as cilia, axopodia and the mitotic apparatus. High pressures are known to immobilize eukaryotes (as well as prokaryotes), to cause loss of cell shape and to “freeze” the mitotic apparatus. It is thought that these effects are related to pressure effects on microtubules. As early as 1884, Regnard discovered that pressure immobilized protozoa, and Ebbecke (1935) reported that 400 atmospheres completely stopped the movement of Paramecium sp. and caused cells to be transformed into spheres. Marsland and Brown (1936) found that pressures of 400 to 600 atmospheres resulted in reversible retraction of pseudopodia and sphering of Paramecium sp., as well as inhibition of protoplasmic streaming. They related the change in form and mobility to large, measured, decreases in relative viscosity

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of the cells. Pressure also has been found to stop pinocytosis (Zimmerman and Rustad, 1965). Kitching (1957b) observed that pressure caused a collapse of the axopodia o f heliozoans. Collapse pressures for A c t i n o p h r y s sp. were 136 t o 204 atmospheres at 5 t o 1O”C, and 272 t o 340 atmospheres at 1 5 t o 20°C. Axopodia are characterized by a patterned array of microtubules (Kitching, 1957b; Tilney e t al., 1966), and presumably loss of axopodial structure is a direct result of loss of microtubules due t o pressure-induced disaggregation. After decompression of the cells, microtubules reform, and axopodia re-appear. Kitching (1957a) observed that pressure immobilized a whole range of flagellate and ciliate protozoa; movement was reversibly stopped by pressures of 272 to 953 atmospheres, and somewhat higher pressures caused structural damage to the cells. Electron microscopic studies by Kennedy and Zimmerman (1970) of immobilized Tetrahymena pyriformis cells, fixed under pressure, revealed that pressurization caused disintegration of microtubules. Two minutes of pressurization at 5 10 atmospheres caused dissolution of the proximal portions of central ciliary microtubules, and 680 atmospheres, for 1 0 minutes, caused damage to the basal body of the cilium. Pressure also inhibits ciliary movements of the gill filaments of the marine mussel Mytilus sp. (Flugel and Schlieper, 1970). It can immobilize sea urchin embryos and cause loss of cilia (Young e t al., 1972). Pressurization of embryos of Arbacia sp. at 408 atmospheres results in complete loss of cytoplasmic microtubules (Tilney and Gibbins, 1969), but cilia may be retained. However, Young e t al. (1972) found that 408 atmospheres completely blocks formation of new cilia by deciliated embryos. Recovery of motility was possible at lower pressures. Microtubules are major structural elements in the eukaryotic mitotic spindle, and it has been known for some years that pressures of about 400 atmospheres can completely stop cell division of eukaryotic cells (Marsland, 1970). Compression of dividing eggs that have already formed a cleavage furrow results in recession of the furrow and rounding-up. Pressures of 272 to 476 atmospheres also have been found t o cause a type of “freezing” of the mitotic apparatus, and cessation of chromosome movement, as first observed by Pease (1941, 1946). The “freezing” involved structural damage to the linear pattern of the spindle and the radial pattern of the aster. The damage caused by high pressures of about 544 t o 680 atmospheres, for more than five minutes, is irreversible; damage caused by

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lower pressures has been found t o be reversible. It appears that the basic effect of pressure here is a dissolution of the microtubules of the spindle-aster complex (Marsland, 1970), and agents such as DzO, that stabilize microtubules, antagonize pressure inhibition. Agents such as colchicine, which inhibit microtubule aggregation, are potentiated in their action by pressure. Pressures of some 340 atmospheres for six minutes can also cause nuclear anomalies such as triploidy in eggs of the leopard frog (Dasgupta, 1962). Although many structures that depend on microtubules for their integrity are destroyed by high pressure, there are others that are relatively unaffected. For example, Hinsch and Marsland (Marsland, 1970) found that 680 atmospheres, for ten minutes, caused little or no damage t o microtubules present in the flagella of certain sperm cells. Recently, O’Conner et al. (1974) found that neuronal microtubules, in uivo and in vitro, were not affected by pressures as high as 680 atmospheres applied for 10 to 45 minutes. They concluded that “the depolymerization of microtubules in several types of nonneuronal cells, which has been reported, may have been overgeneralized with regard t o the direct action of pressure on microtubule stability”. Thus, it seems that some caution is needed in interpreting pressure effects in terms of disaggregation of microtubules. In fact, there may be more than one type of microtubule, at least as far as pressure sensitivity is concerned, and accessory factors or elements may contribute to the stabilities in uivo of microtubules (and bacterial flagella) exposed to high pressures. Another type of protein interaction, which may be at the basis of many barophysiological effects, is the association of enzyme monomers t o form active multimers. This sort of association occurs also with regulatory proteins. For example, the functional form of the lac repressor is a tetramer, and individual monomer units are inactive. Penniston (1971) made the provocative proposal that multimeric enzymes are inhibited by pressures below the denaturing pressure because of dissociation into subunits while, in contrast, monomeric enzymes are stimulated by low pressures. Penniston (1971) presented his own data, and that from the literature, for a series of monomeric and multimeric enzymes. The monomeric ones included peroxidase, myokinase, trypsin, amylase, lysozyme and chymotrypsin, while the multimeric ones were mitochondrial adenosine triphosphatase (ATPase), erythrocyte membrane ATPase, sarcotubular vesicle ATPase, pyruvate carboxylase, argininosuccinase,

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alkaline phosphatase and creatine kinase. In his experiments, Penniston (1971) used saturating concentrations of substrates so that the pertinent AV* values were those for formation of activated enzyme-substrate complexes rather than those for initial substrate binding. He concluded, in regard to the adaptation of deep-sea organisms to pressure, that “Their .enzyme systems either must be monomeric, or must have stronger non-covalent interactions between multimers, in order to operate at the high pressures under which these organisms live”. He also pointed out that there is not a correlation between cold lability and sensitivity to pressure inhibition as there is between the multimeric state and pressure sensitivity. Although Penniston (1971) clearly identified a mechanism by which pressure can affect enzymes, it seems that his conclusions cannot be generalized. There are many multimeric enzymes that are stimulated by pressure rather than inhibited by it. For example, we have found that the membrane ATPase of Strep. faecalis is stimulated at high substrate concentrations. Also, Ikkai and Ooi (1971) found that the ATPase activity of myosin A, heavy meromyosin or subfragment I was stimulated by pressures up to about 1000 atmospheres and inhibited by higher pressures. Moreover, there are monomeric enzymes that axe inhibited by pressure. For example, Becker and Evans (1969) found that the AV* value for lysozyme activity was negative when the enzyme was activated by Na+ but positive when it was activated by K + . However, it is still clear that subunit dissociation must be taken into consideration when interpreting the effects of pressure on multimeric enzymes. Such dissociation has been demonstrated unequivocally by Dicamelli e t al. (1973) who showed that the tryptophan synthetases of E. coli and Salmonella typhimurium dissociated into isolatable subunits in the pressure field generated by the ultracentrifuge.

2. Rib osom es Dissociation due to pressure in the ultracentrifuge occurs with ribosomes as well as with multimeric enzymes. The dissociation was studied in detail by A. A. Infante and his coworkers (Infante and Baierlein, 1971; Infante and Krauss, 1971). They used ribosomes isolated from unfertilized sea-urchin eggs, which are mainly o f the 75s or monosome type, and found that the sedimentation coefficient of the structures varied considerably depending on just how

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they were centrifuged. They showed that the major perturbing element, at constant ionic strength, was the hydrostatic pressure generated in the centrifuge tube. Pressure favoured dissociation of the 75s monosomes into 56s and 35s subunits. The equilibrium was extremely sensitive to pressure, and the apparent volume change for dissociation was calculated to be 500 k 100 ml/mole. These observations made it necessary t o review previous findings of what were thought t o be conformational changes in ribosomes reflected in changes in sedimentation patterns. A similar dissociation under pressure has been demonstrated for bacterial ribosomes (van Diggelen et al., 1971, 1973). Previously, i t had been proposed that there are two types of ribosomes in bacterial cells-native and derived. The native ones are obtained as subunits from bacterial extracts in solutions containing 0.01 M Mg2+. These extracts also contain 705 monomers which can be salt-dissociated to yield derived subunits. When native subunits are made to combine, they form 61s ribosomes, whereas derived subunits combine to yield 7 0s ribosomes. van Diggelen et al. (1971, 1973) concluded, however, that these ribosomes do not differ in true sedimentation rate, but rather in their pressure sensitivity. So-called native ribosomes are more sensitive to pressure and so dissociate more readily with resultant apparent lowering of their sedimentation coefficient. It appears that native ribosomes may have been damaged by hydrolytic enzymes during extraction, and, presumably, their greater sensitivity t o the dissociating action of pressure is related to this damage. van Diggelen et al. (1971, 1973) pointed out that, in 10 to 30% linear sucrose gradients, maximum pressures that may be attained are about 134 atmospheres with a Beckman SW27 rotor at 9,000 revolutions per minute, about 667 atmospheres with a Beckman SW27 rotor at 41,000 revolutions per minute, and about 1,950 atmospheres with a Beckman SW27 rotor at 22,000 revolutions per minute. In other words, pressures in the kilobar range can be attained with the ultracentrifuge in routine use, and the possibility of denaturation or polymer dissociation must be considered when using the instrument. 3. Lipid- Lipid In t erac tio ns

Pressure perturbs lipid-lipid interactions of the type that occur, or are thought to occur, in natural membranes. Melchior and Morowitz (1972) found that there were increases in volume associated with the

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gel-to-liquid crystal transitions of micellar or vesicular aggregates of chmyristoyl-, dipalmitoyl- or distearoyl-r. -a-lecithins. The specific increases were, respectively, 11.8, 14.1 and 20.5 ml/mole. Subsequently, Srinivasan e t al. (1974) showed that increased pressures up t o about 1 0 0 atmospheres resulted in increased transition temperatures for dipalmitoyllecithin bilayers, but that the volume change accompanying the transition did not vary with pressure. Trudell and coworkers (1974) have found that pressures of about 136 atmospheres, established with helium gas, cause 3 t o 5°C increases in the solid-to-liquid phase transitions for mixed phosphatidylcholine bilayers. They found also that pressure decreased the fluidity of vesicle bilayers as indicated by changes in electron spin resonance signals from spin-labelled probes. They concluded that pressure tends t o convert fluid lipid phases in bilayers to more tightly packed gel phases. The striking finding from studies of pressure effects on lipid-lipid interactions, at least from the point of view of barophysiology, is that the apparent volume changes are s o small and are not suggestive of much in the way of co-operative interactions. However, there is certainly the possibility that, in fact, the lipid phase transitions are highly co-operative but that compensatory volume changes lead t o only small net volume changes of the magnitude of those encountered in studies of pressure denaturation of protein. 4. Conclusions

The studies reviewed in this section lead t o some generalizations that are helpful in approaching microbial barophysiology in search of molecular interpretations of pressure effects on various cell functions. First, pressure seems, if anything, to stabilize the native states of DNA, but to denature proteins, even though low pressures will protect both DNA and proteins against thermal denaturation. Volume changes for denaturation of both DNA and proteins are generally small, and the processes are not highly sensitive to pressure. However, as pointed out, other factors may affect the processes, and denaturing pressures for proteins can be less than 1000 atmospheres when, for example, the pH value, or the temperature, is close to the limits that are compatible with native states. Moreover, it is also clear that biopolymer denaturation is co-operative, and that the small volume

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changes measured are net changes made up of compensatory positive and negative volume changes of component reactions in the overall denaturation process. Phase transitions of lipids in bilayers or other micellar arrangements also tend t o be small, and so very high pressures would be needed t o force the equilibrium very much in favour of the more condensed states. In contrast, the associations of microtubules, of ribosomal subunits, of flagellar subunits, of certain multimeric enzymes and other functional proteins appear to have very large volume changes and t o be highly sensitive to pressure. The exquisite barosensitivities of some of these processes are clearly reflected in the effects of pressure on living organisms. However, the delineation of the molecular bases for the barosensitivities of complex processes such as cell growth still represents a major challenge.

V. Effects of Pressure on Some Specific Microbial Cell Functions A. PERMEABILITY AND TRANSPORT REACTIONS

As indicated in Section IV, there is some basis for thinking that pressure might affect membrane functions because of its effect on lipid bilayers, primarily in shifting the phase equilibrium in the direction of more condensed gel phases. However, the relatively few studies that have been carried out with microbial cells have not indicated very dramatic effects. Non-lethal pressures can slightly enhance potassium leakage from cells (Matsumura et al., 1974), but the losses are not major except at extreme values of pH. Moreover, Johnson et al. (1973) found that pressure could actually reverse the cation leakage from liposomes caused by anaesthetic agents, and pressure can stimulate certain transport reactions. For example, it was found by Schlamn and Daily (1971) and by Daily and Schlamn (1972) that low pressures of about 68 atmospheres, established with compressed helium, stimulated uptake of P-methylthiogalactoside and 0-methylgalactoside at 37OC by E. coli cells with constitutive 0-galactoside permease systems. The effect involved an increase in the maximum velocity of some 20 t o 4876, but n o change in the Michaelis constant for the transport reaction. Marquis and Keller (1975) subsequently extended these observations by showing that a hydrostatic pressure of 400 atmospheres, at 3OoC, somewhat stimulated uptake of thiomethylgalactoside by E. coli. Previously Landau (1967) had concluded that a pressure of 670 atmospheres, a t 37OC,

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did not block entry of isopropylthiogalactoside into E. coli. Matsumura (1975) found just the opposite for Strep. faecalistransport of 0-galactosides was markedly inhibited by pressure, and it appeared that, in media with lactose as a main catabolite, the slowing of lactose uptake under pressure resulted in a slowing of growth rate. These seemingly contradictory results are not surprising when one considers that 0-galactosides are taken up by E. coli via a galactoside permease system, whereas they are taken up by Strep. fueculis via a phosphotransferase system to yield phosphorylated 0-galactosides. The permease system seems to be stimulated by pressure, whereas the phosphotransferase system is inhibited. Shen and Berger (1974) determined the effect of pressure on another phosphotransferase system-that for a-methylglucoside uptake by E. coli. They found that uptake was inhibited by pressure. In fact, uptake over 20 minutes was significantly decreased by pressures of only 200 atmospheres, and was completely stopped by a pressure of 680 atmospheres. Pressure stimulated the efflux of a-methylglucoside as well as inhibiting its uptake. Schwarz and Landau (1972a) found that concentration intracellularly of L-amino acids by E. coli was enhanced by 670 atmospheres pressure, at 3 7” C, even though this pressure effectively stopped protein synthesis. In their experiments, the net flux of amino acids into the cells was actually greater at one atmosphere than at 670 atmospheres, but at one atmosphere most of the amino acids were funnelled into protein synthesis. Paul and Morita (1971) had proposed that inhibition of amino acid uptake by pressure was the basis for the inability of a facultatively psychrophilic marine bacterium (called MP-38) to grow under pressure. However, in their experiments they fixed cells with 0.1 N sulphuric acid before assessing the extent of uptake. Therefore, pool amino acids would have been released and only those incorporated into acid-insoluble polymers would remain. Thus, it seems that, in this case, “uptake” refers to both transport and incorporation into polymers, and inhibition of growth by pressure probably was not due to inhibition of amino acid transport. However, Albnght and Hardon (1972) did report a major inhibition by pressure of uptake of labelled phenylalanine by E. coli B/r. Recently, Schlamm e t al. (1974) have found that 68 atmospheres of pressure, established with compressed helium, enhanced iron

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uptake by E. coli and that this enhancement could speed growth by decreasing the lag phase in minimal medium. However, the effect appeared to be due to the helium rather than to pressure. B. CATABOLIC PROCESSES

The effects of pressure on microbial catabolism of various classes of compounds have been studied so extensively that it is reasonable here to present only a few examples and generalizations. If we were to survey generally the pathways for degradation of organic compounds, the first reactions to be considered would be those that occur outside the cell. Bacteria and fungi are osmotrophic or holophytic in their basic nutrition, and only those molecules which are sufficiently small to pass through the porous network of the cell wall can be transported across the cell membrane to interact with intracellular enzymes. When polymers are used as nutrients by osmotrophic organisms, the first step in catabolism is hydrolysis outside the cell catalysed by exo-enzymes. Most exo-enzymes that have been studied have been found to be relatively insensitive to pressure, possibly because of their small sizes and relatively simple tertiary structures. For example, Kim and ZoBell (19 72) and ZoBell and Kim (1972) found that a bacterial chitinase was fully active at 1,000 atmospheres and at either 4 or 25°C. A fungal chitinase was found to be slightly inhibited by pressure, while a bacterial protease and an agarase were more severely inhibited. The latter was slowly inactivated by pressure. However, all of these enzymes were active at 1,000 atmospheres, and Kim and ZoBell (1972) proposed that they may serve as agehts for the degradation of biopolymers in the deep sea. They found also that 1,000 atmospheres, at 25"C, had no effect on the dimeric amylase of Bacillus subtilis, at pH 6 in phosphate buffer, but was inhibitory when the enzyme was in sea water at pH 8.2. Previously, ZoBell and Hittle (1969) had shown that a-amylases from 20 different species of marine bacteria were active at 1,000 atmospheres, and 4°C. Higher pressures do denature a-amylase, and it has been found that calcium ions are effective in antagonizing the denaturing action of high pressure (Miyagawa et al., 1965). Weimer and Morita (1974) found that pressures of up to 600 atmospheres, at 25 to 4OoC, had relatively little effect on a gelatinase produced by a marine Vibrio. Previously, Kriss et al. (1969) had

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found that pressure does not adversely affect isolated gelatinase of Bacterium candicans, but that a pressure of 500 atmospheres, at 27”C, selectively, and almost completely, inhibited gelatinase production, even though cultures grew at this pressure to nearly the same bacterial density as cultures grown at one atmosphere pressure. Neville and Eyring (1972) found that pressure stimulates the action of lysozyme on walls of intact Micrococcus luteus cells. The AV* values they reported were all negative, with a minimum value of -9.7 ml/mole at low ionic strength, except for one positive value of 1.5 ml/mole for hydrolysis in 0.125 M sodium chloride solution. There is one report (Berger, 1959) of pressure inhibition of cell-wall synthesis. It is possible that the apparent inhibition of synthesis could have been the result of enhanced autolytic activity. The study of the effects of pressure on catabolism of monomeric compounds began before the turn of the century with the work of both Regnard and Certes. For example, Certes and Cochin (1884) found that yeast cells could carry out glycolysis under pressures of 300 t o 400 atmospheres, but that rates were slower than the rate at one atmosphere pressure. Later, E. Buchner (1897) and H. Buchner (1897) reported that yeast juice also could catalyse fermentation under pressures of 400 to 500 atmospheres. More recent studies (Morita, 1965; Marquis et al., 1971) have shown that bacterial or yeast glycolysis is inhibited by pressure, but that barosensitivity is not extreme in that AVt values calculated from rate data are 50 ml/mole or less at pressures below 1,000 atmospheres. Thus, even though glycolysis is a complex sequence of coupled reactions, there must be compensating effects that result in relatively low AVS values for the net process. It is also possible that the rate of substrate flow through the glycolytic system is controlled by a single “bottleneck” step, and that the AVt value for the whole system is primarily that for the rate-limiting step. There is essentially no information on the effects of pressure on alternative pathways for gIucose degradation, o r on the partitioning of glucose among the pathways in versatile bacteria such as E. coli that can form enzymes for the Embden-Meyerhof, the hexose monophosphate shunt and the Entner-Doudoroff pathways. With Strep. faecalis cells, we could detect no effect of pressure on yields of lactic acid per mole of glucose used. Chumak and Blokhina (1964) found

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that a pressure of 500 atmospheres nearly stopped the production of carbon dioxide from glucose, by Pseudomonas desmolyticum, and resulted in greater acid production. Subsequent analyses (Chumak et al., 1964) indicated that the acids that were produced in excess, under pressure, were mainly formic and acetic acids, but not lactic acid. In these experiments, care was taken to exclude air from all of the cultures. Similar behaviour was noted for another marine pseudomonad by Kriss et al. (1967). Stupakova et al. (1974) found that certain marine bacteria could be induced to produce keto acids and amino acids, and t o excrete them into the culture medium, by pressures from 200 to 500 atmospheres. No such production, or only minimal production, occurred at one atmosphere pressure. There is a reasonable amount of information on the barosensitivities of enzymes that catalyse reactions that yield substrates for glycolysis. For example, Becker and Evans (1969) found that pressure stimulated the tetrameric P-galactosidase isolated from E. coli, when Na+ was used as activator, but it inhibited the enzyme when K + was used. However, the AVS values they calculated were relatively small (-12.0 ml/mole and +4.5 ml/mole at pressures as high as 1,500 atmospheres). Berger (1958) found that the glycosidase of a streptomycete was inhibited by pressures in the range of 250 to 1,000 atmospheres, but that the inhibition could be decreased by raising the incubation temperature. Eyring et al. (1946) had previously found that yeast invertase was markedly stimulated by pressures as high as 680 atmospheres, andJohnson et al. (1948) later showed that this stimulation could be enhanced by phosphate ions. In addition, Williams and Shen (1972) found that ribonuclease was stimulated by pressures as high as 1,362 atmospheres and they calculated a AVS value of -20 ml/mole. On review, it appears that catabolic, hydrolytic reactions are affected by pressure, but that AVS values tend to be relatively small and that pressure is often stimulatory rather than inhibitory. The effects of pressure on reactions of the tricarboxylic acid cycle, and o n oxygen or nitrate reduction by microbes, are generally inhibitory, although Morita (1957b) did find that endogenous respiration of E. coli, with methylene blue as terminal electron acceptor, was stimulated by pressure. However, succinate, formate and malate dehydrogenase activities of intact cells, at about 27OC with exogenously added substrates, were decreased under pressure. At 1,000

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atmospheres, enzyme activities approached zero, and at 600 atmospheres, AVS values were only some 2.3 ml/mole for malate dehydrogenase, 27.0 ml/mole for succinate dehydrogenase and 3.8 ml/mole for formate dehydrogenase. Previous studies (Morita and ZoBell, 1956) had indicated that succinate dehydrogenase is slowly inactivated by 600 atmospheres pressure. Morita (195 7a) also investigated the effect of 600 atmospheres pressure on ammonia production from various substrates by E. coli cells at 30°C. He measured increased ammonia release from aspartate, glutamate or alanine, but decreased release under pressure from serine, cysteine or histidine. Macdonald (1965) measured oxygen consumption by Tetrahymena cells in a pressure chamber by use, in situ, of a vibrating platinum electrode. He found that 262 atmospheres pressure decreased oxygen consumption by about 14%, while 544 atmospheres pressure caused a 54% decrease. ZoBell and Budge (1965) assessed the nitrate-reducing capacities, under pressure, of a series of marine bacteria. They found that reduction was inhibited by pressure in all cases, but that, even at 600 atmospheres, the rates were generally decreased by less than 50%. There are relatively few bacteria that can grow at pressures as high as 600 atmospheres, and yet catabolic activities are not extensively inhibited by this high pressure, and may even be stimulated by it. This variance in barosensitivity between growth and catabolism led Pope and Berger (1973b), and Albright (1975), to propose that pressure inhibits microbial growth primarily by inhibiting biosynthetic reactions, particularly protein synthesis, rather than catabolic reactions. In a previous study of growth inhibition of Strep. faecalis by pressure (Marquis e t a!., 1971), it was noted that the barotolerance of the bacterium could be affected in a major way by changing the fuel source for growth. Cells inoculated into a tryptone-yeast-extract medium, with pyruvate as the fuel source, were completely inhibited in their growth by pressures of only about 200 atmospheres. If pyruvate was replaced by ribose, growth occurred at pressures up to about 450 atmospheres; while if glucose, galactose, maltose or lactose was used as fuel source, growth was possible at pressures up t o about 550 atmospheres. It was also found (Marquis and ZoBell, 1971) that supplementation of glucosecontaining medium with 50 mM Mg2+ or Ca2+ allowed growth to proceed at pressures as high as 750 atmospheres. In other words, the

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barotolerance of this single species varied tremendously in response to changes in the fuel source provided for growth. Adjunct experiments carried out with non-growing suspensions of Strep. faecalis cells indicated that pressure effects on sugar degradation were relatively small and were not related, in any consistent way, to growth effects. How then can the barotolerance of Strep. faecalis be so dependent on catabolites if pressure affects catabalism less severely than it affects growth? The recent work of Matsumura (1975) indicates that the critical factor in barotolerance of Strep. faecalis is the rate at which ATP can be supplied for growth. For example, the bacterium can degrade glucose relatively rapidly, and the flow of ATP from glycolysis is relatively high. The growth rate at one atmosphere is relatively high in glucose-containing medium, and barotolerance is relatively high. The bacterium degrades ribose less rapidly, the flow of ATP to biosynthetic reactions is slower, growth at one atmosphere is slower and barotolerance is less. It has been found (Marquis et al., 1971; Matsumura, 1975) that growth under pressure is relatively inefficient, as indicated by low Y A T P values (grams dry weight of cells synthesized per mole of ATP produced). For example, the YA value for Strep. faecalis, grown in a tryptone-yeast-extractglucose medium, was found to be about 1 5 at one atmosphere, but only about 1 0 at 408 atmospheres pressure. At both pressures, exponentially growing cultures were used t o avoid problems due to the greater pH sensitivity of growth, as compared with glycolysis, in cultures approaching the stationary phase. Streptococcus faecalis degrades glucose to lactic acid via the Embden-Meyerhof pathway. Presumably, pressure could not decrease the ATP yield per mole of glucose used, since ADP is an obligatory reactant, and ATP an obligatory product of glycolysis. What then happens to the ATP that is somehow diverted from biosynthesis? Part of it may be used to maintain ion gradients under pressure. However, Strep. faecalis cells, grown under pressure, have lowered levels of potassium ions at all stages in the culture cycle, and so they seemed to carry out less, rather than more, uptake and concentration of K'. Of course, it is possible that the energetic expense of maintaining ion gradients is greater under pressure. Part of the extra ATP required for growth under pressure appeared to be used for polymer replacement. Matsumura (1975) found that

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turnover o f stable RNA and protein, per unit time, was the same at 408 atmospheres as at one atmosphere. Since growth under pressure was slower, turnover per generation was greater. However, calculations indicated that the net investment of ATP in polymer replacement was minor compared with the apparent ATP loss. Matsumura (1975) proposed that much of the ATP loss was due to pressure activation of the membrane ATPase of the bacterium. This multimeric enzyme, which is attached to the cell membrane by means of a protein called nectin, was inhibited by pressure when the ATP concentration was less than 0.5 mM, but was stimulated at higher ATP concentrations. This behaviour can be interpreted, along the lines proposed by Laidler (1951), in terms of an ATP-binding reaction that has a positive volume change and an activated-complex forming reaction that has a negative volume change. At low substrate concentrations, binding determines the rate of reaction, while at high substrate concentrations, activated complex formation sets the rate. Mohankumar and Berger (1974) have described a similar situation with regard to the barosensitivity of the malate dehydrogenase of a marine Vibrio sp. Pressure was inhibitory at low concentrations of substrate but was stimulatory at high ones. Moreover, the Michaelis constant for the reaction decreased with increasing pressure, and the malate concentration for optimal enzyme activity increased with increasing pressure. Matsumura (1975) found that the ATP pool of Strep. fueculis cells, growing at one atmosphere, rose during the culture cycle from 5 t o about 1 5 pmoles/g dry weight, and then declined sharply a t about the time exponential growth stopped. The maximum attainable pool size at 408 atmospheres was only about 11 pmoleslg dry weight. These values indicate maximum intracellular ATP concentrations of about 1.5 t o 4.5 pmoles/ml cell water (i.e. concentrations above the threshold for pressure stimulation of the ATPase of isolated cell membranes). Certainly, there could be debate regarding the effective concentration of ATP in the neighbourhood of the cell membrane. However, the case for inefficiency being due to enhanced ATPase activity under pressure was strengthened by the finding that the efficiency of growth at 408 atmospheres could be increased to the one-atmosphere level by adding t o the growth medium 0.01 mM dicyclohexylcarbodiimide, a known inhibitor of membrane ATPase. In addition, it was found that 50 mM Mg2+, which enhances barotolerance, also enhances ATP pool levels in pressurized cells.

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Pressure activates ATPases of other organisms, including the myosin ATPase of the benthic, marine, rat-tail fish (Dreizen and Kim, 1971), and it is possible that one of the major problems of organisms growing under pressure is to maintain adequate ATP supplies. The problem may not be one of synthesizing sufficient ATP but of effectively utilizing for biosynthesis that which is produced. Studies of enzymes from deep-sea fish have led Hochachka and his coworkers (Hochachka et al., 1972) to some provocative notions regarding the design of proteins that would function at high pressures. They feel that the most pertinent information regarding the effects of pressure on an enzyme is not the AVt value, or volume of activation, but rather the changes at increased pressures in the affinities of the enzyme for substrates, cofactors and modulators. There is need here to realize that Hochachka et al. (1972) use the term “volume of activation” to refer specifically to the volume change for conversion of the enzyme-substrate complex t o an activated form and not necessarily the AV$ of equation 2 (p. 168). The binding reactions for substrates, co factors and modulators also involve transitions states or activated complexes. Therefore, a volume of activation can be determined for each of these reactions, as well as for activation of the enzyme-substrate complex. In fact, as Laidler (1951) showed, at low substrate concentrations, it is very often the substrate-binding reaction that determines barosensitivity. Thus, in an enzyme-catalysed reaction, the net effect of pressure may be primarily due to effects on initial substrate binding, on activation of the enzyme-substrate complex, on decay of the complex, or on any combination. The situation is even more complex for enzymes that react with allosteric modulators or with activating, inorganic ions. However, it does seem important when considering AV$ values to be aware of just how they were obtained and what precisely they mean. To date, no studies of bacterial enzymes have been undertaken that are equivalent to those of Hochachka et al. (1972) with enzymes from deep-sea fish. In fact, as mentioned previously, there is some doubt that bacteria can actually adapt to growth under high pressure and that there are truly barophilic enzymes in prokaryotes. There are many prokaryotic enzymes that work better under elevated pressure, but they do not seem specifically to be adapted to a barophilic existence. Hydrostatic pressure has been used to a limited extent for basic studies of photosynthesis. The primary interest in this sort of work is

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obviously not ecological since photosynthesis cannot take place below the photic zone, which extends some 50 metres below the surface where the pressure is only about five atmospheres. However, Vidaver (1972) has pointed out that some photosynthetic organisms do spend part of their life cycles at greater depths, and their photosynthetic apparatus may be subjected at times to pressures greater than five atmospheres. Also, pressures of only a few atmospheres can affect the buoyancy of those photosynthetic organisms with gas vacuoles. In his extensive reviews of the barophysiology of photosynthetic organisms, Vidaver (1969, 1972) has described the details of pressure inhibition of photosynthetic oxygen generation, assessed with platinum electrodes in situ. He concluded that it is necessary to control concentrations of oxygen under pressure in order to obtain readily interpretable information on pressure effects. Pope and Berger (1973a, c) have designed apparatus that has an oxygen-detecting electrode couple plus another couple with a separate, large, oxygen-consuming, platinum anode. With their apparatus, which electrochemically reduces the oxygen produced by photosynthesis, they were able to control the dissolved oxygen concentration to within 0.5%. They studied a number of algae, but concentrated their attention on Anacystis nidulans for which the assimilation of carbon dioxide was more pressure sensitive than was the evolution of oxygen at pOz values close to zero. Growth and uptake of carbon dioxide showed nearly the same sensitivity to pressure, as one might expect. At about 450 atmospheres, growth was almost completely stopped, but the production of oxygen was essentially unchanged from that in the control culture at one atmosphere pressure. Thus, it appears that, in a photosynthetic organism, under pressure, there is a diversion of energetic equivalents t o processes other than biosynthesis, and that growth of A . nidulans under pressure is therefore inefficient, as is that of Strep. faecalis. Pressure has been found to inhibit a number of other exergonic metabolic processes. For example, sulphate reduction by deep-sea bacteria was found by ZoBell (1964) to proceed more rapidly under pressures as high as 1,000 atmospheres than at one atmosphere. However, a pressure of 1,400 atmospheres was inhibitory, and the reaction was completely stopped by 1800 atmospheres. There has been some interest in the effects of pressure on anaerobic corrosion of metals, catalysed by the activities of sulphate-reducing marine

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bacteria. Willingham and Quinby (1971) found that three isolates of sulphate-reducing bacteria were more corrosive for iron test pieces at 200 atmospheres than at one atmosphere during incubation in a culture medium at 20°C. However, a pressure of 600 atmospheres markedly decreased corrosion. Tests with aluminium or stainless steel pieces indicated that the particular bacteria used had little effect on the metals at any pressure. Ehrlich (1974) has recently summarized work carried out in his laboratory on the effects of pressure on bacteria associated with ferromanganese nodules from the ocean floor. The flora of these nodules includes bacteria that are capable of oxidizing o r reducing manganese. Both these activities can occur under deep-sea conditions of high pressure and low temperature. At 4"C, Mn(I1) oxidation was detected at 476 atmospheres, but not at 544 atmospheres, in 17 hours with a Gram-negative motile rod, previously isolated from a nodule dredged from the Pacific Ocean floor at a depth of about 5,000 metres. Manganese dioxide reduction by another Gram-negative motile rod, isolated from the same nodule, occurred at 408 atmospheres, but not 476 atmospheres. Growth of the bacteria in a variety of media was found t o be more sensitive t o pressure than were manganese oxidizing and reducing activities. Again, growth seemed to be more sensitive to pressure than were catabolic processes. Even though they may be less severe than growth inhibitory effects, there is still a major concern that the inhibitory effects of pressure on catabolic processes may limit degradation of waste materials in the depths of the oceans. Large quantities of biological materials from the productive zones settle to the ocean floor, or to the floors of other bodies of water. The microbial degradation of these materials, which is necessary for the natural cycling of their constituents, seems to be a very slow process. Moreover, there is now a great desire to use the marine environments of the world as a general dump for waste materials that are difficult to dispose of at land-based facilities. A major question, especially in regard t o toxic wastes, concerns whether or not normal biological degradative systems are sufficiently active in the deep to bring about detoxification, and t o avoid massive build-up of poisons in the ocean. Certainly, there are also other matters for concern regarding the excessive enrichment of ocean waters, and upsets t o ecological balances in the ocean.

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Central to these issues is the question of whether or not there are populations of micro-organisms specifically adapted to life in the deep; that is, organisms that are psychrophilic or cold-tolerant, and barophilic or barotolerant. As indicated previously, samplings of the ocean floor at all depths have yielded living micro-organisms, and there is at least some preliminary evidence that some of them may be barophilic. However, estimates of microbial degradation of organic matter in the deep have indicated only very low activity. Perhaps the most dramatic, and presumably unplanned, experiment came from the sinking of the Woods Hole research submarine, the Alvin. As Jannasch et al. (1971) described, the vessel contained the crew's lunch when it sank on the 16th day of October 1968 to a depth of about 1,540 metres (ambient pressure of about 150 atmospheres, temperature of 3 to 4'C). After recovery of the vessel nearly a year later, the lunch was opened and found to be in a remarkably well-preserved state, although it was thoroughly soaked. The results of planned experiments with radio-actively labelled substrates, conducted subsequent t o the sinking of the Alvin, have led to the conclusion that rates of degradation of organic materials in the deep sea are extremely slow. Thus, there do not seem to be large numbers of micro-organisms specifically adapted t o function well in the cold and compressed environment of the deep sea. Certainly, this apparent paucity of microbial activity calls for at least some hesitation in the use of the ocean as a dumping site. However, because of the relatively sparse experimental evidence, it also seems wise t o keep an open mind, especially regarding degradative rates in marine sediments where nutrient concentrations may be high, where surface effects may be important and where there may be microenvironments with high concentrations of inorganic ions. C. BIOPOLYMER SYNTHESIS

There is a general feeling in the field of microbial barophysiology that the inhibitory effects of pressure on the growth of microorganisms are due primarily to pressure inhibition of polymer synthesis. ZoBell and Cobet (1964) determined the gross polymer composition of E. coli B, grown a t various pressures. They found that the cells became greatly elongated and grew as filaments with relatively few cross-walls. Therefore, the dry weight per cell increased

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considerably under pressure, and both protein and RNA mass per cell increased. For example, cells grown at 450 atmospheres (a pressure near the maximum growth pressure) weighed about 2.8 times more than cells grown at one atmosphere pressure. They contained about 2.7 times more protein and about 3.5 times more RNA than control cells. However, their DNA content per cell was not elevated, so that the amount of DNA per unit of cell mass was significantly decreased. These responses to pressure are similar to responses of E. coli B to agents such as certain amino-acid analogues, penicillins or excess magnesium ion. In contrast to these results for E. coli B, the results of Kriss et ul. (1969) with a marine Pseudomonas organism indicated that there was little reduction in DNA content per unit mass, or of protein content, but that RNA and polysaccharides (anthronereactive materials) made up a smaller fraction of the weight of cells grown under pressure. They also indicated that growth under pressure resulted in defects in the cell wall that were apparent in electron micrographs. Pollard and Weller (1966) assessed the effects of pressure on the incorporation of radio-actively labelled precursors of RNA, protein and DNA by E. coli 1 5 cells in minimal medium at room temperature. Their data, obtained with cultures grown at 450 atmospheres, seem most pertinent since this pressure is approximately the maximum growth pressure for E. coli in minimal medium. Incorporation of labelled uracil was unaffected by 450 atmospheres pressure, as was incorporation of labelled thymine. However, incorporation of labelled proline or valine was decreased to only about 70% of the value at one atmosphere pressure. Thus, it seemed that DNA synthesis was not usually sensitive to pressures in the range permitting growth. Yayanos and Pollard (1969) extended these initial experiments, and came to the conclusion that a pressure of about 500 atmospheres specifically stops initiation (or termination) of chromosome replication. At this pressure, bacteria could complete previously initiated rounds of chromosome replication but could not initiate new ones. Thus, the extreme barosensitivity of DNA synthesis appeared to be related to inhibition of initiation processes rather than to polymerization itself. Yayanos (1975) has found subsequently that pressurization of E. coli at about 600 kg/cm2 initially stimulated cell separation.

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Landau (1966, 1967) investigated in more detail the effects of pressure on the incorporation of labelled amino acids into trichloroacetic acid-precipitable material by E. coli K-12. He found, somewhat surprisingly, that pressures within most of the growth range for this bacterium were not inhibitory for incorporation. In fact, a pressure of 272 atmospheres, at 37OC, was actually stimulatory for cells in nutrient broth, a pressure of 408 atmospheres had essentially no effect, and pressures of 544 or 680 atmospheres were inhibitory, the latter markedly so. Landau's experiments were relatively shortterm ones which lasted only about half an hour. He found that the adverse effects of the higher pressures that were tested were fully and quickly reversible, and so did not appear to involve any major damage to the biosynthetic system. As part of the same study, pressure effects on the incorporation of labelled adenine and uridine were also assessed. Again, 272 atmospheres stimulated incorporation, while 680 atmospheres was inhibitory. However, Landau pointed out that RNA synthesis did occur at 680 atmospheres, even though it was slowed, whereas synthesis of DNA or protein was completely stopped. It is somewhat difficult to interpret these findings, regarding the incorporation of labelled precursors, in terms of pressure inhibition of the growth of E. coli since growth is very much slowed at 37OC by pressures of about 400 atmospheres, which seem to have little or no effect on incorporation of labelled precursors. Indeed, even in rich media, E. coli will not grow at pressures much above 500 atmospheres, and so there are dramatic effects of pressure on growth with only marginal effects on incorporation. However, it is certainly possible that synthesis of some particular type of polymer (e.g. initiation proteins) is unusually sensitive to pressure and that this barosensitivity largely determines the growth response to pressure. Moreover, it also seems reasonable to suppose that the complete inhibition of DNA or protein synthesis, by 680 atmospheres pressure, would pose a barrier to growth at higher pressures in the absence of any specific adaptation of the biosynthetic machinery to pressure. There is also a very important effect of temperature change on the barosensitivity of protein synthesis. Landau (1970) found that 2 72 atmospheres inhibits incorporation of labelled leucine into intact E. coli cells at temperatures below about 26OC; it is only at higher temperatures that this pressure is stimulatory. He also found

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that a pressure of 408 atmospheres was inhibitory only below about 37°C while, at 37"C, incorporation rates under pressure and at one atmosphere were about equal. Albright (1969) has determined the effects of pressure on polymer synthesis by Vibrio marinus, a marine, obligate psychrophile that can grow at pressures up to about 420 atmospheres. He found that this marine bacterium behaved in much the same way as did E. cob in that protein synthesis was more pressure sensitive than was nucleic acid synthesis. The application of 544 atmospheres pressure to cultures of V. marinus caused a rapid halt in cell growth or division and in synthesis of RNA or protein. Synthesis of DNA was decreased in a more gradual manner. Decompression of the cultures after sixty to ninety minutes resulted in rapid recovery of growth and biosynthetic activities similar to those extant at one atmosphere pressure. Three separate groups of investigators have undertaken detailed studies of the effects of pressure on the various steps in the process of protein synthesis. Arnold and Albright (1971) used a cell-free protein-synthesizing system isolated from E. coli B/r. They found that the polyuridylic acid-directed binding of charged phenylalanine tRNA to polysomes was greatly decreased by pressure, and was essentially completely inhibited by 600 atmospheres pressure at about 20°C. A pressure of 1,000 atmospheres caused release of previously bound phenylalanyl-tRNA. However, the inhibitory effects of even 1,000 atmospheres pressure were found t o be readily reversible. It was found also that peptide-bond formation was more barosensitive than binding, as indicated by increased ratios of phenylalanine bound to phenylalanine polymerized as pressure was increased from one to 600 atmospheres. Pressure appeared also to cause polysome dissociation, and again the effect was readily reversible. Subsequent work by Hardon and Albright (1974) with E. coli extended these observations. They found that pressure appeared to inhibit the activation of amino acids in that protein synthesis was more sensitive to pressure when amino acids and tRNA were substrates than when previously charged tRNA was used. In fact, a pressure as low as 100 atmospheres caused slightly more than 50% inhibition of amino-acid incorporation, but only about a 3% decrease in transfer of amino acids from tRNA to polypeptides. This extreme sensitivity was demonstrated also with an isolated tRNA-charging

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system, and it was found that charged tRNA was degraded under pressure. The net conclusion of this work was that pressure inhibits all of the steps in protein synthesis, but that the initial tRNAcharging reactions are most sensitive and can be substantially inhibited by pressures in the growth range for E. coli. Schwarz and Landau (1972a, b). used whole cells and a cell-free protein-synthesizing system from E. coli K-12 in a similar study that led to somewhat different conclusions. They found that, at 3 7"C, pressure was inhibitory to the cell-free system and that 680 atmospheres completely stopped synthesis with either poly-LJ message or messenger RNA for MS2 phage. Low pressures, below 400 atmospheres, were not stimulatory for the cell-free system, as they are for the incorporation of labelled amino acids into intact E. coli cells. Their studies with whole cells indicated that 670 atmospheres inhibits peptide elongation, but has no inhibitory effects on amino-acid uptake, tRNA charging or polysome integrity. Subsequent work by Pope et at. (1975b) confirmed that polysomes are stable under 680 atmospheres in whole cells. Studies with extracts indicated that the most barosensitive step was the binding of tRNA to polysomes, and the calculated AV* value of 100 ml/mole for this reaction was essentially equal to the AV$ value for overall synthesis of polypeptide. The puromycin reaction was relatively insensitive to pressure and was decreased by only about 16% at 680 atmospheres in the presence of GTP. Thus, peptidyl synthetase did not appear to be very barosensitive. The net conclusion from this work was that the binding of charged tRNA, probably to the promotor site on the ribosome, is the most barosensitive step in protein synthesis, while tRNA charging, and peptide bond formation, are relatively insensitive to pressure. It seems that at least some of the variance between the results of Arnold, Hardon and Albright o n the one hand, and of Schwarz and Landau on the other, reflects different experimental temperatures, 20°C for the former and 37°C for the latter. In other words, the charging reaction may be the most barosensitive reaction at low temperature, while binding of charged tRNA to the ribosome may be the most barosensitive at higher temperature. Yet another study of the effects of pressure on cell-free protein synthesis is that of Hildebrand and Pollard (1972) with E. c o k W3110 at 22°C to 24°C. They also found that the rate of p d y -

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uridylic acid-directed synthesis of polyphenylalanine was almost completely stopped by pressures above 600 atmospheres. Their studies indicated that tRNA charging is inhibited by pressure, but that some step later in the synthetic process is more barosensitive. They suggest that the ribosome is the most pertinent structure for barosensitivity. They found that pressure caused dissociation of the polysome complex with charged tRNA, release of the tRNA and possibly some hydrolysis. Interestingly, they also found that increased Mg2+ concentrations reversed some of the adverse effects of pressure. Polyphenylalanine synthesis was less barosensitive in the presence of 16.8 compared with 10.8 mM Mg2+. The latter concentration was nearly optimal at one atmosphere pressure. Changes in the concentration of potassium ions in the reaction mixture had relatively little effect on barotolerance. Hildebrand and Pollard (1972) found that the apparent activation energy for synthesis of polyphenylalanine changed from 35 Kcal/mole, for the temperature range from 11 to 26"C, to a value of 1 3 Kcal/mole over the range 26 t o 37°C. Again, it seems that differences in conclusions among the various groups may simply be due to differences in the experimental conditions which they used. In general, it seems that nearly every stage of protein synthesis in E. cok, except perhaps the transport of amino acids, is inhibited by pressure, and that the relative barosensitivities of the reactions may depend on experimental conditions, especially temperature and magnesium-ion concentration. However, as noted previously, none of these cell-free systems showed the stimulation of synthesis by low pressures that occurs in whole cells. Since there are baroduric bacteria that can grow at pressures greater than 680 atmospheres, it seems clear that protein synthesis, in at least some organisms, can take place at pressures above those which completely inhibit the cell-free systems of E. coli. The interesting question here concerns whether or not there are specific adaptations of the protein-synthesizing machinery for operation at high pressure, as there are for operation at high temperature. Pope et al. (1975a) have undertaken to answer this question by comparing the effects of pressure on protein synthesis by P. bathycetes that can grow at pressures as high as 800 t o 1,000 atmospheres with effects on the less barotolerant E. coli and Pseudomonas fluorescens. Initial work reported by Swartz et al. (1974) indicated that incorporation

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of labelled amino acids, or uridine, into the trichloroacetic acidinsoluble fractions of P. bathycetes or P. fluorescens cells was less sensitive to pressure than was incorporation by E. coli cells. The work of Pope e t al. (1975a) indicated, however, that the responses of cell-free systems differed from those of whole cells. At 37"C, the cell-free system for E. coli showed essentially the same response to pressure as did that from P. bathycetes-both were totally inhibited by 800 atmospheres pressure. At 25"C, the P. bathycetes system was more barotolerant, while at 15"C, the E. coli system was more barotolerant. At both temperatures and 800 atmospheres pressure, the cell-free system isolated from P. fluorescens was inhibited by pressure the least. The E. coli system at 15OC and the P. bathycetes system at 25 or 37°C were stimulated by pressure instead of being inhibited, as was the case with whole cells in Landau's experiments. Pope et al. (1975a) found that the response t o pressure depended primarily on the source of ribosomes and not on the soluble factors. Their subsequent work (Smith et al., 1975) has indicated that barosensitivity is associated specifically with the 3 0s subunit of the ribosome. On the basis of these detailed studies, they proposed that the major locus of pressure effects on protein synthesis is in the ribosome, as Hildebrand and Pollard (1972) had proposed. They also mention work to be described later that shows that a pressure of 670 atmospheres prevents dissociation of polysomes in growing E. coli cells, and thus blocks the protein-synthesizing machinery. Their work suggests that there is no specific adaptation of the proteinsynthesizing machinery of P. bathycetes t o life at high pressure. In fact, the finding that synthesis by intact cells is significantly more resistant to pressure inhibition than is that by the cell-free system suggests that other factors within the intact cell ameliorate the adverse effects of pressure. Also, it seems noteworthy that the incorporation of amino acids can take place at pressures as high as 1,000 atmospheres in both intact cells and cell-free preparations. It also seems pertinent t o point out again that P. bathycetes has an optimum growth temperature of about 37OC, and so is not truly adapted to life at the bottom of the ocean. Hence, the question of how bacteria grow at high pressures still seems an open one. Do they have enzyme systems that are inherently barotolerant, or do they manage to protect their enzyme systems from adverse effects of pressure in some other way such as by concentrating divalent cations?

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The effects of pressure on biopolymer synthesis by eukaryotic Tetruhymena cells, and by HeLa cells, are very similar to those for E. coli (Landau, 1970; Zimmerman and Zimmerman, 1970; Zimmerman, 1971). Nucleic-acid synthesis is less sensitive to pressure than is protein synthesis. However, Zimmerman (1971) concluded from studies with synchronized Tetrahymena cells that pressure sensitivities of synthetic processes change with time during the cell cycle. Reports on the effects of pressure on synthesis of polymers other than DNA, RNA and protein are essentially non-existent, except for the preliminary studies of Kriss et at. (1969). Some years ago, Berger (1959) did report in an abstract that pressure inhibits the formation of cell walls in E. coli, and Kriss et al. (1969) saw morphological aberrations in the walls of the bacteria they examined in the electron microscope. It seems that there should be some follow-up of these preliminary observations, especially since pressure does cause some bacteria to grow in snake form, with relatively few cross-walls. D. CELL DIVISION AND MORPHOLOGICAL DIFFERENTIATION

ZoBell and Oppenheimer (1950) observed that some of the bacteria they grew under pressure became extremely elongate. For example, Serratiu marinorubra grew very slowly in nutrient broth at 600 atmospheres, and 23"C, to form filaments up to 100 nm in length, with very few cross-walls. Subsequent observations (ZoBell, 1970) have revealed that many bacteria grow under pressure to become elongate forms, and so it appears that cell division of these organisms is slowed by pressure more than is cell growth. However, other bacteria undergo no such change in morphology, and division does not appear to be more barosensitive than is growth. Moreover, ZoBell and Cobet (1962, 1964) found that the extent of differential barosensitivity varies, even among strains of the same species. The B strain of E. coli is particularly liable to form filaments under pressure, and also under other adverse circumstance, whereas other E. coli strains show much less tendency to become filamentous. Boatman (1967) carried out detailed electron-microscope examinations of a number of types of bacteria grown under pressure. He found that Gram-negative E. coli, Aerobacter sp. and a Vibrio sp. all became elongated under pressure, and some of the Vibrio cells were grossly deformed or converted to sphaeroplasts. In contrast, a

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Gram-negative Pseudomonas organism showed n o morphological aberrations when grown under pressure. This same variance was apparent also with the Gram-positive bacteria studied by Boatman (1967). Bacillus mycoides and a Corynebacterium organism became ~ not. We have filamentous under pressure, while Bacillus sp. 8 7 1 did found that cocci, including streptococci, micrococci and staphylococci, generally do not show major morphological aberrations when grown under pressure. Boatman (1967) noted that filament formation could be suppressed by culturing the bacteria in media supplemented with 1% (w/v) glucose but did not determine the basis for the effect of the sugar. It seems from these results that pressure can selectively inhibit septation or cross-wall formation in some bacteria. Many other adverse factors have the same sort of differential action. For example, sublethal levels of penicillin or p-fluorophenylalanine induce filament formation with E. coli B. However, it seems that there must be more than one site of action because we have found that pressure does not potentiate the action of these other agents. In fact, there is evidence (Schlamm et al., 1969; Schlamm and Daily, 1972) that some bacteria are less sensitive t o penicillin under pressure. Perhaps the use of synchronized or synchronous bacterial cultures would help t o identify more closely the pressure-sensitive reaction in cell division. Yayanos and Pollard (1969) mentioned that pressure itself may be a phasing or synchronizing agent for E. coli in that the cultures they grew under pressure became at least partly phased. Zimmerman and Laurence (1975) have recently found that pressure can be used for synchronizing cultures of Tetra h y mena p yrifo rm is. There is only very scanty, preliminary information on the effects of pressure on the types of cell division that occur during microbial differentiation. It was mentioned earlier that pressure can act as a germinating agent for bacterial endospores. ZoBell (19 70) indicated that the process of sporulation is more sensitive to pressure than is ‘growth, except for Bacillus abysseus which appeared t o form spores more readily at 400 atmospheres than at one atmosphere. AIso, Boatman (1967) observed that cultures of B. mycoides, Bacillus 871M or Bacillus 874M yielded small numbers of spores at 21°C under pressures of 272 t o 544 atmospheres. It is well known that sporulation is sensitive to many environmental factors, including Eh and oxygen supply, and there is a need to separate pressure effects

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from effects due to changes in other factors in enclosed pressure vessels. At present, there is no information concerning barosensitivities of other types of spore formation, or other types of morphological differentiation of microbial cells.

E. REGULATORY FUNCTIONS

The long-term survival of any organism depends on its adaptive capacities. Free-living bacteria have highly developed adaptive mechanisms, many of which are related to the need of small unicellular organisms to cope with limited and varying nutrient supply in highly competitive natural situations. Landau (1967) has carried out a thorough study of the best known bacterial adaptive system-the lac operon of E. coli. He found that adaptive synthesis of 0-galactosidase was remarkably sensitive to pressure and was completely inhibited by pressures greater than 265 atmospheres over a 10-minute period at 37°C. An analysis of the system revealed that the exquisite barosensitivity of induced enzyme synthesis was due to pressure inhibition of derepression rather than of the processes of transcription or translation. These findings have very restrictive implications for marine ecology in that survival under pressure in natural environments must involve synthesis of adaptive enzymes for many or most bacteria. We have recently extended Landau’s study to include longer term experiments and experiments with growing cultures (Marquis and Keller, 1975). When unadapted E. coli B cells were used to inoculate minimal medium with lactose as the sole source o f fuel and carbon, there was a short lag of two to three hours at one atmosphere, and 3OoC, before growth began. However, at 400 atmospheres pressure, this lag was greatly prolonged to as much as twelve to sixteen hours, but in time, the bacteria did become derepressed, they did synthesize P-galactosidase and they were able to degrade lactose and grow. Under pressure, the bacteria never did become fully derepressed, even after a number of subculturings, and their growth rates were slower than those of bacteria that had been previously derepressed at one atmosphere pressure, or of a constitutive mutant strain. Moreover, the maximum growth pressure for cultures in lactose-minimal medium inoculated with unadapted cells was lower than that for

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similar cultures inoculated with adapted cells or cells of a constitutive mutant. Thus, it did appear that the extreme barosensitivity of the derepression process acts t o compromise the bacteria in their capacities to grow under pressure. However, the barosensitivity of derepression in long-term experiments was not as great as that indicated by the results of Landau's short-term experiments. It was found that induction of exopenicillinase synthesis by Bacillus licheniformis was also unusually barosensitive and that unadapted cells were much more sensitive to penicillin under pressure than they were at one atmosphere. A number of other adaptive processes proved to be less sensitive to pressure. For example, previously adapted or unadapted E. coli cells grew equally well at 400 atmospheres in a minimal medium with arabinose as the sole source of fuel and carbon. Similarly, adaptation of Strep. faecalis to catabolism of lactose, ribose o r maltose was less sensitive to pressure than was growth, and there was essentially n o difference in response to pressure between cultures inoculated with previously adapted versus unadapted bacteria. There is almost a total lack of information concerning the effects of pressure on other re'gulatory functions, although we did find that catabolite repression in E. coli is not altered by pressures as high as 400 atmospheres. Also, Izui (1973) has found that the allosteric phosphoenolpyruvate carboxylase of E . coli can be protected against pressure inactivation by L -aspartate, an allosteric inhibitor, but is made more pressure sensitive by the allosteric activator, lauric acid. Certainly one might expect that some of the most barosensitive sites in bacterial cells would be regulatory proteins that generally undergo co-operative con formational changes or subunit dissociation during activation-deactivation cycles. However, a recent dilatometric estimation of the volume change associated with allosteric binding of threonine by the aspartokinase-homoserine dehydrogenase-1 complex of E. coli (Wampler and Katz, 1974) indicated a rather low AV of only 40 ml/105 g protein. This low value suggests relative insensitivity to pressure changes, but our findings (Marquis and Keller, 1975) regarding derepression indicate that, although some regulatory processes are relatively insensitive to pressure, others are highly sensitive. The process studied by WampIer and Katz (1974) may simply be one of the former.

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F. MOTILITY

The inhibitory effects of pressure on the motility of eukaryotic micro-organisms that contain microtubules were discussed previously. Pressure also inhibits movement of prokaryotes that do not contain microtubules. Inhibition of movement of flagellate bacteria by pressure was first described by Regnard (1891) and subsequently, in more detail, by ZoBell (1970). Recent work (Meganathan and Marquis, 1973) has shown that hyperbaric pressure slows the movements of bacteria with flagella formed during growth at normal atmospheric pressure, and also inhibits formation o f new flagella. The latter process is extremely barosensitive and can be completely suppressed in E. coli B275 by pressures as low as 100 to 200 atmospheres, even though the bacterium has a maximum growth pressure of about 550 atmospheres. Similar observations were made for a series of other bacteria, including both Gram-positive and Gram-negative types. It remains to be determined whether pressure inhibits synthesis of flagellin, o r other flagellar components, or inhibits assembly of the components. There have been no studies of the effects of pressure on bacterial chemotaxis. The extreme barosensitivity of motility may in itself preclude tactic responses under pressure and may compromise survival in deep-sea environments where chemotaxis has selective advantage. G. LUMINESCENCE

The effects of pressure and temperature on bacterial luminescence have been studied extensively by F. H. Johnson, H. Eyring and their colleagues. Their work has been reviewed a number of times, most recently by Johnson and Eyring (1970). Much of our current perspective of the molecular aspects of barophysiology is due to their efforts, particularly their application of the theory of absolute reaction rates to pressure-temperature studies of biological processes. They interpreted the effects of pressure and temperature on bioluminescence in terms of a light-yielding process that has a positive volume change for activation, and a heat “denaturation” process that is also accompanied by an increase in volume. At low temperatures, the action of pressure is simply to inhibit the light-yielding process. At temperatures above the denaturing temperature, pressure still

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inhibits the light-yielding process, but it also reduces denaturation. Therefore, at high temperatures, the net effect of pressure is to enhance luminescence. Under pressure, the optimum temperature for luminescence is shifted upward, although the actual optimum depends on the test organism. In other words, there is an individuality in the luminescent system of e.ach organism, and presumably, this individuality is related to the peculiarities of the tertiary and quaternary structures of the proteins involved. A major job ahead for microbial barophysiologists is to decipher the details of the relationships between the structural peculiarities of biopolymers and the physiological effects of pressure on bioluminescence and other functions. The work of Johnson and Eyring (1970) has set a general framework for interpretation, but the current need is for molecular details t o add substance to this framework.

VI. Acknowledgement The author’s work was supported by the U.S. Office of Naval Research. I thank Diana Marquis for critically reading the manuscript. REFERENCES

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The Regulation of Respiration Rate in Growing Bacteria DAVID E. F. HARRISON Woodstock Laboratories, Shell Research Ltd, Sittingbourne, Kent

I. Introduction . . 11. Response of Respiration Rate t o Environmental Changes . A. Response to Dissolved Oxygen Tension . . B. Response to Temperature . . C. Response to pH Value. . D. Growth Rate . . 111. Substrate Control of Respiration IV. Adenosine Phosphates as Regulators of Respiration . A. Steady-State Contents of Adenosine Phosphates in Growing Cells B. Concept of Energy Charge . . C. Transient-State Studies V. Role of NADH in the Regulation of Respiration . A. Measurement of Nicotinamide Nucleotides . B. Response of NAD(P)H Content to Perturbations of the Steady . State C. Oscillations in NAD(P)H Fluorescence . . D. Regulatory Role of NADH Dehydrogenase . VI. Cytochromes as Regulators of Respiration A. The Inducibility of Bacterial Cytochromes . B. Branched Electron-Transport Systems . VII. Energy Conservation . VIII. General Conclusions References . .

.

.

. .

.

.

. .

.

.

. . .

. . .

243 245 246 256 257 258 259 263 265 267 269 277 278 281 285 290 290 291 297 303 306 309

I. Introduction The regulation of respiration in mitochondria has been extensively investigated and was reviewed as early as 1956 by Chance and Williams. Recent studies (Chance et al., 1972) have elucidated further the regulation of mitochondria1 respiration, but the accepted 243

244

D.

E. F. HARRISON

mechanisms remain more or less as proposed by Chance (1957) and are summarized in Table 1. In contrast, the regulation of respiration in prokaryotic organisms has been, until recently, a somewhat neglected study. One reason for this n o doubt has been the greater difficulty, in prokaryotic organisms, of isolating the respiratory components from the rest of the cell’s metabolic system; respiration in the bacterial cells is, perhaps, more intricately linked with growth, ion transport and other functions than in the eukaryotic cell where the respiratory components are restricted t o the mitochondria. An added problem in relating regulation of respiration in bacteria t o that in mitochondria lies in identifying the physiological “state” of the TABLE 1. The characteristics of steady-states of mitochondria1 respiration and the identity of rate-limiting c o m p o n e n t s State

Oxygen status

Substrate

1. 2.

Endogenous Starved

Excess Excess

3.

Active

Excess

Endogenous Low approaching zero Excess

4.

Resting

Excess

Excess

5.

Anaerobic

Absent

Excess

ADP concentration

Respiration Limiting rate component

Low High

Slow

High

Fast

Low High

Slow Zero

Slow

ADP Substrate Respiratory chain ADP Oxygen

~~

(After Chance and Wi!!iams, 1956).

bacterial cell. The term “logarithmic state” and “stationary state” have been commonly employed t o describe the physiological condition of bacterial cells, but neither of these terms is really definitive. “Logarithmic” cells may, for instance, be substrate-limited or have excess substrate, and the “stationary state” of cells will depend both on the environmental factor that caused cessation of growth and on the period of time in which the cells have been held in the non-growing state.’ Bacteria in a state of active growth and division present particularly interesting problems of regulation. The respiration rate must be appropriate t o the conditions of growth in an environment subject to rapid and radical changes. This review will attempt to rationalize the available date relevant t o understanding the type of regulatory mechanisms involved in control of respiration in growing bacteria.

T H E REGULATION OF RESPIRATION R A T E IN GROWING BACTERIA

245

Bacteria growing on a compound which serves as both carbon and energy source are faced with the problem of apportioning the substrate between catabolic and anabolic processes. The extent to which the energy requirements are balanced by the rate at which energy is made available will determine the efficiency of conversion of sybstrate into cell biomass. It would seem reasonable t o assume that maximization of substrate conversion into cell mass would have some selective advantages (although not perhaps under all conditions) and therefore that efficient regulatory mechanisms would have been evolved in heterotrophic bacteria. This would, of course, apply equally to the eukaryotic organism. However, bacteria differ from eukaryotic organisms in one important respect: the respiratory system of the eukaryotic organism is restricted to the mitochondrial membrane which is surrounded by the cell cytoplasm and is therefore cushioned from the more extreme and rapid changes in the environment to which the bacterial respiratory system, located on the cell membrane, must be subject. Probably it is for this reason that the bacterial cytochrome systems are not only more varied, but demonstrate much greater adaptive responses to environmental changes.

11. Response of Respiration Rate to Environmental Changes

Before considering mechanisms for the regulation of respiration, it is necessary to examine the way in which the regulatory responses are manifested in terms of response to environmental change. It is important to reco
246

D. E. F. HARRISON

A. RESPONSE TO DISSOLVED OXYGEN TENSION

1. Steady-State Systems

The effect of dissolved oxygen on respiration rate has been studied more than any other single environmental factor. The type of response to dissolved oxygen tension, described by early workers for non-growing cells, was one in which respiration rate was independent of dissolved oxygen at oxygen tensions over a certain “critical” value, and decreased with oxygen tension below this value in a pseudo first-order relationship (Gerard and Falk, 1931). Recent studies on growing cells have mostly confirmed that there is, indeed, a range of dissolved oxygen tension (generally between 1 5 and 150 mm Hg) over which the respiration rate is insensitive to dissolved oxygen. But there is at least one notable exception to this, namely the nitrogen-fixing bacterium, Azotobacter (Drozd and Postgate, 1970). Although the response of some growing micro-organisms t o oxygen tension below the critical level has been described as complying with Michaelis-Menten-type kinetics (Harrison e t al., 1969; Button and Garver, 1966;Johnson, 1967) in many cases the response is much more complex. The response of a chemostat culture of Klebsiella aerogenes to dissolved oxygen tension was found to fall into three main phases (Fig. 1).At oxygen tensions above a value of about 10 mm Hg there was no effect of dissolved oxygen tension on respiration rate or metabolism. As the dissolved oxygen tension was lowered t o just below 10 mm Hg, the culture entered an unsteady-state condition with oscillatioiis in oxygen tension reflecting an oscillating respiration rate (Fig. 2). When the oxygen supply was lowered further, and the dissolved oxygen tension fell below 0.5 mm Hg (the lower limit o f the sensitive range of the hlackereth (1964) oxygen electrode that was used), then a steady-state of respiration was again established but the Qo, was actually higher than that expressed in the presence of excess oxygen (Table 2). Furthermore, this increase in QoZ was associated with a decreased yield of organisms from the substrate. Lowering the oxygen supply further eventually caused a diminution in the respiration rate. Harrison and Pirt (1967) examined in some detail the oscillations obtained in the transition phase between excess and limiting concentrations of oxygen. They showed that when the measured oxygen tension was at the minimum detectable, the Qo, of the culture was

THE REGULATION OF RESPIRATION RATE IN GROWING BACTERIA 110-

Oxygen- limited phase

100-

- 90-

-I1 0 Trans"'on (oscillating q value1 '2 phase

Excess-oxygen phase

I

0

247

J- 100

I

80-

p

40-

-40

20-

-20

10 0 0

5

-30 ?

>

-

0"

-I 0 1

1

1

/ I l l

I

l

l

I

I

I

I

0

10 20 30 40 50 60 70 00 90 100 110 120 130 140 Partial pressure ofoxygen in thegas phase (mm Hg)

FIG. 1. Effect o f dissolved oxygen tension on respiration rate in a chemostat culture of Klebsiella aerogenes. Organisms were grown at a dilution rate o f 0.20 h-' (pH 6.0) in a nitrogen-limited medium containing excess glucose. The culture Qo, value (a)and dissolved oxygen tension ( 0 ) are plotted as a function of the partial pressure of oxygen in the gas phase. (Data of Harrison and Pirt, 1967.)

higher than that extant in the "excess oxygen" steady-statey and when the oxygen tension reading was at a maximum, the Qoz was about the same as that of the "excess oxygen" steady-state (Fig. 1, Table 2). In order to ascertain whether these changes in Qo, were a direct response to the dissolved oxygen tension, sudden changes in the dissolved oxygen tension were induced by temporarily altering the oxygen partial pressure in the gas phase. When the dissolved oxygen tension was above 10 mm Hg (i.e. the Qo, was at a

Time (hours)

FIG. 2. Oscillations in oxygen tension observed during the transition phase between excess and limited oxygen supply in a chemostat culture of Klebsiella aerogenes (D = 0.20 h-' , pH = 6.0, nitrogen-limited, glucose in excess). At times indicated by arrows, the culture was sampled. The points (i) and (ii) are referred to in Table 2. (Data o f Harrison and Pirt, 1967.)

hl P

03

TABLE 2. Respiration rate and cell yield in relation to the changes in dissolved oxygen tension in a chemostat culture of Klebsiella aerogenes -Condition

Excess oxygen Oscillating Transition phase' (i) (Max oxygen tension) (ii) (Min oxygen) Limited oxygen More oxygen limited Anaerobic

Dissolved oxygen tension (mm Hs)

15'

Qo, Value

Yglucose value

5.05

0.34

YO, value

Glucose carbon in fermented products (%)

(m moles g-' h - l )

1.28

35

Q rn

n I

D

10-13 0.2 (0.2

90.2 0.0

4.65 7.17 6.45 1.98 0.00

0.34 0.25 0.15 0.13 0.09

1.37 0.89 0.99 3.20 0.0

I

48 45 58 73 78

a See Fig. 2 (p. 247) for points (i) and (ii) on oscillation. Organisms were grown at a dilution rate of 0.20 h-1 (pH 6.0) in a nitrogen-limited chemostat culture supplied with an excess of glucose. (Data oPHamson and Pirt, 1967.)

lJ

z

v)

0

z

THE R E G U L A T I O N OF RESPIRATION R A T E IN GROWING BACTERIA

249

minimum), a sudden decrease in the dissolved oxygen tension to 6 mm Hg had n o effect on the oxygen uptake rate; when the oxygen supply rate was returned to its previous value the dissolved oxygen tension increased again. However, when the dissolved oxygen tension was temporarily decreased to 4 mm Hg, or less, and then the oxygen supply rate restored, the dissolved oxygen reading did not increase in unison, but continued to decrease until it reached a zero reading. The oxygen uptake rate had increased and the oxygen tension then remained low for about 1-5 hours before showing a sudden spontaneous increase to about 1 3 mm Hg, like that found in the cyclic fluctuation shown in Fig. 2. This response was obtained with cultures challenged at any point on the high part of the cycle of oscillations in oxygen tension, whether the oxygen tension had just increased from zero or whether it had remained at the high part of the cycle for more than two hours. It was noticed, however, that the longer the culture was maintained in the oscillating state, the lower was the threshold oxygen tension at which the respiration was stimulated. Thus, whereas initially this threshold value was about 4 mm Hg, after 24 hours it had fallen to 2 mm Hg. When the oscillating dissolved oxygen tension was challenged at a minimum value (that is, the oxygen uptake rate was at a maximum), by increasing the oxygen supply rate until the dissolved oxygen tension rose to 5 mm Hg, there was no effect on oxygen uptake rate and the oxygen tension fell at once to zero on restoring the oxygen supply to its previous value. However, when the dissolved oxygen tension was allowed to increase to 10 mm Hg, the oxygen tension did not return to zero on restoring the oxygen supply to its previous level, but continued to increase. The respiration rate was found t o have failen; and the oscillations underwent a phase-change to the maximum part of the cycle. From these, and other experiments carried out by Harrison and Pirt, it would seem that the culture responded to a fall in dissolved oxygen tension below a certain threshold value (which varied between 4 and 2 mm Hg) by increasing the respiration rate so that the dissolved oxygen tension decreased further. Similarly, when the respiration rate was at a maximum, a sudden increase in oxygen tension to above 7 mm Hg caused a decrease in the respiration rate to the level extant in the presence of excess oxygen. Thus, not only is the steady-state respiration rate increased at low oxygen tension (a shown in Fig. l), but the respiration rate reacts rapidly and

250

D. E. F. HARRISON

spontaneously to sudden changes in oxygen tension around a certain “critical” level. The oscillations obtained by Harrison and Pirt (1967) with continuous cultures of Klebsiella aerogenes have been modelled by Degn and Harrison (1969). Their model showed that such oscillations could be generated provided that three criteria were met: (1) there must be a region of negative slope in the relationship between respiration rate and oxygen tension in the liquid. This type of relationship was shown clearly to exist with K . aerogenes (see Fig. 1); (2) there must exist a dependence of respiration rate of the culture on time. A mechanism for such a time dependence was not demonstrated in the work of Harrison and Pirt (1967), but one could postulate several mechanisms which might give such a dependence, for example, the build-up of an inhibitor of respiration which is removed during the phase of low respiration rate, or the fall in cell concentration during the high respiration phase caused by the diminished yield coefficient; ( 3 ) there must be a limitation of the transport of oxygen from the gas to the liquid phase. This latter condition is not related to any physiological or biochemical mechanism o f the cells. Resistance to diffusion of the gas liquid interface is well known and is usually represented by the constant KLa. This model of Degn and Harrison (1969) only demonstrates that such oscillations can be explained by the known response of the organism, but does not in any way establish the actual mechanisms by which the organism responds to a low dissolved oxygen tension. However, the existence of such osscillations, and the rapid response o f respiration rate to changes in oxygen tension demonstrated by Harrison and Pirt (1967), are a clear indication that the respiration rate in this bacterium is under some mode of feedback regulation. The seemingly anomalous response by K . aerogenes to low dissolved oxygen tensions (that is, an increased respiration rate accompanied by a decreased yield of cell mass) is not, however, restricted to this organism. Similar responses have been reported in the obligate aerobe Haemophilus parainfluenzae (White, 1963) and with Escherichia coli (Harrison and Loveless, 1971a). An increased Qoz at dissolved oxygen tensions below 30 mm Hg was also reported with Pseudomonas AM1 growing on methanol (Maclennan et al., 1971), but with this organism there was no reduction in the yield coefficient and the response does not appear to be analogous to that

251

THE REGULATION OF RESPIRATION RATE I N GROWING BACTERIA

found with K . aerogenes. The increased Qo, values found with Pseudomonas AM1, growing at dissolved oxygen tensions between 7 and 30 mm Hg, could be expIained by the oxidation of some accumulated product of metabolism (Harrison, 1973a). However, at dissolved oxygen tensions below 7 mm Hg, the Qo, value of Pseudomonas AM1 cultures increased further and, although n o steady-state could be maintained, this did appear t o be associated with a depressed cell yield. The response of nitrogen-fixing Azotobacter organisms t o changes in the dissolved oxygen concentration is in direct contrast to the examples given above. With this organism there appears to be no distinct “critical” oxygen tension; the Qo2 value increases with respiration rate over the whole range of oxygen concentrations up t o air saturation (Drozd and Postgate, 1970). The increase in Qo2 value is accompanied by a diminished yield coefficient and the Qo, values attained by this organism at air saturation are extraordinarily high (Dalton and Postgate, 1968). Some confusion has arisen in the past literature over the term “Qo2”;different rates can be obtained with the same cells depending on how respiration is measured. The Qo2 value is defined simply as the number of millimoles of oxygen taken up by lg dry weight of cells per hour, but this does not in itself indicate whether it has been TABLE 3. Effect of oxygen availability on the ‘‘in situ” Qo, and “potential” Qo, values of glucose-limited chemostat cultures of Escherzchia coli and Klebsiella aerogenes

Qo, value (m mole g-’ h-’ )

Oxygen status

in situ

Potentiai

Escherichiu coli (D = 0.15 h - ’ , pH 6.5, t = 35OC) Excess

5.0

5.8

Limited

8.8

16.0 9.0

Anaerobic Klebsiella uerogenes (D = 0.2 h-’ ,pH = 6.5, t = 3OoC) Excess Limited

5.6

7.2

9.1 12.7

Anaerobic

-

22.4

(After Hamson and Loveless, 1971a.)

252

D. E. F. HARRISON

-

15 -

-

c -

‘m

0“

: 10E u)

.. 80

0 c

-60

a”

c

& x 0 x

-40

I

I

I

-20

: 5: V

:

Partial pressure of oxygen in gas feed(mm Hg)

FIG. 3. Effect on both the Qo2in situ, and “potential” Qo2,of altering the dissolved oxygen tension in a chemostat culture of Beneckea natrie ens. Organisms were grown in a glucose-limited chemostat culture (D = 0.2 h- ; pH 7 . 2 ; t = 31.5OC) in which the dissolved oxygen tension (A) was varied by altering the oxygen partial pressure in the gas feed. The Qo, in situ (m) and the “potential” Qo, ( 0 ) were determined and correlated with the dissolved oxygen tension. From Linton et al. (1975).

B

obtained under optimal conditions. The respiration rate o f organisms growing under growth-restricted conditions in a continuous culture will, generally, be lower than that of the same organisms removed from the culture and supplied with excess of substrate and oxygen. The terms “in situ” Qoz and “potential” Qoz have been coined to express, respectively, the respiration rate as measured directly from growing cultures and that measured with cell suspensions supplied with excess oxygen and substrate. Previously we have discussed only the response of the in situ respiration rate, but in Table 3 both the << potential” and in situ respiration rate responses to dissolved oxygen tension are compared (with cultures of K. aerogenes and E. coli). The potential respiration rate was higher throughout, but reached a

THE R E G U L A T I O N OF RESPIRATION R A T E IN G R O W I N G BACTERIA

253

maximum, with K. aerogenes, in cultures grown under an atmosphere of “white spot” nitrogen. With cultures of Beneckea natriegens the response of both the “potential” Q, and QO, in situ varied considerably (Fig. 3 ) . Unlike the rate in situ, the “potential” 20, was affected at dissolved oxygen tensions above the apparent critical value of 8 mm Hg. In fact, the “potential” Qo, showed one increase as the dissolved oxygen tension was decreased below 100 mm Hg, and a second larger increase as the oxygen supply was decreased with the dissolved oxygen tension reading zero (i.e. <0.5 mm Hg). These changes were accompanied by changes in the cellular cytochrome content; this will be discussed more fully later (p. 301). 2. Transient Responses Although studies on steady-state cultures are most useful in identifying the aspects of microbial metabolism which are under feedback regulation, it is only by perturbing the steady-state, and examining the response, that regulatory mechanisms can really be elucidated. Thus, the oscillating transient state between the fully aerobic and oxygen-limited phases, observed in chemostat culture of K. aerogenes (Harrison and Pirt, 1967) and as discussed above (p. 250), is most revealing of the regulation of respiration in these organisms. Unfortunately there have been few careful studies of disturbed steady-states of respiration in growing bacteria. One such study examined the response of the facultative organism, K. aerogenes, t o aeration after a period of anaerobic growth (Harrison and Loveless, 197 lb). A culture of Klebsiella aerogenes was grown anaerobically at a growth rate of 0.15 h-’ (pH 6.8) during which time a steady-state was achieved. The culture was then made aerobic by replacing the nitrogen gas supply with air. In Figs. 4a, b and c are shown the effects of this change on the culture. After an initial decrease, production of carbon dioxide increased during the next two hours (Fig. 4a). Oxygen uptake also increased (as shown by the fall in oxygen tension), accompanied by a steep rise in the concentration of organisms. The potential Qo, value increased to about three times that the aerobic state level, and the in situ Qo, value increased similarly, perhaps indicating that at this point the oxygen uptake by the cells

254

D. E. F. HARRISON

Time of oerobiosis ( h )

FIG. 4. Response, on re-aeration, of a chemostat culture of Klebsiella aerogenes, grown anaerobically for 74 h at a growth rate of 0.15 h-I. (a) ( 0 ) bacterial concentration (dry weight); solid line, recorder trace of dissolved oxygen tension; broken line, recorder trace of C02 output. (b) (A) Y glucose; (A) Y O , ; (0) Qo2 (potential); ( 0 ) Qo, (in situ). (c) (0) acetate; ( 0 ) ethanol; ( 0 ) butanediol; (m) pyruvate. Points shown are the results of duplicate estimations. The limits shown are the standard, technical errors. (From Harrison and Loveless, 1971b.)

was not limited by the concentration of substrate, but by the respiratory potential of the cells. During this period the accumulated metabolites rapidly disappeared, presumably being oxidized (Fig. 4c). However, in spite of the extra carbon available from these metabolites, in addition to the glucose fed to the culture, the calculated yield coefficient from glucose was lower (0.3 to 0.4) than that of the steady-state aerobic value (0.43). The yield from oxygen

THE REGULATION OF RESPIRATION RATE IN GROWING BACTERIA

255

was also lower than that of an aerobic culture, and during this period, therefore, growth seemed to occur with a decreased efficiency. The identity of the substrate associated with the subsequent rise in oxygen uptake is not known; there was no increased utilization of acetate at this point. Both the in situ and potential Qo2 values decreased over the period from 2.5- 6.5 hours after re-aeration until the in situ Qo, value had returned very nearly to that of the aerobic steady-state value. The potential Qo, took rather longer to decrease to the steady-state level, as indicated by its value 24 hours after re-aeration (Fig. 4b). The cause of the high value of the yield from oxygen (Y 02) obtained between 5 and 6 hours after re-aeration (Fig. 4b) is not apparent; possibly it was caused by an accumulation of storage products, but this would not be expected to occur during glucose-limited growth. Eight hours after re-aeration the culture was virtually back to a steady-state condition. The response of Klebsiella aerogenes to a change from anerobic to aerobic conditions can be divided into three stages: a short period of increased acetate production and low carbon dioxide production, immediately following re-aeration; a subsequent period of high respiration rate with low yield coefficients for glucose (Ys) and oxygen (YO,); and finally, after about 8 hours, the attainment of an aerobic steady state. The stimulation of acetate production and the lag in carbon dioxide production on re-aerating anaerobic cultures reveal an interesting aspect of the metabolic control of the cell. Clearly, the bacteria are potentially capable of oxidizing more glucose (there is a substantial difference between the potential Qo, value and Qo2 value in situ) but, in the culture, fermentation to acetate is stimulated before the alternative oxidative metabolism takes over. As ener,gy was derived from both fermentative and respiratory pathways, the calculated yield from oxygen at this point was high. The high respiration rate and low cell yield during the period between 0.5 to 2.5 hours after re-aeration seem to represent a degree of uncoupling between growth and energy conserving mechanisms. This may be a direct result of the change from anaerobiosis, or a consequence of the high growth rate of the bacteria. Bacteria grown at a high rate in a chemostat culture may utilize glucose less efficiently than cells grown at a low rate (Harrison and Loveless,

256

D. E. F. HARRISON

197la). Whatever the mechanism, this “uncoupling” process reveals a weakness in the control system of the cells, which can lead to a diminished efficiency of conversion of substrate into cells. The observed response of anaerobically grown organisms t o aeration indicated regulation of respiration at several levels. Firstly, there was an initial rapid response during which respiration appeared to be inhibited and fermentation predominated; a second response during which there seemed to be no feedback regulation of respiration, this being limited only by the potential respiration rate; and the more long-term response mediated by induction and repression of enzyme synthesis, as evident from the change in 6 6 potential” respiration rate. To these could, perhaps, be added yet another level of response, that of selection of a strain better fitted t o the environment. The genetic variation in heterotrophic bacteria is extremely great and radical changes in growth conditions can be expected t o favour selection of a mutant form of the organism. B. RESPONSE TO TEMPERATURE

Temperature and pH value may affect both the growth rate and the yield of bacteria (Senez, 1962; Harrison and Loveless, 1971a; Topiwala and Sinclair, 1971), and therefore must also influence respiration rate. The response of steady-state growth rate to temperature has been found to follow a simple Arrhenius relationship (Ingraham, 1958; Ng e t al., 1962; Topiwala and Sinclair, 1971). From this relationship, a sudden change in temperature in a growing culture might be expected t o produce an immediate change in the maximum growth rate, and therefore a change in the potential Qo, value. There is, however, a lag in the growth rate, before it accelerates to the expected new value, on subjecting bacteria to a sudden increase in temperature (Ryu and Mateles, 1968; Topiwala and Sinclair, 1971). That there should be delay in the response of cultures t o a stepwise change in temperature is possibly not surprising; an increase in growth rate requires the synthesis of greater amounts of RNA and/or some other regulatory molecules whose concentrations are limiting growth and respiration rate at the lower temperature. The response of a chemostat culture of K. aerogenes to a sharp decrease in temperature was a fairly smooth transition to a lower concentration of organism, with only a small lag period being

THE REGULATION OF RESPIRATION RATE IN GROWING BACTERIA

257

apparent. Thus, although the steady-state relationships t o temperature may be of a simple Arrhenius type, the transitionary response indicates a more complicated underlying regulatory mechanism. Senez (1962) reported that at temperatures far below the optimum for growth the yield coefficient of bacteria was diminished; this would be reflected in less efficient respiration. Harrison and Loveless (1971a), however, found no effect of temperature on the yield coefficient, over a wide range of temperatures, in glucose-limited chemostat cultures of Escherichiu co li. C. RESPONSE TO pH VALUE

The effect of pH value on Qoz and yield values, in chemostat cultures of E. coli, is shown in Table 4 (Harrison and Loveless, 1971a). It can be seen that only at a very low pH value (5.3)was the respiration rate affected; at this point there was a sharp decrease in the efficiency of aerobic growth. The pH value can have a more profound effect on respiration rate where the substrate is one whose metabolism is much affected by the pH value of the culture. For instance, addition of formate to glucose-limited chemostat cultures of Beneckea nutriegens caused a sharp increase in respiration rate at a pH value above 7.0, but a much lower increase in respiration rate at a pH value below 7.0 (J. Linton, personal communication). The pathway of dissimilation TABLE 4. Effect of pH value on the growth yield and respiration rate of a glucose-limited chemostat culture of Escherichiu coli pH Value

8.2

7.5 7.0 6.6 5.4

Yield constant (g cells g glucose-' )

0.35 f O.OZb 0.39 f O.OZb 0.37 O.OZb 0.37 f. O.OZb

4.9 f 0.4

0.32 f O . O Z b

8.3 f 0.3

*

5.5 f 0.3 5.5 f 0.3 n.d.

a d . indicates that the value was not determined: =that each value is the mean o f duplicate estimations; bthat the value was not significantly different at the 95% confidence level. Organisms were grown at a di!ution rate of 0.15 h-' and at 35°C. (Data of Hamsonand Loveless, 1971a)

258

D. E. F. HARRISON

of formate in this organism seems t o be very dependent on whether the pH value of the environment is above or below neutrality. Similar results t o this were obtained on addition of formate to methanollimited cultures of Pseudomonas extorquens. D. GR0WT.H RATE

The influence of growth rate on the respiration rate of bacteria can only be studied in a meaningful way by the use of chemostat culture techniques whereby the rate of growth can be varied, independently of other environmental parameters, by changing the dilution rate. The effects of dilution rate on the respiration rate in situ can be predicted, assuming a constant yield coefficient for oxygen ( Y o ) ,from the relationship:

where Qo, is the specific oxygen uptake rate (mmole g-’ h-’), p is the specific growth rate (h-’ ;equal to the dilution rate D), Yo is the true molar yield coefficient for oxygen (g cells synthesized per mole 0, consumed), and m, is the maintenance respiration rate (mmoles g-’

1-

From equation 1, a plot of Qo, value against the dilution rate, D, should give a straight line cutting the Qo, axis at a value equivalent t o the “maintenance” respiration rate, mo . This relationship appears to hold true for cultures where the energy-source is growth-limiting (Tempest and Herbert, 1965), but the response obtained in a glucose-limited chemostat culture of K. aerogenes deviated somewhat from that which was expected (Harrison and Loveless 1971a). At dilution rates between 0.05 and 0.06 h-’ the Qoz value in situ increased linearly with dilution rate while the potential Qo2 value increased less steeply, and was greater than the value Zn situ. At a dilution rate of 0.60 h-’ , the potential Qo, value and that in situ converged. When the dilution rate was increased to 0.65 h-’ , the cell concentration began to fall but complete washout of culture did not occur. Instead the cell dry weight concentration settled t o a new low level and a steady-state was maintained. This steady-state was accompanied by a high extracellular glucose concentration, and both the potential Qo, value and that in situ were much higher than a t a

T H E REGULATION OF RESPIRATION RATE I N GROWING BACTERIA

259

dilution rate of 0.6 h-' . The physiological state of the cells would appear to have changed on increasing the growth rate from 0.60 to 0.65 h-' . The results obtained with a batch culture of K. aerogenes are compared with those of a chemostat culture in Table 5 of Harrison and Loveless (1971a). In the batch culture the cells grew a t a rate of 0.87 h-' , but did not exhibit the high respiration rate and the low yield that had been observed in continuous culture grown close to the maximum obtainable growth rate (0.75 h-'). The reason for the low maximum growth-rate and the diminished yield at high growthTable 5. Effect of initial methanol concentration on the maximum yield value of batch cultures of Pseudomonas extorquens, compared with methanol-limited chemostat cultures Initial methanol cone (g 1-1)

Yield (g cell dry wt g methanol-' )

2.5 5.0 10.0 25.0 Continuous culture

0.33 0.27 0.20 0.15 0.40

Data of Hamson et al. (1973).

rates was not ascertained. A medium constituent other than glucose may have become limiting at high dilution rates under these conditions, although the concentrations of all medium constituents apart from glucose were doubled without having any effect on cell concentration at adilution rate of 0.75 h-l ,and the addition of casarninoacids and yeast extract had no effect on the respiration rate or growth rate. Possibly the cells produced a growth regulating substance which could be maintained at an optimal concentration in batch culture, or in continuous cultures operated at low dilution rates, but which, at higher dilution rates, washed out faster than it was formed.

I11 Substrate Control of Respiration It is obvious that respiration rate will depend, to some extent, on the availability of oxidizable substrate to the cell. We have already stated that respiration rate is a function of growth rate (equation 1); this implies that the same type of Michaefis-Menten relationship that

260

D. E. F. HARRISON

was proposed by Monod (1950) for the response of growth rate to substrate concentration would also apply to respiration rate. Such a relationship has been shown to hold for the potential respiration rate of methanol-utilizing or methane-utilizing organisms (Harrison, 1 9 7 3 ~ ) Figure . 5 shows the Lineweaver-Burk plot for respiration by Pseudomonas extorquens, with methanol as substrate. Similar relationships have been obtained with more conventional substrates, for example with glucose oxidation by Beneckea natriegens (Linton et al., 1975). 1-

35r

l/S(mM-’l

FIG. 5. A Lineweaver-Burk plot of the response of respiration rate to methanol concentration in cultures of Pseudomonas extorquens. (From Harrison, 1973c.)

The rate of respiration depends not only on the concentration of substrates but also on their chemical nature. Figure 6 shows the results of experiments by Harrison and Maitra (1969) in which pulses of glucose, fumarate, succinate or acetate were added to a glucoselimited chemostat culture of Klebsiella aerogenes. In each case respiration rate was stimulated to a new level, represented by a fall in dissolved oxygen tension. The rate of respiration is reflected by the height of the plateau in the plot of dissolved oxygen tension. The addition of excess glucose elicited the fastest respiration rate, and succinate, fumarate, and acetate were each respired at different rates, in descending order of magnitude. Also, on exhaustion of glucose, fumarate or succinate, the dissolved oxygen tension returned to its previous value only after going through an intermediate plateau, indicating the respiration of some substance that had accumulated during the oxidation of the added substrate. The level of the

THE REGULATION OF RESPIRATION RATE IN GROWING BACTERIA

261

dissolved oxygen tension during this plateau was almost identical with that obtained on the addition of acetate to the culture, and so it would seem most likely that the substance accumulated was, in fact, acetate. Similar studies have recently been carried out by Degn et al. (1973). From the trace of dissolved oxygen tension subsequent to adding glucose to a resting cell suspension of K. aerogenes, in an

15minl

FIG. 6. Stimulation of respiration rate, indicated by a fall in oxvgen tension, on adding (a) glucose, (b) fumarate, (c) succinate, or (d) acetate to a glucose-limited chemostat culture of Klebsiella aerogenes. Addition of each substrate (to a concentration of 1 mM) is indicated by the arrow. (From Harrison and Maitra, 1969.)

open oxygen electrode system, they could demonstrate the presence of no less than six intermediates of glucose metabolism (Degn and Wohlrab, 1971). In the case of a substrate for which an inducible permease is required (for example, citrate uptake by K. aerogenes), a lag of about one half-hour was observed, after adding the substrate, before the respiration rate increased to its new level (unpublished data). From the phenomena discussed above there can be no doubt that respiration rate may be regulated by the rate at which substrate is

262

D. E. F. HARRISON

taken up by the bacterial cell. However, it is inconceivable that all of the substrate which is taken up by growing cells is oxidized, for if that were so there would be no growth on single-carbon substrates. The relative proportion of substrate oxidized to that incorporated into cell material will determine the yield coefficient of the organism. The yield coefficient of bacteria such as K. aerogenes, growing aerobically on glucose, has been found to represent efficient use of energy derived from oxidative phosphorylation (Harrison and Loveless, 1971a) and to be more or less constant over a wide range of growth conditions. However, growth may become less efficient under certain conditions such as low oxygen tension, high dilution rate or extremes of temperature and culture pH value. So it would seem that there must be a regulatory mechanism operating, in addition to simple substrate availability, which determines the amount of the substrate entering the cell which is oxidized via catabolic pathways and the proportion of substrate which is assimilated for synthesis of cell material via anabolic pathways. Not all organisms have well developed regulation processes for substrate utilization. Table 5 shows the effect on yield coefficient of methanol concentration, for the methanol-utilizing bacterium Pseudom o m s extorquens. With this organism, the maximum yield of cells from methanol is achieved only under strict methanol-limiting conditions (Harrison et al., 1973). Whenever methanol is in excess, it is oxidized to a greater extent and the cell yield is much depressed. Pseudomonas extorquens, growing on methanol, would appear to possess much looser regulation of respiration than K. aerogenes growing on glucose. This difference in regulation of substrate oxidation is also reflected in the proportion of added substrate which is oxidized when a pulse of substrate is supplied to cells. From the experiments discussed on p. 270 it can be concluded that when a pulse of glucose was added to a glucose-limited chemostat culture of K. aerogenes, only 17% of the added glucose was oxidized, the remainder presumably being incorporated into cell material. With non-growing cells, incubated in the absence of a nitrogen source, Degn et al. (1973) found a much higher proportion (37%) was oxidized. Methanol-oxidizing bacteria, however, utilized one molecule of oxygen for each molecule of methanol added, which would indicate that nearly all of the methanol was oxidized to formate (Harrison, 1 9 7 3 ~ )Methanol . is a highly toxic substance, and so there might be

T H E REGULATION OF RESPIRATION RATE IN GROWING BACTERIA

263

more advantage to the organism in removing this compound as rapidly as possible, by oxidation, rather than in regulating respiration in order to maximize the growth efficiency. In summary, the respiration rate of bacteria is a function of substrate uptake rate, and has been shown, in many cases, to follow Michaelis-Menten-type kinetics in its relationship to substrate concentration. However, respiration rate is also a function of other environmental parameters, and a simple model for control of respiration by substrate supply alone would not allow for balanced energy metabolism, and growth, in substrate-limited environments. Therefore, superimposed on the regulation of respiration by substrate uptake rate must be other regulatory mechanisms which would allow for modification of respiration rate in response to environmental factors, and which serve to balance anabolic and catabolic use of substrate. The extent to which such regulatory mechanisms have evolved will undoubtedly vary between different bacterial species.

IV. Adenosine Phosphates as Regulators of Respiration The adenosine phosphates, ATP, ADP and AMP (including cyclicAMP), are known to be important as regulators of metabolism in many in vivo systems. They can function as inducers and repressors of enzyme synthesis, allosteric regulators, and as substrates in key enzyme reactions. In isolated “coupled” mitrochondna, respiration rate can, in the States 1 and 4 (Table l),be limited by the rate of supply of ADP for oxidative phosphorylation. The term “respiratory control” is used specifically for this phenomenon. Furthermore, ATP is the major currency of available energy required by enzyme reactions of living organisms. Therefore it is to be expected that adenosine phosphates play an important role in the regulation of respiration rate in growing bacteria. The estimation of ATP, ADP and AMP in cell extracts offers no great problems. These compounds can be estimated with a high degree of accuracy (Estabrook and Maitra, 1962; Dhople and Hanks, 1973). However, there is considerable difficulty in obtained meaningful estimations of the “pool” sizes of adenosine phosphates in actively growing bacteria because of the very high turnover rates of these compounds in vivo (Harrison and Maitra, 1969). The turnover rate for ATP cannot be measured directly, but a minimum

264

DAVID E. F. HARRISON

turnover rate can be calculated. Thus there must be a certain minimum number of ATP molecules required to synthesize one gram of cell material (this is the YATP constant of Bauchop and Elsden, 1960). Therefore, for x grams of cells to be produced, the number of moles of ATP that must be “turned over” is: x. YATP.During growth, the rate of synthesis of cell. material is given by: p. x, where p is the specific growth rate (h-’ ), and x is the equivalent dry weight concentration of organisms (g I-’) at any specific point in time. Therefore, as the ATP content of a cell does not increased infiniturn, but reaches a steady-state, the rate of ATP turnover (in moles g-’ h-’ ) is given by: ___/J

YATP Taking, as an example, a culture with the modest growth rate of 0.2 h-’ and a value for YATPof 1 0 g mole-’, the minimum turnover rate for ATP will be 5.6 x l o w 6 mole g-’ sec-’. This takes no account of a maintenance requirement. The ATP content of K. aerogenes, growing at a rate of 0.2 h-’ , is about 1 0 x mole g-’ (Harrison and Maitra, 1969) and therefore the turnover time for the ATP pool must not be more than 1.6 seconds. Thus, in order t o obtain a meaningful estimation of the adenosine phosphate levels in actively metabolizing cells, the cell reactions must be quenched within a few-tenths of a second after being removed from the culture. Thus, results based on procedures for the estimation of ATP concentrations in growing micro-organisms in which delays of the order of a few seconds occur subsequent to sampling (Wimpenny, 1967; Chapman et al., 1971; Cole et al., 1967) must be interpreted with caution. Studies in which cells were subjected to a delay of several minutes, or which were filtered from the medium prior to the extraction of ATP (Polakis and Bartley, 1965; Forrest and Walker, 1964; Smith and Maal$e, 1963), cannot be taken as representative of the ATP levels in the growing state of the cell. Holms e t al. (1972) used a sampling procedure which entailed a threet o five-second delay, but they ensured that the environmental conditions prevailing in the growing culture did not change significantly while the organisms were being transferred t o perchloric acid for extraction; thus their confidence in the results being representative o f the actual “pool” sizes of the adenosine phosphate seems

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justified. Harrison and Maitra (1969) overcame the problem by developing a technique for the rapid quenching of samples from a chemostat culture. The culture was removed b y a narrow port fitted flush with the base of the fermenter vessel, and samples (10 ml) were collected in three-quarter inch diameter test tubes containing 0.86 ml of 72% (w/v) perchloric acid. The sampling procedure was filmed with a cine camera at 67 frames/second, and it was found that approximately 0.08 seconds was the time required for mixing. The total time for collection of a sample was 0.5 seconds, and the time for the first drop to reach the acid after leaving the vessel was less than 0.1 seconds. Therefore, the maximum time interval between the cell leaving the culture vessel and being immersed in the acid was 0.2 seconds. The time required t o inactivate most of the enzyme systems of a cell is not known, although in yeast it is less than 0.5 seconds (P. K. Maitra, unpublished observations). However, even though the enzymes may not be completely destroyed within 0.2 seconds they will have little activity at such a low pH value. A. STEADY-STATE CONTENTS OF ADENOSINE PHOSPHATES IN GROWING CELLS

The adenosine phosphate contents of Klebsiella aerogenes, grown under a wide variety of different conditions in chemostat culture, are shown in Table 6. Under no conditions did the ADP content fall far below 1.0 mole/g dry weight, and under no conditions did the ATP/ADP ratio rise above 4.0. Compartmentalization of adenosine diphosphate in bacterial cells seems unlikely; it can be concluded therefore that under no conditions does the ADP content fall t o a value which is likely to restrict oxidative phosphorylation; “respiration control”, as understood in the mitochondrial system (States 1 and 4, Table 1) cannot have applied to any of these cells. In non-growing cells it may be possible for respiration rate to be limited by ADP concentrations, as studies with yeast by Maitra and Estabrook (1967) indicate. However, most reliable reports of adenosine phosphate content of micro-organisms during growth (Atkinson, 1970; Chapman et al., 1971; Holms et al., 1972) are in general agreement with the findings that the steady-state content of ATP and ADP does not change radically with most growth conditions. In no case where reliable measurement of the ADP content of growing bacterial cells, has been made, does the content appear t o fall low

N

m m

TABLE 6. Steady-state concentrations of adenosine phosphates and energy charge in Klebsiella aerogenes organisms grown in chemostat cultures that were glucose-limited and nitrogen-limited, respectively, with different oxygen concentrations Growth conditions

Concentration of adenosine phosphates (/*mole g dry wt.-l) 7

ATP

ADP

Energy charge

7

p rn

AMP

I

Glucose-limited, /* = 0.18 hExcess oxygen (> 10 mm Hg) Transition phase' (0.5-10 mm Hg) Limited oxygen (<0.5 mm Hg) Anaerobic (under gaseous nitrogen)

6.5 6.2 3.7 3.7

2.2 2.3 2.9 3.9

Nitrogen-limited, p = 0.2 h-' Excess oxygen (> 10 mm Hg) Limited oxygen (<0.5 mm Hg)

4.5 5.5

-

'The respiration rate oscillated under these conditions; see Fig. 2. Data of Harrison and Maitra (1969).

Ratio ATP/ADP

2.7

0.4

0.5 0.8 1.1 1.o

-

3.0 2.7 1.3 0.9

1.7

-

0.84

0.82 0.70 0.64 0.7 1 -

D

3

9

T H E REGULATION OF RESPIRATION RATE I N GROWING BACTERIA

267

enough to give the equivalent of the “State 4” of isolated mitrochondria. The very high turnover rate of ATP within the cells makes it obvious that the so-called ATP “pool” cannot function in any way as a reserve for energy. Nor does the content of ATP, nor even the ATP/ADP ratio, indicate the state of energy surplus or deficiency in the cells. From Table 6 it can be seen that, under conditions of nitrogen-limitation and excess glucose, when the cells contained an abundance of stored energy source (Harrison and Pirt, 1967), the ATP content and the ATP/ADP ratio were depressed. The ATP and ADP content of the cell is clearly very crucial; if either ATP or ADP contents were to fall to near zero, then the cell would experience a catastrophic situation from which metabolism could probably not recover. Such a collapse of control over ATP content appears to occur in Acetobacter aceti. Bachi and Ettlinger (1973) demonstrated that when this organism grew on ethanol, in batch culture, the ATP content was constant and the ratio: ATP + 0.5 ADP/total adenosine phosphate (energy charge) was 0.87; but immediately the ethanol became exhausted and growth ceased, the ATP content fell precipitously, almost to zero, and the energy charge ratio dropped to 0.06. It would be interesting to know whether under these conditions the cells could recover, and recommence growth, on addition of more substrate, or whether they entered a lag phase. Unfortunately, the authors did not report the results of such an experiment. B. CONCEPT OF ENERGY CHARGE

According to the theory expounded by Atkinson (197 I), the regulation of adenosine phosphates in all living cells is effected in a similar way. The allosteric control of vital anabolic and catabolic enzymes by adenosine phosphates is arranged so that when there is a high level of “energy chal\ge”, anabolic pathways are stimulated and catabolic pathways are inhibited. A low energy charge, on the other hand, would give the opposite effect. The energy charge is defined as : [ATP] + 0.5 [ADP]/[ATP]

+ [ADP] + [AMP]

This balancing of catabolic and anabolic activities by the relative contents of ATP and ADP in the cell has been widely accepted for

268

D. E. F. HARRISON

some years, but Atkinson took the theory further and stated that the value of the energy charge for balanced healthy growth had to be in excess of 0.8, and that growth was impossible when the energy charge fell below this value. A table o f values for the energy charge, taken from studies of a great variety of cells and tissues, was compiled by Chapman et al. (1971.) and showed convincingly that for most actively metabolizing cells the value was indeed between 0.80 and 0.90. For micro-organisms, there were several exceptions which Chapman et al. (1971) dismissed on the grounds that the extraction procedures used were insufficiently rapid. This was undoubtedly a fair criticism, but there are very few studies of adenosine phosphate contents in growing micro-organisms that employed sufficiently fast extraction procedures to reliably determine the energy charge of the growing cell. In fact, this criticism could be applied to the work of Chapman et al. (1971). Results of Harrison and Maitra (1969), for which great care was taken to extract the growing cells in less than 0.2 seconds, gave values for “energy charge” (Table 6) of above 0.80 for aerobic glucose-limited cells; but for nitrogen-limited cultures, o r those grown under anaerobic conditions, the energy charge was considerably lower. These results are incompatible with Atkinson’s energy charge hypothesis if it is rigorously interpreted, since, according t o Atkinson (1971), no growth should be possible at such low levels of energy charge. Possibly the results of Harrison and Maitra represent an atypical situation; on the other hand, the tendency for the reported energy charge in growing cells t o be estimated as 0.80 may have been a result of delays, albeit of a few seconds or so, in the extraction o f the adenosine phosphate pool. Studies on isolated enzymes (Atkinson, 1970) indicate that allosteric control by adenosine nucleotides operates in such a way that the energy charge of 0.80 favours either activation or inhibition (Fig. 7). However, Atkinson stated that the activation and inhibition curves can be modified by various factors (Fig. 7), and this should be considered in combination with the fact that, in an actively growing and metabolizing system, the enzymes may be operating far from an equilibrium situation, and far below their maximum rate of turnover. Thus, although the energy charge of 0.80 may be the appropriate regulation value for isolated enzymes, and for many situations in metabolizing cells, it would seem not unreasonable t o expect that under certain conditions within the cell

T H E REGULATION OF RESPIRATION R A T E I N GROWING BACTERIA

0

0 2

04 06 08 Energy charge

10

0

02

04 06 Energy charge

08

269

10

FIG. 7. Generalized interaction between energy charge and end-product concentration in the control o f a regulatory enzyme in (a) an anabolic pathway, and (b) a catabolic pathway. The curves correspond to low (l), normal (n) and high (h) response. (From Atkinson, 1968.)

the energy charge at which regulation is most sensitive could be below this value. To decide whether this is correct, or whether the Atkinson energy charge theory can be applied rigorously to all cells, in whatever state, requires many more studies to be made of the contents of adenosine nucleotides in growing bacteria, studies which take into account the very high turnover rate of ATP. The weight of evidence would suggest that the values of the energy charge in a growing cell is a result of the regulatory mechanisms operating in the cell rather than a representation of its energy status. Thus, estimations of steady-state values of adenosine phosphates are of only limited value in studying control mechanisms. More revealing of the underlying feedback loops which operate in the cell are the transient responses of the adenosine phosphate pools to perturbations in the steady state. C. TRANSIENT-STATE STUDIES

The simplest way to produce a step change in respiration rate in a chemostat culture is to add a pulse of extra substrate. Harrison and Maitra (1969) studied the effect on the cellular ATP level of pulsing a small amount ( 2 mmole) of extra glucose to a glucose-limited chemostat culture of Klebsiella aerogenes growing aerobically under steady-state conditions. On addition of the glucose, the dissolvedoxygen tension fell rapidly, indicating a rise in respiration rate. The ATP content, after going through a small oscillation, fell to a value that was 70% of that of the steady-state culture. At first consideration,

270

D. E. F. HARRISON

this fall in ATP content with an increased availability of substrate, and increased respiration rate, seems anomalous, but it can be explained by an increased rate of utilization of ATP for the synthesis of polysaccharides and other cell components that would be stimulated by the increased glucose 6-phosphate content which occurred at this time (Segal et al., 1963). In fact, by integrating the oxygen-uptake and carbon dioxide-production curves it was found that only about one-sixth of the excess of glucose added was oxidized, with an overall respiratory quotient of about 1.0. The rest may have been incorporated into cell material or converted into a non-oxidizable product, although the former seems the more likely. When the excess o f glucose was exhausted, the glucose 6-phosphate content fell and the ATP content immediately returned t o its previous steady-state value. The return of the ATP content to its previous value, after addition of glucose, and the relatively small extent of its deviation while respiration was increased by addition of glucose, is an indication of a high degree of control over the ATP content of the cell. A similar experiment was carried out with the addition of 1 mmole of succinate, which, it was thought, might be less readily converted into storage products. Figure 8 shows the results obtained.

0

'

b I ;

: d,

k

;

e

0

;

Ib

Time after addition of succinate ( m i n )

FIG. 8. Effect of the addition of succinate on the dissolved-oxygen tension and ATP content of cells in a glucose-limited culture of Klebsiellu uerogenes

(dilution rate = 0.2 h-'). Solid line, dissolved-oxygen tension; ( 0 ) ATP concentration. The arrow marks the time of adding 1 mmole of succinate. A fall in dissolved-oxygen tension denotes an increase in respiration rate, and a rise in dissolved-oxygen tension denotes a decrease in respiration rate. (From Harrison and Maitra, 1969.)

THE REGULATION OF RESPIRATION RATE I N GROWING BACTERIA

271

On addition of succinate, the respiration rate immediately increased, causing a fall in dissolved-oxygen tension, then maintained a new steady-state before decreasing abruptly (presumably due to the exhaustion of succinate). An intermediate plateau was obtained before the dissolved-oxygen tension finally returned to the aerobic steady-state value. The increased respiration rate, on addition of succinate, was accompanied by a small increase in the cellular content of ATP which then fell to a value just below that extant in the steady-state culture. When the respiration rate fell again, there was a sharp fall in ATP content, but this quickly recovered. The glucose concentration in the culture did not rise after addition of succinate, although the supply of glucose was maintained throughout; clearly, glucose utilization was not affected by the presence of succinate. It seems that addition of succinate caused an increased production of ATP, but that this was rapidly followed by a higher rate of turnover of ATP which served to depress the cellular ATP content again. As with the addition of glucose, the higher turnover of ATP may be caused by utilization of ATP by anabolic pathways, as in this case also only one sixth of the added succinate was oxidized and the remainder was presumably incorporated into the cell material. The fall in ATP content, when the succinate was exhausted, presumably was caused by the high turnover rate of ATP, but this turnover rate was quickly decreased allowing the ATP content to return to its previous value. Thus it would seem that the ATP content is controlled in such a manner that any change in its value causes reactions that tend to bring it back to the steady state value, but there are time delays in the system that cause “overshoots” to occur. These results are a further indication that respiration rate is not ADP-limited in these cells since the increase in respiration rate is accompanied by a rise in ATP content, and therefore, presumably, by a decrease in ADP content. From these studies we conclude that not only is the steady-state cellular content of ATP regulated to a constant value, but that feedback regulation operates rapidly to maintain the cellular ATP content within narrow limits, despite large changes in respiration rate. That these changes in respiration rate are accompanied by changes in the rate of ATP production is indicated by the sharp

272

0.E. F. HARRISON

change in ATP content on addition, and exhaustion, of succinate (Fig. 8). Previously (p. 249). I described how lowering the dissolved oxygen tension to a certain critical value caused an increase in the respiration rate of a growing culture of K. aerogenes. This phenomenon was investigated further by Harrison and Maitra (1969) who studied changes in the adenosine phosphate contents during the switch-over from low to high respiration rate. In these experiments, the transition from low to high respiration rate was achieved by first obtaining steady-state conditions of respiration and dissolved oxygen tension (with a glucose-limited chemostat culture in which the dissolved oxygen tension was held just above the critical value) and then cutting off the oxygen supply for a short period so as to cause the oxygen tension to fall below the critical value when the oxygen supply was resumed. In response to this procedure, the respiration rate of the culture increased, as indicated by a low reading of the dissolved oxygen tension (Fig. 9). The respiration rate remained high for a time, well after the partial pressure of oxygen in the gas phase had been restored to its previous value, but then spontaneously reverted to its previous steady-state value (Fig. 9). During this cycle, samples of culture were taken for the analysis of pool levels of adenosine phosphates and other metabolic intermediates. The results are shown in Fig. 9. The extremely rapid fall in ATP content, as the dissolved oxygen tension fell to a low value, emphasized the need for the rapid extraction of the samples. The total adenine nucleotide content (as calculated from the sum of ATP, ADP and AMP concentrations) appeared to fall, with the fall in ATP content, on cutting off the oxygen supply. This was due to incomplete extraction causing the ADP and AMP concentrations to be underestimated. After the initial fall, the ATP content recovered slightly. Other experiments were carried out in which the culture was left growing anaerobically for over 10 minutes; in this case the ATP content recovered to a steady-state value of about one-half that extant in the aerobic steady-state culture. The rapid fall in ATP content, on cutting off the oxygen supply, was no doubt caused by the sudden imbalance between ATP production and turnover rates since the respiration rate of the cells abruptly fell. The subsequent establishment of a constant ATP content indicated that the cell could correct this imbalance. On resumption of the oxygen flow, the ATP content

T H E REGULATION OF RESPIRATION R A T E IN GROWING BACTERIA

273

(cl

Oxygen off

//-'

,

b'

-

-

0 -30

0

30

60

,o-

,.oo---o

90

d

120

290 3 2 0

350 380

Time ( s 1

FIG. 9. Concentrations of metabolic-intermediates and adenine-nucleotides in cells during the stimulation of respiration caused by interruption of the oxygen supply. The culture was glucose-limited and grown at a rate of 0.2 h-' The solid line represents the dissolved-oxygen tension; (a) ( 0 ) phosphoenolpyruvate; ( 0 ) citrate. (b) (A) glucose 6-phosphate; (0)fructose 1,6-diphosphate plus triose phosphate, expressed as C3 units; (c) (0) ATP, ( 0 ) ADP; (m) AMP. Arrows mark the times of cutting off and resumption of the oxygen supply, The low dissolved-oxygen tension reading after resumption of the oxygen supply indicates the period of stimulated respiration, and the rise in dissolved-oxygen tension after 300 seconds indicates the spontaneous reversion to the steady-state respiration rate. (From Harrison and Maitra, 1969.)

.

274

D. E. F. HARRISON

rose rapidly, overshot and then fell back t o a steady-state value just below the fully aerobic steady-state value. When the dissolved oxygen tension rose again, representing the spontaneous reversion t o the lower respiration rate, the ATP content increased slightly to the previous fully aerobic steady-state value. The changes in ATP content were accompanied by opposite changes in the cellular content of AMP and ADP. The extent t o which the concentration of AMP changed reflected the changes in ATP content, and the small extent of changes in ADP concentration indicated the presence of an active adenylate kinase reaction. This enzyme catalyses the reaction: ATP + AMP

* 2 ADP,

and so, during a change in energy charge, it tends t o buffer the ADP concentration. Although the respiration rate had increased o n resumption of the supply of oxygen to the culture, the fact that the ATP content did not rise above the previous aerobic steady-state value, but was slightly lower, suggests either that the ATP turnover rate had increased or that the number of ATP molecules produced per molecule of oxygen taken up had decreased during the stimulation of respiration. An increased respiration rate in the absence of an elevated ATP content argues further against direct control of respiration by ADP concentration (that is, of the type demonstrated by State 4 mitochondria; Chance and Williams, 1956). These results cannot, of course, distinguish between the roles of ATP, ADP and AMP in feedback control mechanisms, since the concentrations of all three are interconnected by the high adenylate kinase activity present in the cells. Harrison and Maitra also followed the changes in concentration of key intermediates of the Embden-Meyerhof pathway (Fig. 9). The glucose 6-phosphate concentration was found t o fall rapidly, with the fall in dissolved-oxygen tension, and then t o recover. On resumption of the supply of oxygen to the culture, the glucose 6-phosphate concentration returned to approximately its initial value. This pattern was reversed for the concentration of fructose 1,6-diphosphate plus triose phosphate; this rose when that of glucose 6-phosphate fell, and fell, after a short delay, when that of glucose 6-phosphate rose. This is consistent with the cross-over pattern reported for the Pasteur effect in yeast (Ghosh and Chance, 1964)

T H E REGULATION OF RESPIRATION RATE IN GROWING BACTERIA

275

where it is interpreted as indicating a site of glycolytic control at the level of the phosphofructokinase reaction. When oxygen was turned off and on, the concentrations of phosphoenolpyruvate and citrate changed in an opposite direction to those of fructose 1,6-diphosphate and triose phosphate. This would indicate a reverse cross-over between fructose 1,6-diphosphate, triose phosphate and phosphoenolpyruvate that in itself cannot be interpreted as a site of interaction (Chance et al., 1958), but does indicate that there is another positive cross-over further down the pathway. The flux of glucose moieties through phosphoenolpyruvate increases on a change to anaerobiosis because of an increased rate of reaction at pyruvate kinase, or at a stage beyond, or both. These interpretations assume that the net flux of glucose through the system is in fact increased when it becomes anaerobic (i.e. that the Pasteur effect holds for growing bacterial cells); evidence for this was given by Harrison (1965). Also it is assumed that glycolysis is by far the most important pathway of glucose metabolism under these conditions. Summarizing these studies on the stimulation of respiration rate at low dissolved oxygen tensions, it can be said that an increased rate of glucose oxidation was not accompanied by an elevated ATP content. Therefore either the P/O ratio had decreased, or the rate o f ATP turnover had increased to compensate for an increased rate of ATP synthesis arising from the increased rate of respiration. Harrison and Pirt (1967) had shown that, under these conditions of stimulated respiration, the yield coefficient of the cell was diminished, and so any increase in the rate of ATP production was not offset by a stimulation of anabolic pathways. Possibly there was an increased ATP turnover rate not coupled with anabolic reactions but representing a wastage of ATP, although a more likely explanation would seem to be that, at low dissolved oxygen tension, the coupling of ATP production to respiration became less efficient (i.e., the PI0 ratio fell). This would give rise to a transient fall in ATP content which would feed back to stimulate catabolism; in particular, to affect the regulation of glycolysis via phosphofructokinase. Thus the glucose dissimilation rate would increase and, assuming respiration to be limited by the availability of reduced co-enzyme, give rise to an increased respiration rate and diminished yield coefficient. Such an uncoupling of oxidative phosphorylation might, as suggested by Harrison and Maitra (1969), be brought about by the operation at

276

D.

E. F. HARRISON

low dissolved oxygen tensions of an alternative pathway for electron flow to oxygen, bypassing one or more sites of oxidative phosphorylation. The mechanism for feedback control of glycolysis suggested above is the same as that which has been shown t o operate in yeasts (Pye, 1969; Chance e t al., 1965) and in higher organisms (Williamson e t al., 1967) and so the existence of such a mechanism in bacteria would not seem surprising. However, Thomas et al. (1972) have recently found that, for E. coli organisms grown on glucose, there appear to be two different types of phosphofructokinase synthesized depending on whether the cell is grown under fully aerobic or anaerobic conditions. The phosphofructokinase present under anaerobic conditions was subject to allosteric control by ATP, while that formed under aerobic conditions was found to be insensitive to the presence of adenosine phosphates. From this it is implied that there is no feedback regulation of glycolysis by adenosine phosphates in aerobically grown E. c o b organisms. It would seem that, at least when grown at oxygen tensions just above the critical value, K. aerogenes possesses the “anaerobic” type of phosphofructokinase since regulation of this enzyme by adenosine phosphates was indicated by the results of Harrison and Maitra (1969). One reservation that might be voiced over the results of Thomas e t al. (1972) follows from the observation that the curve of activity of the aerobic phosphofructokinase which they presented is remarkably similar to that of the anaerobic enzyme in the presence of AMP. This suggests that possibly the aerobic enzyme, as isolated, may already have been in the inactivated state owing, perhaps, to incomplete removal of AMP from the preparation. The only other reported study of changes in adenosine nucleotide contents in growing bacteria during perturbations of the growth conditions is that of MiijviC and Gibson (1973) who studied the photosynthetic bacterium, Chromatium. These workers obtained a delay time, between sampling and extraction, of less than 200 milliseconds and so were able to follow rapid changes in adenosine phosphate contents. In this study, the growing culture was subjected to changes in light intensity. When the culture was growing under steady-state conditions, the intracellular ATP level was very constant. Under conditions of illumination, the energy charge was 0.81, but under dark conditions it was higher (0.85), even though the growth

THE REGULATION OF RESPIRATION RATE I N GROWING BACTERIA

277

rate and energy flux were decreased. This confirms the finding of Harrison and Maitra (1969) that the ATP content and enerm charge does not reflect the energy status or metabolic rate of the cell. When a Chromatium culture was subjected to a step-down in light intensity 1i.e. a decrease in energy flux in the cell), the ATP content and the energy charge fell initially, before recovering to their former levels; this took only 40 seconds. A step-down to completely dark conditions gave a larger and longer-lasting fall in energy charge. The changes in ATP and energy charge could not be correlated with any of the physiological changes resulting from the step changes in light intensity. Here, as in K . aerogenes, it would seem that feedback control functions to regulate the adenosine phosphate content. In spite of many contradictions in the experimental data, it would seem to emerge that, under conditions of active growth, bacteria tend to control their intracellular content of adenosine phosphates. Any environmental change which alters the rate of ATP generation or turnover is likely to elicit a response in the metabolic rate which would tend to restore the balance between ATP generation and its utilization. V. Role of NADH in the Regulation of Respiration The central roIe played by nicotinamide nucleotides in oxidative reactions of organisms inevitably implicates them in the regulation of respiration; NADH is, after all, the main immediate electron donor to the respiratory chain. As a substrate for respiration, NADH must exert some influence on its rate. In isolated mitochondria, NADH is limiting for respiration rate in State 2 (Table 1). This would be equivalent t o the aerobic, substrate-starved condition in whole resting cells. The questions that are begged are these: does this state arise in the actively growing bacterium? What other influences does NADH content exert on respiration rate of the whole organism? Studies on the oxidation-reduction state of the nicotinamide nucleotides in a wide variety of animal cells and tissues have shown that this can be a good indication of the metabolic and respiratory state of the cells (Chance et al., 1964). Fortunately, the reduced form of nicotinamide nucleotides is not only a useful indicator of respiratory control mechanisms, but is also quite conveniently monitored in living cells.

278

D. E. F. HARRISON

A. MEASUREMENT OF NICOTINAMIDE NUCLEOTIDES

Nicotinamide nucleotides (NAD+,NADH, NADP', NADPH) can be measured by extraction from whole cells followed by chemical or enzymic analysis (Estabrook and Maitra, 1962; Takebe and Kitahara, 1963; Cartier, 1968). As with adenosine nucleotides, however, there is a problem in extracting the coenzyme from the cells sufficiently rapidly. Not only is the turnover rate of NADH, like that of ATP, very high compared with the total pool size, but also there are very many enzymes and substrates which can react with nicotinamide nucleotides so that the rate of change in concentration of NAD(P)H and NAD(P)+ is likely to be even faster than that of ATP. An added complication in the chemical analysis of nicotinamide nucleotides is that the oxidized form, NAD(P)+,must be extracted in acid, but the reduced form, NAD(P)H, in alkali. The rapid, and often complex, changes in nicotinamide nucleotide that can occur really necessitates a method of continuous monitoring. One such method is the dual beam spectrophotometric system of Chance (1951) which monitors NAD(P)H by recording the absorption of light at 340 nm. This technique has been applied successfully to studies of resting microorganisms (Chance, 1954; Hempfling et al., 1967; Maitra et al., 1963). Clearly, for studies on growing cells it would be ideal if NAD(P)H levels could be monitored directly in continuous cultures o f organisms. Attempts were made to use spectrophotometric techniques for this purpose by circulating a culture through a flow cell (B. Chance, unpublished data). However, any such circulation of culture of necessity entails a delay between the time that an organism leaves the culture vessel and arrives at the measuring cell; this difficulty can be circumvented by the use of reflectance fluorimetry. The characteristic fluorescent property of the reduced from of nicotinamide nucleotides was first exploited for studies of enzyme preparations (Boyer and Theorell, 1956), and later of isolated mitochondria (Chance and Baltscheffsky, 1956) and the whole yeast cells (Duysens and Amesz, 1956). The great advantage of fluorimetry is that, being a reflectance technique in which the detector can be at an angle of 60° to the illumination, it can be applied to nontransparent material. The adaptability of the technique is illustrated by the variety of material to which it has been applied, for example,

THE REGULATION OF RESPIRATION RATE IN GROWING BACTERIA

279

cell suspensions (Betz and Chance, 1965; Chance, 1964), whole animal organs (Chance et al., 1965), and individual animal cells (Kohen, 1964). Fluorimetry was used by Schon and Drews (1968) to follow NAD(P)H changes in batch cultures of bacteria, using a flow-cell technique, but the disadvantages of flow-ccIIs have already been stated. A more direct way of applying fluorimetry t o fermenter-grown micro-organisms was developed by Harrison and Chance ( 1 97 0).

Optical filters

vapour Mercury+ lamp

Photornultipller

and detector

EGl

r-

Recorder

FIG. 10. A schematic drawing of the apparatus used for monitoring nicotinamide nucleotide fluorescence in a continuous culture. (From Harrison and Chance, 1970.)

A diagram of a fluorimeter applied to a culture vessel is shown in Fig. 10. The phototube and light source are applied to the glass sides of the fermenter vessel, but there are certain limitations to its use in stirred vessels (Harrison and Chance, 1970; Harrison and Harmes, 1972). Optimal sensitivity is obtained with cell densities in excess of 4 g 1-' and with an even distribution of small sized bubbles in the vessel. By using a large surface area (approximately 1 0 c m 2 ) for illumination and collection of light, the fluorescence can be integrated and noise, due t o small bubbles in the liquid, minimized. The technique is extremely sensitive to changes in NAD(P)H (Fig. 11) and has a very short response time (<0.5 seconds for 90% response).

D. E. F. HARRISON

280 30

-Stirring o f f

Stirring on

pH value

-605

T

0-

__

-

-~

~

-_

--

-

.

FIG. 1 1 . An NAD(P)H fluorescence trace o f a chemostat culture o f Klebsiella aerogenes showing the response to an aerobic-anaerobic transition, and to re-aeration. The culture was glucose-limited and grown at a rate of 0.2 h-’ .

The limitations are: (1) it is not an absolute method and can indicate only relative changes in the reduced form of nicotinamide nucleotide, not the actual concentration; to calibrate, samples must be taken and analysed chemically. (2) The method does not distinguish between the NADH and NADPH. (3) The calibration of the fluorimeter system, as presently used, is not stable over periods of time longer than a few hours. Attempts are being made to improve this stability. Fluorimetry makes use of the property, apparently unique to reduced nicotinamide nucleotides, to absorb radiation at 340 nm and emit radiation at 460 nm wavelength. Flavoproteip fluorescence should give negligible interference if the wavelength bands used are sufficiently narrow. Use of fluorimetry is open to criticism (Wimpenny and Firth, 1972) on the grounds that it is indirect, and that some of the fluorescence changes may be due to changes in the degree of binding of reduced nicotinamide nucleotide t o proteins or membranes. Bound NADH has a higher fluorescence yield than has the unbound form (Estabrook, 1962). However, Chance et al., (1964) demonstrated with yeast cells the validity of fluorimetric determination of NAD(P)H by simultaneously monitoring the changes in 340 nm light absorption. They found no evidence of changes in fluorescence that could be attributed to changes in binding of nicotinamide nucleotide, and demonstrated a linearity of response of fluorescence to NAD(P)H concentration. Also very many of the data accumulated from many different types of material (e.g., Betz and Chance 1965; Chance, 1964; Chance et al., 1964; Chance et

T H E REGULATION OF RESPIRATION RATE IN GROWING BACTERIA

281

al., 1965; Pye, 1969) are consistent with fluorimetry measurements giving indications of real changes in the content of reduced nicotinamide nucleotide. B. RESPONSE OF NAD(P)H CONTENT TO PERTURBATIONS OF THE STEADY STATE

In order to determine whether induction of bacteriochlorophyll was associated with the redox state of NAD(P)+/NAD(P)H couple, Schon and Drews (1968) monitored changes in the fluorescence emission of reduced nicotinamide nucleotides in cultures of photosynthetic facultative organisms of the genus Athiorhaceae. They found that there was no change in the state of NAD(P)H until the dissolved oxygen tension fell below 0.2-0.5 mm Hg; then the concentration of NAD(P)H increased very rapidly. On increasing the oxygen tension again NAD(P)H was rapidily oxidized. Small bursts of aeration to anaerobic cultures gave incomplete oxidation of NAD(P)H which may have been simply a reflection of a time delay in circulating the culture through the flow-cell. No correlation was found to suggest that reduced nicotinamide nucleotide was responsible for induction of bacteriochlorophyll. The direct fluorimetric technique was used more recently to follow, in a glucose-limited chemostat culture of K. aerogelzes, the changes in reduced nicotinamide nucleotide fluorescence following small step-changes in oxygen tension (unpublished result). A steadystate of growth was first obtained under fully aerobic conditions, and then the oxygen supply was decreased in small steps, allowing the oxygen tension and fluorescence to settle to a new steady-state level (i.e. changing by less than 5% in 1 0 minutes) before changing the oxygen supply again. It should be noted that these readings represented quasi steady-states of respiration but not steady-states of growth. Figure 1 2 shows the pattern of response obtained. The largest response in NAD(P)H was obtained when the dissolved oxygen reading had fallen below the lower limit of sensitivity of the electrode. However, it can be seen that there was a small shift to reduction of nicotinamide nucleotide when the dissolved oxygen tension fell to 10 mm Hg. This is at an oxygen tension above the apparent critical level at which respiration rate begins to respond. Thus it would seem that the internal redox state of the cell may respond to changes in oxygen concentration well above the K, for the terminal oxidases, and before respiration is restricted.

D. E. F. HARRISON

282 120

-2 5

I10 I00

G 90-

-2 0

I

-> E

-15; c -

U

60

0

50

a

40-

30 20 100 0

P - ' ; I

10

20

5-

I

l

l

80 90 100 Oxygen partial pressure in gas phase (mm Hg)

30

40

50

60

70

FIG. 12. Changes in NAD(P)H fluorescence, in a chemostat culture o f Klebsiella aerogenes, in response to stepped changes in oxygen partial pressure. The culture was glucose-limited and grown at a rate of 0.2 h-' (m) NAD(P)H response to decreased Po2; (0) NAD(P)H response to increased Po2; ( 0 ) dissolved O2 tension response to reduced Po2; (0)dissolved 0 2 tension response to increased Po2.

.

The switch from low to high respiration rate, triggered by a fall in oxygen tension below the threshold value, is denoted in Fig. 1 2 by a fall in dissolved oxygen reading to zero when the partial pressure of oxygen in the gas phase was 42 mm Hg. This was accompanied by a sharp increase in NAD(P)H fluorescence. The fact that the increased respiration rate was accompanied by an increased level of reduced nicotinamide nucleotide would indicate that the increased respiration rate is caused by, or at least is accompanied by, an increased electron flux from the substrate via a rapid feedback control. If the sole regulatory point in the electron transport chain lay between NAD+/NADH and oxygen then an increase in respiration rate would be accompanied by an oxidation of theNAD+/NADH couple. As the

T H E REGULATION OF RESPIRATION RATE I N GROWING BACTERIA

283

partial pressure of oxygen in the gas phase (Po,) was lowered further the NADH content showed little response, as indicated by the plateau in Fig. 12, but as the PO, approached zero there was a large increase in NAD(P)H level. On increasing the oxygen supply again, a large amount of hysteresis was obtained in the response. The reason for this may be that the enzyme content o f the cells altered during the period of anaerobiosis; also, some fermentation products may have accumulated in the culture. In a recent study, Wimpenny and Firth (1972), employing rapid sampling techniques and very sensitive enzyme assays for NAD+ and NADH, found very rapid changes in the concentrations of total nicotinamide nucleotides during shifts from anaerobic t o aerobic conditions in cultures of E. coli. The measured concentrations of NAD' fell by some 60% within a minute of shifting from aerobic t o anaerobic conditions, while the NADH content increased little. This result contrasts sharply with those studies of Harrison, discussed above, and with all previous studies of nicotinamide nucleotide changes in micro-organisms. If correct, and applicable t o all bacteria, this phenomenon would indicate that measurement of NADH would not reflect changes in the redox potential of the NAD+/NADH couple at all, the largest influences being the change in total nicotinamide nucleotide concentration. Wimpenny and Firth's results are at sharp variance with those obtained by Harrison and Chance (1970), and by London and Knight (1966), using similar techniques. A comparison of the results is given in Table 7. The high turnover rate of total nicotinamide riucleotide content, proposed by Wimpenny and Firth (1972) was not found by Lundquist and Olivera (197 1) who studied the synthesis of nicotinamide nucleotides, during growth of E. coli, with the aid of actively labelled precursor substances. A likely explanation of the anomalies of the results of Wimpenny and Firth is that the acid extraction procedure which they employed only removed NAD+ slowly, and that, under anaerobic conditions (though not aerobic conditions), some NAD' was reduced t o NADH. As NADH would be destroyed by the acid environment, the reduction of NAD+ would go much further than in the whole intact cells. The concentration of acid (0.03 N) used by Wimpenny and Firth (1972) for the extraction of NAD+ was shown by London and Knight (1966) to give incomplete recovery of NAD'.

TABLE 7. Reported levels of pyridine nucleotides in facultative anaerobes grown under aerobic and anaerobic conditions Organism

Aerobic r

NAD* Escherichia coli Klebsiella aerogenes Escherichia coli Klebsiella aerogenes

Anaerobic

A

NADH

-

-

3.10 4.8 5.5

0.45 1.4 1.4

\

Total

7

NAD+

Reference

NADH

Total

0.78

2.67

0 rn -n

I

D

2.44 3.55 6.2 6.9

-

2.80 2.9 3.9

1.9 1.6

3.58 4.8 5.5

London and Knight (1966) Harrison and Chance (1970) Wimpenny and Firth (1972) Wimpenny and Firth (1972)

XI

T H E REGULATION OF RESPIRATION RATE IN GROWING BACTERIA

285

C. OSCILLATIONS IN NAD(P)H FLUORESCENCE

Biochemical oscillations are of great interest for the information they can provide on metabolic control systems. For instance, in recent years extensive studies of oscillations in reduced nicotinamide nucleotide concentrations in yeast organisms have contributed much t o our understanding of the regulation of glycolysis (Ghosh and Chance, 1964; Betz and Chance, 1965; Pye, 1969).

I 0

a z

FIG. 13. Damped oscillations in the nicotinamide nucleotide fluorescence following an anaerobic shock to an aerobic chemostat culture of Klebsiella aerogenes. Calibration of fluorescence is in units of NADH per millilitre culture, based on enzymic assay. The delay between turning off the oxygen supply and the increase in fluorescence represents the time required for the culture to become anaerobic. Growth was glucose-limited at a rate of 0.2 h-'. The steady-state oxygen tension was 43 mm Hg. (After Harrison, 1970.)

Oscillations in the nicotinamide nucleotide fluorescence were observed after an anaerobic shock had been applied t o a steady-state aerobic culture of Klebsiella aerogenes (Harrison, 1970). When the air supply to the culture was restored, the fluorescence fell and then underwent a few damped oscillations (Fig. 13). This phenomenon was found to be repeatable. After several such anaerobic shocks the damping became less and the number of oscillations increased until, eventually, undamped oscillations were obtained (Fig. 14). The high frequency of these oscillations precludes, as an explanation, synchronization of the cell division cycle since the growth rate of the culture was much slower (0.2 h-' ). The precise number of anaerobic shocks required to produce continuing oscillations varied with the state o f

D. E. F. HARRISON

286

Joxygen tension -

-

~

-~

,

$7 0 6

FIG. 14. Oscillations in nicotinamide nucleotide fluorescence, and corresponding oscillations in dissolved oxygen tension found with a chemostat culture o f Klebsiella aerogenes. Calibration o f the fluorescence is in units of NADH per millilitre of culture, based on enzymic assay. The time scales are synchronized The culture was glucose-limited and grown at a rate of 0.2 h-' (After Harrison, 1970.)

.

the culture, but generally about six shocks over a period of twentyfour hours produced continuing oscillations. When the culture was maintained aerobic, these oscillations continued for over two days without a change in amplitude or frequency. No changes in these oscillations, or the mean fluorescence reading, were detected when the oxygen tension was varied between 2 and 100 mm Hg, that is, over the range where respiration rate and metabolism are independent of dissolved oxygen tension in K . aerogenes (Harrison and Pirt, 1967). This would seem to eliminate the possibliity that the oscillations in nicotinamide nucleotide were caused by small fluctuTABLE 8. Effect of oxygen tension on the frequency of oscillations in oxidized and reduced nicotinamide nucleotide levels in a continuous culture of Klebsiella aerogenes Oxygen tension (mm Hg) 20-150

< 0.5

0.0 (anaerobic) 'Varied with degree of oxygen deficiency. Data of Harrison (1970).

Period of oscillations (min)

2.4 3.0-5.0" 12.0

T H E REGULATION OF RESPIRATION RATE IN GROWING BACTERIA

287

ations in oxygen tension. At oxygen tensions below 1 mm Hg, when the oxygen supply becomes limited, the frequency and amplitude of oscillations were affected (Table 8). Close attention was given to other parameters of the culture in order to see whether any of these also oscillated. No oscillations could be detected in the rate of acid-production in the culture, even though the pH recording was sensitive to changes of less than 0.01 pH units. However, when the sensitivity of the oxygen tension recording instrument was increased to 10 mm Hg full scale, it was found that oscillations in oxygen tension of the same frequency as those of the nicotinamide nucleotide were occurring (Fig. 14). Also oscillations of the same frequency were detected in the rate of production of carbon dioxide. The amplitude of these oscillations in oxygen tension and carbon dioxide production amounted t o only 1% of the total respiration rate of the culture. The traces shown in Fig. 14 are interpreted as follows. The sharp fall in fluorescence indicates a rapid partial oxidation of the nicotinamide nucleotide while the oxygen tension was at a minimum (i.e. respiration rate was at a maximum). A few seconds later, the oxygen tension began to rise (i.e. respiration rate fell) while the nicotinamide nucleotide remained oxidized. After about 70 seconds the nicotinamide nucleotide trace showed a rapid shift back towards reduction and, about 30 second later, while the nicotinamide nucleotide was still in the reduced phase, the oxygen tension began to fall, indicating an increase in respiration rate. This phase relationship was maintained. When comparing the phase relationships it must be borne in mind that the oxygen probe had a much slower response time (90% in 30 seconds) than the fluorimeter (90% in 0.5 seconds) and that a further delay in oxygen tension response would be caused by the time required to equilibriate the gas and liquid phases in the culture vessel. Therefore, although the oscillations in oxygen tension and nicotinamide nucleotide appear to be 20-30 seconds out of phase, the changes in respiration rate may coincide with the changes in the state of the nicotinamide nucleotide. That a shift to reduction of nicotinamide nucleotide should be followed by increased respiration rate, and a shift to their oxidation by a fall in respiration rate, would be consistent with control of respiration rate by the concentration of substrate, reduced nicotinamide nucleotide being the main substrate for respiration.

288

D. E. F. HARRISON

A series of samples were taken from the culture while it was oscillating to see whether any oscillations in the metabolic intermediates could be correlated with those in NAD(P)H. Figure 15 shows some of the results obtained with samples from an aerobic glucoselimited culture. There were no changes in glucose 6-phosphate, fructose 1,6-diphosphate, triosephosphates o r phosphoenolpyruvate which could be correlated with changes in nicotinamide nucleotide. However, the ATP content gave a saw-tooth fluctuation with a similar lncreasmg NADH

1

-

Q nrnoles NADH

FIG. 15. Changes in levels o f intermediates during oscillations in nicotinamide nucleotide fluorescence of a chemostat culture of Klebsiella aerogenes. Calibration of fluorescence is in units of NADH per millilitre culture, based on enzymic assay. Other intermediates were determined enzymically on samples removed from the culture. The culture was glucose-limited and grown at a rate of 0.2 h-' The oxygen tension was 46 mm Hg. (After Harrison, 1970.)

.

frequency to the nicotinamide nucleotide oscillations. In a glucoselimited culture, which did not demonstrate oscillations in nicotinamide nucleotide, the ATP content showed no such fluctuations and the standard deviation for ATP estimations on 20 samples was only k0.4 mpmole/mg dry weight. Therefore the fluctuations obtained in ATP level in an oscillating culture would seem to be significant. The standard deviation for glucose 6-phosphate measurements in a nonoscillating culture was k l . 0 mPmole/mg dry weight so that the fluctuations in glucose 6-phosphate were within the error for the estimation. From Fig. 15 it can be seen that the sharp decrease in NADH was accompanied by a sudden increase in the ATP level, indicating that the oxidation reactions involved in the oscillations may

T H E REGULATION OF RESPIRATION RATE I N GROWING BACTERIA

289

be accompanied by phosphorylation. A similar experiment was carried out on an oscillating culture which was nitrogen-limited, with glucose in excess. In this case no oscillations in the ATP level were detected. Although oscillations in nicotinamide nucleotide may be accompanied by oscillation in the level of ATP, this does not appear to be an essential feature of the oscillations. Thus it would seem that, in the case of glucose-limited cells, the ATP fluctuations are a result, rather than a cause, of the nicotinamide nucleotide oscillations. Continuing oscillations in nicotinamide nucleotides, of similar frequency to those reported here, have been obtained in cell-free extracts of yeast (Pye and Chance, 1966) and these were shown to be of glycolytic origin. However, the fact that the oscillations in the concentration of reduced nicotinamide nucleotides in K . aerogenes organisms, growing in a chemostat culture, could be maintained with succinate as sole carbon supply (Harrison, 1970) would seem to exclude glycolysis as the source of the oscillations. Also, the oscillations were maintained under anaerobic conditions, albeit with a much lower frequency, so that the main oscillator cannot be associated directly with the respiratory system. The fact that oscillations were observed under anaerobic conditions indicates that the oscillations in respiration rate were most probably an effect rather than a cause of oscillations in NADH. The amplitude of the oscillations in oxygen tension and carbon dioxide production were very small, representing only about 1%of the total respiration rate of the cell. Possibly this represents a minor pathway for oxygen uptake which does not involve the normal electron-transport chain. Alternatively, the oscillations in oxygen uptake may reflect the dependency of the total respiration rate on the level of NADH, which is the main substrate for respiration via the electron transport chain. It would appear that the respiration rate is quite insensitive to NADH concentration in this range as a 10% change of NADH caused only a 1% change in respiration rate. The phase relationships between the oscillations in NADH and those in oxygen tension are consistent with the control o f respiration rate by NADH concentration. Positive identification of the primary oscillator reaction is not possible from the data obtained so far. Indeed, in such a complex system as a whole growing bacterial cell it might be very difficult to decide which particular feed-back control caused the oscillations, since the observed manifestation of the oscillations may be quite remote from their origin. However, such oscillations are surely

290

D. E. F. HARRISON

indicative of feed-back loops of metabolism involving NADH; moreover the results also show that respiration rate is at least partially under the control of NADH. D. REGULATORY ROLE O F NADH DEHYDROGENASE

The oxidation of NADH to NAD’, linked to the cytochrome chain via NADH dehydrogenase, is undoubtedly a key step in oxidative metabolism. The rate of this reaction will depend both on the relative concentrations of NAD+ and NADH and also on the amount and activity of the dehydrogenase enzyme present. If NADH dehydrogenase were the sole rate-limiting enzyme for respiration, then an increased rate of respiration could result only from induction or activation of the enzyme, and would be accompanied by a fall in the NADH concentration. This is certainly not what is found with whole cell preparations. Increased respiration rate, caused by addition of substrate to substrate-limited cells, was accompanied by a small increase in NADH fluorescence (Harrison e t al., 1969). Also, as shown in Fig. 12, the stimulation of respiration-rate at low dissolved oxygen tension in K. aerogenes was accompanied by an increase in NADH. The phenomena discussed above would suggest that it is the rate of reduction of NAD’ by substrate dehydrogenases which governs the respiration rate in the growing ceI1. Possibly NADH dehydrogenase could become the limiting enzyme when its substrate is present in excess (Le., for the potential Qo 2 ) . However, the extent of reduction of the NAD’INADH couple in the presence of oxygen is not very high, even when substrate is in excess (London and Knight, 1966; D. E. F. Harrison, unpublished data). Further evidence against the role of NADH (or succinate) dehydrogenase as a regulating enzyme is that its concentration in E. coli remains almost constant during adaptation from anaerobic to aerobic growth (Cavari e t al., 1968).

VI. Cytochromes as Regulators of Respiration Early workers (Baumberger, 1939; Winzler, 1941) interpreted the first-order response of resting cells to dissolved oxygen, at oxygen concentrations below the “critical” value, as indicating limitation of respiration rate by a single enzyme reaction, namely that o f oxygen with the terminal oxidase. To account for the sharp break in this relationship at the “critical” oxygen concentration, it was assumed

T H E REGULATION OF RESPIRATION RATE I N GROWING BACTERIA

291

that the terminal oxidase reaction only became limiting at oxygen concentrations below the “critical” value, and some other, unspecified, reaction limited respiration rate at higher oxygen concentrations. This simple model failed t o explain both the variation in “critical” oxygen concentrations found among organisms with similar terminal oxidases and the variation in the “critical” oxygen concentration with the metabolic state of the cell (Winzler, 1941). Longmuir (1954) attempted to explain the variation in K, value for oxygen between various organisms by proposing that the oxygen uptake rate was limited by diffusion o f oxygen through the cell. This, however, still did not explain variations in apparent K, value with the metabolic state of the cell. Extensive studies of the electron transport system by B. Chance and his colleagues (Chance and Williams, 1955a, by c; Chance e t al., 1955) led t o the construction of a model system which took into account the interaction of all the components of the electron transport chain (Chance, 1957). This model explains the sharply defined “critical” value, and allows for small variations in this with the metabolic state of the cell without postulating any unusual enzyme stoichiometry or significant diffusion effects. As far as suspensions of discrete bacterial cells are concerned, it is highIy unlikely that diffusion of oxygen from the bulk of the Iiquid to the cytochromes, which are situated on the membrane o f the cell, could have any significant effect on cell respiration and response to oxygen. In the case of a flocculated culture, or bacteria growing in films, however, there is likely to be an important contribution t o the regulation of oxygen uptake rate by diffusion (Atkinson, 1974). This will have the effect of raising the apparent K, value for oxygen, and making the “critical” dissolved oxygen concentration very dependent on parameters such as particle size, film thickness, and temperature. According to the model of Chance (1957), only in State 3 (that is, under conditions of high substrate, oxygen and ADP levels; Table 1) does the cytochrome chain of mitochondria become limiting for respiration rate. This is not necessarily to say, of course, that it is the terminal member of the electron transport chain that is the ratelimiting step. However, at very low oxygen tensions (State 5 ) , the rate of reaction between oxygen and the respiratory chain will be limiting.

A. THE INDUCIBILITY OF BACTERIAL CYTOCHROMES

Two aspects of bacterial cytochrome systems set them apart from

TABLE 9. Effect of oxygen availability on cell cytochrome oxidase content and oxygen uptake kinetics in various micro-organisms

h,

W

h,

~

Oxygen status

Cytochrome oxidase

Respiration rate (mmole g-’ h-’ ) :‘int”potential’:

“Critical” oxygen

JS,,,

value

Organism

Reference

(/AM)

(/AM)

content Excess Limited Excess

d d d 0

Limited

d 0

Excess

aa3 0

Limited

aa3 0

Excess

d a1 0

Limited

d a1 0

Excess Limited Excess

d d d a1 0

+ +++ + + +++ +++ +++ ++ ++ +++ + + + ++ ++ ++ + + ?

+++

++ +++ -

75

-

-

}

110

Escherichia coli

Moss (1952)

Haemophilus

White (1963) U

-

-

4.5

m 7

-

-

-

-

45

-

17

-

+++

2.2 J

+

80 20

-

-

I

Micrococcus denitrificans

Sapshead and D ’ Wimpenny (1970)

Azotobacter vinelandii

Ackrell and Jones (1971)

Nishizawa et aL

16

vinelandii Achromobacter sp. Arima and Oka (1965)

I

0

I

I

99

o m 0 3

3

h

3

07

.r i

v

P, YI

u

4

0

.-C 0 .-

M

ti

293

i+ i+.ii

THE REGULATION OF RESPIRATION RATE IN GROWING BACTERIA

I

v"

h

I

3

0

c\I

hl

9

0

2 3

3

:

2

I

I

I

+I +I

++ +i +i ++ ++ ++ ++ ++ ++ +

+ + ++ ++ ++ % G O

m e, V

wx

-u:o

ti

3

B 3

294

D.

E. F. HARRISON

those of eukaryotic cells: (l), bacteria possess a great variety of different cytochromes compared with the extremely conservative cytochrome complement of eukaryotic organisms; for example, many bacteria possess three recognizably different terminal oxidases, and (2), bacterial cytochromes tend to be highly inducible, with both qualitative and quantitative changes occurring in response to variations in growth conditions. It seems reasonable to suppose that these aspects of the bacterial respiratory system, which set them apart from higher organisms, facilitate adaptation to an everchangeable environment, and as such may play a role in regulating respiration. The State 3 of mitochondria (Chance, 1957) would correspond most closely to bacteria growing at the maximum growth rate, or to the “potential” respiration rate. If, in this state, the terminal oxidase is limiting for respiration rate, then changes in the cytochrome content should be reflected in changes in the “potential” respiration rate. Table 9 contains collected data for several organisms (including the yeast Saccharomyces carlsbergensis), of changes in the content of terminal oxidase and in “potential” Qoz and respiration rate in situ. While, in some cases, an increase in cytochrome oxidase is accompanied by an increased potential respiration rate (e.g. Haemophilus parainfluenzae; White, 1963), in Arotobacter vinelandii (Nishizawa et al., 1971), Klebsiella aerogenes (Harrison, 1973b) and Beneckea natn’egens (Linton et al., 1974) changes in potential respiration rate are unrelated to changes in the cytochrome oxidase content. A further argument against limitation of respiration rate by the terminal oxidase reaction, in the presence of excess oxygen, is that the bacterial cell usually appears to have a vast excess of terminal oxidase over its respiratory requirements. The turnover number of bacterial cytochrome oxidase has been quoted (Smith, 1961) as being as high as 620 per second, and for yeast (Chance and Williams, 1955) as being 120 per second. If one assumes a bacterial terminal oxidase to have a turnover number of 100 per second, and an extinction coefficient of 14 x lo3 at 626 nm, then the oxygen uptake rate per unit change in absorbance could be as high as 1.8 x mole per second. Now, cytochrome oxidases are usually detected by their reduced-minus-oxidized difference spectra in the visible region of the spectrum. A sensitive difference spectrophotometer (Chance, 1951) might permit the detection of a peak o f absorption of no less than AA = 0.0001 in a bacterial suspension of

THE REGULATION OF RESPIRATION RATE IN GROWING BACTERIA

295

1.0 mg ml-l, contained in a 1.0 cm cuvette. For a peak of absorption just at the limit of detection, the oxygen uptake rate via the oxidase could be as high as 6.4 mmoles oxygen per gram dry weight bacteria per hour. This would represent a very respectable Qoz value for the bacteria. Thus, even when the terminal oxidase content is at or below the sensitivity of the most modern spectrophotometers, there may be sufficient present to support a high rate of respiration. This is borne out in results obtained with action spectra (Castor and Chance, 1959). This technique, in which relief of respiration rate by specific wavelengths of light is measured in cells exposed to mixtures of carbon dioxide and oxygen, gives a more direct estimation of the cytochromes involved in the terminal reaction with oxygen. Using the action spectrum technique, Weston and Knowles (1974) detected a large contribution by cytochrome d to respiration in Beneckea natriegens, even though the absorption band was hardly detectable in a reduced minus oxidized difference spectrum. Most bacteria seem to contain vastly more cytochrome oxidase than that required for respiration under aerobic conditions. Mutants of Bacillus subtilis which were deficient (but not totally lacking) in cytochrome a , showed no significant decrease in oxygen consumption rate compared with the parent strain (Taber, 1974). To draw inferences regarding the importance of any particular cytochrome oxidase t o respiration from the amount of the cytochrome present is therefore unsound. For most aerobically-growing bacteria it would seem very doubtful whether the respiration rate can be limited by the terminal oxidase. Possibly there are exceptions to this as, for instance, in the case of Beneckea natriegens, which contains very low concentrations of cytochrome d , although this cytochrome appears t o be a major oxidase in this organism (Weston and Knowles, 1973). In many cases the terminal oxidases of bacteria are maximally induced at low dissolved oxygen tensions (Harrison, 1973b). From Table 9 it can be seen that this applies to Escherichzh coli, Haernophilus parainfluenzae, cytochrome o of Micrococcus dentrificans, and to Ktebsiella aerogenes. Possibly, this would enable the bacteria to maintain a high respiration rate as the dissolved oxygen tension fell below the K, value for the cytochrome. A good correlation between the cellular content of cytochrome oxidase and the K, value for oxygen was found with Haemophilus parainfluenzae by White (1963) and Sinclair and White (1970); and certainly, in K . aerogenes, both the in situ respiration rate and

296

D. E.

F. HARRISON

cytochrome a , and d contents are higher under oxygen-limited conditions (Harrison, 1973b; Harrison and Loveless, 1971a). Meyer and Jones (1973a) found a correlation between the K, value for oxygen and the nature of the cytochrome oxidase possessed by bacteria (Table 9), although they made the assumption that the only functioning terminal oxidases were those detected by difference spectra, which, for reasons outlined above, may not be a sound assumption. The method used by most workers to determine the K, value for oxygen is to obtain a desaturation curve using a closed oxygenelectrode system, and to plot tangents of this curve against the oxygen concentration. This method is open t o two large sources of error: (1) the response time of the electrode may be significant; and (2) there may be back diffusion of oxygen into the solution from air. The response time of a membrane electrode can be quite different at low oxygen tensions to that at near air-saturation (Harrison, 1965), a fact not always appreciated by authors. None of the authors cited above gave sufficient details of their methods for the results to be assessed critically, and one cannot be certain that all sources of error were eliminated. Accurate measurement of K, values for oxygen is, in fact, quite difficult. Undoubtedly, the best method for estimating this constant is the respirograph, an open oxygen electrode cell with the facility to linearly increase the Po This instrument (Degn and Wohlrab, 1971) 2: enables steady-state respiration rates to be measured under changing oxygen tensions. No systematic study of the K, value for bacterial systems, using the respirograph, has yet been published, but work with K. aerogenes (H. Degn and D. E. I;. Harrison, unpublished data) indicates that the K, for oxygen of this organism is less than 2.0 pM, a value which is below the range of sensitivity of membrane oxygen probes. In the absence of direct evidence for a decreased K, value for oxygen with an increase in terminal oxidase content, we are left only with the circumstantial evidence that, in K. aerogenes and E. coli, an increased respiration rate at low dissolved oxygen tension (Harrison and Loveless, 1971a) is accompanied by an increased content of cytochromes a l and d (Moss, 1952; Harrison, 1973b). However, in K. aerogenes, synthesis of cytochromes a 1 and d was also induced at very low growth-rates in the presence of excess oxygen (Harrison, 1973b), although both the respiration rate in situ and the “potential” respiration rates were lower than for cells grown at higher rates. If

THE REGULATION

OF RESPIRATION RATE I N GROWING BACTERIA

297

there is a common factor that causes induction of cytochrome a 1 and d under both low oxygen and low growth-rate conditions, it obviously cannot be the state of reduction or oxidation of the terminal oxidase, since this will be greatly different under the two conditions. Nevertheless, perhaps the increased cytochrome oxidase content is an adaptation which prevents the oxidase step becoming limiting at low dissolved oxygen levels, and the overproduction of cytochromes a 1 and d at low growth-rates is just an accidental aberration in the mode of action of the regulator(s) of cytochrome content.

C. BRANCHED ELECTRON-TRANSPORT SYSTEMS

The fact that many bacteria appear to possess up to three different terminal oxidases suggests that electron transport in bacteria can proceed along a number of alternative routes to oxygen. If this is so, then the following questions arise: (1) what are the functions of these different paths of electron transport; and (2) how are they regulated? A branched respiratory pathway for bacteria was suggested as long ago as 1956, by Lenhof and Kaplan, and Harrison and Pirt (1967) proposed that the high, uncoupled, rate of respiration in K. aerogenes at low dissolved oxygen tension was a result of a change to an alternative pathway of electron flow t o oxygen. Lenhof et al. (1956) found a cytochrome c peroxidase in Pseudornonas fluorescens which was functional only in cells grown at low oxygen tensions. This system, it was suggested, could by-pass the respiratory system at low dissolved oxygen tensions, and would be cyanide-insensitive. An alternative respiratory system, which was flavin-linked and insensitive t o cyanide, has been found in Neurospora crassa (Colvinetal., 1973);this becomes the dominant pathway of oxygen uptake in some mutants (Lambowitz and Slayman, 1971; Drabikowska et al., 1974). In plants, cyanide-resistant alternative electron-transport pathways have been found which, in certain circumstances, play a predominant role in oxygen reduction (James and Elliot, 1955). These alternative respiratory systems in plants are mediated through non-haem iron proteins, and are effectively “uncoupled” in relation to oxidative phosphorylation (Bendall and Bonner, 1971). This confers a definite advantage on some plants, such as Symplocarpus foetidus, in which the increased heat production allows germination when the ambient temperature is low.

298

D. E. F. HARRISON

There is generally a small proportion of respiration in bacteria which is insensitive to cyanide, and this doubtless represents oxygen uptake by peroxidase and oxygenase reactions. However, some bacteria demonstrate a much greater tolerance t o cyanide. Recently the possibility that tolerance to cyanide represents alternative respiratory pathways has received much attention. The most extensively studied branched electron transport system in bacteria is that of Azoto bacter vinelundii. This bacterium possesses the cytochromes 6 , , c4 + c s , a l , d and o (Jones and Redfearn, 1966). Cyanide sensitivity studies (Jones and Redfearn, 1967) suggested that there were at least two separate pathways by which electrons flow t o oxygen, one that is cyanide sensitive and the other that is very insensitive to cyanide (Fig. 16). These pathways were distinguished by the

Site I

NADH-

I

SitelI

Fp-

I

Cyt C4+5 quinone

/ \CY+

b

,'+i

cyta,o

-

cytd

FIG. 16. Branched electron transport system of Azotobacter as suggested by Ackrell and Jones (1971).

substrates which would act as electron donors in cell-free preparations. Respiration by small membrane particles, using NADH or succinate as substrate, was not inhibited by 50 pM cyanide, a concentration which inhibited completely the high respiration rate obtained with ascorbate-tetramethyl phenylenediamine (TMPD) as electron donor. The use of ascorbate-TMPD as an electron donor in cell-free systems was also the technique employed by Bendall and Bonner (1971) to demonstrate the branched pathway in plant mitochondria. Jones and Redfearn (1967) employed dual-beam spectrophotometry to identify the cytochrome pigments invoIved in the two pathways of Azotobucter. Because the absorption attributed to cytochrome 6, was less developed than that of cytochromes c4 and cs, in the steady-state of aerobic respiration, Jones and Redfearn concluded that these pigments were oxidized through different terminal oxidases. Inhibition of the cyanide-insensitive pathway was relieved by red light, but not by blue light, which led Jones and Redfearn (1967) to conclude that it was cytochrome d (which has no absorption band in the Soret region) that was the cyanide insensitive

T H E REGULATION OF RESPIRATION RATE I N GROWING BACTERIA

299

terminal oxidase, and that cytochromes a , and a were inhibited by low cyanide concentrations. From these considerations they constructed the branched electron-transport system shown in Fig. 16. Ackrell and Jones (1971) attempted to relate this branched electron-transport system to the high respiration rates observed with Azotobacter cultures when the oxygen tension was increased above the “critical” value (Dalton and Postgate, 1968; Drozd and Postgate, 1970; Nagai e t al., 1971). They reported that, under oxygen-limited conditions, the activity of the ascorbate-TMPD oxidizing pathway increased nine- t o 13-fold over that found under excess oxygen conditions. Also they found higher P/O ratios for membrane particles oxidizing ascorbate-TMPD than when either NADH or succinate was the substrate. From these results they concluded that the two pathways had different phosphorylation sites, as shown in Fig. 16. At high oxygen tensions the cytochrome d pathway would predominate (though no direct evidence was provided for this) and, because it is deficient in oxidative phosphorylation, would cause a high respiration rate and low yield coefficient (Yo). This would serve to protect the nitrogenase system against the inhibitory effect of oxygen (Drozd and Postgate, 1970). At low dissolved oxygen tensions, when protection of nitrogenase is not required, the alternative, cyanide sensitive cytochrome a / o pathway would predominate, both because there is a higher activity present, and because cytochrome a l has a low K, value for oxygen (Meyer and Jones, 1973a). This is a more “coupled” pathway and would lead to the higher yield coefficients (Ys and Yo) and lower Qo, values reported (Drozd and Postgate, 1970). More recently, C. W. Jones and J. Drozd (unpublished data) have confirmed, using the proton pulse technique of Mitchell and Moyle (1967), that on increasing the oxygen concentration, the number of sites of oxidative phosphorylation decreases from three to one. This is a very plausible mechanism for the observed response o f Azotobacter sp. to oxygen but, as yet, the two pathways have only been demonstrated in cell-free systems. However, the proposed branched electron-transport pathway, and its relationship to the increased respiration rates under conditions of excess oxygen, does not adequately explain the very large increases in Qo, vaIues, and decrease in yield coefficients, that have been reported (Drozd and Postgate, 1970; Nishizawa et al., 1971). The loss of only one site of

300

0.E. F. HARRISON

oxidative phosphorylation, which would result from the switching from the cytochrome a l o to the cytochrome d pathway (Fig. 16), could only result in a fall in yield (and concomitant increase in Qo, value) of about 33%, and the loss of two sites would cause only a 60% change. In fact ten-fold changes in yield and Qo, value have been reported (Dalton and Postgate; 1968; Nishizawa et al., 1971; Drozd and Postgate, 1970). These changes would suggest a much greater degree of uncoupling than the loss of one or two sites of oxidative phosphorylation. C. W. Jones (personal communication) suggests that, at high oxygen tensions, the ATP turnover rate is increased by an “uncoupling” of the nitrogenase reaction; this would lead, via a feedback mechanism akin to that postulated by Harrison and Maitra (1969), to a large increase in respiration rate. The anomalous switch-over response of Klebsiella aerogenes and Escherichia coli to lower oxygen tensions bears some resemblance to the response of Azotobacter sp. to elevated oxygen tensions. The increased respiration rate in K. aerogenes and E. coli is accompanied by a diminished yield (Harrison and Loveless, 1971a), which is the result of a diminished phosphorylation efficiency (Harrison and Maitra, 1969). An alternative electron transport system, deficient in one site of oxidative phosphorylation and operating only at low dissolved oxygen tensions, was postulated to explain this phenomenon (Harrison and Maitra, 1969; Harrison and Loveless, 1971a). A mechanism somewhat similar to that postulated for Azotobacter sp. could be used to explain the behaviour of K. aerogenes. In this latter case there would be a switch in the electron-transport chain, at low oxygen tensions, from a fully coupled branch, terminating in cytochrome 0 , to a branch terminating in cytochrome a , and/or d (these, according to Meyer and Jones, 1973a, having lower K, values for oxygen) which is deficient in one site of oxidative phosphorylation. The decrease in yield coefficient and the increase in respiration rate in K. aerogenes, on lowering the oxygen tension below the “critical” value, are consistent with the loss of about one site of oxidative phosphorylation (Harrison and Loveless, 1971a). The induction of cytochromes a l and d at low dissolved oxygen tensions could represent an attempt by the organism to maintab a high respiration rate at low dissolved oxygen tensions at the expense of energy coupling. This, of course, implies a completely different involvement of the three terminal oxidases ( a l , d and 0 ) to that

T H E REGULATION OF RESPIRATION RATE IN GROWING BACTERIA

301

postulated for Azotobacter sp. As yet there is no direct evidence for the involvement of any alternative electron pathway in the switchover mechanism, so the above hypothesis is very speculative. Harrison (1973b) reported kinetic changes of a pigment in K. aerogenes (absorbing light at 500 nm), which indicates that this pigment changes its state of oxidation/reduction only at low dissolved oxygen tensions. Possibly this suggests the presence of a pathway of oxidation specific to low dissolved oxygen tensions. A branched system of electron transport similar to that proposed for Azotobacter sp. has been claimed for the marine facultative bacterium, Beneckea natriegens (Weston et al., 1974). This organism appears to have four terminal oxidases in its complement o f cytochromes (Weston and Knowles, 1973). The cytochromes which have been identified in this organism are a , , d, o and cco. A cyanideinsensitive pathway for ascorbate-TMPD oxidation was reported to operate in cell-free particles of this organism, but NADH oxidation showed a biphasic response to cyanide. This was interpreted as indicating the existence of a pathway which would be inhibited by low cyanide concentrations (up to 50 pM), and a cyanide-resistant pathway which was only inhibited by cyanide concentrations above 1 mM (Weston et al., 1974). Studies on whole B. natriegens organisms have failed to demonstrate this biphasic response to cyanide. Instead, the sensitivity to cyanide was found to be highly dependent on the respiration rate of the cells, as shown in Fig. 1 7 (J. Linton, unpublished data). This response was most unusual and did not represent any of the normal relationships for enzyme inhibition. A similar response was obtained whether respiration rate was lowered by varying substrate concentration, the nature of the substrate, or the temperature. The pattern of response suggests that cyanide titrates against a component which is rate-limiting at maximum respiration rate. At maximum respiration rate even low concentrations of cyanide inhibit but, at sub-maximum rates, some cyanide can bind with the component decreasing its availability until it once more becomes the rate-limiting step. This explanation would imply that the maximum potential respiration rate is limited by the cytochrome chain. AS we have seen above, the weight of evidence indicates that there is no relationship between the potential respiration rate and the cellular content of terminal oxidase. Indeed, it has been shown with B. natriegens that an increase in the carbon monoxide-binding c-type

302

D. E. F. HARRISON

:

OO

20

40

60

a0

100

Potassium cyanide concentration ( p M )

FIG. 17. Effect of cyanide on the respiration rate of Beneckea natriegens organisms that were respiring at different rates. The initial respiration rate was varied by changing the glucose concentration present in an oxygen electrode cell containing the bacterial suspression. ( 0 ) 133 pM glucose; (A)44 pM glucose; (0) 22 p M glucose; (m) 11 pM glucose. The respiration rates are expressed as a percentage o f the maximum rate obtained in the presence of excess glucose (34 m o l e g-' h-' ).

cytochrome was not reflected in an increased potential respiration rate, although the cells were more resistant to cyanide (Linton et aE., 1974). In the scheme proposed by J. Linton and his coworkers, only one cyanide-binding pigment limits the potential respiration rate, although other cytochromes also bind cyanide. The rate-limiting cytochrome oxidase could be cytochrome d , which is known t o be present in very low amounts even though a major part of respiration is routed via this cytochrome (Weston and Knowles, 1974). Increasing the content of the carbon monoxide-binding cytochromes c and o would render the cell less sensitive to cyanide without increasing the respiration rate, and this would fit the observed data. It is difficult to imagine, however, why organisms should have an excess of terminal oxidases that have no apparent direct respiratory function. Possibly these cytochromes serve as a type of redox buffer for the membranes, so that localized high-reducing conditions can be prevented. In B. natriegens, unlike K . aerogenes, no sharp increase in Qoz value was observed at low dissolved oxygen tensions, and the yield

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coefficient for oxygen (Yo) was constant over a wide range of growth conditions (Linton e t al., 1974). No particular physiological conditions have been found which suggest that an alternative electron-transport pathway is used. The presence of such a multiplicity of carbon monoxide-binding pigments in this organism is, therefore, something of an enigma. The three organisms, A z o t o bacter vinelandii, Klebsiella aerogenes and Beneckea nitriegens, are all similar in possessing, as terminal oxidases, cytochromes a , , d and 0. Beneckea natriegens, in addition, possesses a carbon monoxide-binding cytochrome c (Weston and Knowles, 1973). All these organisms respond quite differently to changes in oxygen tension and are adapted to quite different modes of life. As already discussed (p. 297), branching of the electrontransport chain to different terminal oxidases has been postulated to explain the behaviour of these organisms. If these hypotheses are all correct then, clearly, such branching must be extremely adaptable to give rise t o such varied response to environmental change. However, the involvement of these cytochrome oxidases in branched electrontransport chains, while a reasonable hypothesis, is far from proven. A convincing reason for the existence of several cytochrome oxidases, and the inducibility of cytochromes in bacteria, remains t o be established. VII. Energy Conservation

The main purpose of respiration in bacteria is the production of utilizable energy to drive growth processes. By discussing the regulation of respiration we have implied that this process is reasonably efficient in growing bacteria, although this has been doubted by some workers (Gunsalus and Schuster, 1961; Wimpenny, 1969). However, careful estimation of growth yield values (Y, and Y o ) in aerobic chemostat cultures that were limited in their growth by energy-source, indicated that aerobic growth in bacteria could be just as efficient as that in eukaryotic organisms such as yeast (Harrison and Loveless, 1971a; D. E. F. Harrison and E. K. Pye, unpublished data). The YATP concept of Bauchop and Elsden (1960) has been wideIy used for estimating the efficiency of bacterial &owth (Stouthamer, 1970). This method has several shortcomings, one of

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which is the somewhat arbitrary assumption that the YATp value is a constant for all organisms at about 10.5. However, the use of YATp values does enable an estimate to be made of the efficiency of energy conservation in growing bacteria. The criticisms of Stouthamer and Bettenhaussen f1973), that maintenance energy is not accounted for in calculations based on YATp values, do not apply to the work of Harrison and Loveless (1971a) who used the relationship:

where Qoz is the specific respiration rate (mole g-’ h-’), p is the specific growth-rate (h-I), YATP is the Bauchop and Elsden constant (g cells per mole ATP) and MATp is the maintenance ATP requirement (mole g-’ h-’). Assuming a YATPvalue of 10, Harrison and Loveless concluded that K. aerogenes must possess at least two sites of oxidative phosphorylation. Stouthamer and Bettenhaussen (1973) proposed that true YATP value should be about 27 g mole-’, which would indicate much lower phosphorylation efficiencies. This figure was based partly on studies using an auxotrophic strain of K. aerogenes that was grown tryptophan-limited, with glucose in excess, and was derived by correcting for a maintenance energy value that was seemingly some ten times greater than that normally found in the glucose-limited, wild-type organisms. It would seem that this, and possibly other cases cited by Stouthamer and Bettenhaussen (1973), represents atypical situations in which the energy source was not growthlimiting, and in which the “maintenance” ration was large. Under such circumstances, the assumption that “maintenance” energy is a constant (Pirt, 1965) can be questioned, and the correction made for such large maintenance energy coefficients is of doubtful validity. Direct estimations of P/O ratios using bacterial membrane particles have usually produced values of less than 1.0. These low values can probably be attributed t o damage caused t o the integrity o f the system during the disruption of the cells. Attempts to measure P/O ratios in whole bacteria (Hempfling, 1970a, b; Van der Beek and Stouthamer, 1973) may be criticized on the grounds that they did not take into account the possibility that the rate of ATP turnover could be linked to the rate of its production, and so they most probably understimated the P/O ratio (Harrison, 1973a). The most promising

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technique so far developed for estimating true P/O ratios in whole bacteria is the proton pulse technique (Mitchell and Moyle, 1967), as modified for application to bacteria by Scholes and Mitchell (1970). This technique was employed by Meyer and Jones (197313) to a variety of bacteria, and they concluded that organisms respiring via cytochromes aa3 possessed three sites of oxidative phosphorylation, but those respiring via cytochrome d were deficient in site 3. However, this method, too, is not uncontroversia1 since the evidence for a direct relationship between proton extrusion and ATP formation in whole bacterial cells is indirect. At present, therefore, there is seemingly no irrefutable evidence that the P/O ratio of bacteria can alter in response to the growth environment, as has been suggested by Harrison and Maitra (1969) and by Ackrell and Jones (1971) t o explain a lowering of growth efficiency under certain conditions. However, for the yeast Saccharomyces it has been demonstrated that site 1 of oxidative phosphorylation, while being absent from cells grown in the presence of excess glucose, can be induced by aerating non-growing starved cells (Ohnishi, 1970). Growth yields suggest that a P/O ratio of 3 obtains when this yeast is grown in a glucose-limited chemostat culture, but that the efficiency falls when glucose is present in excess (D. E. F. Harrison and E. K. Pye, unpublished data). Site 1 oxidative phosphorylation is also lost by the yeast Candida utilis when it is grown under conditions of iron-limitation (Light et al., 1968). If some sites of oxidative phosphorylation can be lost or induced in yeast cells, depending on the environmental conditions, there would seem to be no reason to suppose that this cannot occur also in bacteria. This would provide one mechanism for changes in the efficiency of aerobic growth. As discussed above, branching of the electron-transport chain could provide an alternative mechanism for a loss or gain in phosphorylation efficiency which could give rise to a more rapid response than the induction or repression of, for instance, site 1 formation. From these considerations, a reasonable hypothesis would be that bacterial respiration is normally regulated t o provide optimum efficiency of energy conservation but that, under certain growth conditions, it is advantageous to the organism to increase its respiration rate at the cost of some loss in the efficiency of energy conservation. For example, when fixing nitrogen, A z o t o b a c t e r vinelandii organisms express a high respiration rate which serves to

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remove oxygen that would otherwise inhibit the nitrogenase system (Postgate, 1971); and in Klebsiella aerogenes, an increased respiration rate at low oxygen tensions might favour the growth of a facultative organism over obligate aerobes. VIII. General Conclusions In the foregoing account I have reviewed studies which relate to the control of respiration during growth of bacteria. Much of what can be said of regulation of respiration in the growing organism would apply equally to non-growing or “resting state” bacteria, and no attempt has been made to delineate those effects which apply only to the non-growing condition. The main difference in regulatory mechanisms betweefi growing and non-growing states is, of course, that the growing state allows for responses which involve changes in the enzyme constitution of the bacteria, while induction and repression of enzyme synthesis are unlikely to contribute to regulation in non-growing cells. Otherwise, the differences between the more rapidly-responding regulatory systems (i.e. those not involving repression and induction of enzymes) in growing and non-growing bacteria will be a result of differences in the environment and physiological state. For example, growing bacteria will have access to substrate, while resting-state bacteria may be starved. Also the responses of bacteria while growing logarithmically will undoubtedly be different from those of bacteria which have entered a “resting” state. An availability of nitrogen source is one aspect which might distinguish a growing state from a non-growing one. There seem to be no reports which suggest that uptake of a source of assimilable nitrogen per se has any effect on the regulation of respiration, but perhaps this has not been studied specifically. Synchronous growth of the yeast Schizosaccharomyces p o m b e has been found to be accompanied by peaks of respiratory activity at two separate points in the division cycle (Poole et al., 1973). It w a ~ suggested that these peaks of respiratory activity are caused by changes in the concentration of a rate-limiting enzyme (Poole et al., 1973), but oscillations in possible rate-limiting enzyme activities could not be found which were in phase with the respiratory oscillations. Possibly these oscillations, which involve only a portion of the total respiration, are a result of changes in the concentration

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of regulatory molecules during the division cycle. The regulation of

respiration rate by ADP in the classical State 3 sense (Table 1) was eliminated by the failure of uncouplers to stimulate respiration but, presumably, it is still possible that adenine nucleotides o r other coenzymes could have been involved via feedback control of substrate catabolism. An alternative mechanism, less plausible but not impossible, would be one in which the cell-division cycle is involved directly in the regulation of respiration. As yet, there have been no careful studies reported of respiration in synchronously dividing bacteria which might reveal any direct influence of cell division on respiration rate. The general picture that emerges from the foregoing account is that no one single step or compound alone can be said t o regulate respiration rate in growing bacteria. Substrate supply and uptake nearly always influences respiration rate but, as this substrate is often used for anabolic pathways as well as catabolic ones, further regulation of the rate of substrate catabolism must be imposed by the cell. This regulation appears t o be mediated via adenine nucleotides o r “energy charge”. The concentration of ADP does not limit respiration at the oxidative phosphorylation stage, as it does in State 3 mitochondria. The feedback control of catabolism alters the rate at which NADH is made available, and this probably provides the ultimate limitation of respiration in the presence of excess oxygen. When oxygen tension is above a “critical” level of about 10- 20 mm Hg, the reaction of oxygen with cytochrome oxidase is probably rarely, if ever, the limiting step for respiration in actively growing bacteria. The “potential” respiration rate may be limited by the terminal oxidase reaction, as appears t o be the case in Beneckea natriegens (Linton et al., 1974) but this is probably not s o in all bacteria. At very low oxygen tensions the rate of reaction of oxygen with the cytochrome chain must undoubtedly become the limiting step in respiration. However, in some organisms such as K. aerogenes and E. coli, there is a condition (as the oxygen tension just becomes limiting) when respiration rate increases above the fully aerobic rate (Harrison and Pirt, 1967; Harrison and Loveless, 1971a). A possible explanation for this, and similar “uncoupling” phenomena observed in Azotobacter at high oxygen tensions (Drozd and Postgate, 1970), is that alternative electron-transport pathways with lower phosphorylation efficiences become functional (Harrison and Maitra,

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1969; Ackrell and Jones, 1971). Alterations of the route by which electrons flow to oxygen, and the loss of sites of oxidative phosphorylation, possibly provide bacteria with additional regulators of respiration and energy metabolism not possessed by higher organisms. In the above, much simplified, scheme I have considered only those rapid and short-term responses which do not involve induction or repression of enzyme synthesis. To ignore the controls exerted over respiration rate via changes in the enzyme content of bacteria would present a grossly distorted picture. In the foregoing account, enzyme induction has been discussed in relation to the various regulatory mechanisms, but has not been covered under a separate heading since it cannot be considered in isolation from other, more rapidly responding, regulatory systems. In the extreme case, respiration may be almost completely repressed in some facultative organisms by growth under strictly anaerobic conditions (Cavari et al., 1968). This is to be contrasted with the high potential respiration rate in organisms grown under extremely low oxygen tensions (Harrison and Loveless, 1971a). And Cavari et al. (1968) demonstrated that protein synthesis, but not cell division, was essential for the development of respiratory activity in E. coli organisms that had been grown strictly anaerobically. The development of respiratory activity, on aerating anaerobic bacteria, parallels the synthesis of respiratory enzymes (Hino and Maeda, 1966; Takahashi and Hino, 1968). Clearly, extreme anaerobiosis prevents the formation of key enzymes of respiration, either through limitation of a necessary precursor or by repression of protein synthesis. Under these conditions of transition between anaerobiosis and aerobiosis, the activity of certain enzymes may limit respiration rate. Takahashi and Hino (1968) suggest that the uptake of substrate is altered by anaerobiosis, and this may limit respiration as the cells are re-aerated. On aerating an anaerobically-grown chemostat culture of Klebsiella aerogenes there was an initial period of up to one hour when the respiration rate in situ in the growth vessel was limited by the potential respiration rate of the bacteria (Harrison and Loveless, 1971b). After eight hours, however, the respiration rate in situ had fallen below that of the potential rate, indicating a shift in the regulatory step. In addition to the effect on cytochrome content, which was discussed previously, (p. 290), the activities of many other enzymes

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have been found t o depend on the oxygen concentration prevailing during the growth of the bacterium. These include enzymes associated with fermentation pathways which are repressed in the presence of oxygen (Brown and Johnson, 1971). The Krebs-cycle enzymes were found to be maximally induced under low oxygen tensions in Escherichia coli, and t o decrease both under anaerobic and fully aerobic conditions (Wimpenny and Necklen, 1971). Enzymes of the main catabolic pathways are also susceptible t o change in activity; for instance activity of phosphofructokinase, a key enzyme of glycolysis, was found to increase in E. coli as the oxygen tension fell below 28 mm Hg (Riechelt and Doelle, 1971). The increase in the content of a specific enzyme associated with respiration does not imply that this enzyme represents a rate-limiting step, although this is often tacitly assumed by authors. In the complex system which constitutes bacterial metabolism, with its branchedchain enzyme systems and feedback regulation, it is doubtful whether any one enzyme reaction can become the sole rate-limiting factor, except if it occupies a terminal position in metabolism (i.e. if it is an oxidase or substrate-permease). Rather, the effect of changing enzyme content will be to alter (or redress) the balance of metabolism in the face of environmental changes. For instance, increasing the activity o f Krebs-cycle enzymes at low oxygen tensions might enable facultative bacteria to maintain a high level of respiration rate although the fermentation pathways begin to compete for the available pyruvate. The balance between catabolism and anabolism may, of course, be affected by changes in the activity of enzymes associated with synthetic pathways, as well as enzymes more directly concerned with respiration. Thus regulation by induction and repression of enzymes is superimposed c n the more direct feedback regulatory systems to alter the balance, or even direction, of the overall control system. REFERENCES Ackrell, B. A. C. and Jones, C. W. (1971). European Journal of Biochemistry 20, 29 Arima, K. and Oka, T. (1965). Journal of Bacteriology 90, 734. Atkinson, B. (1974). Proceedings of the Society for General Microbiology 1 , 58. Atkinson, D. E. (1968). Biochemistry, New York 7 , 4 0 3 3 . Atkinson, D. E. (1970). In “The Enzymes”, 3rd edition, Vol I., (P. D. Boyer, ed.) p. 461. Academic Press, New York.

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Atkinson, D. E. (1971). In “Metabolic Regulation”, (H. J. Fogel, ed.) p.1. Academic Press, New York. Bachi, B. and Ettlinger, L. (1973). Archivfur Mikrobiologie 9 3 , 155. Baumberger, J. P. (1939). Cold Spring Harbour Symposia o n Quantitative Biology 7, 195. Bauchop, T. and Elsden, S. R. (1960). Journal of General Microbiology 2 3 , 4 5 7 . Bendall, D. S. a n d Bonner, W. D. (1971). Plant Physiology 4 7 , 236. Betz, A. and Chance, B. (1965). Archives of Biochemistry and Biophysics 109, 585. Boyer, P. U. andTheorel1, H. (1956). Acta Chemica Scandinavia 10, 447. Brown, C. M. a n d Johnson, B. (1971). Antonie van Leeuwenhoek 3 7 , 4 7 7 . Button, D. K. and Gamer, J. C. (1966). Journal of General Microbiology 4 5 , 195. Cartier, P. H. (1968). European Journal of Biochemistry 4, 247. Castor, L. N. and Chance, B. (1959). Journal of Biological Chemistry 2 1 7 , 4 5 3 . Cavari, B. Z., Avi-Dor, Y. a n d Grossowicz, N. (1968). Journal of Bacteriology 96, 751. Chance, B. (1951). Review of Scientific Instruments 22, 634. Chance, B. (1954). Science, N e w York 1 2 0 , 7 6 7 . Chance, B. (1957). Federation Proceedings. Federation of American Biological Societies 16, 67 1. Chance, B. (1964). Acta Union Internationale Contre le Cancer 20, 1028. Chance, B. and Baltscheffsky, H. (1956). Journal of Biological Chemistry 233, 736. Chance, B., Erecinska, M. a n d Chance, E. M. (1972). In “Oxidases and Related Redox Systems”. Vol 2 , (King, T. E., Mason, H. and Morrison, M., eds.) p. 851. University Park Press, Baltimore. Chance, B., Estabrook, R. W. and Ghosh, A. ( 1 9 6 4 ) .Proceedings of t h e National Academy of Sciences of the United States of America 51, 1244. Chance, B., Holmes, W., Higgins, J. J. and Connelly, C. M. (1958). Nature, London 1 8 2 , 1 1 9 0 . Chance, B., Schoener, B. and Elsaesser, S. (1965a). Journal of Biological Chemistry 240, 3170. Chance, B., Schoener, B. and Schindler, F. (1964) “Oxygen i n the Animal Organism”. Pergamon Press, Oxford. Chance, B. and Williams, G. R. (1955a). Journal of Biological Chemistry 217, 383. Chance, B. and Williams, G. R. (1955b). Journal o f Biological Chemistry 217, 409. Chance, B. and Williams, G. R. ( 1 9 5 5 ~ )Journal . of Biological Chemistry 217, 429. Chance, B., Williams, G. R., Holmes, W. F. and Higgins, J. (1955). Journal of Biological Chemistry 21 7, 439. Chance, B. a n d Williams, G. R. (1956). Advances in Enzymology 17, 65. Chance, B . , Williamson, J. R. Jamieson, D. and Schoener, B. (1965b). Biochemische Zeitschrift 341, 357. Chapman, A. G., Fall, L. and Atkinson, D. E. (1971). Journal o f Bacteriology 108,1072. Cole, H. A., Wimpenny, J. W. T. and Hughes, D. E. (1967). Biochimica e t Biophysics Acta 143, 445.

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Colvin, H. J., Sauer, B. L. and Munkres, K. D. (1973). Journal of Bacteriology 116,1314. Dalton, H. and Postgate, J. R. (1968).Journal of General Microbiology 5 4 , 4 6 3 . Degn, H. and Harrison, D. E. F. (1969). Journal of Theoretical Biology 22, 238. Degn, H., Lilleer, M. and Iversen, J. J. L. (1973). Biochemical Journal 136, 1097. Degn, H. and Wohlrab, H. (1971).Biochimica et Biaphysica Acta 245,347. Dhople, A. M. and Hanks, J. H. (1973).Applied Microbiology 26, 399. Drabikowska, A., Kosmakos, F. C. and Brodie, A. F. (1974). Journal of Bacteriology 117, 733. Drozd, J. and Postgate, J. R. (1970). Journal of General Microbiology 46, 193. Duysens, L. N. M. and Amesz, J. (1956). Biochimica et Biophysica Acta 24, 19. Estabrook, R. W. (1962).Analytical Biochemistry 4, 231. Estabrook, T. W. and Maitra, P. K. (1962). Analytical Biochemistry 3 , 3 6 9 . Forrest, W. W. and Walker, D. J. (1964). Nature, London 201,49. Gerard, R. W. and Falk, I. S. (1931). Biological Bulletins of the Marine Biological Laboratory, Wood’s Hole 60, 213. Ghosh, A. and Chance, B. (1964). Biochemical and Biophysical Research Communications 16, 1 7 4 . Gunsalus, I. C. and Shuster, C. W. (1961). In “The Bacteria” Vol. I (Gunsalus I. C. and Stanier, R. Y., eds.) Academic Press, New York and London. Harrison, D. E. F. (1965). Ph.D. Thesis: University of London. Harrison, D. E. F. (1970).Journalof Cell Biology 415, 514. Harrison, D. E. F. (1973a). Critical Reviewsin Microbiology 2, 182. Harrison, D. E. F. (1973b). Biochimica et Biophysica Acta 275, 83. Harrison, D. E. F. (1973c),Journal of Applied Bacteriology 36, 301. Harrison, D. E. F. and Chance, B. (1970). Applied Microbiology 19,446. Harrison, D. E. F. and Harmes, C. S. (1972).ProcessBiochemistry 7 , 13. Harrison, D. E. F. and Loveless, J. E. (1971a).Journal of General Microbiology 68, 35. Harrison, D. E. F. and Loveless, J. E. (1971b). Journal o f GeneralMicrobiology 68, 45. Harrison, D. E. F., Maclennan, D. G. and Pirt, S. J. (1969). In “Fermentation Advances” (D. Perlman, ed.) pp. 117- 144. Academic Press, New York. Harrison, D. E. F. and Maitra, P. K. (1969). Biochemical Journal 112, 647. Harrison, D. E. F. and Pirt, S. J. (1967). Journal of General Mioobiology 46, 173. Harrison, D. E. F., Topiwala, H. H. and Hamer, G. (1973). In “Fermentation Today” (G. Terui, ed.) p. 491. Society of Fermentation Technology, Japan. Hempfling, W. P. (197Oa). Biochimica et Biophysica Acta 205, 169. Hempfling, W. P. (1970b). Biochemical and Biophysical Research Communications 41, 9. Hempfling, W. P., Hofer, M., Harris, E. J. and Pressman, B. C. (1967). Biochimica e t Biophysica Acta 141, 391. Hino, S. and Maeda, M. (1966). Journal of General and Applied Microbiology 12, 247. Holms, W. H., Hamilton, I. D. and Robertqon, A. G. (1972). arc hi^ fur Mikrobiologie 83, 95. Ingraham, J. L. (1958). Journal ofBacteriology 76, 75. James, W. 0. and Elliot, D. C. (1955). Nature, London 175, 89.

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Biochemistry and Genetics of Nitrate Reductase in Bacteria A. H. STOUTHAMER Biologisch Laboratorium der Vrce Universiteit, de Boelelaan 1087, Amsterdam-Buitenveldert, The Netherlands

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315 I. Introduction 316 Properties of Nitrate Reductase A. Differentiation of Nitrate- and Chlorate-Reducing Enzymes . 316 318 B. Purification and Properties of Nitrate Reductase A 325 C. Role of Molybdate in the Formation of Nitrate Reductase . 329 D. Role of Metals in Nitrate Reductase Activity 332 111. Regulation of the Formation and Activity of Nitrate Reductase 332 A. Regulation of the Formation of Dissimilatory Nitrate Reductase B. Influence of Oxygen on the Activity of Dissimilatory Nitrate 339 Reductase C. Regulation of the Formation and Activity of Assimilatory Nitrate 34 1 Reductase IV. Electron-Transport Chain to Nitrate and Energy Conservation During 343 Nitrate Respiration A. Electron-Transport Chain to Nitrate 343 350 B. Energy Conservation During Nitrate Respiration . V. Genetics of Nitrate Reductase Formation 356 A. Methods for the Isolation of Mutants Blocked in Nitrate 356 Respiration . B. Genetic Mapping of Mutations Affecting Nitrate Reductase 357 Formation. 3 60 C. Physiological Properties of Chlorate-Resistant Mutants . D. Protein Composition of Membranes of Chlorate-Resistant 362 Mutants . E. In uitro Complementation Between Chlorate-Resistant Mutants . 3 64 367 VI. Concluding Remarks and Future Prospects 369 VII. Acknowledgements . 370 References

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I. Introduction Micro-organisms can utilize nitrate for two distinct purposes. In the first place, nitrate can be used as the sole source of nitrogen for 31 5

31 6

A. H. STOUTHAMER

the synthesis of all the nitrogen-containing compounds of the cell. This process, called nitrate assimilation, can occur under both aerobic and anaerobic conditions. In the second place, nitrate can be used under anaerobic conditions as a terminal hydrogen acceptor; this process is called nitrate respiration. In both cases the first step in nitrate utilization is a reductian of nitrate to nitrite and in a number of micro-organisms nitrite may also be used under anaerobic conditions as a terminal hydrogen acceptor. In this latter case nitrate is converted to gaseous products such as nitrogen or nitrous oxide, a process called denitrification. The literature on the metabolism of nitrogen oxides by micro-organisms has been reviewed recently by Payne (1973) and by Pichinoty (1973), and some aspects of nitrate metabolism were treated in a review b y Brown et aZ. (1974). This review will therefore be devoted t o some new developments, and t o a number of aspects which were not treated extensively in the earlier reviews. 11. Properties of Nitrate Reductase A. DIFFERENTIATION O F NITRATE- AND CHLORATE-REDUCING ENZYMES

Reduction of nitrate is accomplished by nitrate reductase. A very useful manometric method for the assay of this enzyme, in bacterial cell-free extracts, has been developed by Pichinoty and Pikchaud (1968). In this method, reduced benzylviologen is used as a hydrogen donor for nitrate reduction, and the reduction of benzylviologen, with hydrogen, is mediated by a hydrogenase isolated from Desulfovibrio vulgaris. Thus, nitrate reduction can be followed by measuring hydrogen consumption. This assay has been used for measuring the reduction of nitrate and chlorate in a large number of bacteria (Pichinoty et aZ., 196913). Three enzymes acting on nitrate and chlorate, which differ in substrate specificity and in their sensitivity towards inhibitors, could be distinguished. The properties differentiating these enzymes are given in Table 1. Nitrate reductase A has always a role in nitrate respiration, but the function of nitrate reductase B is different in different bacteria. In a number of facultative anaerobic bacteria, nitrate reductase B has a respiratory function (Pichinoty, 1970) and it is therefore questionable whether this enzyme is always a cyto-

31 7

BIOCHEMISTRY A N D GENETICS OF NITRATE REDUCTASE

TABLE 1. Differentiating properties of the various enzymes that reduce nitrate and chlorate Property

Enzyme Nitrate reductase

Reduction of nitrate Interaction with chlorate Inhibition by azide Localization in cell-free extract

Chlorate reductase

A

B

C

+

+

-

Substrate

Inhibitor

+

+

Cytoplasmic membrane

Cytoplasm

Substrate Cytoplasmic membrane

plasmic enzyme, since it is difficult to envisage a respiratory function for an enzyme not bound to the cytoplasmic membrane. Pseudomonas putida, which can perform nitrate assimilation but which is unable t o perform nitrate respiration, forms nitrate reductase constitutively under both aerobic and anaerobic conditions; in this organism, the enzyme has a nutritive function. In Micrococcus denitrificans, nitrate reductase A is formed under anaerobic conditions in the presence of nitrate. However this organism is able to form, in addition, nitrate reductase B under both aerobic and anaerobic conditions. In both Ps. stutzeri and M. denitrificans the formation of nitrate reductase B is not repressed by ammonia, which is different from the situation with the assimilatory nitrate reductase in Klebsiella aerogenes (van 't Riet et al., 1968); this will be dealt with later in this review (p. 341). The only known activity of chlorate reductase C is the reduction of the non-physiological chlorate ion. This enzyme was originally detected in a Hafnia sp. (Pichinoty, 1965), but later it was found along with nitrate reductase A and B in Proteus mirabilis (de Groot and Stouthamer, 1969). The specific activities of the nitrate- and chlorate-reducing enzymes, after growth with various hydrogen acceptors, are shown in Table 2. From these results it was concluded that, after growth without a hydrogen acceptor, nitrate reductase B and chlorate reductase C are formed. Proteus mirubilis is unable to assimilate nitrate, and therefore the function of nitrate reductase B in this organism is unknown. During growth with nitrate, formation of nitrate reductase A, which reduces both nitrate and chlorate, is induced, and the synthesis of nitrate

31 8

A. H. STOUTHAMER

TABLE 2. Formation of nitrate reductase A and B, and of chlorate reductase C, under various growth conditions, in Proteus mirubilis organisms. After de Groot and Stouthamer (1969, 1970a) Hydrogen acceptor present during growth

None Oxygen Nitrate

Specific activity (nmoleslmin mg protein) for the reduction of Nitrate

Chlorate

47 7 512

393 11 633

Inhibition (%) of chlorate reduction by 1 mM NaN3

0 not tested 80

reductase B and of chlorate reductase C is repressed. Under aerobic conditions the formation of the three enzymes is repressed. Up t o the present time, only the nitrate reductase A (of several bacteria) has been obtained in a pure state. Consequently only with this enzyme are a number of properties of the molecule known. However, a comparison of the molecular properties of the various nitrate- and chlorate-reducing enzymes may be of considerable importance since the possibility might be considered that, in organisms in which two different enzymes can be formed, some subunits are common to several enzymes. Only after detailed comparative studies have been performed can a theoretical background of the differentiation of nitrate reductase A and B, and of chlorate reductase, be given. B. PURIFICATION AND PROPERTIES OF NITRATE REDUCTASE A

1. Methods f o r Solubilization of Nitrate Reductase As mentioned previously, nitrate reductase A is associated with the cytoplasmic membrane. Therefore, in the purification procedure, one of the earliest steps must be the solubilization of the enzyme. This has been accomplished in various ways as shown in Table 3. It is evident that the preferred method is that which gives a good selective solubilization of the enzyme. Thus, by treatment of the membrane particles from K. aerogenes with 1.5% deoxycholate, 85% of the nitrate reductase activity was solubilized, whereas only 30% of the total membrane protein became soluble (van 't Riet and Planta, 1969).

TABLE 3. Solubilization methods used for the iso1a:ion of bacterial nitrate reductase Treatment

Organism

Reference

-<

1 -

Alkali-acetone Deoxycholate

Sodium dodecylsulphate Triton X-100, Triton 144 Heat ( 7 min at 6OoC) t-Amy1 alcohol

Micrococcus denitrificans Micrococcus halodenitrificans Escherichh coli Escherichia coli Klebsiella aerogenes Micrococcus denitrificans Pseudomonas denitrificans Bacillus stearothermophilus Escherichia coli Escherichia coli Pro teus m ira bilis

0 Forget (1971) GI Rosso et al. (1973) rn 2 Forget (1974) rn I! Enogh and Lester (1974) 0 van 't Riet and Planta (1969) cn Lam and Nicholas (1969b) $ Radcliffe and Nicholas (1970) Kiszkiss and Downey (1972) II] MacGregor (1975a, b) D MacGregor et al. (1974) -4 rn Oltmann et al. (1974) n

$

iv

rn 0

C

0 -I

Lrn

320

A. H. STOUTHAMER

It is evident that in this way not only is a good solubilization achieved, but also a considerable purification of the enzyme over the membrane fraction. In most cases detergents have been used to accomplish solubilization, but a procedure avoiding the use of detergents has been worked out for the enzyme for Proteus mirabilis. This procedure is promising since tetrathionate reductase, chlorate reductase C, hydrogenase and formate dehydrogenase also can be solubilized (Oltmann et al., 1974). The whole purification procedure worked out for purification of membrane-bound enzymes of P. mirabilis consists of: (a) isolation of the cytoplasmic membrane by centrifugation of a crude cell-envelope preparation, obtained by osmotic shock from penicillin sphaeroplasts, on discontinuous sucrose gradients (Oltmann and Stouthamer, 1973); (b) solubilization by repeated extraction with tertiary amyl alcohol; and (c) separation of the solubilized enzymes by isoelectric focussing (Oltmann et al., 1974). The nitrate reductase of Escherichia coli was solubilized by heat treatment at alkaline pH values (MacGregor et al., 1974). Here, solubilization was shown to be due to activation of a membranebound protease (MacGregor, 1975a), and the amount of nitrate reductase solubilized was greatly decreased by the presence of protease inhibitors (e.g. p-aminobenzamidine hydrochloride) during the heat treatment at alkaline pH values. Solubilization was attended by proteolytic cleavage, and one of the subunits of the nitrate reductase was found to be especially susceptible to proteolytic cleavage. The properties of purified nitrate reductases obtained from a number of different micro-organisms are shown in Table 4. For the enzyme of E. coli, three different molecular weights have been reported: 1 x l o 6 daltons (Taniguchi and Itagaki, 1960); 8 x l o 5 daltons (MacGregor et al., 1974) and 3.2 x l o 5 daltons (Forget, 1974). As detailed below, some of these discrepancies may be explained by the observations of van 't Riet and Planta (1969, 1975), with the nitrate reductase of Klebsiella aerogenes, and the observations of MacGregor et al. (1974), Enogh and Lester (1974) and MacGregor (1975a; b) with the enzyme from Escherichia coli.

2. Nitrate Reductase of Klebsiella aerogenes Nitrate reductase of K. aerogenes can be obtained in different forms, dependent on the isolation procedure that is used; these have

E

'TABLE 4. Properties of nitrate reductases isolated from various micro-organisms Property

Escherichia coli

Escherichia coli

K l e bsiella aerogenes

0 0 I

Micrococcus denitrificans

Micrococcus halodenitrificans

- - I

Molecular weight (daltons)

3.2 x 1 0 5

8 x lo5

1 x 106

1.6 x 105

1.65 x 105

Subunit structure

not determined

4 x 142.000 4 x 58.000

4 x 117.000 4 x 57.000 8 x 52.000

not determined

not determined

Isoelectric point Iron content (atoms/mole) Molybdenum content (atoms/mole) Labile sulphide (moles/mole) Michaelis constant for nitrate (mM) Reference

4.25 20 1.5 18.6 1.5 Forget (1974)

3.2

MacGregor e t al. (1974) I

32 0.1 van 't Riet and Planta (1969) van 't Riet e t al. (1975)

7.53

a

< D

2

0 Q rn 2

rn

4.2 48 0.96

I

v)

-I 2

0.3 1 10 4 0.25 1.3 Forget (197 1 ) Rosso e t al. (1973)

G

41 5 -I

a D

a

rn

0

C

0

-4

Lrn w N

322

A. H. STOUTHAMER

been designated nitrate reductase I and nitrate reductase 11. The normal enzyme is nitrate reductase I, which can be converted into nitrate reductase I1 by treatment at pH 9.5 in the presence of deoxycholate and 0.5 M sodium chloride. Ageing has the same effect. Nitrate reductase I has a sedimentation coefficient of 22.1 S, while the sedimentation coefficient of nitrate reductase I1 usually was found to be 21.7 S. In the presence of detergents, both nitrate reductase I and nitrate reductase I1 are changed t o slower-sedimenting forms that also have a higher electrophoretic mobility. Deoxycholate-treated nitrate reductase I has a sedimentation coefficient of 9.8 S and a molecular weight of about 2.6 x l o 5 daltons. This indicates that nitrate reductases I and I1 are present as tetramers, which dissociate into monomers in the presence of deoxycholate. This dissociation is reversible, since removal of deoxycholate resulted in the re-appearance of enzymes sedimenting at 22 S. An intermediate form of nitrate reductase 11, with a sedimentation coefficient of 13.9 S, has also been observed. This form is thought to be a dimer. Determination of the subunit structure of nitrate reductase was performed by studying electrophoresis of nitrate reductase on gels containing sodium dodecylsulphate. Nitrate reductase I of K. aerogenes contained three different subunits, with molecular weights of 1.17 x l o 5 , 5.7 x l o 4 and 5.2 x l o 4 daltons, respectively. The molecular ratio of these three subunits in the native enzyme was found to be 1:1:2. Nitrate reductase I1 was found to consist of only two subunits, having molecular weights of 1.17 x l o 5 and 5.7 x l o 4 daltons, respectively. Apparently the treatment a t pH 9.5, in the presence of 1%deoxycholate and 0.05 M sodium chloride, removed both copies of the smallest subunit from nitrate reductase I, and the remaining subunits were found t o be present in an equimolar ratio. One important consequence of these results is that, although nitrate reductase I1 lacks the two smallest subunits that are present in nitrate reductase I, it is still catalytically active. It has been proposed that the two smallest subunits have a structural role, since nitrate reductase 11, which lacks these subunits, is much more labile than nitrate reductase I. However, a functional role for the smallest subunit in the enzymic activity of whole cells cannot be excluded. It is important to realize that purified preparations have to be assayed for enzymic activity with artificial electron donors. In this respect,

BIOCHEMISTRY A N D GENETICS OF NITRATE REDUCTASE

323

we may mention that the enzyme from E. coli was shown to contain cytochrome b as the smallest subunit; but the presence of this component was not necessary for nitrate reduction with artificial electron donors (see the following section). Therefore, involvement of the smallest subunit of the enzyme of K . aerogenes in its actual activity is, in my opinion, not excluded, especially since electron paramagnetic resonance (EPR) studies (see Section II-D; p. 329) have shown that these subunits contain strongly bound iron atoms. Inactivation of nitrate reductase I1 can be prevented by the presence of deoxycholate; moreover, inactivated nitrate reductase I1 can be re-activated by deoxycholate. These observations suggest that deoxycholate restores the conformation of nitrate reductase 11, which is lost when the subunit with a molecular weight of 5.2 x l o 4 daltons is removed.

3. Nitrate Reductase of Escherichia coli Nitrate reductase from E. coli can be solubilized in such a way that it is complexed with other membrane proteins. A complex of nitrate reductase with cytochrome b has been isolated (Enogh and Lester, 1974; MacGregor, 1975b), as well as a complex of nitrate reductase with cytochrome b and formate dehydrogenase (Iida and Taniguchi, 1959; Itagaki et al., 1962). The nitrate reductase isolated by MacGregor et al. (1974), after solubilization of the enzyme by heat treatment, is not complexed with other proteins, however. Therefore the form in which nitrate reductase is isolated is strongly dependent on the procedure used for the solubilization of the enzyme. The nitrate reductase isolated by MacGregor et al. (1974) was found to consist of equimolar amounts of two subunits with molecular weights of 1.42 x l o 5 and 5.8 x l o 4 daltons, respectively. And, since the molecular weight of the enzyme was estimated to be about 8 x l o 5 daltons, it must be a tetramer. This shows that the nitrate reductase of E. coli resembles the nitrate reductase I1 of K. aerogenes. The molecular weight of the smallest subunit of the nitrate reductase of E. coli is identical with that of the smallest subunit of nitrate reductase I1 of K. aerogenes. The molecular weights of the largest subunits are however, different. By treatment of membranes with deoxycholate, a nitrate reductase was solubilized

324

A. H. STOUTHAMER

containing three subunits whose molecular weights were estimated to be 1.55 x l o 5 , 6.3 x lo4 and 1.9 x l o 4 daltons, respectively (Enogh and Lester, 1974). The purified enzyme was found to contain cytochrome 6 , and the smallest subunit was found to contain the haem. A similar complex was isolated by MacGregor (1975a, b). She used purified nitrate reductase, solubilized by heat treatment at alkaline pH values, to prepare specific antibody which was then used to precipitate nitrate reductase from Triton extracts of the membrane. In this way MacGregor ( 1 9 7 5 ~ )found that the nitrate reductase protein amounted to 15 to 18% of the Triton-extracted membrane proteins. Nitrate reductase, precipitated from Triton extracts, contained three subunits with molecular weights of about 1.42 x l o 5 , 6 x l o 4 and 1.95 x l o 4 daltons, respectively, in a molar ratio of 1 : l : Z (MacGregor, 197513). The smallest subunit, which is absent from the enzyme solubilized by heat treatment at alkaline pH values, was identified as the apoprotein of cytochrome b (MacGregor, 1975b). In a hem A mutant, which cannot synthesize haem in the absence of 6-aminolaevulinic acid, the smallest subunit is absent from Tritonextracted nitrate reductase when ever the mutant was grown without the haem precursor. Furthermore, nitrate reductase was overproduced, and accumulated in the cytoplasm, after growth without 6-aminolaevulinic acid (MacGregor, 1975b). Since the largest part of the nitrate reductase activity, in the hem A mutant, is present in the cytoplasm, after growth without haem precursor, it may be concluded that cytochrome b is involved in the attachment of nitrate reductase to the membrane. The absence of cytochrome was also shown to affect the stability of the membrane-bound form of the enzyme (MacGregor, 197513). Cytochrome b , in the nitrate reductase of E. coli, thus has a number of functions in common with the smallest subunit of the nitrate reductase I of K. aerogenes, since both affect membrane attachment and stability of nitrate reductase. Upon addition of the antiserum for nitrate reductase to Tritonextracted membrane proteins, about 65% of the total amount of cytochrome b coprecipitated with nitrate reductase (MacGregor, 1975b). This indicated that not all of the cytochrome b was complexed with nitrate reductase. Purified formate dehydrogenase also contains cytochrome b (Hager and Itagaki, 1967; Enogh and Lester, 1975), and the enzyme purified by Enogh and Lester (1975),

BIOCHEMISTRY AND GENETICS OF NITRATE REDUCTASE

325

which has a molecular weight of about 6 x lo5 daltons, was found to contain three subunits whose molecular weights were estimated to be 1.1 x lo5, 3.2 x l o 4 and 2 x l o 4 daltons, respectively. The haem in formate dehydrogenase was associated with the smallest subunit. This strongly suggests that the cytochrome b apoprotein in nitrate reductase is identical with that in formate dehydrogenase. Formate dehydrogenase was found to contain, in relative molar amounts, 1.0 haem, 0.95 molybdenum, 0.96 selenium, 14 non-haem iron and 13 acid-labile sulphide. The identification of formate dehydrogenase as a molybdoprotein has important consequences that will be discussed later in this review (p. 368). As mentioned previously, nitrate reductase can be solubilized in such a way that it is complexed with cytochrome b and formate dehydrogenase (Iida and Taniguchi, 1959; Itagaki et al., 1962). These observations are important since, in E. coli, formate is the favoured electron donor for nitrate reduction in vivo (see Section IV-A-1, p. 344), and since nitrate reductase-less mutants are also affected in the formation of formate dehydrogenase and cytochrome b (see Section V-C, p. 360).

C. ROLE OF MOLYBDATE IN THE FORMATION OF NITRATE REDUCTASE

Nitrate reductase, isolated from various bacteria, contains varying amounts o f molybdenum (Table 4). The importance of molybdenum for nitrate reductase becomes apparent from studies that have been carried out both on the influence of molybdate on nitrate reductase formation, and on mutants which have defects in molybdate metabolism. The influence of molybdate, selenite and tungstate on nitrate reductase formation has been studied by Lester and De Moss (1971) and Enogh and Lester (1972). Their results are summarized in Table 5. The specific activity of nitrate reductase activity, as measured with NADH and reduced benzylviologen, is increased by growth in the presence of molybdate. Nitrate reductase activity, with formate as hydrogen donor, appears only after growth with molybdate and selenite. This may be explained by the observation that the presence of selenite is essential for the formation of formate dehydrogenase and formate hydrogenlyase activity (Pinsent, 1954), and is in accordance with the observation of Enogh and Lester

326

A. H. STOUTHAMER

TABLE 5. Influence of the presence of molybdate, selenite and tungstate in the growth medium on the levels of enzyme activities related to nitrate reduction in Escherichia coli. Data of Lester and De Moss (1971)and of Enogh and Lester

(1972) Molar concentration of supplements added to the minimal medium Naz Moo4

Na2 SeO3

10-6

-

-

10-6 10-6 10-6 10-6 10-6 10-6 10-6

10-6 10-7 10-7 10-7 10-7 10-4

Nitrate-reducing activity (nmoles/min mg protein) with various hydrogen donors

Na2 W04 Formate

NADH

Reduced benzy lviologen

4 2

50 130 40 120

260 1900 270 4150 1550 960 750 700 2300

2 1300 -

10-5 10-4 10-3 10-3

not determined

(1975) that formate dehydrogenase contains selenium. In the presence of increasing concentrations of tungstate, the activity of nitrate reductase is decreased, although this effect could be counteracted by increasing the concentration of molybdate. It is known that tungstate is a competitive inhibitor of molybdenum utilization in bacteria; for instance, the formation of formate dehydrogenase in E. coli (Pinsent, 1954), the assimilation of nitrate and nitrogen fixation by Azotobacter (Takahashi and Nason, 1957; Keller and Varner, 1957; Guerrero et al., 1973) and nitrogen fixation by Klebsiella pneumoniae (Brill et al., 1974) are antagonized by growth of the organisms in the presence of tungstate. An influence of tungstate on nitrate assimilation in fungi and higher plants has also been observed repeatedly. The formate dehydrogenase of Clostridium thermoaceticum can be formed, however, during growth in the presence of either molybdate or tungstate (Andreesen and Ljungdahl, 1973). Further evidence for a role of molybdenum on the formation of nitrate reductase has been obtained from an analysis of mutants of E. coli (Glaser and De Moss, 1971) and of Pseudomonas aeruginosa (van Hartingsveldt and Stouthamer, 1973). Some mutants (chl D, from E. coli, and nar D, from Ps. aeruginosa) were found t o form nitrate reductase only during growth in media with high concentrations of

327

BIOCHEMISTRY A N D GENETICS OF NITRATE REDUCTASE

TABLE 6. Formation of nitrate reductase activity by chl D mutants of Escherichia coli and nar D mutants of Pseudomonas aeruginosa during growth with high concentrations of molybdate. Data of GIaser and De Moss (1971), and van Hartingsveldt and Stouthamer (1973) Organism

Nitrate reductase activity (nmoles/min mg protein) Molybdate concentration in minimal medium

M Escherichia coli wild type Escherichio coli chl D

2015 35

5 x 10-4 M

2060 2340

Molybdate concentration added to complex medium

Pseudomonas aeruginosa wild type Pseudomonas aeruginosa nar D

0

5 x 10-4

600 6

560

M

380

molybdate (Table 6). In the chl D mutants of E. coli, formate dehydrogenase and formic hydrogenlyase are formed only during growth in the presence of high concentrations of molybdate. This is in accordance with the identification of formate dehydrogenase as a molybdoprotein (Enogh and Lester, 1975). The molybdenum content of the chl D mutant of E. coli was about the same whether growth occurred in the presence or absence of molybdate. However, the amount of molybdenum in particulate matter of cell-free extracts was much lower in cells grown without high concentrations of molybdate. Consequently the mutation seems to affect the partition of molybdenum between the soluble and particulate fractions. These observations seem to exclude the possibility of the mutants having a lesion in their uptake mechanism for molybdate, and it is reasonable to suppose, therefore, that the conversion of molybdate to a molybdenum cofactor of nitrate reductase is affected in these mutant organisms. In Aspergillus nidulans, mutations in five loci ( c n x loci) can result in the pleiotropic loss of two molybdoproteins, namely assimilatory nitrate reductase and xanthine dehydrogenase (Arnst et at., 1970). In these mutants, the formation of a molybdenum-containing cofactor,

328

A. H . STOUTHAMER

common to nitrate reductase and xanthine dehydrogenase, is supposed to be affected. The nar D mutant of Ps. aeruginosa can grow only with nitrate or hypoxanthine (which is oxidized by xanthine dehydrogenase) as sole nitrogen source, when a high concentration of molybdate is present in the medium (van Hartingsveldt and Stouthamer, 1973). Furthermore, nar B and nar E mutants are affected in assimilatory and dissimilatory nitrate reductase and in xanthine dehydrogenase. However, in the latter mutants, the lesion cannot be repaired by growth in the presence of high concentrations of molybdate. Thus in Ps. aeruginosa, as in A. nidulans, several loci are involved in the formation and attachment of the molybdenum-containing cofactor. Interesting observations on the nature of this molybdenumcontaining cofactor from Neurospora crassa have been made by Nason and his coworkers. In vitro assembly of a hybrid assimilatory nitrate reductase was attained by mixing an extract of a nitrateinduced N. crassa mutant (nit 1)specifically with acid-treated (pH 2.5) bovine milk or intestinal xanthine oxidase, rabbit liver aldehyde oxidase, chicken liver xanthine dehydrogenase, nitrogenase from Azotobacter vinelandii or bovine sulphite oxidase (Ketchum et al., 1970; Nason et al., 1971; Lee et al., 1974a). Acid treatment of a large number of molybdoproteins can thus liberate a cofactor which can activate the inactive nitrate reductase of the nit 1 mutant of N. crassa. Upon incubation of the acid extract of xanthine oxidase, the activating effect on the nit 1 extract is lost. A very good reconstitution of nitrate reductase activity was obtained, however, with pre-incubated acid extract of xanthine dehydrogenase in the presence of lo-* M molybdate. From these results it may be concluded that the molybdenum-containing cofactor is labile, and can be protected against inactivation by the presence of molybdate. The molybdenum-containing cofactor does not contain protein, since it is not inactivated by treatment with proteolytic enzymes. When reconstitution is performed in the presence of NaZ9MoO4, molybdate is incorporated into the reconstituted nitrate reductasc. After sucrosegradient centrifugation of the reconstituted enzyme, radio-activity and enzyme activity show the same distribution over the various fractions (Lee et al., 1974a). The reconstitution process is strongly inhibited by the presence of tungstate and vanadate, both of which can be incorporated into the enzyme (Lee et al., 1974b). In the

BIOCHEMISTRY A N D GENETICS OF NITRATE REDUCTASE

329

discussion of one of their papers, the authors refer to unpublished results on the purification of the cofactor, which is reported to be a compound with a molecular weight of about 1 x l o 3 daltons, or less. More definite date are not yet available. Clearly, this system will be of great value in the identification o f the molybdenum-containing cofactor of nitrate reductase and other molybdoproteins. Similar studies on the activation in uitro of tungsten-containing inactive nitrogenase of A . uinelandii, with acid-extracted molybdoproteins, have only just started (Nagatani e t id.,1974). D. ROLE O F METALS IN NITRATE REDUCTASE ACTIVITY

The results given in Table 4 show the presence o f molybdenum, iron and labile sulphide in nitrate reductase. A monomer of nitrate reductase I from K. aerogenes contains eight iron- sulphur groups and four tightly bound non-haem iron atoms; nitrate reductase I1 lacks the tightly bound iron atoms (van't Riet et al., 1975). Consequently the tightly bound iron is associated with the low molecular-weight subunit, which is absent from nitrate reductase 11. The non-haem iron and acid-labile sulphide content of monomeric nitrate reductases I and 11 equals that of the nitrate reductase of M . denitrificans (Forget, 1971), and is about double that of the nitrate reductase of E. coli (Forget, 1974). The role of molybdenum and iron in the functioning of nitrate reductase becomes evident from studies with inhibitors. Thiocyanate, which is known to chelate molybdenum, is a strong inhibitor of nitrate reductase activity of M. denitrificans (Lam and Nicholas, 1969a), Ps. denitrificans (Radcliffe and Nicholas, 1970) and K. aerogenes (van 't Riet et al., 1975). The enzyme from the first two organisms was also inhibited by another molybdenum-chelating agent, dithiol. Iron-binding agents, such as bathophenthroline, also have an inhibitory effect on nitrate reductase activity in K. aerogenes (van 't Riet et al., 1975) and Ps. denitrificans (Radcliffe and Nicholas, 1970). These results prove that molybdenum and iron are integral components of the enzyme and are essential for enzyme activity. However they do not give any indication of whether these metals are directly involved in electron transfer to nitrate by nitrate reductase. The participation of these metals in nitrate reductase activity has been studied by electron paramagnetic resonance (EPR)

330

A. H. STOUTHAMER

spectrometry of the enzyme from M. denitrificans (Forget and Der Vartanian, 1972), E. coli (Der Vartanian and Forget, 1975) and K. aerogenes (van 't Riet et al., 1975). Measurements of molybdenum absorption were determined at 80-97"K, whereas iron-sulphide resonances were determined at 12- 18'K. The oxidation state of molybdenum may vary from (I) t o (VI), and of these states, the uneven ones can be paramagnetic. The results of these studies, which are almost identical for the three organisms that have been studied, are shown in Table 7. The oxidized enzyme shows signals at g = 1.98-1.99 and at g = 2.02-2.045, and since this signal has the same characteristics (saturation behaviour and temperature dependence) as that of the well studied molybdenum-containing enzymes xanthine- and sulphite-oxidase, the signal in the former enzyme was assigned to Mo(V). Reduction of the enzyme of E. coli and M . denitrificans results in the appearance of a new type of resonance whereas reduction of the enzyme of K. aerogenes causes the lines to increase in intensity, especially that at g = 2.02. The new signals of the reduced enzyme of E. coli and M. denitrificans were tentatively attributed to Mo(II1) by Forget and Der Vartanian (1972), and by Der Vartanian and Forget (1975). Although this hypothesis is most likely, van 't Riet et al. (1975) have pointed out another possibility, namely that the change in the intensity of the signals upon reduction of the enzyme may be due to an uncoupling of spin-spin interaction in a diamagnetic Mo(V) dimer. The change in the shape of the signal relative to that in the oxidized enzyme may then be due to a change in the ligand field of molybdenum, possibly induced by reduction of an iron- sulphur centre. If this explanation is correct, then molybdenum does not participate in electron transfer. However, the interpretation of the EPR studies is difficult, and they do not yet prove or disprove the participation of molybdenum in electron transfer. The interpretation of the results of EPR studies a t low temperature is easier. The signal of oxidized enzyme can be assigned to non-haem ferric ion. Reduction of enzyme results in the appearance of two different iron-sulphur centres with the enzyme from E. coli and M. denitrificans, but not with that of K. aerogenes. Since the signals in the EPR spectra of the enzyme undergo changes upon reduction by dithionite, and re-oxidation by nitrate, we may conclude that these iron- sulphur centres participate in electron transfer to nitrate.

TABLE 7 . Electron paramagnetic resonance signals of oxidized and reduced nitrate reductase at high temperature (80-97" K) and at low temperature (12-18'K) Temperature

Redox state of enzyme

g values OF electron paramagnetic resonance signals of nitrate reductase From Escherichia coli

High Low Reference

Oxidized Reduced Oxidized Reduced

1.988 2.008; 2.032 2.005; 2.074 1.861; 1.889; 2.047 1.948; 2.030 Der Vartanian and Forget (1975)

Micrococcus denitrificans

Klebsiella aerogenes

1.985; 2.045 1.999; 2.023 2.016 1.881; 1.947; 2.057 1.926; 2.031 Forget and Der Vartanian ( 19 7 2)

1.90; 2.02 1.98; 2.02 2.015; 2.03; 2.10 1.88; 1.95; 2.05 -I

van 't Riet et al. (1975)

n

3

rn Jl rn 0

C

0 -I

Rrn

332

A. H. STOUTHAMER

111. Regulation of the Formation and Activity of

Nitrate Reductase k REGULATION OF THE FORMATION O F DISSIMILATORY NITRATE REDUCTASE

In general, nitrate reductase A is an inducible enzyme which is formed only in the absence of oxygen and in the presence of nitrate, although in some organisms anaerobic conditions are sufficient to obtain formation of this enzyme. Such occurs in Bacillus licheniformis (Schulp and Stouthamer, 1970) and in Haemophilus influenzae (Sinclair and White, 1970) but even then, the amount of nitrate reductase formed is increased when nitrate is present. Under anaerobic conditions, not only can nitrate induce the formation of nitrate reductase, but so can nitrite and azide (Hackenthal and Hackenthal, 1966; de Groot and Stouthamer, 1970a, Chippaux and Pichinoty, 1970). The formation of nitrate reductase, and of a number of other enzymes, during growth of P. mirabilis with various hydrogen acceptors in the presence of nitrate, nitrite o r azide, is shown in Table 8. The specific activity of nitrate reductase, after growth with azide, is about four-fold higher than after growth with nitrate. The highest specific activity is found after anaerobic growth with azide and nitrate. During growth with oxygen and nitrate, benzylviologen-linked formate dehydrogenase, hydrogenase and formate hydrogenlyase are repressed. Furthermore, formate hydrogenlyase was shown t o be inhibited by nitrate, nitrite and azide. It was therefore proposed that one of the components of the formate hydrogenlyase system is involved in the regulation of the formation of nitrate reductase (de Groot and Stouthamer, 1970a). The reduced form of one of the intermediate electron carriers, in an operative formate hydrogenlyase system, might repress the formation o f the nitrate reductase complex. When this component is oxidized, and its reduction prevented, the repressive effect is lost. This hypothesis can also provide an explanation for the observation that, when a culture of P. mirubilzs, growing aerobically without nitrate, is shifted to anaerobic conditions, nitrate reductase synthesis is temporarily depressed (de Groot and Stouthamer, 1970a). This effect, which is shown in Fig. l ( a ) , indicates that aerobic cells do not contain a repressor for nitrate reductase synthesis. The results in Table 8 show that, in aerobically-grown cells, formate

I

C .0,

c

2 n

600

m I OO( E \

.-C E

h

v)

c .-

2

c

3

Q

b

0 3

e

U

+ .-

Y

400

0) +

$ Y

.-c 60(

A

c ..->

-

v)

Q

c

0

-00

0

E

c

v

c

A

c .> .c

200

0 0

Q

20( 3

0

P

Q +

Q)

c

*ze c

(-

I

2

3

0

2e

L

oo

Time after transfer (h)

I

2

3

010

m 0

rn

U

C

0 -I

5

v)

FIG. 1. The influence of a shift from aerobic to anaerobic conditions, and vice versa, on the nitrate reductase present in Proteus rn mirabilis organisms. (a) A culture growing aerobically for two hours in complex medium without nitrate was made anaerobic by sparging with nitrogen gas containing 5% carbon dioxide. (b) Bacteria growing anaerobically for two hours in complex medium with nitrate were shifted to aerobic conditions by vigorous aeration. In both experiments, samples were taken during subsequent growth for determination of nitrate reductase activity and eventual nitrite release into the medium. 0-0, nitrate reductase; ~ - m , total nitrate reductase activity; x--x, nitrite concentration; 0-0, growth.

W

W

P

TABLE 8. Influence of the presence of hydrogen acceptors, nitrite and azide in the growth medium on the formation of nitrate reductase, and several components of the formate hydrogenlyase complex, in Proteus mirabilis (after de Croot and Stouthamer, 1970a). Formate dehydrogenase activity is given with methylene blue and benzylviologen as hydrogen acceptor Hydrogen acceptor

Oxygen Oxygen Nitrate Nitrate Nitrite Nitrite None None

Azide

Specific activity (nmoIes/min mg protein) of Nitrate reductase

7 8 512 2627 429 1443 47 1863

Formate dehydrogenase

Hydrogenase

Methylene blue

Benzylviologen

187 215 308 313 484 276 46 7 393

0 0 3 14 103 86 154 146

Formate hydrogenlyase

? ? v) -I

0 C

0 0 0

83 236 302 364 348

0

0 0 0

114 5 226 65

BIOCHEMISTRY A N D GENETICS OF NITRATE REDUCTASE

335

hydrogenlyase activity is absent. When formate hydrogenlyase has been formed, further nitrate reductase synthesis is prevented. The induction of nitrate reductase synthesis by nitrate, nitrite and azide, in Bacillus cereus (Hackenthal and Hackenthal, 1966) which does not contain the formate hydrogenlyase system, can be explained by assuming the existence of an electron carrier reacting in a similar way to the presence of nitrate, nitrite and azide. In the presence of oxygen, nitrate reductase A cannot be formed (Sacks and Barker, 1949; Pichinoty and d’Ornano, 1961; Pichinoty, TABLE 9 . Organisms in which the influence of oxygen on nitrate reductase synthesis and activity has been studied Organism

Reference

Escherichia coli

Showe and DeMoss (1968)

Klebsiella aerogenes

Pichinoty and d’Ornano (1961); Pichinoty (1965); van ’t Riet e t al. (1968)

Proteus mirabilis

de Groot and Stouthamer (1969, 1970a)

Bacillus stearothermophilus

Downey (1966); Downey et al. (1969)

Bacillus licheniformis

Schulp and Stouthamer (1970)

Hae m op h ilus parainflu enzae

Sinclair and White (1 970)

Pseudomonas aeruginosa

van Hartingsveldt et al. (1971); van Hartingsveldt and Stouthamer (1974)

Achromo bacterium fischeri

Prakash and Sadana (1973)

1965). The organisms in which the influence of oxygen on the formation, and the activity, of nitrate reductase has been studied in detail are listed in Table 9. To understand the effect of oxygen on the formation and activity of nitrate reductase it must be realized that the formation of nitrate reductase is needed t o enable nitrate to function as an alternative electron acceptor to oxygen. The standard oxidation-redox potential, and the energy-yielding potential, of a number of systems involved in aerobic and anaerobic respiration are given in Table 10. In P. mirabilis, the formation of nitrate reductase is repressed by oxygen, and the formation of tetrathionate reductase by both oxygen and nitrate (de Groot and Stouthamer, 1969, 1970a, b). In E. coli, the formation of fumarate reductase is repressed by oxygen and by nitrate (Wimpenny and Cole, 1967; Cole and Wimpenny, 1968). These results suggest that the formation of a

336

A.

H. STOUTHAMER

TABLE 10. Oxidation-reduction potentials, and standard free energy changes, of aerobic respiration, and of anaerobic respiration with nitrate, tetrathionate and fumarate. The electron donor for the various kinds of respiration is NADH

AG;~

Redox couple

(kcal/mole) 0 2 /H2 0

NOs-/N02S406--/2Sz03-succinate/ fumarate

+0.82 +0.42 +0.17

-52.5 -34.1

+0.03

-15.9

-22.5

reductase for a certain substrate is prevented when a hydrogen acceptor with a higher energy-yielding potential is also present. This regulatory phenomenon therefore ensures that the largest amount of energy will be released during catabolism. However, these observations do not give any indication about the nature of the processes which regulate the formation of reductases. It has been suggested that the redox potential of the medium, rather than the presence of a specific terminal electron acceptor, regulates the formation of reductases (Wimpenny and Cole, 1967; Wimpenny, 1969). Showe and DeMoss (1968) have suggested, however, that nitrate reductase synthesis is not only controlled by a repressor that is sensitive t o nitrate, but also by a redox-sensitive repressor. The effective intracellular redox potential would be the controlling factor. This intracellular redox potential is influenced by several factors such as the identity and concentration of the electron acceptor (which determines the extracellular redox potential) and the capabilities for electron flow in the cell. Both explanations seem highly unlikely on the basis of newer results of Wimpenny and Firth (1972) who measured the influence of oxygen on the levels of NAD+ and NADH in various bacteria. The NADH level was about the same during aerobic and anaerobic growth; however, the NAD' level was much higher after aerobic growth. During change-over between aerobic and anaerobic conditions, NADH showed a temporary increase, but then returned to a constant level, whereas NAD' changed from a high level aerobically to a low level anaerobically. Such changes were found to be remarkably rapid. On the assumptions that the cell is a single compartment, and that the cofactor is soluble, the intracellular redox potential can be calculated. The intracellular redox potential varied between about -330 mV anaer-

BIOCHEMISTRY AND GENETICS OF NITRATE REDUCTASE

337

obically and -300 mV aerobically. Under these conditions, the redox potential of the medium can vary from -350 mV to +15 mV. The intracellular redox potential is thus fairly constant, and quite precisely controlled. The results of de Groot and Stouthamer (1969, 1970a, b) with P. mirabilis show that, in this organism, various combinations of a number of enzymes involved in anaerobic metabolism are formed under different growth conditions (see also Table 8). If synthesis of nitrate reductase is controlled by a redox-sensitive repressor, as suggested by Showe and DeMoss (1968), we must then assume the existence of several redox-sensitive repressors. Therefore de Groot and Stouthamer (1970a, b) have proposed that the factor regulating synthesis of reductases is the oxidation-reduction state of the components of the respiratory chain. The way in which the oxidation-reduction state of the components of the respiratory chain exerts its effect is not clear at this moment, but is is possible that nitrate reductase (and other reductases) cannot be complexed in an active membrane-bound form with other electron-transport components when electron flow to these enzymes is impeded. Furthermore, it was assumed that, under all conditions in which electron flow to a redox enzyme is impossible, repression of its formation occurs. As a possible mechanism to achieve this repression, it was proposed that oxidized nitrate reductase would act as a repressor for its o w n synthesis. This is a regulation mechanism which is now called “autogenous regulation of gene expression” (Goldberger, 1974). In this connection it must be mentioned that Cove and Pateman (1969) have proposed that nitrate reductase, in Aspergillus nidulans, is involved in the regulation of its own synthesis. At the present moment, the hypothesis of de Groot and Stouthamer (1970a, b) is in accordance with most data on the influence of oxygen on the regulation of nitrate reductase formation. A mutant of E . coli has been isolated which has a non-identified defect in the electrontransport chain (Simoni and Shallenberger, 1972). Membrane vesicles, isolated from this strain, exhibit greatly decreased respiratory activity with NADH, succinate and D -lactate. Nitrate reductase can be formed in this mutant under aerobic conditions, and therefore it is also sensitive towards chlorate under aerobic conditions. This shows that nitrate reductase can be formed, and can function, in the presence of oxygen, if electron transport to oxygen is restricted.

338

A. H. STOUTHAMER

When the supply of oxygen is limited, electron transport t o oxygen, and to nitrate, can also occur simultaneously. This was observed for the first time, in Ps. stutzeri, by Gilmour et al. (1964). The results obtained with glucose-limited chemostat cultures of K. aerogenes (A. H. Stouthamer and C. W. Bettenhaussen, unpublished results) are given in Table 11. In this experiment, the supply of oxygen to the organisms was limited by varying the stirring rate of chemostat culture. With fully-aerated cultures, the dissolved oxygen tension in the culture was higher than 70% of the saturation level, whereas in the experiments in which a limited oxygen supply was maintained TABLE 1 1 . Influence of aerobic growth, anaerobic growth and growth at suboptimal levels of oxygen supply (of glucose-limited chemostat cultures of Klebsiella aerogenes) on the specific rates of consumption of glucose, oxygen and nitrate, the specific rate of acetate production and on the molar growth yields for gIucose Growth condition

Aerobic Limited oxygen supply Anaerobic Anaerobic (no nitrate)

Specific growth rate (h-' )

0.514 0.525 0.516 0.519

Specific rates of metabolism (mmoleslg dry weight/h) glucose oxygen nitrate

8.05 9.05 12.27 19.07

13.95 4.02 0 0

0 9.56 10.64

0

acetate

Molar growth yield (g/mole)

0.28 9.47 15.11 12.52

63.9 58.0 42.0 27.2

the dissolved oxygen concentration was undetectably low. The molar growth yield for glucose is highest for aerobic growth, and lower for anaerobic growth. These results indicate that the highest ATP yield per mole of glucose is achieved under aerobic conditions. In the oxygen-limited cultures, oxygen and nitrate were used simultaneously as hydrogen acceptors, and a large amount of acetate was formed. The molar growth yields for aerobic and for oxygen-limited growth were about the same. This result is in accordance with previous conclusions that the efficiency of oxidative phosphorylation in K. aerogenes is the same with oxygen or with nitrate as the terminal electron acceptor (Hadjipetrou and Stouthamer, 1965; Stouthamer, 1967a). The fact that respiration with oxygen and nitrate can occur simultaneously gives support to the idea that electron transport to oxygen is of great importance for the regulation of nitrate reductase formation. That the oxidation-reduction state of

BIOCHEMISTRY A N D GENETICS OF NITRATE REDUCTASE

339

the components of the electron-transport chain is involved in the regulation of reductase formation is also indicated by the observation that, in P. mirubilis, repression o f tetrathionate reductase formation did not occur by nitrate when nitrate reduction was prevented by azide (de Groot and Stouthamer, 1970b). The mechanism by which nitrate, nitrite and azide induce the formation of nitrate reductase, and the mechanism by which oxygen prevents synthesis of this enzyme, have, according to the theory proposed by de Groot and Stouthamer (197Oa, b), one aspect in common. In both mechanisms, the oxidation-reduction state of electron-transport carriers plays a vital role in induction or repression. However, we are far from an understanding of the molecular mechanism of the regulation of reductase formation, and cannot at this moment provide a detailed description of the mechanism by which the formation of reductases is regulated. B. INFLUENCE OF OXYGEN ON THE ACTIVITY OF DISSIMILATORY NITRATE REDUCTASE

In general it can be stated that the effect of oxygen on the synthesis of nitrate reductase occurs in parallel with an effect of oxygen on the activity of the enzyme. When a culture of K. aerogenes, growing anaerobically with nitrate as the hydrogen acceptor, is shifted to aerobic conditions three effects generally are observed: (i) an immediate cessation of the formation of nitrate reductase (discussed in the previous section); (ii) an immediate cessation of the production of nitrite; and (iii) a partial inactivation of the nitrate reductase already present (van ’t Riet et al., 1968). The effect of oxygen on the activity of nitrate reductase was observed previously by Pichinoty and d’Ornano (1961); the effects of oxygen on the activity of nitrate reductase and on the total activity of the enzyme in P. mirabilis are shown in Figure l ( b ) (p. 333). However, in a number of cases it has been observed that oxygen represses synthesis of nitrate reductase without affecting the activity of that enzyme already present. Thus, when a culture of Bacillus Zicheniformis was grown anaerobically with nitrate and then shifted to aerobic conditions, nitrate reductase synthesis was immediately arrested but nitrite formation cotltinued for another 45-60 minutes (Schulp, 1972).

340

A. H. STOUTHAMER

During anaerobic growth with nitrate, cytochrome a was not formed in B. stearothermophilus (Downey and Kiszkiss, 1969) and in B. licheniformis (Schulp and Stouthamer, 1970). In B. licheniformis, which can obtain energy from glucose fermentation, cytochrome a was not formed during anaerobic growth without nitrate, either (Schulp and Stouthamer, 1970). This indicates that the disappearance of cytochrome a is due to anaerobiosis, and not to a specific effect of nitrate respiration. After a shift of a culture of B. licheniformis, growing under anaerobic conditions in the presence of nitrate, to aerobic conditions, a rapid synthesis of cytochrome a occurred. Nitrite formation continued as long as the maximal amount of cytochrome a had not been formed, and the rate of oxygen uptake increased concomitantly with synthesis of cytochrome a . These results suggest that nitrate respiration can continue after a shift to aerobic conditions so long as the electron-transport chain to oxygen had not been adapted to' its normal aerobic functional state. A similar observation has been made with Pseudomonas aeruginosa (van Hartingsveldt and Stouthamer, 1974). A mutant was isolated in which the aerobic synthesis of haem was blocked. Thus, the mutant could grow only under anaerobic conditions with nitrate. When a culture of this mutant, growing anaerobically with nitrate, was shifted to aerobic conditions, nitrite production continued, but nitrate reductase was slowly inactivated. When the same experiment was performed with the wild-type strain, nitrite formation was rapidly arrested and a rapid increase in respiratory activity was observed. Evidently, in the mutant, nitrite formation continued for a long time because the change to the normal aerobic respiratory activity couId not take place. Inhibition by oxygen of the activity of nitrate reductase in vivo was thus, just as with the effect of oxygen on the synthesis of the enzyme, dependent on electron transport to oxygen. Inhibition of nitrate reductase by oxygen is consequently not due to a direct effect of oxygen on the enzyme molecule. As mentioned before (p. 339), a gradual inactivation of nitrate reductase (assayed with reduced benzylviologen) is observed when a culture of K . aerogenes, gowing anaerobically with nitrate as hydrogen acceptor, is shifted to aerobic conditions (van 't Riet e t al., 1968). The effect is also observed with P. mirabilis (de Groot and Stouthamer, 1970a) as shown in Fig. l ( b ) (p. 333). With both organisms, the enzyme is not completely inactivated but reached a

BIOCHEMISTRY A N D GENETICS O F NITRATE REDUCTASE

341

minimum value after some time. An explanation for this observation is still lacking. In some experiments with K. aerogenes inactivation of nitrate reductase by oxygen was not observed (Pichinoty, 1965; Pichinoty and d'Ornano, 1961). The inconsistency between these observations and those of van 't Riet et al. (1968) must be explained by differences in incubation and assay conditions. Inactivation of nitrate reductase has also been observed in B. s t e a r o t h e r ~ 5 ~ h i l u s (Downey et al., 1969), but does not occur in B. Licheniformis (Schulp, 1972). It has been proposed that inactivation of nitrate reductase is due to withdrawal of electrons from the enzyme via other components of the electron-transfer system (de Groot and Stouthamer, 197Oa); thus, inactivation is due to oxidation of the enzyme. This hypothesis is strengthened by the following observations. Inactivation of nitrate reductase is also observed when cell-free extracts of K. aerogenes are treated with oxygen (van 't Riet et al., 1968). The purified enzyme however was not inactivated by exposure to oxygen (van 't Riet, 1970). When a hem A mutant of P. mirabilis, grown anaerobically with nitrate but without 6-aminolaevulinic acid, was shifted to aerobic conditions, inactivation of nitrate reductase was not observed (de Groot and Stouthamer, 1970a). In this case, nitrate reductase is present in cells which do not contain cytochromes. These observations indicate that the enzyme is only sensitive to oxygen when complexed with a functional respiratory chain. C. REGULATION OF THE FORMATION AND ACTIVITY OF ASSIMILATORY NITRATE REDUCTASE

Only very few studies have been performed on assimilatory nitrate reductases of bacteria, presumably because of their extreme lability. In K. aerogenes, the enzyme was very sensitive to sonic disintegration (van 't Riet et al., 1968). When whole cells of K . aerogenes that had been grown aerobically with nitrate were aerated, no significant inactivation of the assimilatory nitrate reductase was observed. This is in contrast to the situation with whole cells that had been grown anaerobically with nitrate, where the dissimilatory nitrate reductase was inactivated upon aeration. Formation of the assimilatory nitrate reductase was repressed by ammonia, under both aerobic and anaerobic conditions. The assimilatory and dissimilatory nitrate reductases

342

A. H. STOUTHAMER

do not differ in enzymic properties in that they have the same kinetic parameters and pH optimum. Therefore it was proposed that the assimilatory nitrate reductase complex has a different composition from the dissimilatory complex, but that the nitrate reductase is, in both cases, the same protein. Verification of the hypothesis that the assimilatory and dissimilatory complexes contain the same nitrate reductase molecule must await purification and characterization of the assimilatory nitrate reductase, which, owing to the instability of the enzyme, may prove to be a very difficult task. Great lability seems to be a general property of assimilatory nitrate reductases of bacteria since we have also been unable to detect nitrate reductase activity in cell-free extracts of cells of Ps. aeruginosa and B. licheniformis after aerobic growth with nitrate as a nitrogen source. Consequently, some doubt remains as to whether nitrate assimilation is always carried out by nitrate reductase B (compare Section 11-A, p. 317). Recently, the assimilatory nitrate reductase of Azotobacter chroococcum has been characterized by Guerrero et al. (1973) who found it to be a soluble molybdoprotein with a molecular weight of about 1 x l o 5 daltons. The enzyme was formed during aerobic growth with nitrate as nitrogen source and was repressed by the presence of ammonium ions in the medium. The enzyme, which was assayed by measuring nitrite formation with reduced viologens as electron donor, showed very peculiar kinetic properties. Nitrite formation was proportional to time only during the first two or three minutes, after which a rapid and striking inactivation occurred and the reaction rate considerably decreased. This inactivation could be prevented by cyanate, which also was able to re-activate the enzyme once inactivation had occurred. Furthermore, the enzyme was inactivated by the simultaneous presence of low concentrations of nitrate and a reducing agent such as dithionite. The explanation offered was that nitrate reductase can exist in two interconvertible forms, either active or inactive. Under reducing conditions, the enzyme is supposed to be converted into the inactive form, but cyanate, which is a competitive inhibitor for nitrate reduction by nitrate reductase, can protect against inactivation, possibly by reoxidation of the enzyme. This explanation may also account for the regulation of the enzyme function in uiuo. The properties of nitrate reductase of A . chroococcum are similar in a number of respects to those of the assimilatory nitrate reductase in algae, such as Chlamy-

BIOCHEMISTRY A N D GENETICS OF NITRATE REDUCTASE

343

domonas reinhardi (Losada et al., 1973) and Chlorella fusca (Maldonado et al., 1973), and in yeasts (Pichinoty and MCttnier, 1966; Rivas e t al., 1973). The existence of an active and an inactive form, depending on the redox state, thus seems to be a general property of assimilatory nitrate reductases, and has important functional consequences. Thus, inactivation of assimilatory and dissimilatory nitrate reductase occurs by opposite mechanisms; the dissimilatory enzyme is inactivated by oxidation whereas the assimilatory enzyme is inactivated by reduction. That information on assimilatory nitrate reductases of bacteria is still very scanty may be due t o the peculiar properties of the enzyme which cause difficulties in finding the correct conditions for its assay.

IV. Electron-Transport Chain to Nitrate and Energy Conservation During Nitrate Respiration A. ELECTRON-TRANSPORT CHAIN TO NITRATE

In Section 11-A (p. 324) it was shown that the nitrate reductase A of E. coli is complexed with cytochrome b. In h e m A mutants of E. coli (MacGregor, 1975b) and of P. mirabilis (de Groot and Stouthamer, 1970a) nitrate was not reduced in the absence of 6-aminolaevulinic acid during anaerobic growth with nitrate. Similar observations had been made previously with Staphylococcus epidermidis (Jacobs et al., 1963) and with Staphylococcus aureus (Chang and Lascelles, 1963), which have blocks in the biosynthesis of haem. In all these cases, nitrate reductase activity can be detected with artificial electron donors. Thus during growth without haem precursors, nitrate reductase was formed in these organisms, but did not function since the enzyme was not complexed with a functional respiratory chain. The composition and function of bacterial respiratory chains has been reviewed recently by White and Sinclair (19 7 1),and by Harold (1972). In general it can be said that bacterial respiratory chains often appear to be branched, consisting of several distinct chains which are connected at particular sites. However, with only a few organisms has the respiratory chain to oxygen been compared with that to nitrate. The discussion in this section will be limited t o these organisms.

344

A. H. STOUTHAMER

1. Escherichia coli The electron-transport chain to nitrate in E. coli has been studied by Ruiz-Herrera e t al. (1969) and Ruiz-Herrera and DeMoss (1969). These authors have concluded that nitrate reduction was exclusively carried out by an organized complex containing formate dehydrogenase, cytochrome b , and nitrate reductase. Mutants which lack formate dehydrogenase but possess nitrate reductase consequently neither form nitrite nor remove nitrate from the medium during growth. Electron transport from reduced nicotinamide adenine dinucleotide (NADH) to nitrate was therefore considered to be unimportant in growing cells. On the other hand, a number of authors have demonstrated that NADH may serve as an electron donor for nitrate reduction in cell-free extracts (Cole and Wimpenny, 1968; Gray e t al., 1966; Iida and Taniguchi, 1959; Nicholas and Nason, 1955). These contradictory results have been partly resolved by Lester and DeMoss (1971), who also detected nitrate reduction with NADH as electron donor in cell-free extracts. The specific activity of the formate-nitrate reductase activity was about ten times higher than the NADH-nitrate reductase activity, however. Furthermore the highest rates of NADH-nitrate reductase activity observed in cell-free extracts were five to ten-fold lower than the rates of nitrite accumulation in v i m . We may conclude that, in uivo, both formate and NADH can act as an electron donor for nitrate reduction, but that formate is the more important of the two. It must be mentioned that fermentation balances for glucose utilization during anaerobic growth in the presence of nitrate clearly indicate that, in K. aerogenes, electron transport from NADH t o nitrate does occur (Forget and Pichinoty, 1964; Hadjipetrou and Stouthamer, 1965). In this case, NADH seems to be the more important electron donor for nitrate reduction. By growth in the presence of nitrate, synthesis of a nitrate-specific cytochrome b is induced. This cytochrome may be distinguished from the cytochrome b component of aerobic cells by its spectral properties, and by its genetic control. At the temperature of liquid nitrogen, the absorption maximum of the a-band of the nitratespecific cytochrome b was at 555 nm, whereas aerobic cells exhibited two major peaks with absorption maxima at 555 and 562 nm. It could be concluded from the properties of mutants defective in

BIOCHEMISTRY A N D GENETICS O F NITRATE REDUCTASE

345

nitrate reduction that the cytochrome 6 5 5 5 found in cells grown anaerobically with nitrate is distinct from the cytochrome 65 j 5 found in similar cells grown aerobically. The cytochrome b-555, induced during anaerobic growth with nitrate, is undoubtedly identical with the cytochrome b l apoprotein which was found to be associated with purified nitrate reductase (Enogh and Lester, 1974; MacGregor, 197530). Ruiz-Herrera and DeMoss (1969) have concluded that the cytochrome b555 is composed of two distinct cytochromes which possess identical spectral characteristics. This conclusion was based on the observation that the re-oxidation of formate-reduced cytochrome b5 5 5 showed biphasic kinetics. According to the authors, this would point to the presence of two cytochrome b 5 5 5 components of which one is directly oxidized by nitrate and the other only after a definite time lag. In my opinion, however, this explanation of the experimental results is erroneous. Formate was used to reduce cytochrome bl and, when full reduction of the cytochrome 61 was obtained, nitrate was added. The amount of nitrate added was much larger than the amount of formate present; therefore the first phase of the observed biphasic kinetics of the oxidation of cytochrome b555 represents a steady state, in which there is an equilibrium between cytochrome bl reduction by formate and cytochrome bl oxidation by nitrate. As soon as all of the formate is used, the second phase of cytochrome b l oxidation by nitrate is observed. Consequently it is reasonable to conclude that only one cytochrome b555 component is present. It may be mentioned that this cytochrome can also be re-oxidized by oxygen. On the basis of these resuIts a scheme for the nitrate reductase complex of E. coli may be proposed (Fig. 2).

2. Klebsiella aerogenes In K. aerogenes, a cytochrome b 5 , is involved in nitrate respiration (Knook e t al., 1973; van 't Riet et al., 1972). However, in nitrate assimilation, cytochromes are not involved as intermediate electron carriers (van 't Riet e t al., 1972). Difference spectra (dithionite-reduced minus oxidized) at the temperature of liquid nitrogen indicated the presence of cytochromes b , , a,, d and 0. With these difference spectra, no evidence was obtained for the presence of more than one cytochrome b. During anaerobic growth in the

346

A.

H. STOUTHAMER Ascorbate

,

Methylene blue Phenazine methosulfate

Reduced methylviologen

Oxygen

FIG. 2. Suggested scheme for the nitrate reductase complex of Escherichiu coli Based on the results of Ruiz-Herrera and DeMoss (1969).

presence of nitrate, cytochrome b is formed in much larger amounts than during aerobic growth. The relative cytochrome contents, and oxidase activities, of membranes obtained from bacteria grown under various conditions are given in Table 12. The influence of the growth conditions on the oxidase activities is rather limited, but the influence on the cytochrome contents is very pronounced. The large difference in cytochrome b content between cells grown anaerobically with nitrate, and cells grown aerobically, has also been observed with P. mirabilis (de Groot and Stouthamer, 1970a), Haemophilus influenzae (Sinclair and White, 1970), E. coli (Cole and Wimpenny, 1968) and M. denitrificans (Lam and Nicholas, 1969b). Therefore this seems to be a general phenomenon. By studying the effects of nitrate on the reduction levels of NADH-reduced cytochromes 61, a l , and d , as determined by dual-wavelength spectrophotometry , it was concluded that, in K. aerogenes, cytochrome 61 is a carrier in the electron-transport system to nitrate, but that the other cytochromes are not (Knook et al., 1973). Difference spectra, obtained with ascorbate-dichlorophenol-indophenol as an electron donor, indicated the presence of a cytochrome b5 6 3 component, which was not oxidized by nitrate but was rapidly oxidized by oxygen (Knook et al., 1974). This cytochrome b 5 6 3 was not detected previously, since it is estimated to give only a small contribution to the absorbance in the 555 and 565 nm region (about 8% of the contribution by cytochrome b 5 , 9 ) . The oxidase of NADH, and NADH nitrate reductase, were both strongly

m

0 0 I rn

5

v1

-I

n

TABLE 12. Oxidase activities and cytochrome contents of membranes from Klebsiella aerogenes grown under different D conditions (data from Knook e t al., 1973) 2 Q

Growth conditions

Aerobic Anaerobic Anaerobic + nitrate

Oxidase activities with (n-atom &/mg protein min)

Nitrate reductase activities (nmole/mg proteinlmin)

Cytochrome contents

G)

rn 2

b

a1

d

NADH

succinate

lactate

formate

7 00 720

35 60

22 20

15 40

0 80

0.048 0.080

0.001 0.015

0.004 0.015

72 1

60

20

42

464

0.195

0.010

0.013

rn

1

G

% z_ -I n

3rn n

rn 0

C

0 -I

Rrn W

5

348

A. H. STOUTHAMER

inhibited by 2-n-heptyl-hydroxyquinoline-N-oxide (HQNO). Addition of HQNO only slightly affected the aerobic steady-state reduction of cytochrome b1 with NADH, but caused a significantly lower nitrate-reducing steady-state of this cytochrome. Extraction of ubiquinone-8, which is located on the dehydrogenase site of cytochrome 6, 9 , has similar effects on.the nitrate-reducing steady state of this cytochrome (Knook and Planta, 1973). These effects show that the point of inhibition by HQNO is situated at the electronaccepting side of cytochrome b J 59 . The Hill-coefficient of 1.O, obtained in a titration of the NADH-nitrate reductase activity with HQNO, points to the presence of only one HQNO inhibitory site. The lack of an effect of HQNO on the aerobic steady-state reduction of cytochrome b l , with NADH, is explained by the assumption that HQNO inhibits the electron transfer from NADH to oxygen both before and after cytochrome b 5 5 9 . Similarly Cox et al. (1970) assumed several sites of inhibition of the respiratory chain of E. coli by HQNO. Titration of the NADH oxidase activity with HQNO results in a Hill plot with a slope of h = 0.74, which is in accordance with the presence of more than one site of inhibition; the second site of inhibition is supposed to be located between cytochrome b 5 6 3 and cytochrome al. The complete scheme for the electron-transport system of K. aerogenes is shown in Fig. 3. In M . denitrificans (Lam and Nicholas, 1969b), and in B. stearothermophilus (Downey, 1966), nitrate respiration is, in contrast t o oxygen respiration, not Ascorbate DClP Formate-Fp

cyt 0

___f

0 2

Succinate+Fp reductase FIG. 3. Scheme for the electron-transport system of Klebsiellu aerogenes. After b o o k et al. (1974). Abbreviations: Fp = flavoprotein; CoQs = ubiquinone-8; DCIP = dichlorophenol indophenol; HQNO = 2-n-heptyl-4-hydroxyquinolineN-oxide. The sites of inhibition by HQNO are indicated by arrows.

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349

inhibited by HQNO. This may be explained by the absence of an inhibition site at the electron-accepting side of the b-type cytochrome in these bacteria. The influence on electron transport from NADH to nitrate and oxygen of extracting ubiquinone from membrane particles, has been studied by Knook and Planta (1971a, b, 1973). It was established that ubiquinone-8 was the sole quinone present in this bacterium (Knook and Planta, 1971a). Upon extraction of the membranebound ubiquinone-8 with pentane, both NADH oxidase activity and NADH-nitrate reductase activity are lost. Ultraviolet irradiation has the same effect. In pentane-extracted membrane preparations, the activities can be restored by incorporation of ubiquinone-6, -8 or -10, but not by incorporation of menaquinones. In membrane preparations irradiated with ultraviolet radiation, the modified quinone had to be first removed by extraction with pentane before restoration of the enzyme activities, by incorporation of ubiquinone, was possible. Whereas NADH oxidase can be restored to 90% of the original activity by incorporation of ubiquinone-8, only 25% of the original activity of NADH-nitrate reductase can be obtained by these procedures. Possibly ultraviolet irradiation also damages nitrate reductase itself.

3. Bacillus stearothermophilus and Bacillus licheniformis Bacillus stearothermophilus and B. licheniformis also contain c-type cytochromes (Downey, 1966; Schulp and Stouthamer, 1970), which are absent from E. coli and K. aerogenes. Upon addition of nitrate to malate-reduced cytochromes, only cytochrome 61 is re-oxidized, and cytochrome c I not. Thus nitrate reductase transfers electrons from cytochrome 61 to nitrate, and cytochr.ome C I is not involved in the transfer. Oxygen respiration is inhibited by HQNO, but not nitrate respiration, indicating that the site of inhibition of HQNO lies between cytochrome 61 and cytochrome c1 in this organism. Micrococcus denitrificans also contains c-type cytochromes. With this organism, it was also found that reduced cytochrome 61 is oxidized by nitrate but not by reduced cytochrome cl (Lam and Nicholas, 1969b; John and Whatley, 1970). This conclusion was in agreement with the observation that antimycin A

350

A. H. STOUTHAMER

inhibits the NADH oxidase activity much more strongly than the NADH-nitrate reductase activity (John and Whatley, 1970). In B. stearo thermophilus, a menaquinone is involved in oxygen- and nitrate respiration (Downey, 1962). A similar conclusion has been reached for Staph. aureus (Sasarman et al., 1974). 4. Propionate-Forming Micro-Organisms Cytochromes have been detected recently in a number of anaerobic or micro-aerophilic micro-organisms which form propionate as a fermentation product. For example, cytochromes have been found in Propionibacterium freudenreichii and Propionibacterium pentosaceum (de Vries et al., 1972; Sone, 1972), Anaerovibrio lipolytica, Selenomonas ruminantium and Veillonella alkalescens (de Vries et al., 1974). Also cytochromes are present in a number of succinate-forming micro-organisms such as bacteroides (White et al., 1962; Rizza et al., 1968), Vibrio succinogenes (Jacobs and Wolin, 1963), actinomycetes (Taptykova and Kalakoutskii, 1973) and Desulfovibris gigas (Hatchikian and Le Gall, 1972). Indeed, it must be concluded that cytochromes are present in many more anaerobic bacteria than was thought previously. In most of the bacteria listed, cytochrome b functions in electron transport to fumarate (“fumarate respiration”). Nitrate reduction occurs in V. alkalescens (InderIied and Delwiche, 1973; de Vries et al., 1974), S. ruminantium (de Vries et al., 1974). V. succinogenes (Jacobs and Wolin, 1963), actinomycetes (Slack, 1974) and P. pentosaceum (van Gent-Ruyters et al., 1975). With some of these organisms, the participation of cytochrome b in electron transport to nitrate has been clearly demonstrated, and in this connection, an interesting observation was that P. pentosaceum is able to denitrify (van Gent-Ruyters et al., 1975). It is evident that, in many cytochrome-containing bacteria which form succinate or propionate as a fermentation product, the ability to reduce nitrate is present. B. ENERGY CONSERVATION DURING NITRATE RESPIRATION

In Section 111-A (p. 336) the AGb of the reaction NADH + NO3 - + H + NAD’ + NO2 - + H,O was given (Table 10). This reaction can be accompanied by oxidative phosphorylation, since the AGb value for hydrolysis of ATP is -7.53 kcal/mol (Rosing and Slater, 1972). The +

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351

efficiency of oxidative phosphorylation during nitrate respiration may be studied in two ways: (i) by determination of P/Ze- ratios for oxidative phosphorylation coupled to nitrate respiration; and (ii) by measurement of molar growth yields.

1. Determination of the Efficiency of Oxidative Phosphorylation During Reduction of Nitrate by Cell-Free Extracts and Whole Bacteria In cell-free preparations generally, low P/O ratios are obtained (Harold, 1972) and it has been concluded that these are not representative of the P/O ratio extant in intact s o w i n g cells (Stouthamer, 1969a). The same applies t o the P/O ratios determined in intact, resting cells (van der Beek and Stouthamer, 1973). The above remarks, which apply to oxidative phosphorylation coupled to respiration with oxygen, also apply to P/2e- ratios for oxidative phosphorylation coupled to respiration with nitrate. Experimentally determined values of P/2e- ratios for oxidative phosphorylation coupled to nitrate respiration are given in Table 13. For all organisms studied, the P/2e- ratio for oxidative phosphorylation coupled to TABLE 13. Oxidative phosphorylation coupled to reduction of nitrate in cellfree extracts and in whole cells of different organisms Organism

Hydrogen donor

P/Ze-

Reference

Cell-free extracts Pseudomonas denitrificans

succinate

0.25

lactate

0.3

Pseudomonas aeruginosa

lactate

0.3

Escherichia coli

NADH

0.55

Yamanaka et al. (1?62) Ota e t al. (1964)

Micrococcus denitrificans

NADH succinate

0.9 0.06

John and Whatley (1970) van der Beek and Stouthamer (1973) J. van Hartingsveldt and A. H. Stouthamer (unpublished results)

Ohnishi (1963) Ohnishi and Mori (1960)

Whole cells Proteus mirabilis

endogenous

0.37

Pseudomonns aeruginosa

endogenous

0.50

352

A. H. STOUTHAMER

nitrate respiration was lower that the P/O ratio for the same organism; for example, with M . denitrificans the P/O ratio with NADH was about 1.5, and with succinate about 0.5 (John and Whatley, 1970). Similarly, for intact cells of P. mirabilis, a P/O ratio of about 1 was found (van der Beek and Stouthamer, 1973). These findings are in agreement with the data in Table 10, which indicate that the energy-yielding potential for oxygen is higher than that for nitrate. For the reasons given above, these P/2e- ratios d o not give any indication regarding the value of the P/2e- ratio in growing cells.

2. Influence of Nitrate Respiration on Molar Growth Yields The amount of bacterial growth is directly proportional to the amount of ATP which can be obtained from the catabolism of the energy-yielding substrate (Bauchop and Elsden, 1960). These authors introduced the term Y A T P , which represents the dry weight of organisms (in grams) produced per mole of ATP generated. Originally it was supposed that Y A T P is a general biological constant, with a value of 10.5 g per mole of ATP (Bauchop and Elsden, 1960). Later work has shown, however, that Y A T p is dependent on the growth rate, on the composition of the medium and on the nature of the energy-yielding substrate (Stouthamer and Bettenhaussen, 1973; Stouthamer, 1973, 1976). The influence of nitrate on molar growth yields has been studied in a number of organisms and some results are shown in Table 14. In some cases, the influence of oxygen is included to facilitate comparison. With all the organisms that have been studied, the molar growth yield was found to be greatly increased by the presence of nitrate, but remained lower than the values obtained with oxygen. With the enterobacteria, the large increase in molar growth yield in the presence of nitrate is due to two effects: (i) occurrence of oxidative phosphorylation; and (ii) formation of more acetate. During anaerobic growth, glucose in these organisms is fermented according to the equation: 1 glucose + 1 acetate + 1 ethanol + 2 formate. In the presence of nitrate, ethanol is not formed by reduction of acetyl-CoA with NADH. Accordingly, the net ATP production by substrate phosphorylation is increased from three to four moles of ATP per mole of glucose, during anaerobic glucose fermentation and during glucose fermentation in

TABLE 14. Influence of nitrate on molar growth yields of various micro-organisms Organism Klebsiella aerogenesa

___-

Hydrogen acceptor

Glucose

none nitrate oxygen none nitrate oxygen

26.1 45.5 72.7 21.8 50.6 95.5

Hadjipetrou and Stouthamer (1965); Hadjipetrou e t al. (1964)

Mannitol

Molar growth yield (glmole)

ReEerence

Energy source

Proteus mirabilisa

Glucose

none nitrate oxygen

14.0 30.1 58.1

Stouthamer and Bettenhaussen (1972)

Citrobacter ~ p . ~

Glucose

none nitrate oxygen

45.0 65.8 96.1

Kapralek (1972)

Lactate

none nitrate none nitrate

b

Propionibacterium pentosaceum

Glycerol Pseudomonas aeruginosa

Clostridium perfringens

minimal sdts medium complex medium for explanation, see text

Succinate

Glucose

12.9 22.0-52' 26.3 62-8 1'

van Gent-Ruyters et al. (1975)

nitrate oxygen

22.5 34.0

J. van Hartingsvelt and

none nitrate

38.3 45.0

Hasan and Hall (1975)

A. H. Stouthamer (1974 and unpublished observations)

W

ul W

354

A. H . STOUTHAMER

the presence of nitrate, respectively. The molar growth yield in these organisms is much lower for anaerobic growth with nitrate than for aerobic growth. This is due to the absence of an active citric acid cycle during anaerobic growth in the presence of nitrate. Thus, nitrite, produced by reduction of nitrate, has been shown to inactivate aconitase and fumarase (Wi-mpennyand Cole, 1967; Forget and Pichinoty, 1967; Wimpenny and Warmsley, 1968). Comparison of the molar growth yields for glucose and mannitol, with K. aerogenes, gives an interesting result. Mannitol is more reduced than glucose and, during the breakdown of mannitol to pyruvate by the glycolytic pathway, three moles of NADH are produced, whereas with glucose only two moles are produced. In the absence of a hydrogen acceptor, the molar growth yield for mannitol is lower than for glucose. The net ATP production from mannitol is 2.5 moles/mol mannitol (fermentation balance: 1 mannitol+ 1.5 ethanol + 0.5 acetate + 2 formate). In the presence of hydrogen acceptors, however, the molar growth yield for mannitol is higher than that for glucose because, with mannitoI, more NADH is available for reduction of the hydrogen acceptor. These data clearly show that, in growing K. aerogenes organisms, NADH is an important hydrogen donor for nitrate reduction (compare Section IV-A-1, p. 344). From the results in Table 14 the P/2e- ratios were calculated, based on the following equation: Total ATP production =

yglu = 2 (part of the glucose dissimilated) YATP

+ (acetate production/mole glucose) + (amount of hydrogen acceptor used/mole of glucose) x P/2e-. In this equation, a Y A T P value was used which was found for cultures growing anaerobically without an external hydrogen acceptor. In the presence of nitrate, however, the specific growth rate was much higher than in its absence (Table 15). Since the YA T p value is dependent on the specific growth rate (Stouthamer and Bettenhaussen, 1973), it is clear that in previous papers the P/2eratio has been over-estimated. On the basis of new results, a P/2eratio of 1.8- 1.9 has been calculated for oxidative phosphorylation coupled to nitrate respiration in K . aerogenes (Stouthamer and Bettenhaussen, 1973). With P. pentosaceum, several values for the

BIOCHEMISTRY A N D GENETICS OF NITRATE REDUCTASE

355

molar growth yields are given (Table 14). The growth curve obtained with anaerobic cultures growing in the presence of nitrate could be differentiated into several phases (van Gent-Ruyters et al., 1975). In the first period, a Iarge part of the Iactate was converted into pyruvate, which was subsequently utilized. It could be shown that the citric-acid cycle functioned under anaerobic conditions in the presence of nitrate. The molar growth yields indicated a P/2e-ratio of two for electron transfer from NADH to nitrate and of one for electron transfer from lactate to nitrate. TABLE 1 5 . Influence of the presence of nitrate on the specific growth rate of Proteus mirabilis and of Citrobacter sp. growing anaerobically Organism

Hydrogen acceptor

Specific growth rate (h-' )

Reference

Proteus mirabilis

none nitrate oxygen

0.17 0.35 0.69

Stouthamer and Bettenhaussen (1972)

Citro bacter sp.

none nitrate oxygen

0.5 1 0.75 0.99

Kapralek (1972)

With Clostridium perfringens cultures, the presence of nitrate in the medium also caused an increase in the molar growth yield (Hasan and Hall, 1975). This is due completely to an increase in the proportion of fermentation products which can participate in substrate-level phosphorylation. During growth in the presence of nitrate, more acetate and less ethanol and butyrate are formed from glucose than during growth in its absence (Ishimoto et al., 1974; Hasan and Hall, 1975). Chiba and Ishimoto (1973) have shown that reduced ferredoxin is the direct hydrogen donor for nitrate reduction in this organism, which explains the absence of oxidative phosphorylation during nitrate respiration in this organism. From the results in Table 14 it is clear that, with all the organisms studied (with the exception of C1. perfiingens), oxidative phosphorylation occurs during nitrate respiration. But the real efficiency of oxidative phosphorylation coupled to nitrate respiration is generally not known with certainty.

356

A. H. STOUTHAMER

V. Genetics of Nitrate Reductase Formation A. METHODS FOR THE ISOLATION OF MUTANTS BLOCKED IN NITRATE RESPIRATION

Several methods have been .used for the isolation of mutants unable to effect the reduction of nitrate. These methods are as follows: (a) Selection for resistance against chlorate. When several bacterial species are incubated with perchlorate, they lose the ability to reduce nitrate (Hackenthal et al., 1964). Later work has shown that better results are obtained with chlorate (F'ikchaud et al., 1967; Stouthamer, 1967b). Perchlorate and chlorate are reduced by nitrate reductase A (see Section II-A, p. 316) and are then converted to the toxic chlorite which kills the nitrate-reducing bacteria (Pichinoty et al., 1969a; Newman e t al., 1973). By incubating nitrate-reducing bacteria with chlorate, mutants are therefore selected which have lost the ability to reduce nitrate. The gene symbol chl is used for these mutants. (b) Penicillin enrichment of mutants blocked in nitrate respiration or assimilation. It has been shown by Stouthamer (1967b) that mutants of I(. aerogenes blocked in aerobic nitrate assimilation can be enriched by penicillin treatment. Some of the mutants isolated were also deficient in nitrate respiration. Mutants of E. coli blocked in the ability to grow anaerobically on a medium with lactate as carbon and energy source, and nitrate as hydrogen acceptor, were enriched by Venables and Guest (1968). Mutants of Ps. aerup'nosa blocked in the ability to grow anaerobically on minimal medium with glucose as carbon- and energy source, and nitrate as hydrogen acceptor, were enriched by ampicillin treatment by van Hartingsveldt et al. (1971). For mutants isolated in this way, the gene symbol n a y is mostly used. (c) By the above procedures, mutants are isolated which have completely, or nearly completely, lost the ability to synthesize nitrate reductase. Even when only small amounts of enzyme are left in the mutants, they would be counter-selected by incubating with chlorate, or by penicillin enrichment. Consequently these methods are not suitable for the isolation of mutants which form decreased amounts of nitrate reductase. Methods for the isolation of such

BIOCHEMISTRY A N D GENETICS OF N I T R A T E REDUCTASE

357

mutants have been developed by Ruiz-Herrera et al. (1969) and Glaser and DeMoss (1972). I n this method, mutagen-treated cells were plated on nutrient agar. When the colonies had developed, an imprint of them was made on papers which had previously been dampened with a solution of nitrate and formate, and dried. The papers were then incubated and sprayed with nitrite reagent. Colonies able to reduce nitrate appeared as deep red spots, whereas those that could not were colourless or slightly yellow in appearence. By using this isolation method, new mutant classes could be recovered. B. GENETIC MAPPING OF MUTATIONS AFFECTING NITRATE REDUCTASE FORMATION

Genetic mapping of mutations affecting nitrate reductase formation has been performed using the normal methods for transfer of genetic material, that is, conjugation, transduction and transformation. With all organisms that have been studied, a number of genes have been found t o affect nitrate reductase formation. The discussion here well be limited t o the organisms which have been studied in most detail, and inevitably E. coli heads the list. The latest version of the linkage map of E. coli has been given by Taylor and Trotter (1972). The genetic map of this organism then contained 460 gene loci, and the circular linkage map bears a time scale from 0 t o 90 minutes, which is the time needed for the transfer of the complete chromosome by conjugation between a donor and an acceptor strain. Thus, the location of each genetic marker may be given in minutes. The map position of chl genes and the gene-order in some relevant chromosome segments are given in Table 16. The map position of the chl genes was mainly determined by conjugation experiments, but the more precise location was determined by measuring cotransduction frequencies. Up to the present time, seven chl genes have been mapped in E. coli. However a fine-structure analysis of the chl A and chf E loci suggested that both loci may be functionally divisible into two conti'guous genes (Venables, 1972), and the chl B locus was shown t o comprise at least three complementation groups. It is thus evident that the actual number of chl genes in E. coli is greater than seven, and this view is strengthened by the observation that, in other Enterobacteriaceae which have chromosome maps homologous to that of E. coli, other chl genes have been

w

m 03

TABLE 16. Map position a n d gene order of chl genes i n Escherichia coli Gene symbol

chl A chl B chl C chl D chl E chl F chl G Segment

1 2 3

References

Map position (minutes)

18' 773 26' 17' 18'

28' 0

Venables and Guest (1968); Adhya et al. (1968) Puig and Azoulay (1967); Casse (1970) Puig and Azoulay (1967); Guest (1969); Ruiz-Herrera e t al. (1969) Adhya e t al. (1968); Venables and Guest (1968) Venables and Guest (1968) Glaser and DeMoss (1972) Glaser and DeMoss (1972) Gene order

nad A aro G gal chl D att h bio uur B chl A chl E hem A chl C att 1p80ton B trp cys B chl F glp K rha chl B met E ilv

Explanation of gene symbols (Taylor and Trotter, 1972). nad, bio, trp, cys, met, ilv requirement for nicotinic acid, biotin, tryphophan, cysteine, methionine and isoleucine + valine, respectively gal, rha fermentation of galactose and rhamnose, respectively att A, att lp80 attachment sites for the phages X and 980,respectively ton B resistance against bacteriophage TI aro G 2-keto-3-deoxy-7-phospho-heptonate synthetase glP K glycerolkinase hem A deficiency in haemine biosynthesis

? I v)

-I

0 C

-I

s

5

1)

BIOCHEMISTRY A N D GENETICS OF N I T R A T E REOUCTASE

359

identified. The genetics of nitrate reductase formation are thus very complex, which undoubtedly reflects the complexity of the nitrate reductase system itself. The genetics of nitrate reduction in Salmonella typhimurium have been studied by Stouthamer (1969b), Stouthamer and Bettenhaussen (1970), Casse et al. (1972, 1973) and Enomoto (1972). The genetic maps of E. coli and S. typhimurium show a very close homology (Taylor and Trotter, 1972; Sanderson, 1972), and the genetics of nitrate reduction in S. typhimurium, in particular, is interesting since the effects of mutation to chlorate resistance are more severe (see Section V-C, p. 360). In general, the same genes are found as in E. coli and at the same position on the linkage map. In the chl A region, three genes (designated chl A, chl E and chl F) were identified by complementation tests (Stouthamer, 1969b). Also in Citrobacter freundii three chl genes were identified at this position of the chromosome map (de Graaff et al., 1973). In S. typhimurium, a chl G gene has been identified in the 79 t o 82 minutes region of the genetic map (Stouthamer, 196913; Stouthamer and Bettenhaussen, 1970),which is equivalent t o the 52 minutes region of the E. coli linkage map. Consequently this is a chl gene which has not been detected so far in E. coli. The chl G gene o f S. typhimurium is present in a segment with a gene order nic By chl G , thi B, in1 By which is deleted in some chlorate-resistant mutants. A similar segment was found t o be deleted in some chlorate-resistant mutants of K. aerogenes (Stouthamer and Pieterman, 1970). In C. freundii, a chl gene was found at a similar position (de Graaff et al., 1973), and since this mutation could be complemented by an F' plasmid from E . coli, we may conclude that this chl gene is also present in E. coli. The extensive homology between the various Enterobacteriaceae is further illustrated by the observation that most chl genes in S. typhimurium and C. freundii can be complemented with F' plasmids from E. coli (Stouthamer, 1969b; de Graaff et al., 1973). We may conclude that, in these organisms, at least 1 2 genes are involved in the formation of nitrate reductase. The genetics of nitrate reductase formation have also been studied in Ps. aeruginosa and B. licheniformis. In Ps. aeruginosa five nar genes have been identified. Their map position is: nar A 6 5 l ; nar B 4 4 l ; nar C 45' ; nar D 65' and nar E 9' of the Ps. aeruginosa linkage map (van Hartingsveldt and Stouthamer, 1973). In B. licheniformis 13 chl mutation have been identified by reciprocal transformation crosses

360

A. H. STOUTHAMER

(Schulp and Stouthamer, 1972). It is thus clear that generally, in bacteria, many genes are involved in nitrate reductase formation. C. PHYSIOLOGICAL PROPERTIES OF CHLORATE-RESISTANT MUTANTS

Mutations to chlorate resistance generally have a pleiotropic effect. With E. cob, the chl genes affect the formation of both nitrate reductase and formate dehydrogenase (Pikchaud et al., 1967; Venables e t al., 1968; Ruiz-Herrera et al., 1969; Guest, 1969). In most chl mutants, these enzymes are completely absent and consequently they do not form gas from glucose. In chl C mutants, varying amounts of formate dehydrogenase are formed and also the extent of gas production varied (Guest, 1969; Ruiz-Herrera et al., 1969). Other effects of some chl mutations in E. coli are, for example, the formation of increased amounts of the soluble cytochrome c 5 5 2 (Venables et al., 1968), and the formation of decreased amounts of cytochrome b (Guest, 1969; Ruiz Herrera e t al., 1969) and the hydrogenase (Guest, 1969). The proteins whose syntheses are affected are, with the exception of the cytochrome c 5 5 2 , all membrane-bound enzymes. With other members of the Enterobacteriaceae, the pleiotropic effects of chl mutations are even more severe. The enzymes affected by chl mutations in various organisms are listed in Table 17. With P. rnirabilis, the largest number of enzymes is affected. Within the Enterobactericeae, all of the enzymes affected are involved in anaerobic metabolism. In B. licheniformus, however, two unrelated functions are affected; furthermore, in a number of chl mutants of this organism, anaerobic growth is extremely slow. This is also in contrast to the properties of the chl mutants in Enterobactenaceae where chl mutations have no effects on the growth rate. It has been shown with P. rnirabilis that the pleiotropic effect of chl mutations depends on the cultivation procedure. A comparison of the specific activities of formate dehydrogenase and of hydrogenase, in two phenotypic classes of chlorate-resistant mutants, is shown in Table 18. When a special method is used to obtain membrane preparations, which consists of growth at controlled pH values and conversion of the cells into sphaeroplasts by penicillin treatment and osmotic shock (Oltmann and Stouthamer, 1973), formate dehydrogenase and hydrogenase are found in chl I and chl 11

TABLE 17. Enzymes present in various micro-organisms that are affected by chl mutations Escherichia coli

nitrate reductase, formate dehydrogenase

see text (p. 360)

Salmonella typhimurium

nitrate reductase, formate dehydrogenase, chlorate reductase, tetrathionate reductase, thiosulpiiate reductase

Stouthamer (196913)

Proteus mirabilis

de Groot and nitrate reductase, formate dehydrogenase, chlorate reductase, tetrathionate reductase, thiosulphate reductase, fumarate reductase, Stouthamer (1 969); L. F. Oltmann and and hydrogenase A. H. Stouthamer (unpublished results)

Hafnia sp.

nitrate reductase A and B, tetrathionate reductase, formate dehydrogenase

Chippaux and Pichinoty (1968)

Bacillus lichen iform is

nitrate reductase and penicillinase

Schulp (1972)

TABLE 18. Influence of cultivation method on enzyme activities in chlorate-resistant mutants of Proteus mirabilis

-

Method Normal growth and sonication pH value-controlled growth and osmotic shock of penicillininduced sphaeroplasts

Organism

Formate dehy drogenase

Hydrogenase

Reference

de Groot and Stouthamer (1969)

wild type chl I chl I1

28.0 0.1 34.0

22.0 11.8

wild type chl I chl I1

131.4 241.8 346.2

167.4 51.0 61.8

--

0.1

L. F. Oltmann and A. H. Stouthamer (1973 and unpublished results)

D

2 0

362

A. H. STOUTHAMER

mutants. In the normal preparation method, formate dehydrogenase is not detected in extracts of chl I mutants, and hydrogenase not in extracts of chl I1 mutants. The influence of the chl mutation on the formation of formate dehydrogenase, and of hydrogenase, in P. mirabilis is thus a secondary effect. The reason for this effect of the cultivation method is not known, and i t is also not known whether the same effects can be found in other members of the Enterobacteriaceae. These observation are very important, however, for the interpretation of the pleiotropic effect of chl mutations. D. PROTEIN COMPOSITION OF MEMBRANES OF CHLORATE-RESISTANT MUTANTS

An attempt to identify the gene products of the various chl genes in E. coli has been performed by MacGregor and Schnaitman (1971), Rolfe and Onodera (1972) and MacGregor ( 1 9 7 5 ~ )These . authors have compared the protein profiles of cytoplasmic membranes of chl mutants with those of wild-type E. coli. For this purpose, solubilized proteins from membranes were subjected to sodium dodecylsulphate treatment and polyacrylamide gel electrophoresis. About 1 5 protein bands could be distinguished in the gels of the wild type, one of which could be identified as the largest subunit o f the nitrate reductase molecule (MacGregor and Schnaitman, 1971). Changes in four protein bands were observed in the profiles of the membranes of the chl mutants. It was an astonishing observation that the largest subunit of the nitrate reductase molecule could also be detected in membranes of chl A and chl B mutants. In membranes of chl C, chl D and chl E mutants, this protein was absent, however, and these mutants also lacked a second protein. In chl A mutants two other proteins were found to be lacking. Rolfe and Onodera (1972) found changes in five protein components in the membranes of cht A and chl D mutants. In this study it was observed that the protein profile of membranes of aerobically-grown E. coli chl A also differed from a similar preparation from the wild type. A great improvement in these studies was achieved by MacGregor ( 1 9 7 5 ~ ) ;nitrate reductase antisemm was added to Triton-solubilized membrane proteins of wild-type E. coli and chl A, chl By chl C, and chl E mutants. The precipitated protein was then subjected to sodium dodecylsulphate treatment and polyacrylamide gel electrophoresis. The membranes of chl A and chl B

BIOCHEMISTRY A N D GENETICS OF NITRATE REDUCTASE

363

mutants contained normal amounts of nitrate reductase, with all three subunits present. Thus, the nitrate reductase in these mutants is thought to be inactive due to the lack of the molybdenum-containing cofactor which is essential for nitrate reductase activity (see Section 11-C, p. 325). In membranes of chl C mutants, much less antibodyprecipitable protein was present. When the antibody-precipitated protein was analysed on gels, this protein was found to consist of peptides ranging in size from intact largest subunit t o very small peptides. This can be explained by assuming that this mutant makes a defective enzyme which is cleaved by proteolysis. Consistent with an earlier suggestion (Guest, 1969), it is postulated that the chl C gene is the structural gene for the largest subunit of the nitrate reductase molecule. Chl E mutants contained either diminished amounts of antibody-precipitable material, or none at all. In all chl E mutants, the subunit of molecular weight 1.95 x lo4 daltons of the nitrate reductase molecule was completely absent. It was therefore tentatively proposed that the chl E gene is the structural gene for the cytochrome b apoprotein. In the chl E mutants that made immuneprecipitable protein, the polypeptides were fragmented by proteolysis, as was also observed with chl C. In the hem A mutant, proteolysis of the subunits of the nitrate reductase molecule was also observed after growth without 6-aminolaevulinic acid (Section 11-B; p. 324). It therefore seems that defective nitrate reductase molecules are very susceptible to proteolysis by a membrane-bound protease. A comparison of the protein profiles of the membranes of chl mutants of B. lichenifomis with those of the wild-type organism has been performed by Schulp and Stouthamer (1972). In the wild type, 11 membrane protein bands were observed and membrane protein patterns of the chl mutants showed the greatest difference from those of the wild type after anaerobic cultivation. Differences included the appearance of a new protein band, disappearance of others, and decreases, doubling and increases in yet other protein bands. For instance, in the protein profile of membranes of a chl K mutant, a difference in four protein bands is observed as compared with the protein profile of the wild-type membrane. Also after aerobic cultivation there are differences between the protein profiles of wild type and chl mutants. It was supposed that the explanation for all these changes may be a wholesale disorganization of protoplasmic membrane biosynthesis.

364

A. H STOUTHAMER

In general it can be concluded than an analysis of the membrane proteins of wild-type and chl mutants has not yielded the expected identification of the gene products of the chl genes. It has led only to a tentative identification of the gene products of the chl C and chl E genes of E . coli, and more evidence in support of this identification is urgently needed. However, the most important results from the analysis of membrane composition of chl mutants was the finding that most mutants still synthesize several components of the nitrate reductase molecule. E. IN VITRO COMPLEMENTATION BETWEEN CHLORATE-RESISTANT MIJTANTS

When high-speed supernantant fluids from cell-free extracts of chl A and chl B mutants of E. coli, grown anaerobically with nitrate, are mixed and incubated under specific conditions, particulate nitrate reductase activity is formed (Azoulay and Puig, 1968; Azoulay et al., 1969). The restoration of nitrate reductase activity, by complementation, occurs only in the absence of oxygen in a narrow range of temperature and pH value (32", pH 7-7.6), and at certain protein concentrations. After two hours of incubation, the increase of nitrate reductase activity is complete. The reconstituted enzymic activity is reported t o be about 10% of the wild-type activity. In the course of complementation, 15- 20% of the total soluble protein becomes particulate. The reconstituted enzyme activity was shown to be present in particles by density gradient centrifugation (Azoulay et al., 1969; Azoulay et al., 1972). The reconstituted particles were shown to be heterogeneous, and to comprise material with densities of 1.10-1.12, 1.18 and 1.22. Only in the two lightest particle fractions was reconstituted nitrate reductase activity shown t o be present. The morphology of the particles formed during the complementation between supernatant extracts of chl A and chl B mutants has also been examined by electron microscopy (Mutaftschiev and Azoulay, 1973). The reconstituted particles were highly heterogeneous and comprised well-organized closed vesicles, filaments and aggregates. Therefore the results of the morphological study are in agreement with the results obtained upon centrifugation of the reconstituted particles on sucrose gradients. The reconstituted particles containing nitrate reductase were also shown to have incorporated cytochrome 6 1 . And ATPase, which

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is present in a soluble state in the cell-free extracts, can be incorporated into the newly formed particles (Giordano et ul., 1973). The ATPase was only present in reconstituted particles with a density of 1.18 (Giordano et al., 1975), which approximate closely in buoyant density, composition and morphology t o the native membrane vesicles. The process of reconstitution has also been studied in detail by MacGregor and Schnaitman (1973) who also obtained reconstitution of nitrate reductase activity when high-speed supernatants of cell-free extracts of chl A and chl B mutants, grown with nitrate under aerobic conditions, were mixed. Furthermore they obtained reconstituted nitrate reductase activity when a high-speed supernatant of a cell-free extract of a cht B mutant was mixed with a similar preparation from either a chl A, chl C or chl E mutant. No reconstitution could be obtained, however, with mixtures of the high-speed supernatants of the cell-free extracts of the latter chl mutants. Thus the chl B gene product seems t o be essential in order to obtain reconstituted activity. Earlier, MacGregor and Schnaitman (1972) showed that, in the cytopIasmic fraction of chl B mutants, a factor is present (called “Neurosporu complementation factor”), which can restore NADPH-nitrate reductase activity t o an extract of Neurosporu crassa nit 1 mutant. This mutant produces a defective enzyme, which can be re-activated in various ways (see Section 11-C; p. 328). The restoring activity was found only in preparations from chl B mutants and not in preparations from chl A, C, D or E mutants. The Neurospora complementation factor is most probably a molybdenum-containing cofactor which is bound t o a protein. MacGregor and Schnaitman (1973) have studied the protein composition of reconstituted particles from C-labelled cell-free extracts of chl A mutant and 3H-labe11ed supernatant proteins from a chl B mutant. In this way, they could show that the membrane proteins that were present in the particles were contributed by both mutants. Furthermore, the proteins present in the particles were shown to be the same as the proteins in the cytoplasmic membrane fraction of the two mutants. The membrane proteins in the cytoplasmic fraction, which can be incorporated into the reconstituted particles, are not considered to be newly formed precursors of the cytoplasmic membrane, but are believed to be formed from preexisting cytoplasmic membrane during cell breakage.

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In similar experiments Azoulay et al. (1974) studied the incorporation of phospholipids into the newly formed particles. It was shown that, in supernatants of cell-free extracts of chl mutants, obtained by high-speed centrifugation, 1 5 to 20 times more phospholipids (mostly phosphatidylethanolamine and cardiolipin) were present than in similar supernatants .from wild-type E. coli. A large part of these phospholipids was shown by chromatography to be present as strongly aggregated lipid-protein complexes. Also, in this case, the lipid-protein complexes are thought to be formed during cell breakage. After complementation of the soluble extracts of chl A and chl B mutants, and sedimentation of the newly formed particles by ultracentrifugation, the combined supernatants contain the same amount of phospholipids as did the wild-type supernatant. The phospholipids present in the newly formed particles were contributed by both mutants. The reconstituted particles of various buoyant densities were shown to be different with respect to the ratio of protein to lipid and with respect t o lipid composition (Azoulay et al., 1975). As mentioned previously, the component contributed by the “high-speed supernatant” of a cell-free extract of the chl B mutant, to the reconstituted nitrate reductase activity, is supposed to be a molybdenum-containing cofactor linked to a protein. The high-speed supernatants of cell-free extracts of the other chl mutants must contribute the chl B gene product for the complementation. Recently, the product of the chl B gene has been purified by chromatography on DEAE and sephadex G75 (Rivitre et al., 1975). The purified protein gave a single band when treated with sodium dodecylsulphate and separated by polyacrylamide gel electrophoresis. The molecular weight was estimated to be 3.5 x lo4 daltons. Restoration of nitrate reductase activity, by complementation, can be achieved by addition of the purified protein to a “high-speed supernatant” of a cell-free extract of chl B mutant. During complementation, the purified protein is only incorporated into the particles with buoyant density of 1.18. Therefore the reconstituted particles with a density of 1.10-1.12, which have the highest nitrate reductase specific activity, do not contain the chl B gene product. The chl B gene product can be released from either native or reconstituted membranes in various ways without affecting the nitrate reductase activity. This indicates that the chl B gene

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product is only necessary for assembly of an active membrane-bound nitrate reductase complex, but has no role in the complex itself. The chl B gene product was present in membranes of wild type E. coli, whatever the growth conditions (that is, aerobiosis or anaerobiosis, and in the presence or absence of nitrate).

VI. Concluding Remarks and Future Prospects From the results discussed in this review it becomes clear that nitrate reductase is a non-haem iron protein, containing molybdenum. Furthermore, it is clear that several genes are involved in the formation and the attachment of the molybdenum-containing cofactor to the nitrate reductase apoprotein. The reconstitution of active nitrate reductase (by incubation of the apoprotein of nitrate reductase from the nit-1 mutant of N . crassa with molybdenumcontaining cofactor, obtained by acid extraction of other molybdoproteins) seems promising for the identification of the nature of this cofactor. This is not only of importance for our understanding of the role of molybdenum in the nitrate reductase reaction, but also for our understanding of molybdoproteins in general. No molybdenumcontaining cofactor has yet been identified with any molybdoprotein. It is evident that a definitive conclusion on the role of molybdenum in nitrate reductase cannot yet be given since the electron paramagnetic resonance studies cannot be interpreted unequivocally. In this connection, it may be mentioned that in a recent review on nitrogenase it has been concluded that “nothing is known on the role of molybdenum” (Zumft and Mortenson, 1975). Another approach that may be of importance for the elucidation of the role of molybdenum in nitrate reductase is the study of co-ordination complexes of molybdenum, as models. Such studies have been performed by Stiefel (1973), Garner et a / . (1974) and Garner et al. (1975). Clearly, a great effort will now be given by workers in various fields to elucidate the role of molybdenum in molybdoproteins. Mutations t o chlorate resistance have been shown to have a pleiotropic effect. Such mutations can occur in 113 to 15 different genes. It has been shown that membranes of chl A and chl B mutants contain the nitrate reductase protein, and that all three subunits are present (MacGregor, 1 9 7 5 ~ ) . Combination of the results of

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MacGregor and Schnaitman (1972), MacGregor ( 1 9 7 5 ~ and ) Riviere et al. (1975) leads to the following conclusions. The nitrate reductase protein in chl A and chl B mutants is inactive due to the lack of the molybdenum-containing cofactor, which is essential for nitrate reductase activity. The chl B gene product is involved in the attachment of the molybdenum-containing cofactor to the membrane-bound nitrate reductase apoprotein. The chl A (and chl D) gene product(s) are involved in the formation of this cofactor. The observation of Enogh and Lester (1975) that formate dehydrogenase is also a molybdoprotein is of great importance for the interpretation of the pleiotropic character of chl mutants. If the molybdenum-containing cofactor in formate dehydrogenase is the same as that in nitrate reductase, it is evident that chl A, chl B and chl D mutants cannot form both enzymes. In a number of chl mutants, the formation of tetrathionate reductase, hydrogenase and chlorate reductase is also affected, and it is tempting to speculate that these enzymes are also molybdoproteins. But the verification of this hypothesis must await the purification and characterization of these enzymes. It does not seem, however, that all chl genes are involved in the formation of the molybdenum-containing cofactor, or in the formation of the various subunits of the nitrate reductase molecule. Therefore another explanation must be sought for the pleiotropic character in these mutants. The original hypothesis invoked to explain the pleiotropic effect in these chl mutations may be the best at this moment. It was proposed that, in chl mutants, the structure of the membrane is altered in such a way that the nitrate reductase complex cannot be incorporated in an active state (Azoulay et al., 1967; Stouthamer, 1967a). Alternatively, the mutants might be blocked in the assembly of the components of the nitrate reductase complex. In this respect, it is important to recall that MacGregor ( 1 9 7 5 ~ has ) shown that the inactive nitrate reductase molecule, present in membranes o f chl A and chl B mutants, is very susceptible to proteoIysis. We may suggest that, due to a change in one of the proteins involved in anaerobic metabolism, either the incorporation of the other proteins in the membrane is affected, or that the whole complex becomes susceptible to proteolysis. These conclusions are re-inforced by the observations of L. F. Oltmann and A. H. Stouthamer (unpublished results) that the absence of formate dehydrogenase and hydrogenase

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from membranes of some chl-mutants of P. mirubilis is a secondary effect; these enzymes are present when controlled methods are used for the cultivation of the organisms and the preparation of cell-free extracts. Nitrate reductase has been shown to be an enzyme composed of various subunits. The enzyme can be isolated in different forms, depending on the method which was used for its solubilization; thus, it may be isolated with a different number of units and as a monomer, dimer or tetramer. The occurrence of various subunits seems to be a general property of membrane proteins, and the same has been found for formate dehydrogenase (Enogh and Lester, 1975), tetrathionate reductase (Oltmann and Stouthamer, 1973) and ATPase (see review of Salton, 1974). When the subunit structure of more membrane proteins has been established, the complex protein profiles seen in sodium dodecylsulphate polyacrylamide gel electrophoresis will become interpretable in terms of specific enzyme subunits. This will enable a better characterization to be made of the changes observed in the membrane protein patterns of chi mutants. An important point, which still has to be resolved, is the location of nitrate reductase which might be located at either the outer or inner surface of the cytoplasmic membrane. This question will, most probably, be answered in the near future by a combination of electron microscopy and immunological methods. The reconstitution of active nitrate reductase, by mixing extracts of chl A and chl B mutants of E. coli, is very interesting. It is evident that the studies on the incorporation of proteins and lipids into the reconstituted particles may have a great similarity to the normal process which membrane-bound enzymes undergo during biosynthesis and assembly in vivo. Therefore this system holds great promise for our future understanding of membrane assembly.

VII. Acknowledgements The author is highly indebted t o his colleagues, E. A. Azoulay,

C. H. MacGregor, R. L. Lester and J. van 't Riet, for making available manuscripts prior to publication. Stimulating discussions with Dr. L. F. Oltmann and Dr. J. van 't Riet on a provisional draft of this review are gratefully acknowledged.

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1.

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

A

B

Ackrell, A. C., 21, 31, 88 Ackrell, B. A., 74,88 Acrell, B. A. C., 30, 84, 292,298, 299, 305,308,309 Adelberg, E. A., 29,84 Adhya, S., 358,370 Aiba, S., 292, 294, 299, 300,312 Akazawa, T., 6 3 , 8 4 Akiba, T., 136, 153 Albright, L. J., 185, 212, 216, 225, 234,236,238 Aleem, M. I. H., 77, 78, 8 0 , 8 8 Allen, M. B., 176, 234 Amelunxen, R. E., 1 7 6 , 2 3 5 Ames, G., 99,153 Amesz, J., 278, 311 Anderson, L. E., 6 3 , 8 4 Anderson, R. J., 8 1 , 8 4 Andreesen, J. R., 326,370 Ankel, H., 150, 151, 156, 1 5 7 Antalis, C., 106, 107, 108, 111, 113, 115,117,127,153,157 Archibald, A, R., 129, 153 Arima, K., 292, 293, 309 Arnold, R. M., 225,234 Arnold, W. N., 95, 116, 153 Arnon, D. I., 57, 61, 8 5 , 8 6 Arnst, H. N., Jr. 327,370 Asao, K., 1 0 1 , 1 5 6 Asato, R. N., 74, 7 6 , 8 8 Ashwell, G., 121, 1 5 4 Aston, P. R., 3 2 , 8 8 Atkinson, B., 291,309 Atkinson,D. E., 14, 88, 264, 265, 267, 268,269,309,310 Avi-Dor, Y., 290, 308,310 Azoulay, E., 356, 358, 360, 364, 365, 366, 368,370, 3 7 1 , 3 7 3 , 3 7 4

Babcock, G. E., 1 3 1 , 1 5 7 Babczinski, P., 144, 151,154, 1 5 7 Bachi, B., 267,310 Baddiley, J., 129, 153 Bahl, 0. P., 9 6 , 1 5 4 Baierlein, R., 203, 208, 236 Balbinder, E., 208, 235 Ballou, C. E., 94, 95, 96, 97, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 110, 111, 112, 113, 115, 116, 117, 121, 122, 123, 124, 126, 127, 128, 129, 132, 133, 134, 137, 138, 141, 144, 147, 148, 153, 153, 154, 155, 1 5 6 , 1 57 , 1 5 8 Baltscheffsky, H., 278, 310 Bangham, A. D., 211,236 Bannister, J. V., 43, 8 4 Bannister, W. H., 43, 8 4 Barabas, I., 28, 2 9 , 8 8 Barendsen, J. W.,359,371 Barker, H. A., 335, 374 Barrett, J., 3 3 , 8 9 Bartley, W., 264,312 Barton, L. L., 7 2 , 8 4 Bartsch, R. G., 3 0 , 8 4 Basset, J., 187, 234 Bauchop, T., 264, 303, 310, 352,370 Bauer, S., 121, 140, 145, 146, 154, 156 Bauer, W., 194, 195, 234 Baumberger, J. P., 290,310 Baynes, J . W., 144, 1 5 1 , 1 5 5 Beall, J. A., 196, 237 Becker, R., 3 5 6 , 3 7 1 Becker, R. R., 189, 203,237, 238 Becker, V. E., 208, 215, 234 Beek, E. G., van der, 351, 352, 370

377

378

AUTHOR INDEX

Behrens, N. H., 140, 143, 144, 145, 146,153,154 Beinert, H., 15, 90 Belser, W. L., 175,240 Benbough, J. E., 42,84 Bendall, D. S., 297,298,310 Bender, R. A., 25, 27,89 Benemann, J. R., 8 , 8 4 Benson, A. A., 5 7 , 8 4 Beren, K., 94, 95, 123, 154 Berger,L. R., 165, 166, 212, 214, 216, 218,220,229,234,238,239 Bergersen, F. J., 21,84 Bernstein, S., 80, 85 Beteta, P., 146, 154 Bettenhaussen, C. W., 304, 313, 352, 353, 354,355,359,374 Betz, A., 279, 280, 285,310 Bhatt, R. P., 338,371 Bhattacharjee, S. S., 105, 155 Biely, P., 145, 154 Bigliardi-Rouvier, J., 316, 356, 373 Black, C. C., 60, 64, 84 Blas, G. S., 131, 154 Blokhina,T. P., 178, 214, 215,235 Blumsom, N. L., 129, 153 Boatmas, E. S., 229,230,234 Bodanszky, A., 1 7 1 , 172, 200,235 23 7 B@je,L., 201, 235 Bosch, L., 209, 240 Bone, D. H., 7 8 , 8 0 , 8 4 , 8 5 Bongers, L., 77, 78,85 Bonner, W. D., 297,298,310 Boyer, P. U., 278,310 Brandts, J. F., 196, 197, 199, 235 Brauer, R. W., 160, 165,235 Bray, R. C., 15, 17, 43, 84, 85, 86, 91 Brenchley, J. E., 25, 27,89, 90 Bretthauer, R. K., 131, 149, 150, 153, 154,156 Brice, J. M., 21, 31,88 Bridgman, P. W., 195, 202, 235 Brill, W. J., 15, 23, 69, 86, 90, 91, 326, 329,370,373 Brinkley, S. R., 172, 238 Brintzinger, H., 17, 18, 85 Brock,T. D., 95, 131, 135,155 Brodie, A. F., 297, 311

Brown, C. M., 9, 25, 26, 85, 89, 309, 310,316,370 Brown, D. E. S., 205,237 Brown, W. P., 171, 177, 214, 216, 217, 23 7 Bruschi-Heriand, M., 72, 73, 75, 8 9 Bryant, M. P., 56, 80,85, 350, 375 Buchanan, B. B., 58, 61, 6 4 , 8 5 Biichner, E., 214,235 Buchner, H., 214,235 Budge, K. M., 177,178, 21 6, 241 Bull, A. T., 252, 260, 293, 294, 302, 303,307,312 Burn, V. J., 25, 26,85 Burns, R. C., 15, 1 9 , 8 5 , 8 7 Burris, R. H., 15, 1 7 , 18, 20, 73, 74, 85,87,88,90 Bush, F. E., 293,312 Button, D. K., 246, 310

C Cabib, E., 140, 143, 144, 145, 146, 153,154 Caiger, P., 293,312 Calabrese, L., 43,85, 86 Caldwell, D. R., 350,375 Calvin, M., 57, 84 Cambier, H. V., 328, 372 Campbell, A., 358,370 Cannon, F. C., 2 4 , 8 5 Carlsen, R. B., 96, 154 Carnahon, J. E., 1 0 , 8 9 Cartier, P. H., 278, 310 Casse, F., 358, 359,370 Cassel, J. M., 204, 235 Castor, L. N., 295,310 Cattanea, J., 270, 313 Cattell, M., 160, 235 Cavari, B. Z., 290, 308,310 Cawley,T. N., 108,110,111, 127,154 Certes, A., 214, 235 Chance, B., 37, 38, 52, 85, 87, 243, 244, 274, 275, 276, 277, 278, 279, 280, 283, 284, 285, 289, 291, 294, 295, 310, 311, 312, 313 Chance, E. M., 243, 310 Chang, J. P., 343,310 Chapman, A. G., 264, 265, 268,310

AUTHOR INDEX

Chapman, R. E., 194,235 Chen. J.-S., 15, 22, 85 Chen, C. H., 15, 1 7 , 1 8 , 8 7 Chen, G. C. C., 29,84 Cheniae, G. M., 28,85 Cherni, N. E., 213, 223, 229, 237 Cheung, W. Y., 276,313 Chiba, S., 355,370,372 Chin, J. H., 210, 240 Chippaux,M., 316,332, 359,361,370, 3 73 Christensen, R. G., 204, 235 Chumak, M. D., 178, 214, 215, 235, 237 Chun, P. W., 195, 235 Cifonelli, J. A., 140,154 Clark, L. C., 166, 235 Clarke, P. H., 81, 85 Cleary, P., 358, 370 Clegg, R. A., 305, 312 Cleland, W. W., 18, 85 Clouston, J. G., 187, 235 Cobet, A. B., 222, 229, 241 Cochin, D., 214, 235 Cockle, S. A,, 43, 85 Cohen, E. N., 210, 240 Cole, H. A., 264,310 Cole, J. A., 13, 16, 17, 28, 33, 37,40, 48, 49, 53, 71, 85, 86, 92, 335, 336, 344, 346, 354, 360, 370, 375 Coleman, K. J., 28,85 Coles, H., 37, 3 8 , 8 5 Coles, H. S., 276, 313 Collen, D., 205,235 Collins, P. A., 301,313 Colonna, W. J., 104, 108, 110, 121, 154 Colvin, H. J., 297, 3 1 1 Colwell, R. R., 190, 239 Compton, B. E., 28,85 ConneIly, C. M., 275,310 Connors, N. T., 226,239 Cook, K. A,, 1 5 , 2 2 , 8 6 Cortat, M., 146, 154 Costilow, R. N., 4 5 , 8 5 Cotton, N. T., 250,312 Couchoud-Beaumont, P., 356, 360, 364,3 70,3 73 Cove, D. J., 25, 90, 327, 337, 370

379

Coykendall, A. L., 230,239 Cox, G. B., 348,370 Crabb, J. W., 176,235 Crandall, M. A., 95, 131, 135,154 Criddle, R. S., 6 2 , 8 5 Crissman, J. K., 196, 23 7 Crofts, A. R., 5 9 , 9 2 Crowe, J., 166, 237 Cunningham, W. L., 131,154 Cuotant, P. R., 350,3 74

D Daily, 0. P., 211,230,235,239 Dalton,H.,8, 15, 16, 21, 86, 251, 299, 300,311 Dasgupta, S., 207,235 Davis, D. H., 2, 86 Degn, H., 250,261,262,296,311 Deheo, A. B., 2 5 , 2 7 , 8 9 Deibel, R. H., 343, 3 72 Deleo, A. B., 27, 91 De Ley, J., 20,86 Delwiche, E. A., 350,372 De Maeyer, L., 205,235 DeMoss, J. A., 33, 34, 50, 51, 53, 91, 325, 326, 327, 335, 336, 337, 344, 345, 346, 357, 358, 360, 371,372,374 Denend, A. R., 63,89 Der Vartanian, D. V., 72, 73, 74, 75, 89,330, 331,370,371 De Vries, J., 28, 90, 328, 373 Dhople, A. M., 263,311 Dicamelli, R. F., 208, 235 Diehl, H. S., 187,237 Disteche, A., 1 7 1 , 235 Dixon, R. A., 23, 24, 2 5 , 8 5 , 8 6 Doelle, H. W., 276, 309,313 Donawa, A., 8 0 , 8 8 Donze, M., 21,86 Doudoroff, M., 2 , 8 6 Douglas, M. W., 71,86 Dowben, R. M., 176,235 Downey, R. J., 319, 335, 340, 341, 348, 349,350,370,372 Drabikowska, A., 297,311 Dragoni, N., 73,89 Dreizen, P., 219, 235 Drews, G., 279, 281,313

380

AUTHOR INDEX

Feigenblum, E., 76, 86 Fenn, W. O., 36, 86, 160, 165, 170, 171, 1 7 7 , 214, 216, 217, 235, 23 7 Ferenci, T., 66, 67, 86 Fida, A., 356,373 Fielden, E. M., 4 3 , 8 4 , 8 5 , 86 Firth, A., 48, 52, 92, 280, 283, 284, 313, 336,373 Fisher, R. J., 2 3 , 8 6 E Fleet, G. H., 95, 155 Eady, R. R., 8, 15, 16, 1 7 , 2 2 , 8 6 Fliigel, H., 185, 206,236, 239 Ebbecke, U., 205,235 Fluharty, A. L., 3 7 , 9 1 Edwards, T. E., 94, 99, 156 Fogel,S., 108, 1 1 3 , 1 1 6 , 1 5 3 , 1 5 7 Ee, J. H.,van, 321, 329, 330, 331, 374 Forget, N., 7 3 , 8 7 Ehret, A., 14, 88 Forget, P., 319, 320, 321, 329, 330, Ehrlich, H. L., 221, 235 331, 3 4 4 , 3 5 4 , 3 7 0 , 3 7 1 , 3 7 4 Eimhjellen, K., 177, 2 2 2 , 2 3 6 Foreman, H. J., 43, 86 Eisenberg, H., 194,235 Forrest, W. W., 171, 236, 264,311 Elliot, D. C., 297, 311 Foster, R. A. C., 186, 236 Elorza, M. V., 145, 1 5 4 Frazier, W. A., 111. 328, 372 Elsaesser, S., 276, 279, 280,310 Fredricks, W. W., 69, 86 Elsden, S. R., 264, 303,310, 352,370 Fridovich, I., 43, 44, 45, 46, 47, 86, Enogh,H. G., 319, 320, 323, 324, 325, 87, 88, 89, 92 326, 327, 345, 368, 369, 370, Friedman, A. E., 3 4 , 9 1 3 71 Friis, J., 115, 155 Enomoto, M., 359, 3 7 1 Fry, B. A., 1 4 , 8 6 Erecinska, M., 243, 310 Fujimoto, K., 101, 156 Erickson, R., 328, 372 Fujita, T., 323, 325,372 Erkama, J., 41, 86 Fukuyama, T., 7 1 , 8 6 Estabrook, R. W., 265, 278, 280, 310, Fuller, R. C., 63, 8 4 311,312 Estabrook, T. W., 263, 278, 311 G Ettlinger, L., 267,310 Evans, H. J., 15, 2 8 , 8 5 , 8 8 , 208, 215, Garewell, H. S., 141, 155 Garland, P. B., 305,312 234 Garner, C. D., 367,371 Evans, M. C. W., 6 1 , 8 5 Garver, J. C., 246, 310 Eylar, E. H., 121,154 S., 94, 121, 146,154, 155 Gascon, Eyring, H., 160, 168, 171, 186, 202, 214, 215, 233, 234, 235, 236, Gehring, U., 57, 6 1 , 8 6 Gelder, B. F. van. 321, 329, 330, 331, 23 8 374 Gel’man, N. S., 50, 86 F Gendre, J., 316,373 Gender, R. L., 215, 2 3 5 , 2 3 6 Falk, I. S., 246,311 Gentles, J. C., 1 3 6 , 1 5 5 Fall, L., 264, 265, 268,310 Gent-Ruyters, M. L. W., van 350, 353, F a r k f , V., 121, 140, 145, 146, 154, 355,371 156 Gerard, R. W., 246,311 Farmanfarmaian, A., 177, 222, 236 Gerber, B. R., 203, 204, 236

Drickamer, H. G., 201, 240 Drost-Hansen, W., 172, 235 Drozd, J., 246, 251, 299, 300, 307, 311 Dular, V., 9, 91 Dun, B., 62, 85 Duysens, L. N. M., 2 7 8 , 3 1 1 Dwyer, P., 36, 86

AUTHOR INDEX

Gerrits, J. P., 353,371 Gerschmann, R., 36,86 Gest, H., 54, 67, 68, 69, 72, 73, 86,90 Ghosh, A., 274, 280, 285, 310, 311 Gibbins, J. R., 206,240 Gibson, F., 348,370 Gibson, J., 276,312 Gifford, G. D., 36, 41,86 Gilbert, D. L., 36,86 Gill, S. J., 193,240 Gilmour, C. M., 338,3 71 Giordano, G., 365, 366,368, 371,374 Glaser, J. H., 326, 327, 357, 358, 371 Goldberger, R. F., 337, 371 Gollan, F., 166, 235 Gorbatch, V. I., 101,155 Gordon, G. L., 276,313 Gorin, P. A. J., 96, 97, 101, 104, 105, 113, 127, 130, 137, 155, 157 Gottlieb, J. A., 65, 88 Gottlieb, S. F., 35, 36,86 Gould, G. W., 187,236, 239 Govorchenko, V. I., 101,155 Graaf, J., de, 359,371 Gray, C. T.,48,67, 68,69, 70, 71, 72, 73,86,87,344,371 Gray, J. C., 62,87 Gregory, E. M., 45,46, 8 7 Greiling, H., 122, 155 Grollman, A. P., 136, 137, 157 Groot,G.N. de. 54, 87, 317, 318, 332, 334, 335, 337, 339, 340, 341, 343, 346, 361,371 Grossowicz, N., 290, 308,310 Gruezo, F., 100,156 Guerrero, M. G., 326, 342, 343, 371, 374 Guest, J. R., 356, 358, 360, 363,371, 3 75 Gunsalus, I. C., 303,311 Gunter, K. K., 193, 236 Gunter, T. E., 193, 236 Gurney, E., 23,91 Gurney, E. G., 23,91 Guthrie, R. D., 101, 155

H Hackenthal, E., 332, 335, 356,371 Hackenthal, R., 332, 335, 356.371

381

Hadjipetrou, L. P., 338, 344, 353,371 Hager, L., 324,3 71 Haldane, J. B. S., 4 , 8 7 de la Haba, G., 175,239 Hall, J. B., 353, 355,372 Hamer, G., 259,262,311 Hamilton, J. A., 348,370 Hamilton, I. D., 264, 265,311 Hamilton, W. A., 187,239 Hamilton, W. D., 15,90 Hanks, J. H., 263,311 Hardon, M. J., 212, 225,234,236 Hardy, R. W. F., 15, 1 9 , 8 5 , 8 7 Harmes, C. S., 279, 311 Harold, F. M., 343, 351,371 Harrington, M. G., 108, 110, 111,154 Harrington, W. F., 203, 204,237 Harris, E. J., 278,311 Harrison, D. E. F., 48, 49, 52,87, 246, 247, 248, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 272, 273, 275, 276, 277, 279, 283, 284, 285, 286, 288, 289, 290, 293, 294, 295, 296, 297, 300, 301, 302, 303, 304, 305, 307, 308,311,312 Hart, L. T., 1 0 , 8 7 Hartingsveldt, J., van, 326, 327, 328, 335, 340, 353, 356, 359, 371, 3 72 Hartwell, L. H., 94, 155 Hartzell, T. B., 187,237 Hasan, S. M., 353, 355,372 Hasenclever, H. F., 95, 136, 137,155, 157 Haslam, J. M., 3 0 , 8 9 Hassid, W. Z., 126, 156 Hatch, M. D., 5 9 , 9 1 Hatchikian, E. C., 73,87, 350,372 Haveman, J., 21,86 Hawkins, E. R., 116,155 Hawley,S.A., 196, 197, 198, 201, 236 Haworth, W. N., 94,99, 107,155 Hawthorne, D. C., 129, 138,156 Haystead, A., 21,91 Heath, E. C., 144, 151,155 Heath, R. L., 107,155 Hebert, R. R., 1 5 , 8 7

382

AUTHOR INDEX

Heden, C.-G., 193,236 Heino, K., 41, 86 Hempfling, W. P., 278, 304,311 Henigman, J. R., 185,234 Hensley, D. E., 131,157 Heppel, L. A., 72,87 Herbert, D., 258,313 Herczeg, B. E., 276,313 Herrera, J., 343,3 72 Herschler, W. P., 195,235 Hessler, R. R., 161, 236 Heymann, E., 202,236 Hickman, S., 118,155 Higgins, I. J., 66,87 Higgins, J., 291,310 Higgins, J. J., 275,310 Hildebrand, C . E., 226, 227, 228, 23 6 Hill, S., 20, 8 7 Hino, S., 308,311,313 Hiramoto, Y., 206,240 Hirsch, P., 6 6 , 8 7 Hirst, E. L., 94, 99,155 Hittle, L. L., 167, 184, 212, 241 Hoare, D. S., 1 0 , 9 1 Hochachka, P. W., 219,236 Hodgins, M. G . , 319, 320, 321, 323, 3 72 Hodgson, E. K., 4 3 , 8 7 Hofer, M., 278, 311 Holley, R. A., 147,155 Holmes, W., 275,310 Holmes, W. F., 291,310 Holms, W. H., 264,265,311 Holsten, R. D., 1 5 , 8 5 , 8 7 Holyoke, E. D., 188, 236 Honya, M., 76,80,92 Hornick, C. I., 136,155 Hotchkiss, R. D., 28, 29,88 Houston, L. L., 207,238 Howard, G . A., 81,84 Hsu, A. F., 144, 151,155 Haung, T. C., 17, 8 7 Hubley, J. H., 66,87 Huffaker, R. C., 62,85 Hughes, D., 344,371 Hughes, D. E., 29, 30, 48, 50, 70, 71, 8 7,264,310 Hughes, R. C., 96, 138,155 Hungate, R. E., 8 0 , 8 7

Hvidt, A., 201,235 Hwang, J. C., 15, 1 8 , 8 7 Hyde, M. R., 367,371

I Iida, K., 323, 325, 344,372 Ikkai, T., 205, 208,236 Inderlied, C . B., 350,3 72 Infante, A. A., 203, 208,236 Ingraham, J. L., 256,311,312 Inoue, S., 136,153 Irwin, W. L., 149,154 lsaacs, J. D., 161, 236 lshaque, M., 77, 78, 80,88 Isherwood, F. A., 95, 99,155 Ishida, Y., 80,88 Ishimoto, M., 355,370, 372 Itagaki, E., 320, 323, 324, 325, 371, 372, 375 Iversen, J. J. L., 261, 262, 311 Iverson, W. P., 81, 88 Iwasaki, H., 11, 13, 88,89 Iwata, K., 136,153 Izui, K., 232,236

J Jackson, E. K., 15,87 Jacob, F., 2 5 , 4 8 , 8 8 Jacobs, N. J., 343, 350,372 Jaenicke, R., 202, 205, 236 James, W. O., 297,311 Jamieson, D., 37, 38, 85, 276, 279, 280,310 Jannasch, H. W., 177,222,236 Jansz, E. R., 60,88 Jeanes, A., 131,155 Jeanloz, R. W., 143, 158 Jeffery, E. A., 103,155 Jeng, D., 1 7 , 8 8 Jeng, D. Y., 1 6 , 8 9 Johantges, J., 343,3 72 John, P., 349, 350, 351, 352,372 Johnson, B., 26,85,309,310 Johnson, F. H., 160, 164, 168, 171, 174, 180, 181, 186, 187, 188, 202, 215, 233, 234, 235, 236, 241 Johnson, M., 204,23 7

AUTHOR INDEX

Johnson, M. J., 246,312 Johnson, S. M., 211, 236 Joly, M., 202, 23 7 Jones, C. W., 21, 30, 31, 32, 84, 88, 292, 296, 298, 299, 300, 305, 308,309,312 Jones, D., 4 1 , 8 8 Jones, G., 328,372 Jones, G. H., 103, 107, 122,155 Josephs, R., 203,204,237 Jung, P., 143, 1 4 4 , 1 5 5 , 1 5 8 Jurtshuk, P., 3 2 , 8 8

K Kabat, E. A., 100,156 Kaczorowski, G . J., 131, 150,154 Kadono, H., 200,240 Kadota, H., 80,88 Kalakoutski, L. V., 350,375 Kanegasaki, S., 111,158 Kaplan, N. O., 297,312 Kapoor, V., 356,3 73 Kapralek, F., 353, 355,372 Karush, F., 136,155,156 Katz, S., 196, 232, 237, 240 Kaufmann, H. F., 32, 33, 88, 346, 348,372 Kauppinen, V., 41, 86 Kauzmann, W., 171, 172, 192, 196, 197, 198, 200, 201, 203, 235, 23 7,238,241 Kauzmann, W. J., 215,236 Kavanagh, B. M., 28,85 Kay, R. L., 210,239 Keele, B. B., 44,45, 4 7 , 8 5 , 8 8 , 8 9 , 9 1 Keevil, C. W., 2 8 , 8 5 Kegeles, G., 204, 23 7 Kekwick, R. G. O., 6 2 , 8 7 Keller,D.M., 177, 179, 184, 185, 189, 198,211,231,232,237,238 Keller, R. F., 326,372 Kelly, M., 15, 88 Kemp, J. A., 1 4 , 8 8 Kemp, M. B., 6 5 , 8 8 Kemp, R. B., 306,312 Kennedy, J. R., 206, 237 Kern, K. A., 106, 107, 111, 113, 115, 117, 1 2 7 , 1 5 4 , 1 5 7

383

Ketchum, P. A., 28, 90, 328,327,373 Kettman, M. S., 203, 23 7 Kidby, D. K., 147,155 Kidman, A. D., 7 4 , 7 6 , 8 8 Kim, H. D., 219,235 Kim, J., 212,237,241 Kinne, O., 160, 23 7 Kirikova, N. N., 215, 237 Kisters, R., 122, 155 Kiszkiss, D. F., 319, 335, 340, 341, 3 70,3 72 Kitahara, K., 278,313 Kitching, J. A., 206, 23 7 Kleiner, D., 73, 74,88 Kleinkopf, G. E., 62, 85 Kloepfer, H. G., 151,156 Klucas, R. V., 1 5 , 8 8 Knight, M. K., 283, 284, 290,312 Knobloch, K., 7 7 , 7 8 , 8 8 Knook, D. L., 345, 346, 347, 348, 349, 372,374 Knowles, C. J., 295, 301, 302, 303, 313 Koch, B., 1 5 , 8 8 Kocourek, J., 101, 128, 137,156 Kodama, T., 1 2 , 8 8 Kohen, E., 279,312 Koide, N., 104,156 Kondorosi, A., 28, 29,823 Kopp, F., 146, 254 Kornberg, H. L., 5 6 , 8 8 Kornfeld, R., 118,155 Kornfeld, S., 118, 155 Kosheleva, N. A., 215, 239 Kosinovi,A., 121, 146, 156 Kosmakos, F. C., 297,311 Kovarik, J., 145,154 Kozak, L. P., 149, 150, 153,154,156 Krasna, A. I., 7 6 , 8 6 Kritky, Z., 145,154 Krauss, M., 208, 236 Krebs, H. A., 7 0 , 8 8 Krieg, N. R., 36, 92 Kriss, A. E., 175, 180, 184, 213, 215, 223,229,237 Krista, M. L., 195, 235 Kuehn, G. D., 62, 63, 6 4 , 8 8 KUO,S-C., 146, 156 Kwok, S. C., 9 , 9 1 Kylstra, J. A., 166, 23 7

384

AUTHOR INDEX

L Laidler, K. J., 218, 219,23 7 Lam, Y., 12, 88, 319, 329, 346, 348, 349,3 72 Lamberti, 4., 28, 90, 328,373 Lambowitz, A.M., 297,312 Lampen, J. O., 94, 99, 104, 108, 110, 121, 123, 143, 144, 146, 154, 155,156,157 Landau,J.V., 165,211, 212, 224, 226, 227, 228, 229, 231, 237, 238, 239,240 Lang,G., 15, 17,21,91 Larson, A. D., 10,87 Larson, W. P., 187, 237 Lascelles, J., 343, 370 La Touche, C. J., 136,155 Lauffer, M. A., 205, 236 Laurence, H. L., 230,240 Lawrence, A. J., 65,88 Layng, E. J., 100,156 Lazzarini, R. A., 1 4 , 8 8 Leadbetter, E., 326, 342,3 71 Leadbetter, E. R., 65, 88 Lebeault, J. M., 364,3 70 Lebowitz, J., 208, 235 Lee, J. P., 73, 74, 75,89 Lee, K. Y., 28, 90, 328,372,373 Lee, Y. C., 100, 101, 103, 104, 107, 113,156 Le Gall, J., 72, 73, 74, 75, 84,87, 89, 350,3 72 Lehle,L., 140,141,144, 151,156,157 Lemberg, R., 33,89 Le Minor, L., 356, 360,373 Lenhof, H. M., 297,312 Lester, R. L., 319, 320, 323, 324, 325, 326, 327, 344, 345, 368, 369, 3 70,371,3 72 Letters, R., 108, 110, 111, 154 Lewin, I., 164,236 Li, Y. T., 103,156 Light, P. A., 305,312 Lilleor, M., 261, 262,311 Lindahl, T., 193,236 Lindberg, B., 101,156 Lindmark, D. G., 70, 89 Linke, H. A. B., 80,90 Linnane, A. W., 30,89

Linton, J., 252, 260, 293, 294, 302, 303,307,312 Lipke, P. N., 104, 111, 121, 127, 128, 132,154,156 Lippitt, B., 43, 8 9 Ljones, T., 15, 90 Ljungdahl, L. G., 326,370 LIoyd, D., 306,312 Lloyd, K. O., 100, 122,156 London,J., 283,284,290,312 Longmuir, I. S., 291,312 Lopez, M. D. G., 95,156 Losada, M., 326, 342, 343, 371, 372, 3 74 Loveless, J. E., 250, 251, 253, 254, 255, 256, 257, 258, 262, 293, 296, 300, 303, 304, 307, 308, 311 Lowe,D. J., 15, 17, 43,86, 91 Lukins, H. B., 3 0 , 8 9 Lukoyanova, M. A., 50,86 Lundquist, R., 283,312

M Madge, O., 264,313 McCarthy, J. F., 101,155 McCleskey, C. S., 1 0 , 8 7 McCord, J. M., 43,44,47, 88, 8 9 MacDonald, A. G., 160,216,237,239 MacDonald, D. W., 327,370 MacDonald-Brown, D. S., 9, 85, 316, 3 70 McElroy, W. D., 175,238 McFadden, B. A., 57, 62, 63, 64, 88, 89 MacGregor, C. H., 319, 320, 321, 323, 324, 343, 345, 362, 365, 367, 368, 372,373 Machala, S., 121, 146,156 Macheboeuf, M. A., 187, 234 Mackereth, F. J. H., 246,312 MacLean, F. I,, 60,88 MacLennan,D. G., 246,250,280,311, 312 Mabbs, F. E., 367,371 Madansky, C. H., 328,372 Maeda, M., 308,311 Magasanik, B., 25, 27,89, 90, 91 Magill, C., 10, 89

AUTHOR INDEX

Maitra, P. K., 260, 261, 263, 264, 265, 266, 268, 269, 270, 272, 273, 275, 276, 277, 278, 300, 305, 307,311,312 Maldonado, J. M., 343,372,373 Maley, F., 96, 104, 116, 122,158 Mandel, M., 29,84 Mannheim, W., 356,3 71 Mantner, G. N., 43,86 Marinus, M. G., 335, 356,371 Marquis, R. E., 162, 165, 170, 1 7 1 , 1 7 7 , 179, 184, 185, 189, 198, 204, 211, 214, 216, 217, 231, 232, 233,235,23 7,238 Marr, A. G., 256,312 Marsland, D., 205, 206, 207, 237,240 Mateles, R. L., 256,313 Mathemeier, P. F., 184,238 Matile, Ph., 94, 146, 154, 156 Matsubara, T., 11, 12, 13,89 Matsuda, K., 101,156 Matsumura, P., 177, 179, 184, 185, 189, 198, 211, 212, 217, 218, 237,238 May, F., 328,372 Mayer, R. M., 150, 153,156 Mayeax, J. V., 338,371 Mazza, G., 73,89 Meers, J. L., 9, 26,85,89, 316, 370 Meganathan, R., 162,204, 233,238 Mehta, J. M., 1 0 , 8 9 Melchoir, D. L., 209, 238 Mendershausen, P. B., 101, 103,157 Mendoza, C. G., 95,156 Metenier, G., 343,3 73 Meyer, D. J., 14, 41,88,89, 296, 299, 300,305,312 Meyers, C. E., 230,239 Michalover, J. L., 66,91 Mill, P. J., 108,156 Miller, K. W., 211, 236 Mills, E. L. 161, 236 Miovic, M. L., 276,312 Mishustina, J. E., 213, 223, 229, 237 Mitchell, P., 299, 305,312,313 Mitchell, W. O., 95, 155 Mitskevich, I. N., 180, 184, 213, 223, 229,23 7 Mitton, J. R., 66,87 Miyagawa, K., 213, 238

385

Miyata, M., 11, 13, 89 Miyosawa, Y., 193, 239 Mohankumar, K. C., 165,218,238 Monod, J., 25,48,88,260,312 Moon, T. W., 219,236 Moor, H., 94,156 Mori, T., 11, 12, 13, 88, 89, 351,373 Morita, R. Y., 163, 176, 1 7 7 , 184, 189, 203, 212, 213, 214, 215, 216,23 7,238,240,241 Morowitz, H. J., 209,238 Morrell, A. G., 121,154 Morris, J. A., 1 6 , 8 6 , 8 9 Morris, J. G., 30, 34, 37, 38, 39, 51, 82,89,90 Mortenson, L., 73, 74, 75, 90 Mortenson, L. E., 8, 10, 15, 16, 22, 85,86,87,88,89,367,375 Mortimer, R. K., 129,138,156 Moss, F., 48,89 Moss, F. J., 292, 293, 296,312 Mossman, M., 344,3 71 Mossman, M. R., 48, 70, 71,87 Moustafa, E., 17,89 Moyle, J., 299, 305,312 Muller, M., 70, 89 Multani, J. S., 15, 22,85 Mundkur, B., 95,156 Munkres, K. D., 297,311 Muradov, M., 186, 239 Muramatsu, T., 104,156 Murdock, A. L., 176,235 Mustafa, T., 219, 236 Mutaftschiev, S., 364,3 73

N Nagai, S., 292, 294, 299, 300,312 Nagatani, H., 23, 26,90 Nagatani, H. H., 329,3 73 Nagle, J. F., 210, 239 Nagley, P., 30,89 Nakajima, T., 96, 100, 103, 104, 107, 110, 112, 113, 117, 122, 124, 129, 144, 147, 148, 153, 156, 157 Nakos, G., 73, 74, 75, 90 Nantz, R., 166,237 Nason, A., 28, 90, 326, 328, 344, 732,373,375

386

AUTHOR INDEX

Necas, O., 94, 145,156 Necklen, D. K., 309,313 Neilands, J. B., 173,238 Neuman, R. C., 171,238 Neumann,N. P., 94, 99, 121, 123, 146, 155,156 Neville, W. M., 214, 238 Newman, E. B., 356,373 Newton, N., 1 2 , 3 3 , 9 0 Newton, N. A., 348,370 Ng, H., 256,312 Nicholas, D. J. D., 10, 11, 12, 14,88, 90, 297, 312, 319, 329, 344, 346, 348, 349,372,3 73 Nikaido, H., 126,156 Nishikawa, A. H., 203, 237 Nishizawa, Y., 292, 294, 299, 300,812 Njelsen, S. O., 45,90 Noguchi, H., 200,203,204, 205,236, 238 Normansell, D. E., 319, 320, 321, 323, 3 72 Northcote, D. H., 96, 100. 110, 157 Nuner, J. H., 335, 341,370 Nye, S., 36,86

Orme-Johnson, W. H., 15, 90 Ormerod, J. G., 58, 91 d’Ornano, L., 335,339,341,373 Orosz, L., 28, 29, 88 Orton, W. L., 99, 132, 133, 134, 136, 158 Osterland, C. R., 118,155 Ostrovski, D. N., 50,86 Ota,A., 351,373,375 Ottolenghi, P., 115, 155 Ousby, J. C., 250,312 Ovodov, Yu. S., 101, 155 Owen, B. B., 172,238

P

Painter, H. A., 3, 7, 9, 1 4 , 9 0 Pakman, L. M., 3 5 , 3 6 , 8 6 Palmer, D. S., 185, 238 Palmer, F. E., 175, 238 Palmer, G., 15, 1 7 , 89 Pan, S. S., 28, 90, 328,372,373 Paneque, .4., 343,372,374 Paris, C. G., 25, 27,89 Pascal, M. C., 359,3 70 Paschen, W., 45,90 Pateman, J. A,, 25,90, 337,370 Paterson, A. C., 32, 90 0 Paul, K. L., 212,238 Payan, D. G., 210,240 O’Brien, R. W., 38, 39, 51, 90 Payne, W. J., 10,90, 316,373 O’Conner, T. M., 207,238 Pearson, H. W., 21,91 Ogrinc, M., 175,238 Pease, D. C., 206,238 Ohlenbusch, H. D., 122,155 Peat, S., 94, 99, 156 Ohnishi, T., 305,312, 351, 373 Peat, S. J., 107, 155 Oka, T., 292,293,309 Peck, H. D., 7, 38, 54, 72, 73, 74, 75, Okamoto, B. Y., 201,240 Okunuki, K., 351,373 80,84,89,90 Okunuli, K., 35 1 , 3 75 Penniston, J. T., 207, 208, 238 Old, L., 32,88 Perlin, A. S., 101, 127, 15.5 Oliveira, R. J., 196, 197, 199, 235 Perry, J. E., 212,239 Olivera, B. M., 283,312 Pfitzner, J., 80, 90 Oltmann, L. F., 319, 320, 360, 361, Phaff, H. J., 93, 94, 95, 99, 100, 155, 369,3 73 156 Onodera, K., 362,374 Phillips, K. C., 65, 92 Onodera, M., 299,312 Pichinoty, F., 48, 70, 71, 90, 316, Ooi, T., 205, 208,236 317, 319, 321, 332, 335, 339, Oosterhuis, S. K. H., 350,3 75 341, 343, 344, 354, 356, 360, Oostrom, H., 209, 240 361,368,3 70,3 71,3 7 3 , 3 74 Oppenheimer., C. H., 164, 174, 229, Piechaud, M., 71, 90, 316, 356, 360, 238,241 3 73 Ordal, E. J., 71,86 Pieterman, K., 359,374

AUTHOR INDEX

387

Radcliffe, B. C., l l , 9 0 , 319, 329,373 Radin, D. N., 116, 157 Raeburn, S., 6 9 , 9 0 , 9 1 Ragan, C. I., 305,312 Raizada, M. K., 151, 156, 157 Rajagopalan, K. V., 47, 91 Rank, G. H., 105,113,157 Raschke, W. C., 94, 97, 103, 104, 105, 106, 107, 110, 111, 113, 115, 117, 121, 126, 127, 128, 129, 132,138,154,156,157 Rasper, J., 171, 200, 237 Ratouchniak, J., 359,370 Rautenshtein, Y. I., 186, 239 Redfearn, E. R., 30, 32, 88, 298, 312 Redkina, T. V., 215,239 Regnard, P., 160, 205, 233,239 Rever, B. M., 25,90 Ribbons, D. W., 66, 91 Richard, A. J., 195,235 Rickard, P. A. D., 293,312 Riechelt, J. L., 309,313 Riet, J.,van’t. 317,318, 320, 321, 322, 329, 330, 331, 335, 339, 340, 341, 345, 346, 347, 348, 372, 373,374 Rigano, C . , 356,373 Riklis, E., 76, 91 Rittenberg, D., 76, 91 Rittenberg, S. C., 2, 37, 56, 85, 91 Rivas, J., 343,3 74 Riviire, C., 365, 366, 368, 370, 371, 3 74 Rizza, V., 350, 374 Roberts, P. B., 43,84,85, 86 Robertson, A. G., 264, 265,311 Robinow, C. F., 94,156 Rolfe, B., 362, 374 0 Rosenfeld,L., 100,101,. 102, 103, 104, Quayle, J. R., 55, 56, 57, 58, 61, 64, 106,108,113,124,157 65, 66, 8 7 , 8 8 , 9 0 Rusing, J., 350,3 74 Quigley, M. M., 190, 239 Rosso, J. P., 319, 321,374 Quinby, H. L., 221,240 RotiIio, G., 4 3 , 8 4 , 8 5 , 8 6 Routledge, V. I., 367,3 71 Ruiz-Herrera, J., 33, 34, 53, 91, 344, 345,346,357,358, 360,374 Russell, S. A., 15, 88 R Rustad, R. C., 206,241 Rutberg, L., 186,239 Rabani, J., 45, 90 Ryu, D. Y., 256,313 Rabinowitz, J. C., 69,90,91

Pinsent, J., 325, 326,373 Piotrowski, M., 34,91 Pirt, S. J., 48, 8 7 , 246, 247, 248 250, 253, 267, 275, 286, 290, 297, 304,307,311,312 Planta, R. J., 7, 91, 317, 318, 320, 321, 322, 329, 330, 331, 335, 339, 340, 341, 345, 346, 347, 348, 349,372,373,374 Plummer,T.H.,96, 104, 116, 122, 158 Polakis, E. S., 264,312 Polissar,M. J., 160,171, 186,202,236 Pollard, E.C., 162, 223, 226, 227, 228, 230.236.238.240 Pommier, J., 366, 368,370,374 Ponat, A., 185,238 Poole, R. K., 306,312 Pope, D. H., 175, 216, 220,226,227, 228,238,239 Portelance, V., 350,374 Postgate, J. R., 8, 15, 16, 1 7 , 20, 22, 23, 24, 25, 73, 85, 86, 87, 89, 90, 91, 246, 251, 299, 300, 306, 307,311,313 Prabhakararao, K., 14, 90 Prakash, O., 8, 13, 14, 90, 335, 373 Pressman, B. C., 278,311 Price, G. B., 63, 84 Primrose, S. B., 24,85 Prindaville, F., 195, 234 Pritchard, G. G., 41, 86 Prival, M. J., 25, 27, 8 9 , 9 0 Puig, J., 316, 358, 364, 368,370,373 Purvis, P., 350,374 Pye, E. K., 276, 281, 285, 289,313

388

AUTHOR INDEX

S

Sena, E. P., 116,157 Senez, J. C., 256, 257,313 Sadana, J. C., 8, 13, 14, 90, 335, Sentandreu, R., 96, 100, 110, 143, 3 73 144, 145,154,157 Safranski, M. J., 131, 157 Shah,V. K., 15,90, 326, 329,370,373 Sagan, C., 180,239 Shallenberger, M. K., 53, 91 Sagers, R. D., 69,91 Shanmugan, K. T., 5 8 , 6 1 , 8 5 Saito, T., 105,157 Sharma, C. B., 144,151,157 Sale, A. J. H., 187,236,239 Shen, C., 215, 240 Salton, M. R. J., 369,3 74 Shen, J. C., 212,239 Saltzman, H. A., 166,237 Shidara, S., 11, 12,88 Samson, F., 207, 238 Shirnizo, M., 23, 26,90 Sanadi, D. R., 37,91 Shipp, W. S., 33, 34,91 Sanderson, K. E., 359,374 Short, A. J., 188,240 Sandula, J., 127, 128, 157 Showe, M. K., 50, 51, 53, 91, 335, Sannoe, K., 213,238 336, 337, 344, 357, 358, 360, Sanwal, B. D., 1 3 , 9 2 3 74 Sapshead, L. M., 91, 292,313 Shuster, C. W., 303,311 Sarkar, S., 106, 157 Siehr, D. J., 1 0 , 8 9 Sasarman, A., 350,374 Sigal, N., 270,313 Satchel], D. P. N., 103,155 Sik, T., 28, 29, 88 Sato, K., 63,84 Silver, W. S., 20, 22, 91 Sato, R., 323, 325,372 Simoni, R. D., 53,91, 337, 374 Sauer, B. L., 297,311 Sinclair, C. G., 256,313 Schallenberger, M. K., 337, 374 Sinclair, D. R., 295,313 Scheie, P. O., 180,239 Sinclair, P. R., 33, 92, 332, 335, 343, Schellman, J. A., 200, 239 346, 350,3 74,3 75 Scheraga, H. A., 183,239 Sirevag, R., 58, 91 Schiereck, P., 2 1 , 8 6 Slack, C. R., 59,91 Schindler, F., 277,310 Slack, J. M., 350, 3 74 Schlamm, N. A., 211, 212, 230, 235, Slater, E. C., 350, 3 74 239 Slayman, C. W., 297,312 Schlegel, H. G., 80, 90 Sleigh, M. A., 160, 239 Schlick, XI-J., 3, 91 Slodki,M.E., 100, 129, 130, 131,149, Schlieper, C., 185, 206,239 150,157 Schnaitman,C. A., 319, 320, 321, 323, Smith,B. E., 15, 16, 1 7 , 21, 22, 86, 91 362,365,368,372 Smith, B. R., 15, 17, 91 Schoener, B., 276, 277, 279, 280,310 Smith, F., 140,154 Schoenmaker, G. S., 319, 320,373 Smith, L., 294,313 Scholes, P., 305,313 Smith, R. C., 264, 313 Schon, G., 279,281,313 Smith, W. L., 96, 99, 103, 116, 123, Schulp,J. A., 332, 335, 339, 340, 341, 129, 147,148,153,157 349, 360, 361, 363,374 Smith, W. P., 227, 228, 238, 239 Schurmann, P., 58, 61, 64,85 Snoswell, 4.M., 348,370 Schutzback. J. S., 150, 151, 156, 1 5 7 Sone, N., 350,374 Schwarz, J. R., 190, 212, 226, 227, Spencer, J. F. T., 96, 97, 104, 105, 239, 240 113,137,155,157 Scocca, J. R., 100,156 Sperl, G. T., 10, 91 Scrutton, M. C., 4 6 , 9 1 Spilker, E., 73, 74, 75,89 Segal, I. H., 270,313 Srinivasan, K. R., 210,239 Segal, N., 270, 313 Stadtman, E. R., 69,86

AUTHOR INDEX

389

Stanier, R. Y., 2 , 4 8 , 60, 6 4 , 8 6 , 91 Steiner, A. L., 3 2 6 , 3 70 Stewart, T. S., 101, 1 0 3 , 1 5 7 Stewart, W. D. P., 21, 91 Stiefel, E. I., 367, 374 Stiller, M., 10, 91 Stouthamer, A, H., 7, 54, 8 7 , 91, 303, 304, 313, 317, 318, 319, 320, 326, 327, 328, 332, 334, 335, 337, 338, 339, 340, 341, 343, 344, 346, 349, 350, 351, 352, 353, 354, 355, 356, 359, 360, 361, 363, 368, 369, 370, 371, 3 7 2 , 3 7 3 , 3 7 4 , 3 75 Streicher, S., 23, 91 Streicher, S. L., 23, 25, 27, 89, 91 Stumpf, P. K., 173, 238 Stupakova, T. P., 215,237,239 Sturtevant, J. M., 194,235 Sugiyama, T., 63,84 Summers, D. F., 136, 137, 1 5 7 Sunayama, H., 1 0 5 , 1 3 7 , 1 5 7 Suzuki, H., 11,88 Suzuki, I., 9, 91 Suzuki, K., 183, 193, 200, 213, 238, 239, 240 Suzuki, S., 105, 1 3 7 , 1 5 7 Svab, Z., 28, 29,88 Svoboda, A., 145,154 Swaminathan, N., 96, 154 Swartz, R. W., 227, 228, 238,240 Sacks, L. E., 3 3 5 , 3 74

Tarasova, N. V., 215,235 Tarentino, A. L., 96, 104, 116, 122, 158 Taylor, A. L., 357, 358, 3 5 9 , 3 7 5 Taylor, N. W., 99, 132, 133, 134, 136, 158 Tempest, D. W., 26, 89, 258,313 Teulings, F. A. G., 353,371 Theede, H., 185, 239 Theorell, H., 278, 310 Thibodeau, L., 1 6 5 , 2 3 7 Thieme, T. R., 94, 96, 103, 104, 105, 108,110,122, 1 2 3 , 1 5 8 Thomas, A. D., 276,313 T h e y , L. G., 206,240 Timson, W. J., 188, 240 Topiwala, H. H., 256, 259, 262, 321, 313 Toplin, I., 193,236 Trotter, C. D., 357, 358, 3 5 9 , 3 7 5 Trudell, J. R., 210,240 Tso, M-Y. W., 1 5 , 9 0 Tsuchiya, M., 200,240 Tubb, R. S., 25,91 Turner, P. R., 2 5 , 8 5 Tyler, B., 27, 91 Tyler, B. M., 25, 2 7 , 8 9

T

V

Taber, H., 295,313 Takahashi, H., 326,375 Takahashi, Y., 308,313 Takebe, I., 278,313 Tam, L. Q., 166,234 Tamiya, N., 76, 80, 92 Tanaka, F., 201, 240 Tanford, C . , 1 9 9 , 2 4 0 Taniguchi, S., 320, 323, 325, 344, 372,375 Taniguchi, Y., 183, 193,239 Tanner, W., 140, 141, 143, 144, 151, 154,155,156,157,158 Taptykova, S. D., 350,375

Vacquier, V. D., 175,240 Vagabov, V. M., 140,154 Valentine, R. C., 8, 10, 23, 26, 69,84, 89, 90, 91 Vallentyne, J. R., 174, 240 Vance, R. G., 47, 91 Van der Beek, E. G., 304,313 Vandereycken, G., 205,235 Van der Veen, J. M., 105, 158 van der Walt, J. P., 127, 158 van Diggelen, 0. P., 209, 240 Van Gelder, B. F., 32, 33, 88 vant’ Hoff, J. H., 169, 240 Van’t Riet, J., 7, 91

U Umeyama, M., 355,372 Uruburu, G., 9 5 , 1 5 6

AUTHOR INDEX

390

Varner, J . E., 326,372 Vasey, R. R . , 250,312 Vega, J. M., 326, 3 4 2 , 3 71 Venablrs, W. A., 356, 357, 358, 360, 3 75 Vidaver, W., 220, 240 Villanueva, J . R., 95, 156 Vinograd,J., 194, 195, 234 Vishniac, W., 80, 85 Vogele, P., 122, 155 Vojtkovi-LepSikovi, A., 1 2 7 , 1 2 8 , 1 5 7 Vries, W., de, 350, 353, 355, 371, 3 7 5

W Wagncr, W., 1 6 6 , 2 3 7 Walker, D. A., 59, 92 Walker, D. J., 171,236, 264,311 Walsby, A. E., 1 6 2 , 2 4 0 Wampler, E., 232, 240 Ward, F. B., 7 1 , 86 Ward, M. A., 16, 86 Warmsley, A. M. H., 48, 92, 354, 375 Warren, C. D., 1 4 3 , 1 5 8 Wasserman, A. R., 141, 155 Watanabe, H., 101, 156 Watkins, J., 4 1 , 8 8 Watson, P. R., 131, 155 Weale, K. h., 1 6 8 , 2 4 0 Weber, G., 201,240 Weida, B., 193, 240 Weidenmuller, R., 176, 235 Weimer, M. S., 213, 240 Weise, M. J., 131, 150, 154 Weisiger, R. A., 44, 46, 47, 92 Weller, P. K., 162, 223, 238 Wells, J. S., 36, 92 Weser, V., 45, 90 Weston, J. A., 295, 301, 302, 303,313 Westort, C., 196, 197, 199, 235 Westwood, A. W., 276,313 Wever, R., 321, 329, 330, 331,374 Whalley, E., 170, 240 Whatley, F. R., 349, 350, 351, 352, 3 72 Whelan, W. J., 94, 99, 156 White, D. C., 33, 92, 250, 292, 294, 295, 313, 332, 335, 343, 346. 350,374,375 Whittenbury, K., 65, 92

Wiemken, A., 4 6 , 1 5 6 Wijck-Kapteijn, W. M. C., van, 3 5 0 , 3 7 5 Wild, J. R., 212, 239 Wilkinson, J. F., 65, 66, 87, 92 Williams, G. R., 244, 274, 291, 294, ,710 Williams, R. K., 215, 240 Williamson, J. R., 276, 279, 280,310, 313 Willingham, C. A., 221, 240 Wills, P. A., 187, 235 Wilson, D. F., 34, 92 Wilson, P. J., 1 0 , 9 0 Wimpenny, J., 344,371 Wimpenny, J. W. T., 2, 29, 30, 33,40, 48, 49, 50, 52, 56, 57, 70, 71, 85, 86, 87, 91, 92, 264, 280, 283, 284, 292, 303, 309, 310, 313, 335, 336, 344, 346, 354, 360,370,375 Winzler, R. J., 290, 291,313 Wirsen, C. O., 1 7 7 , 222,236 Wohlrab, H., 261, 296,311 Wolfe, R. S., 55, 5 6 , 8 5 , 92 Wolin, E. A,, 5 6 , 8 5 Wolin, M. J. 56, 85, 3 5 0 , 3 72 Wolk, C. P., 60, 92 Wright, A., 1 1 1 , 1 5 8 Wright, V., 21, 3 1 , 8 8

Y Yagi, T., 65, 66, 73, 74, 76, 80, 92 Yamanaka, T., 3 5 1 , 3 7 3 , 3 7 5 Yanagihara, R., 76, 88 Yang, J. T., 200,238 Yayanos, A. A., 2 2 3 , 2 3 0 , 2 4 0 Yen, P. H., 94, 100, 106, 122, 132, 133, 134,158 Yost, F. J., 45, 47, 92 Young, A. D., 206,240 Young, H. L., 3 6 , 9 2 Young, P. G., 206,240

Z Zaichkin, E. I., 175, 237 Zarowny, D. P., 1 3 , 9 2 Zheleznikova, V. A., 213, 223, 229, 23 7

AUTHOR INDEX

Zimmerman, A. M., 160, 206, 229, 230,237,240,241 Zimmerman, S. B., 229,241 Zipp,A., 171,192, 196,197, 198, 201, 203,238,241 Zobel1,C. E., 161, 163, 164, 167, 174,

176, 177, 178, 179, 180, 184, 185, 187, 188, 189, 216, 220, 222, 229, 230, 236, 23 7 , 2 3 8 , 2 4 1 Zumft, W. G., 15, 17, 8 7 , 89, 3 75

391

181, 212, 233, 367,

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SUBJECT INDEX

A

Actin helices, effects of pressure on aggregation of, 205 Acetate, effect of, on respiration rate Actinomycetes, effect of pressure on, of Klebsiella aerogenes, 260 175 production by Klebsiella aerogenes, Adaptation to tolerance to high 255 pressure, 176 Acetobacter aceti, control of adenoAdenosine nucleotides, allosteric consine triphosphate synthesis in, 267 trol of catabolism by, 268 Acetolysis fingerprints of yeast Adenosine phosphates, as regulators of mannans, 114 respiration in bacteria, 263 Acetolysis of yeast wall mannans, 100 steady-state contents of, in growing N-Acetyl-D-glucosamine, mannan cells, 265 linked to, in the yeast wall, 97 residues in mannan of Kluyuero- Adenosine triphosphatase, effect of m y c e s lactis, 147 pressure on, 178, 207 Acetyl groups in phosphomannans of of chlorate-resistant bacteria, 365 hansenulas, 1 31 Adenosine triphosphate, role of, in Acetylene reduction, to measure nitronitrogen fixation, 1 7 gen fixation, 18 synthesis, microbial, effect of A c h r o m o bacter fischeri, gas producoxygen on, 40 tion from nitrite by, 10 Adenylylation, regulation of glutamate A. thallassius tolerance of pressure by, dehydrogenase by, 27 174 Aero bacter aerogenes, capsular polyAchromobacter sp., cytochrome saccharide of, 126 oxidase activity of, 292 Aerobic hydrogen metabolism, 76 effect of oxygen on, 36 Agents used to detect structures in A c h r o m o bacterium fischeri, synthesis yeast wall mannans, 106 of nitrate reductase by, 355 Agg!utination of mating types of Acid hydrolysis, selective, of yeast Hansenula wingei, 131 wall mannans, 100 Agglutinin of walls of Hansenula Acid lability of exocellular phosphowingei, 9 4 mannans of Hansenula spp., 129 Agglutinins, structure of, from Acidity, effect of, on barotolerance, Hansenula wingei, 132 184 Aggregation of proteins, effects of effect of, on pressure denaturation pressure on, 202 Agromyces ramnosus, effect O f of proteins, 195 on synthesis of formate hydrohydrogen peroxide on, 41 genlyase, 7 1 Alcaligenes faecalis, gas production potentiation of effects of, by presfrom nitrite by, 1 0 sure, 179 nitrite reduction by, 1 3 response of bacterial respiration rate Alcian-blue binding, use of, in studies to, 257 on yeast wall mannans, 106 393

394

SUBJECT INDEX

Alkali stability of yeast wall mannans, 97 Alkaline p-elimination reactions on yeast mannan, 113 Alkaline degradation of mannan from wall of Saccharomyces cerevisiae, 110 Alkaline phosphatase, effect of pressure on, 208 Alkaline phosphomonoesterase, action of, on yeast wall mannans, 104 Allosteric control of metabolism by adenosine nucleotides, 268 Amines, volatile, production of, 56 Amino-acid composition of 5-agglutinin from Hansenula wingei, 135 Amino-acid composition of ribulose diphosphate carboxylases, 63 Amino-acid oxidases in microbes, 29 Amino-acid residues in yeast wall mannans, 98 Amino-acid uptake, effect of oxygen on, in bacteria, 36 L-Amino acids, effect of pressure on transport of, 2 11 Ammonia assimilation, nature of, 8 Amylase, bacterial, effect of pressure on activity of, 2 12 Anacystis nidulans, effect of pressure on photosynthesis by, 220 Anaerobes, obligate, hydrogen production by, 68 occurrence of nitrogenase in, 19 Anaerobic bacteria, effect of pressure on yields of, 178 Anaerobic growth of Klebsiella aerogenes, 253 Anaerobic shock, effect of, on contents of nicotinamide nucleotides in Klebsiella aerogenes, 285 Anaero vibrio lip0 ly tica, cytochrom es in, 350 Anomerization during acetolysis of yeast wall mannans, 100 Antagonism between pressure and temperature responses of microorganisms, 180 Antigenic determinants of yeast wall mannans, 105

Antisera, use of, in studies on yeast wall mannans, 106 Arabinose, effect of pressure on degradation of, 232 L-Arabinose in yeast wall mannans, 97 Argininosuccinase, effect of pressure on, 207 Arthrobacter crystallopoietes, effect of pressure on longevity of, 191 Asparagine residues, in yeast manna1 structure, 122 in yeast wall mannans, 9 8 Aspartate as a product of carbon dioxide fixation in plants, 60 A s p er
SUBJECT INDEX

B Bacillus borborkoites, tolerance of pressure by, 174 B. cereus, effect of pressure on, 187 nitrate reductase synthesis in, 335 B. coagulans, effect ofpressure on, 187 B. licheniformis, effect of pressure on induction of exopenicillinase by, 232 electron transfer to nitrate in, 349 genetics of nitrate reductase in, 359 B. meguterium, absence of glutamate dehydrogenase from, 26 B. mycoides, induction of filament formation in, by pressure, 230 B. popilliae, superoxide dismutase of, 45 B. stearo thermophilus, cytochrome synthesis in, 340 effect of pressure on, 182 malate dehydrogenase of, 184 electron transfer to nitrate in, 349 solubilization of nitrate reductase from, 3 19 B. subtilis, cytochrome mutant of, 295 effect of pressure on amylase of, 212 effect of pressure on transforming deoxyribonucleic acid of, 193 effect of temperature on, 176 synchronous cultures of, 41 Bacteria, growing, regulation of respiration rate in, 243 obligately barotolerant, possible existence of, 176 Bacterial cytochrome oxidases, 30 Bacterial cytochromes, inducibility of, 29 1 Bacterial endospores, effect of pressure on, 186 Bacterial luminescence, effect of pressure on, 233 Bacterial mannanases, nature of, 104 Bacterial ribosomes, effect of pressure on, 209 Bacteria1 superoxide dismutase, 44

395

Bactericidal effect of oxygen on microbes, 42 Bacterium candicans, effect of pressure on gelatinase of, 213 B. formoxidans, carbon dioxide fixation by, 57 Barobiology, physiological aspects of, 159 Baroduric bacteria, 227 nature of, 174 Barokam, construction of, 165 name for a pressure vessel used in microbial studies, 164 Barophilic marine organisms, isolation of, 160 Barophobic bacteria, nature of, 1 7 7 Barotolerances among microorganisms, 174 Base-labile oligosaccharides in yeast wall mannan, 118 Bauchop-Elsden values, effect of nitrate respiration on, 352 effect of pressure on, 177, 217 for bacteria, 303 Beneckea natriegens, branched electron-transport chain in, 301 cytochrome oxidase activity of, 293 effect of glucose concentration on respiration of, 260 effect of temperature on respiration rate of, 257 response of, to dissolved oxygen content, 252 Benson-Calvin cycle of carbon dioxide fixation, 57 Benthos, nature of, in oceans, 160 Benzylviologen, as an electron acceptor in assay of nitrate reductase, 316 effect of, on formation of nitrate reductase, 334 Beta -Galacto sidase, effect of pressure on, 215 effect of pressure on induction of, 231 Beta-Galactosides, permeability of, effect of pressure on, 21 1 Bilayers, vesicle, effect of pressure on fluidity of, 210

396

SUBJECT INDEX

Binding sites on agglutinin from Hansenula wingei, 1 3 4 Biochemical basis of oxygen toxicity, 34 Biochemical reactions, volume changes during, 17 1 Biochemistry of nitrate reductase in bacteria, 31 5 Biomass production by Streptococcus faecalis, effect of pressure on, 179 Biomass yield, effect of pressure on, 177 Biopolymer synthesis, effect of pressure on, 2 2 2 Biopolymers, bonds in, effect of pressure on, 1 8 3 effects of pressure on, 191 Biosynthesis of the mannan of the yeast cell envelope, 93 Biosynthesis of yeast mannans, 138 Biphasic derepression of nitrate reductase in Escherichia c o l i , 5 1 Blood coagulation, effects of pressure on, 2 0 5 Blood, human, and superoxide dismutase activity, 4 3 Bovine erythrocytes, amino-acid composition of superoxide dismutase of, 4 7 Branched electron-transfer chains in bacteria, 30 Branched electron-transport systems in bacteria, 297 Breakage of chemical bonds, volume changes associated with, 173 Bud scars, location of chitin in, on the yeast wall, 9 5

C Candida albicans, structure of mannan from, 1 3 6 C. atrnospherica, structure of mannan from, 137 C. diddensii, structure of mannan from, 137 C. parapsilosis, structure of mannan from, 1 3 6 C. stellatoidea, structure of mannan from., 1 3 6-

C. utilis, oxidative phosphorylation in, 305 Candidas, mannans from, 136 Carbohydrate composition of yeast mannans, 9 6 Carbohydrate in yeast invertase, 121 Carbon compounds, gaseous, formation of, 5 5 reduced, metabolism of, 6 4 Carbon dioxide, fixation pathway, criteria for establishing, 6 0 formation of methane from, 56 in the biosphere, 1 utilization of, 56 Carbon monoxide, action spectroscopy, and bacterial cytochromes, 32 binding cytochromes of bacteria, 302 effect of, on Beneckea natriegens, 301 inhibition of nitrogenase, 18 metabolism of, 6 5 production by microbes, 3 stimulation of oxygen reduction by, in bacteria, 6 6 Carbonic anhydrase activity in microorganisms, 4 Carbowax, use of, in pressure studies, / 165 I Carboxylation of phosphoenol-\ pyruvate, 5 8 Catabolic processes, effects of pressure on, 2 1 2 Catabolism, bacterial, stimulation of, 275 Catalase, and resistance to oxygen, 4 1 as a protective enzyme, 4 1 in micro-organisms, 29 Cell division, effect of pressure on, 229 in yeast mannan mutants, 116 Cell envelope, organization of mannan in, 94 yeast, structure and biosynthesis of mannan of, 9 3 Cell separation, effect of pressure on, 223 Cell yield, relation of, to dissolved oxygen content, 248

SUBJECT INDEX

Cells, growing, steady-state contents of adenosine phosphates in, 265 Cell-wall formation in bacteria, effect of pressure on, 229 Cell-wall mannans of Hansenula wingei, 13 1 Centromere linkage in yeast mannan mutants, I 1 3 Characterization of mannan mutants of Saccharomyces cerevisiae, 11 1 Charged groups, changes in, during denaturation, 200 Chemical bond breakage, volume changes associated with, 173 Chemolithotrophs, nature of, 2 Chemo-organotrophs, cytochrome oxidases in, 30 Chemostat culture of Klebsiella aerogenes, dissolved oxygen tension in and respiration rate, 247 Chicken cytoplasm, amino-acid composition of superoxide dismutase of, 47 Chicken mitochondria, amino-acid composition of superoxide dismutase of, 4 7 Chitin, location of, in the yeast wall, 95 Chitinase , bacterial, effect of pressure on activity of, 212 Chlamydomonas reinhardi, nitrate reductase of, 343 Chlorate, differentiation of reduction of, from that of nitrate, 317 reducing enzymes in bacteria, 3 16 resistance, and nitrate reductase mutants of bacteria, 356 resistance genes, map position of, in Escherichia coli, 358 resistant mutants of bacteria, complementation between in vivo, 364 physiological properties of, 360 proteins in membranes of, 362 Chlorella fusca, nitrite reductase in, 10 Chlorobium spp., carbon dioxide fixation by, 58 Chromatium sp., changes in adenosine nucleotide contents of, 276 properties of hydrogenase from, 76

397

Chromosome movement, effect of pressure on, 206 Chromosome replication, effect of pressure on, 223 Chymotrypsinogen, pressure denaturation of, 196 Citrate lyase activity, and carbon dioxide fixation, 58 Citro bacter freundii, chlorate resistance genes in, 359 Citrobacter sp., effect of nitrate on specific growth rate of, 355 effect of nitrate respiration on molar growth yield of, 353 Clostridium acetobutylicum, effect of oxygen on, 38 effect of redox potential on enzyme synthesis by, 5 1 Cl. butylicum, properties of hydrogenase from, 76 Cl. pasteurianum, nitrite reductase in, 10 nitrogenase of, 15 structure of hydrogenase from, 73 Cl. perfringens, effect of nitrate respiration on molar growth yield of, 353 Cold lability, relation of, to pressure sensitivity, 208 Complementation between chlorateresistant mutants of bacteria, in vivo, 364 Compressibilities of proteins, and pressure, 197 Concept of energy charge, 267 Conjugation, use of, to map nitrate reductase mutants of bacteria, 357 Conservation of energy in bacteria, 303 Constant pressure contours for metmyoglobin denaturation, 198 Continuous culture, monitoring of contents of nicotinamide nucleotides in, 279 Contraction accompanying protein denaturation, 201 Control of enzyme synthesis by oxygen, 4 8 Control of respiration rate by substrate in bacteria, 259

398

SUBJECT INDEX

Conversion of substrate into cell mass, 245 Copper in bacterial hydrogenases, 76 Copper ions and activity of superoxide dismutase, 44 Corrosion of metals, effect of pressure on, 220 Corynebacterium nephridii, nitrous oxide production by, 10 Creatine kinase, effect of pressure on, 208 Cross reactivity of nitrogenases, 20 Cryptococcus laurentii, mannan biosynthesis in, 150 Cyanate, effect of, o n inactivation of nitrate reductase, 342 Cyanide, sensitivity of bacteria to, 298 sensitivity of bacterial cytochromes, 32 Cyanobacteria, carbon dioxide fixation by, 60 nitrogen fixation by, 1 5 Cyanogen as a terminal electron acceptor, 21 Cyclic adenosine monophosphate, regulation of bacterial respiration by, 263 Cycloheximide, effect of, on wall regeneration by yeast protoplasts, 145 Cysteine residues in yeast mannan structure, 123 Cytochalasin A, effect of, on invertase secretion by yeast protoplasts, 146 Cytochrome b , presence of, in formate dehydrogenase, 324 Cytochrome c, and superoxide dismutase activity, 43 peroxidase of Pseudomonas fluorescens, 297 Cytochrome-deficient mutants of bacteria, 33 Cytochrome mutant of Bacillus subtilis, 295 Cytochrome oxidase activities of micro-organisms, 49 Cytochrome oxidase activity of bacteria, effect of oxygen on, 292 Cytochrome oxidase, bacterial, turnover number of, 294

Cytochrome oxidases, bacterial, 30 Cytochrome synthesis, effect of oxygen on, 31 Cytochromes, as regulators of respiration, 290 bacterial, and electron transport to nitrate, 346 effect of chlorate resistance on, 3 60 inducibility of, 29 1 genes for, in Escherichia coli, 34 o l sulphate-reducing bacteria, 73 Cytoplasmic membrane, association of bacterial nitrate reductase with, 318

D Deamination, effect of pressure on, 216 Death of micro-organisms under pressure, 174, 185 Decarboxylases, action of, in carbon dioxide production, 55 Deep ocean as a source of barotolerant micro-organisms, 175 Deep sea, effects of pressure on microbial activities in, 222 Deep-sea fish, effect of pressure on enzymes of, 219 Degradation, enzymic, of yeast wall mannans, 103 of organic compounds, effect of pressure on, 212 Dehydrogenases, bacterial, effect of pressure o n activity of, 215 Deletion mutants of nitrogenase, 23 Denaturation, of deoxyribonucleic acid by heat, 194 of mannan-protein complex from yeast walls, 99 of proteins by pressure, 184, 190, 195 Denitrification, by micro-organisms, 7 nature of, 3 the process, 316 Denitrifying bacteria, properties of nitrite reduction systems in, 1 3

SUBJECT INDEX

399

Deoxycholate, use of, to solubilize Dithiol, effect of, on bacterial nitrate bacterial nitrate reductases, 3 19 reductases, 329 2-Deoxyglucose, effect of, on wall Dithiothreitol, effect of, on agglutinin from Hansenuta wingei, 1 3 3 regeneration by yeast protoplasts, effect of, on extraction of yeast 145 invertase, 1 2 3 Deoxyribonucleic acid, biosynthesis, Diversity of oxygen metabolism in effect of pressure on, 1 9 3 , 223 micro-organisms, 29 Desulfovibrio desulfuricans, metabolism of carbon monoxide by, 6 5 Dolichol phosphates in mannan biosynthesis in yeast, 1 4 4 nitrite reduction by, 1 3 D. gipas, cytochromes of, 3 5 0 D. vulgaris, cytochromes of, 7 3 hydrogenase of, 3 1 6 structure of hydrogenase from, 73 Desulfovibrio hydrogen metabolism,

71 Desulfovibrio spp., tolerance of pressure by, 1 7 4 Desulfovibrios, hydrogen production by, 6 8 Detergents, use of, to solubilize bacterial nitrate reductases, 320 Di-N-acetylchitobiose units in yeast mannans, 1 2 2 Diethyl sulphide, production of, by yeasts, 81 Digestion of mannan from Saccharomyces cerivisiae, 118 Dimethyl sulphide, formation of, by yeasts, 81 production by microbes, 3 Dinitrogen reduction, mechanism of, 17, 1 8 Dinitrogen, transport of, into bacteria, 19 Di-oxygenases, action of, in microbes, 29 Diplococcus sp., operation of the serine pathway in, 6 5 Dissimilatory nitrate reductase, effect of oxygen on activity of, 3 3 9 regulation of formation of, 332 Dissociation of hydrophobic interactions by pressure, 1 7 2 Dissociation of ribosomes, effect of pressure on, 2 0 9 Dissolved oxygen tension, response of respiration rate to, 246 Disulphide bonds in yeast mannan structure, 1 2 3

E

Effects of pressure on biopolymers, 191 Effects of pressure on polymeric interactions, 2 0 2 Egg albumin, effect of pressure on, 195 Electron acceptors, provision in studies on effects of pressure, 1 6 6 Electron paramagnetic signals of bacterial nitrate reductases, 33 1 Electron-transfer chains, rate-limiting action of flavoprotein dehydrogenases in, 5 3 Electron-transport chain to nitrate in bacteria, 3 4 3 Electron-transport systems, branched, in bacteria, 297 Elongation of bacteria, effect of pressure on, 229 Embde n-Mey er ho f path way, changes in control of, affected by dissolved oxygen tension, 2 7 4 Endo-0-acetyl-D -glucosaminidase, use of, in degradation of yeast wall mannans, 1 0 4 Endospores, bacterial, effect of pressure on, 1 8 6 Endosymbiotic origin of mitochondria, evidence for, 48 Energy charge, concept of, 267 of growing Klebsiella aerogenes, 2 6 6 Energy conservation, during nitrate respiration, 303, 343, 3 5 0

400

SUBJECT INDEX

Energy-conserving mechanisms in bacteria, uncoupling of, from growth, 255 Energy metabolism of sulphatereducing bacteria, 72 Envelope, yeast cell, structure and biosynthesis of mannan of, 93 Environmental changes and respiration rate, 245 Environmental factors, effect of, on microbial responses to pressure, 180 Enzyme synthesis, control of, by oxygen, 48 Enzymes, effect of pressure on, 207 gas, evolution of, in microbes, 82 Enzymic defects in yeast mannan mutants, 112 Enzymic degradation of yeast wall mannans. 103 Enzymic protection against oxygen toxicity, 40 Erwinnia spp., absence of glutamate dehydrogenase from, 26 Escherichiu coli, amino-acid composition of superoxide dismutase of, 47 biphasic depression of nitrate reductase in, 51 cytochrome oxidase activity of, 292 effect of acidity o n respiration rate of, 257 effect of molybdate on formation of nitrate reductase by, 326 effect of oxygen on content of nicotinamide nucleotides in, 283 effect of oxygen on enzyme synthesis by, 48 effect of pressure, on biomass production by, 179 on biopolymer synthesis by, 223 on flagella formation by, 162 on permeability of, 2 11 on respiration by, 2 15 on yields of, 180 electron paramagnetic resonance signals of nitrate reductase of, 331 electron transport to nitrate in, 344 gas production from nitrite by, 10

hydrogenase synthesis by, 70 map position of chlorate resistance genes in, 358 oxidative phosphorylation in, 351 properties of hydrogenase from, 76 -properties of nitrate reductase from, 321, 323 response of, to dissolve oxygen tension, 250 solubilization of nitrate reductase from, 3 19 superoxide dismutase from, 45 synchronous cultures of, 41 synthesis of cytochrornes in, 34 synthesis of phosphofructokinase in, 276 toleration of hyperbaric oxygen by, 167 Esterification of veast wall mannans. 104 Ethanol as an energy source for Acetobacter aceti, 267 Evolution of gas enzymes in microbes, 82 Evolution of nitrogenase, 19, 20 Exergonic metabolic processes, effect of pressure on, 220 of Exocellular phosphomannan Hansenula species, 129 Exocellular phosphomannans from species of Hansenula, 104 Exomannanses, use of, to degrade yeast wall mannans, 103 Exopenicillinase, effect of pressure on induction of, 232 Extracellular redox potential, effect of, on growth rate of Clostridium acetobutylicum, 39

F 21-Factor from Hansenulu win& interaction with 5-agglutinin, 135 Facultative anaerobes, formate hydrogenlyase activity of, 70 Feedback control of glycolysis in bacteria, 276

SUBJECT INDEX

401

Ferromanganese residues, effect of G pressure on formation of, 221 Fibrinogen, effects of pressure on Galactomannans from yeasts, strucconversion of, to fibrin, 205 tures of, 137 Filament formation, by bacteria, and D-Galactose in yeast wall mannans, 97 pressure, 222 Gas enzymes, evolution of, in by Serratia marinorubra, and presmicrobes, 82 sure, 229 Gas metabolism, microbial, 1 Fingerprints, acetolysis, of yeast Gas production from nitrate, 9 mannans, 114 Gas vacuoles of halobacteria, effect of Fixation of nitrogen by microbes, pressure on, 162 14 Gaseous carbon compounds, formaFlagella formation, effect of pressure tion of, by micro-organisms, 55 on, 162 Gaseous intermediates in the nitrogen Flagellin, effects of pressure on aggrecycle, 7 gation of, 203 Gases, metabolism in relation to Flavoprotein dehydrogenases, ratevolatility by micro-organisms, 5 limiting effect of, in electronprincipal ones in the biosphere, 1 transfer chains, 53 Gelatinase, bacterial, effect of pressure Flavoprotein oxidases in microbes, on, 212 Gene expression, autogeneous regu29 lation of, 337 Fluidity of vesicle bilayers, effect of Genetic loci in yeast mannan mutants, pressure on, 210 113 Fluorocarbon liquids, use of, in Genetic regulation of nitrogenase, 22 pressure studies, 166 Formaldehyde fixation by bacteria, Genetics of nitrate reductase, formation, 356 65 Formate dehydrogenase, bacterial, in bacteria, 315 solubilization of, 320 Germination of bacterial spores, effect effect of chlorate resistance on, 360 of pressure on, 187 presence of selenium in, 326 Glucanase sensitivity of yeast mannan Formate hydrogenlyase activity, in mutants, 115 aerobic bacteria, 48 Glucanases, action of, on yeast walls, of facultative anaerobes, 70 99 of Proteus mirabilis, 54 location of, in the yeast wall, 95 regulation of, 7 1 Glucose, degradation by bacteria, Formation of gaseous carbon comeffect of pressure on, 178 pounds by micro organisms, 55 limitation, effect of, on energy Fragments, isolation from the yeast charge of Klebsiella aerogenrs, mannan inner core, 119 266 Frequency of recombination between oxidase in fungi, 29 different nif mutation sites, 24 oxidation of, by Klebsiclla aeroL-Fucose in yeast wall mannans, 97 genes, 255 Fumarate, effect of, on respiration 6-phosphate, changes in contents of, rate of Klebsiella aerogenes, 260 during oscillations in nicotinamide reductase, effect of chlorate resistnucleotide fluorescence in ance on, 361 Klebsiella aerogenes, 288 reduction by sulphate-reducing bac- D-Glucose in yeast wall mannans, 97 D-Glucuronic acid in yeast wall teria, 73 Fungal glucose oxidase, 29 mannans, 97

402

SUBJECT INDEX

Glutamate dehydrogenase activity in microbes, 26 Glutamate synthesis by microbes, 26 Glutamine synthetase, role of, in regulation of nitrogenase synthesis, 9 Glycolysis, effect of pressure on, 214 effect of pressure on rate of, 173 intermediates, effect of oxygen tension of contents of, in cells, 275 volume changes during, 170 Glycoprotein nature of agglutinin from Hansenula wingei, 133 Glycosylation sequence in yeast mannan biosynthesis, 152 Glycosyl-serine linkages in yeast wall mannan, 99 Glycosyl-threonine linkages in yeast wall mannan, 99 GOGAT pathway for ammonia assimilation, 26 Growing bacteria, regulation of respiration rate in, 243 Growing cells, steady-state contents of adenosine phosphates in, 265 Growth, bacterial, efficiency of, under pressure, 179 Growth of micro-organisms, in relation to energy charge, 268 Growth phases, bacterial, effect of pressure on, 177 Growth rate, of bacteria, and respiration rate, 258 response of, to change in temperature with bacteria, 256 specific, effect of nitrate on, for bacteria, 355 Growth, stimulation of, under pressure, 180 under pressure, 174 Guanosine diphosphate mannose, incorporation of, into mannan by yeast, 140

H Hadal regions of the ocean, nature of, 160

Haem, as a component of nitrite reductases, 12 Haemo p hilu s parainf l u enzae , cy t ochrome oxidase activity of, 292 response of, to oxygen tension, 250 Haemoproteins of sulphate-reducing bacteria, 73 Hafnia sp., enzymes in, affected by chlorate resistance, 361 Halobacterium spp., effect of pressure on gas vacuoles of, 162 Hansenula capsulata, exocellular phosphomannan of, 130 mannan biosynthesis in, 1 0 phosphomannan of, 131 H. holstii, mannan biosynthe is in, 150 mannobiose phosphates derived from, 30 phosphomannan of, 131 H. wingei, cell-wall mannans of, 131 wall mannan of, 94 Hansenula spp., exocellular phosphomannan of, 129 mannans from, 104 Hatch-Slack-Kortschak pathway of carbon dioxide fixation, 58 Heat denaturation of deoxyribonucleic acid, 1 9 4 Heat inactivation of spores, effect of pressure on, 187 Heat, use of, to solubilize bacterial nitrate reductases, 319 Helix stabilization of deoxyribonucleic acid, by pressure, 193 H e m mutants, properties of nitrate reductases from, 324 Heteropolysaccharides, cell-wall, in Cryptococcus laurentii, 150 Heterotrophic micro-organisms, effect of carbon dioxide on, 56 High-pressure chemistry, 168 High-pressure microbial physiology, 159 Histidase genes, inactivation of transcription of, by glutamine synthetase, 27 Histidase synthesis, repression of, by ammonia, 25 Homo logy between amino -acid co mposition of ribulose diphosphate

t

SUBJECT INDEX

403

carboxylases from various bacteria, Hydrostatic pressure, effect of, on 64 biopolymers, 161 Hydrogen, acceptors, effect of, on Hydroxylamine, and the nitrogen cycle, 7 formation of nitrate reductase by as an intermediate in fungal nitrite bacteria, 334 reduction, 1 0 bonding, decrease in volume during, Hyperbaric oxygen, and induction of 172 synchrony, 41 enthalpy changes in, 183 response of vibrios to, 35 cycle, microbial, 80 Hyperbaric oxygenation, provision of, dehydrogenase, nature of, 80 167 in the biosphere, 1 metabolism, 67 Hyphomicrobium sp., denitrification aerobic, 76 by, 10 by Desulfovibrio spp., 7 1 by hydrogenomonads, pathways I for, 79 peroxide, toxicity of, to microbes, Imidazole groups in proteins, proton41 ation of, 200 production of, by micro-organisms, Immunochemical characteristics of 67 yeast mannans, 128 sulphide, production of, by yeasts, Immunochemical determinants of the 81 yeast wall mannans, 100 Hydrogenase action, and that of Immunochemistry of yeast wall nitrogenase, 67 mannans, 105 Hydrogenase activity, as a metabolic Inactivation of microbial enzymes by valve, 68 oxygen, 35 in cultures of aerobic bacteria, 48 Inducibility of bacterial cytochromes, in micro-organisms, 67 29 1 Hydrogenase, induction of synthesis Inner core in mannan of Saccharoof, 78 myces cerevkiae, characterization molecular structure of, 73 of, 1 1 7 of sulphate-reducing bacteria, 7 1 Inorganic nitrogen metabolism, reguHydrogenases, bacterial, isoenzymes lation of, 25 of, 74 Interactions, polymeric, effects of bacterial, properties of, 75 pressure on, 202 Hydrogenomonads, amino-acid com- Intergeneric transfer of nitrogenposition of ribulose diphosphate fixation genes, 24 carboxylases from, 63 Intermediates, lipid-bound, in mannan metabolism of hydrogen by, 77 biosynthesis in yeast, 143 nature of, 2 Intracellular location of mannan synpathways for hydrogen metabolism thetases in yeast, 146 by, 79 Intracellular redox potentials in micHydrogenomonas eutropha, ribulose robes, 35 diphosphate carboxylase of, Intracellular water activity, effect of pressure on, 162 62 H. ruhlandii, hydrogen dehydrogenase Invertase, excretion of, by yeast protoplasts, 146 from, 8 0 from Saccharomyces spp., 94 Hydrophobic interactions, and presin yeast walls, 99 sure, 183 of yeast mannan mutants, 116 dissociation of, by pressure, 172

404

SUBJECT INDEX

Invertase-continued release of, from mannan mutants, 116 Ionic bond formation, volume changes during, 183 Ionic composition of growth media, and pressure responses of microbes, 185 Ionic strength, effects of, on pressure denaturation of proteins, 195 Ionization of lactic acid, volume change during, in glycolysis, 170 Iron, content in medium, and synthesis of formate hydrogenlyase, 71 role of, in enzyme synthesis, 50 uptake, effect of pressure on, 212 Isolation, of mannan mutants of Saccharomyces cerevisiae, 111 of yeast wall mannan, 99 Isonitriles as terminal electron acceptors, 2 2

K Klehsiella aerogenes, contents of nicotinamide nucleotides in, during transition from anaerobic to aerobic conditions, 280 cytochrome oxidase activity of, 293 effect of nitrate respiration on molar growth yield of, 353 electron paramagnetic resonance signals of nitrate reductase of, 331 electron transfer to nitrate in, 345 energy charge of growing, 266 mutants of deficient, in nitrate reductase, 356 oxidase activities and cytochrome contents of, 347 possible existence of a branched electron-transport chain in, 300 properties of nitrate reductase of, 320 response of respiration rate of, to dissolved oxygen tension, 246 scheme for electron transport in, 34 a

solubilization of nitrate reductase from, 319 steady-state contents of adenosine phosphates in, 265 transient responses of, to dissolved oxygen tension, 253 K.' pneumoniae, nitrogen fixation by, 23,326 nitrogenase from, 1 6 Kloeckera brevis, structure of mannan in wall of, 109 Kluyveromyces dobrhanskii, structure of mannan of, 128 Kluyv. fragilis, antigenici y of, 106 Kluyv. lactis, mannan bi synthesis in, 147 mannan mutants of, 129 structure of mannan of, 128 Kluyv. marxianus, structure of mannan of, 128 Kluyveromyces sp., mannan in the wall o f , 97 Knallgas reaction, nature of, 2

k

L Lactic acid bacteria, superoxide dismutase of, 4 4 Lactic acid production, effect of pressure on, 214 Lacto bacillus casei, protection against oxygen toxicity in, 41 L. plantarum, absence of superoxidase dismutase from, 45 Lake Baykal, microbes from, 161 Leakage of potassium, effect of pressure on, 211 Lecithin aggregates, effect of pressure on, 210 Life under pressure, 174 Light, effect of, on synthesis of adenosine nucleotides by Chromatium sp., 277 Lipid-bound intermediates in biosynthesis of yeast mannan, 143 Lipid-lipid interactions, effect of pressure on, 209 Loci, genetic, of yeast mannan mutants, 113

SUBJECT INDEX

Longevity of Arthro bacter crystallopoietes, effect of pressure on, 191 Long-term survival under pressure, 174 Low temperature spectroscopy in the study of bacterial cytochromes, 3 3 Luminescence, microbial, effect of pressure on, 233 Lysogeny, effect of pressure on, 186 Lysozyme, effect of pressure on, 208 effect of pressure on action of, on Micrococcus luteus. 214

M Macromolecular structure of the mannan of Saccharomyces cerevisiae, 107 Maintenance energy of bacteria, 304 Maintenance respiration rate of bacteria, 258 Malate, as a product of carbon dioxide fixation in plants, 60 dehydrogenase, effect of pressure on activity of, 216 of Bacillus stearothermophilus, effect of pressure on, 184 Maltose, effect of pressure on degradation of, 232 Manganese, bacterial superoxide dismutase and, 44 oxidation by bacteria, effect of pressure on, 2 11 Mannan, Saccharomyces cerevisiae, structure of, 97 Mannan biosynthesis, in Hansenula spp., 149 yeast, model for, 151 Mannan from Saccharomyces cerevisiae, detailed structure of, 120 Mannan inner core of SaccharomycPs cerevisiae, characterization of, 117 Mannan mutants, in yeasts, phenotypes of, 115 of Saccharomyces cerevisiae, isolation of, 11 1 use of, in studies on mannan biosynthesis in yeast, 139 Mannan, organization of, in the yeast wall, 94 protein, and invertase in yeast, 121

405

secretion of, by yeast protoplasts, 145 side chains, yeast, possible sequences in, 123 structure in yeast, alleles for, 111 synthetases, location of, in yeast, 146 yeast, biosynthesis of, 121, 138 yeast cell envelope, structure and biosynthesis of, 9 3 yeast wall, extraction of, 94 Mannanases, effect of, on mannail from Hansenula wingei, 132 use of, to degrade yeast wall mannans, 104 to elucidate mannan structure, 117 Mannans, cell-wall, structure of, from Hansenula wingei, 134 yeast, carbohydrate composition of, 96 properties of, 9 6 structural analysis of, 99 structures of, 107 Mannitol, growth of Klebsiella aerogenes on, 354 Mannobiose residues, release of, from yeast mannans, 124 Mannoaligosaccharides, as side chains in yeast wall mannans, 108 production of, during degradation of yeast wall mannans, 104 Mannose as a component of yeast wall mannans, 96 Mannosidades, use of, in elucidation of structure of yeast wall mannans, 103 Mannosyl dolichol phosphate, role of, in yeast mannan biosynthesis, 144 Mannosyl-lipid, role of, in mannan biosynthesis in yeast, 143 Mannosyltransferases, multiplicity of, in yeast, 139 of Kluyveromyces lactis, 148 role of, in mannan synthesis, 112 Mannotriose residues, release of, from yeast mannans, 124 Mapping of nitrate reductase mutants, 357 Marine mussel, effect of pressure on, 185

406

SUBJECT INDEX

Medium composition, effect of, on response of micro-organisms to pressure, 184 effect of, on synthesis of formate hydrogenlyase, 7 1 Membrane, as a location for the hydrogenase of Desulfovibrio vulgaris, 74 bound cytochromes in bacteria, 33 bound enzymes in mannan biosynthesis in yeast, 140 particles from bacteria, oxidative phosphorylation by, 304 Membranes of chlorate-resistant mutants of bacteria, proteins of, 362 Metabolism, of $s by microbes, importance o solubility, 6 of gases in micro-organisms, 1 of hydrogen, 67 of oxygen by microbes, 29 of reduced carbon compounds, 64 Metalloproteins, and gas metabolism by micro-organisms, 82 Metals in bacterial nitrate reductases, 329 Methane formation by bacteria, 55 Methane hydroxylase, activity of, 67 Methane metabolism by microorganisms, 2 Methane oxidation, mechanism of, 66 Me than o ba cteriu m bar kerii, methane formation by, 56 M. omelianskii, mixed nature of, 56 Methanol, as a source of methane in bacteria, 55 effect of concentration of, on yield value of Pseudomonas extorquens, 259 growth of pseudomonads on, 250 Methanomonas methano-oxidans, operation of the serine pathway in, 65 Methodology in studies on effects of pressure on micro-organisms, 163 Methods for assaying adenosine phosphate contents of cells, 264 Methylation analysis of yeast wall mannans, 107

Methylene blue, effect of, on formation of nitrate reductase, 334 Methylocystis capsulatus, methane fixation by, 65 Methylosarcinus trichsporium, carbon monoxidase oxidation by, 66 Metmyoglobin, pressure denaturation - o f , 191 Microbial gas metabolism, 1 Microbial physiology, high-pressure, 159 Microbial cell functions, specific, effect of pressure on, 21 1 Micrococcus aquivivus, tolerance of pressure by, 174 M. denitrificans, cytochrome oxidase activity of, 291 electron paramagnetic resonance signals of nitrate reductase of, 33 1 metabolism of hydrogen by, 77 nitrate reductase of, 3 1 7 oxidative phosphorylation in, 351 properties of nitrate reductase from, 321 solubilization of nitrate reductase from, 319 M. euryhalis, tolerance of pressure by, 174 M. halodenitrificans, solubilization of nitrate reductase from, 319 M. luteus, effect of pressure on action of lysozyme on, 213 salt concentration and effect of pressure on, 185 M. lysodeikticus, effect of pressure on, 189 Microtubules, effect of pressure on, 206 effects of pressure on formation of, 205 Mitochondria, origin of, and superoxide dismutase, 46 yeast, biogenesis of, 30 effect of oxygen on composition of, 38 Mitochondria1 respiration, identity of rate-limiting components, 244 Mitotic spindles, effect of pressure on, 206

SUBJECT INDEX

407

Mixed function oxidases in microbes, genetic sharing of genetic determi29 nants in, for nitrate reductase and Model for mannan biosynthesis in nitrogenase, 28 yeast, 15 1 nitrate reductase of, 328 Molar growth yields, effect of nitrate Nicotinamide adenine dinucleotide, respiration on, 3 5 2 oxidized and reduced couple, and Molecular barophysiology, nature of, regulation of enzyme synthesis, 52 161 redox potential in microbes, 35 Molecular orbitals and oxygen, 4 2 Nicotinamide nucleotide contents of Molecular oxygen as an effector in cells, effects of environment on, protein synthesis, 4 8 281 Molybdate, role of, in formation of measurement of, 278 nitrate reductase, 325 Nicotinamide nucleotides, role of, in Molybdenum, in bacterial hydrogenregulation of respiration rate, 277 ases, 73 nifgenes, evolution of, 21 in bacterial nitrate reductases, 3 2 1 transference of, 2 4 Molybdoferredoxin, from Clostridium Nitrate, ability of, to induce formation pasteurianum, 15 of dissimilatory nitrate reductase, Molybdoproteins, in Aspergillus nidu332 and the nitrogen cycle, 7 lans, 327 Morphological differentiation, effect as a terminal electron acceptor in of pressure on, 2 2 9 pressure studies, 166 Motility, microbial, effect of pressure as a terminal hydrogen acceptor, on, 233 316 Multimers, enzymic, effect of pressure assimilation, repression of, by ammonia, 2 5 on association of, 207 differentiation of reduction of, from Mutagen, pressure as a, 175 that of chlorate, 317 Mutants, barotolerant, inability to electron transport to, in bacteria, produce, 175 343 mannan, of Saccharomyces cerereductase and nitrogenase, sharing of uisiue, isolation of, 11 1 genetic determinants by, 2 8 nitrate respiration, isolation of, 3 5 6 reductase, bacterial, electron paraof Saccharomyces cerevisiae, varimagnetic resonance signals of, ations in structure of mannan in, 331 107 bacterial, metals in, 3 2 9 Mutation rates, bacterial, effect of properties of, 316 oxygen on, 3 6 complex in Escherichia coli, 3 4 6 Myosin, effects of pressure on aggreformation, genetics of, 3 5 6 gation of, 2 0 3 in bacteria, biochemistry and genetics of, 315 N inactivation of, 3 4 0 mutants, bacterial, mapping of, Native conformations of proteins, 199 357 Nearest neighbour analysis, of side of Proteus mirabilis, effect of chains of yeast mannans, 1 2 4 conditions on synthesis of, 3 1 8 of yeast wall mannan structure, 1 0 3 regulation of synthesis of, in Neurospora complementation factor, Proteus mirabilis, 5 4 nature of, 365 regulation of formation of, 3 3 2 Neurospora crassa, effect of pressure repression in Escherichia coli, 5 0 on, 175

408

SUBJECT INDEX

Nitrate-continued reductase-continued role of molybdate in formation of, 325 synthesis of, in Aspergillus niclulans, 337 reductases, purification and properties of, 318 reduction by microbes, 7 respiration, effect of on molar growth yields, 352 energy conservation during, 343 in bacteria, and nitrate reductase, 316 mutants, isolation of, 356 utilization by bacteria, 31 5 Nitric oxide reductase of pseudomonads, 11 Nitriles as terminal electron acceptors, 22 Nitrite, ability of to induce synthesis of nitrate reductase, 339 gas production from, 9 reductase in fungi, location of, 10 Nitrogen evolution by pseudomonads, 10 Nitrogen fixation, microbial, 8, 14 Nitrogen-fixing bacteria, respiratory protection in, 31 Nitrogen gas in the biosphere, 1 Nitrogen gases, microbial metabolism of, 7 Nitrogen limitation, effect of, on energy charge of Klebsiella aerogenes, 266 Nitrogen oxide production by microbes, 3 Nitrogenase, concentration of, in anaerobes, 2 1 evolution of, 1 9 , 2 0 genetic regulation of, 22 of Azoto bacter vinelandii, presence of tungsten in, 329 protection of, in bacteria, 299 synthesis, inability of dinitrogen to induce, 25 Nitrogenases, bacterial, I5 proDerties of, 15 Nitrosomonas europea, oxidation of

- -

ammonia by, 9 Nitrous oxide, production of, by bacteria, 10 reductases of microbes, 1 2 Nuclear magnetic resonance spectroscopy of yeast wall mannans, 104 Nuclear repair mechanisms, effect of oxygen on, in bacteria, 36 Nucleic acids, effects of pressure on, 192

0 Obligate anaerobes, hydrogen production by, 68 Obligate barophilic bacteria, possible existence of, 176 Oil wells, existence of high pressures in, 161 Operon, possible existence of, for nitrogenase, 25 Origin of mitochondria and superoxide dismutase, 4 6 Oscillations, in nicotinamide nucleotide fluorescence in cells, 285 in oxygen tensicn, response of bacteria to, 246 Osmotic fragility of yeast mannan mutants, 11 5 Outer chain, mannan, structure of, 107 portions of yeast mannan, partial structures of, 142 structure of yeast mannan, 118 Oxidase activities and cytochrome contents of membranes of Klebsiella aerogenes, 347 Oxidases, bacterial cytochrome, 3 0 Oxidative p hosp hory lation, efficiency of, 351 in bacteria, 31 coupled to nitrate respiration, efficiency of, 3 5 5 in Saccharomyces sp., 305 sites in bacteria, 304 Oxides of nitrogen as intermediates in nitrate assimilation, 14 Oxygen, and control of enzyme synthesis, 4 8

SUBJECT INDEX

as an end product of microbial metabolism, 2 deprivation of, effect of, on steadystate levels of intermediates, 273 effect of, in synthesis of nitrate reductase, 339 on activity of dissimilatory nitrate reductase, 339 on formation of dissimilatory nitrate reductase, 332 hyperbaric, effect of, on Escherichia coli, 167 in the biosphere, 1 metabolism, by micro-organisms, 2 9 in micro-organisms, diversity of, 29 partial pressure, effect of, on nicotinamide nucleotide content of Klebsiella aerogenes, 282 toxicity, enzymic protection against, 40 in microbes, biochemical basis of, 34 transport of, into bacteria, 2 5 0

P Paramecium sp., effects of pressure on, 205 Partial structures of outer-chain portions of yeast mannan, 142 Penicillinase, effect of chlorate resistance on, 361 Peptide bonds in yeast wall mannan, 99 Peptide elongation, effect of pressure on, 226 Peptides, rupture of hydrogen bonds in, 200 Peptostreptococcus elsdenii, hydrogen production by, 69 Perchloric acid as a catalyst of yeast mannan acetolysis, 103 Perfluorobutyltetrahydrofuran, use of, in pressure studies, 167 Periplasmic location of hydrogenase in sulphate-reducing bacteria, 72 of mannan in yeast, 95

409

Periplasmic superoxide dismutase in bacteria, 45 Permeability, microbial, effect of pressure on, 21 1 Permeases, effect of pressure on activity of, 21 1 Peroxidase, as a protective enzyme, 29, 41 Phages, effect of pressure on, 186 Phases of growth, microbial, effect of pressure on, 177 Phenotypes of mannan mutants in yeast, 115 Phosphate in yeast wall mannans, 108 Phosphodiester linkages in yeast wall mannan, 99 Phosphoenolpyruvate, carboxylation of, 58 changes in content of, in Klebsiella aerogenes during oscillations in nicotinamide nucleotide fluorescence, 288 Phosphofructokinase, synthesis of, in , Escherichia coli, 276 Phospholipids in reconstituted particles in chlorate-resistant bacteria, 366 Phosphomannans of hansenulas, 131 Phosphorylation, oxidative, efficiency of, 351 Photosynthesis, effect of pressure on, 21 9 Photosynthetic bacteria, as progenitors of nitrogen fixation, 20 reductive carboxylic acid cycle in, 61 Phototrophic origin of nitrogenase, 22 Physical properties of yeast wall mannans, 98 Physiological properties of chlorateresistant mutants of bacteria, 360 Pinocytosis, effects of pressure on, 20 5 Plants, carbon dioxide fixation in, by the Hatch and Slack pathway, 59 Plasma membranes of chlorateresistant mutants of bacteria, proteins in, 362 Polyethylene glycol, use of, in pressure studies, 165

41 0

SUBJECT INDEX

Poly-L-glutamate, transition from helix to coil in, 200 Polymeric interactions, effects of pressure on, 202 Polymerization of amino acids, effect of pressure on, 224 Polypeptide chains in yeast mannan structure, 122 Polypeptide component of yeast cellwall mannan, 9 4 Polyphenylalanine biosynthesis, effect of pressure on, 227 Poly saccharide s, invo Ive m en t of adenosine triphosphate in synthesis of, 270 Poly-L-valyl ribonuclease, effects of pressure on aggregation of, 203 Potential respiration rate, definition of, with bacteria, 252 Potentiation of pressure effects by temperature, 184 Precipitin reaction between antisera against Candida mannans, 137 Preferential barophiles, nature of, 179 Pressure, adaptation to tolerance of, 176 as a mutagen, 175 average, on ocean floors, 160 denaturation of metmyoglobin, 192 denaturation of proteins, 195 dependence of germination of bacterial endospores, 188 effects of on biopolymers, 19 1 on polymeric interactions, 202 %growthunder, 1 7 4 life and death under, 174 optimal, for growth of bacteria, 180 Primitive nature of nitrogenase, 19 Production of hydrogen by microbes, 67 Prokaryotic micro-organisms, regulation of respiration rate in 244 Proline oxidase, synthesis, repression of, by ammonia, 25 Pronase, action of, on yeast mannan, 122 effect of, on agglutinin activity from Hansenula wingei, 133 Propionate-forming micro-organisms, 350

Propionibacteriurn freudenreichii, cytochromes in, 350 P. pentosaceum, effect of nitrate respiration on molar growth yield of, 353 Protease action on membranes of chlorate-resistant mutants of bacteria, 363 Protection, enzymic, against oxygen toxicity, 40 Protein, associations, effects of pressure on, 202 composition of membrane of chlorate-resistant mutants of bacteria, 362 denaturation, by pressure, 195 location of, in the yeast wall, 96 pulse technique for measuring oxidative phosphorylation, 305 synthesis, coupling of, to mannan synthesis in yeast, 139 effect of pressure on, 225 Proteus mirabilis, effect of nitrate on specific growth rate of, 355 effect of nitrate respiration on molar growth yield of, 353 oxidative phosphorylation in, 351 regulation of nitrate reductase synthesis by, 54 solubilization of nitrate reductase from, 319 synthesis of dissimilatory nitrate reductase in, 332 P. vulgaris, properties of hydrogenase from, 76 Proton magnetic resonance of yeast wall mannans, 104 Protoplast particles, yeast, ability of, to catalyse incorporation of mannose residues into mannan in yeast, 140 Protoplasts, yeast, secretion of mannan by, 145 Protozoa, effects of pressure on, 205 Pseudomonads, gas production from nitrite by, 10 metabolism of hydrogen by, 7 7 Pseudomonas aeruginosa, effect of nitrate respiration on molar growth yield of, 353

41 1

SUBJECT INDEX

nitrate reductase of, 3 2 6 oxidative phosphorylation in, 351 Ps. AMI, response of, to dissolved oxygen tension, 250 Ps. bathycetes, effect of hydrostatic pressure on growth of, 161 effect of pressure on protein synthesis by, 2 2 8 pressure tolerance by, 1 7 4 protective effects of salts on, 189 Ps. denitrificans, nitric oxide reductase of, 11 nitrite reduction by, 1 3 nitrous oxide reductase of, 1 2 oxidative phosphorylation in, 351 solubilization of nitrate reductase from, 319 Ps. desmolyticum, effect of pressure on catabolism by, 215 effect of pressure on glucose degradation by, 178 Ps. extorquens, effect of methanol concentration on yield value of, 259 Ps. fluorescens, cytochrome c peroxidase of, 297 Ps. marinopersica, effect of pressure on, 1 7 4 Ps. methanica, methane utilization by, 65 Ps. mirabilis, nitrate reductase of, 317 Ps. oxalaticus, carbon dioxide fixation by, 57 Ps. perfectomarinus, tolerance of pressure by, 1 7 4 Ps. putida, nitrate reductases of, 31 7 Ps. saccharophila, effect of hyperbaric oxygen on, 36 oxidative phosphorylation by, 78 Ps. stutzeri, nitrate reductase of, 317 nitrite reduction by, 1 3 Pulse-chase techniques, use of, in studies on biosynthesis of yeast mannans, 138 Purification of bacterial nitrate reductases, 3 1 8 Pyruvate carboxylase, effect of pressure on, 207

Q Quenching of samples for chemostat cultures in assay of adenosine triphosphate in cells, 265

Random, possible nature of side chains in yeast mannans, 1 2 4 Recessive nature of mannan mutations in Saccharomyces cerevisiae, 1 1 5 Redox couples, microbial, effect of oxygen on, 3 7 Redox potential, in relation to pressure effects, 1 8 4 of a culture, and control of enzyme synthesis, 49 Redox proteins and regulation of enzyme synthesis, 4 9 Reduced carbon compounds, metabolism of, 6 4 Reduced nicotinamide adenine dinucleotide as a protective enzyme, 40 Reduced nicotinamide nucleotide dehydrogenase, regulatory role of, 290 Reduction of nitrate and chlorate, differentiation of, 31 7 Reductive carboxylic acid cycle, and carbon dioxide fixation, 58 Reductive pentose phosphate pathway of carbon dioxide fixation, 57 Reflectance fluorimetry, use of, to measure contents of nicotinamide nucleotides, 2 7 8 Regeneration of wall by yeast protoplasts, 145 Regulation, genetic, of nitrogenase, 2 2 of formate hydrogenlyase activity, 71 of formation of activity and formation of assimiiatory nitrate reductase, 341 of formation of nitrate reductase, 332 of inorganic nitrogen metabolism, 25 of metabolism, effect of pressure on, 231

41 2

SUBJECT INDEX

Regulation-continued of respiration in bacteria by adenosine phosphates, 263 of respiration rate in bacteria, 243 Regulatory genes and nitrogenase, 25 Regulatory role of reduced nicotinamide nucleotide dehydrogenase, 290 Repeating sequences in microbial surface polysaccharides, 126 Respiration, bacterial, substrate control of, 259 effect of pressure on, 215 in bacteria, regulation of, by adenosine phosphates, 263 nitrate, effect of, on molar growth yields, 352 energy conservation during, 350 rate, and environmental changes, 245 in growing bacteria, regulation of, 243 bacteria, effect of growth rate on, 258 of Beneckea natriegens, effect of cyanide on, 302 potential, nature of, with bacteria, 252 rate of, to dissolved oxygen tension, 248 regulation of, in growing bacteria, 243 response of, to dissolved oxygen tension, 246 role of nicotinamide nucleotides in regulation of, 2 7 7 role of cytochromes in regulation of, 290 Respiratory protection in nitrogenfixing bacteria, 3 1 Respiratory rate of Escherichia coli, effect of acidity on, 257 Reversal of tricarboxylic acid cycle, and carbon dioxide fixation, 57 L-Rhamnose in yeast wall mannans, 97 Rhizobium meliloti, nitrate reduction mutants of, 28 Rhodopseudomonas palustris, carbon dioxide fixation by, 57

Rhodospirillum rubrum gas evolution by, 3 ribulose diphosphate carboxylase of, 63 Ribonuclease, pressure denaturation of, 197 Ribonucleic acid, biosynthesis, effect of pressure on, 224 effect of pressure on turnover of, 218 Ribose, effect of pressure on catabolism of, 232 phosphate cycle for metabolism of reduced carbon compounds, 64 Ribosomes, effect of pressure on, 208 effects of pressure on aggregation of, 203 Ribulose diphosphate carboxylase from hydrogenomonads, 62 Ribulose 1,5-diphosphate carboxylase, activity of, 57 nature of action of, 61 Ribulose 5-phosphate kinase, activit-y of, 57 Rotenone, antagonistic action of, 77

S Saccharo my ces carlsbergensis, cytochrome oxidase activity of, 293 hydrogen sulphide production by, 81 structure of mannan of, 127 Sacch. cerevisiae, characterization of inner core of mannan in, 117 detailed structure of mannan of, 120 hyperbaric oxygen and synchronous cultures of, 41 location of chitin in, 95 macromolecular structure of mannan of, 121 mannan, antigenic determinants of, 105 partial structures of outer-chain portions of, 142 properties of the mannan of, 96 structure of mannan in wall of, 97, 109 X2180, use of, as a model yeast, 138

SUBJECT INDEX

41 3

Sacch. cheualieri, structure of mannan Sodium dodecyl sulphate, use of, to of, 1 2 7 solubilize bacterial nitrate reducSacch. italicus, structure of mannan tases, 3 19 of, 127 Solubilization of nitrate reductases, Sacch. rosei, structure of mannan of, 318 127 Specific growth rate of bacteria, effect Sacch. rouxii, structure of mannan of, of nitrate on, 355 127 Specific microbial cell functions, Saccharomyces spp., biosynthesis of effect of pressure on, 211 mannans of, 138 Spectroscopy, nuclear magnetic resonSalmonella typhimurium, genetics of ance of yeast wall mannans, 104 nitrate reduction in, 359 Sperm cells, effect of pressure on, 207 Salmonellae, hydrogen sulphide pro- Sphaeroplast formation, induction of, duction by, 8 1 in bacteria by pressure, 229 Salts, protective nature of against Spinach, amino-acid composition of effects of pressure, 189 ribulo se dip hosp hate carboxylase Secretion of mannan by yeast protofrom, 63 plasts, 145 Spindles, mitotic, effect of pressure Secretory organelles for yeast on, 206 invertase, 147 Spirillum volutans, effect of oxygen on, 36 Selective acetolysis of yeast wall mannans, 102 Spore formation in yeast mannan mutants, 116 Selective acid hydrolysis of yeast wall Spore-forming bacilli, death of, mannans, 100 induced by pressure, 188 Selenite, effect of, on synthesis of Sporulatim, effect of pressure on, 230 nitrate reductase, 325 in yeast mannan mutants, 1 16 Seleno monas ruminantium, cytoStabilization of nucleic acids by chromes of, 350 pressure, 192 Sequences, possible presence of, in side chains of yeast mannans, Staphylococcus aureus, electron transport to nitrate in, 343 123 Serine attachment in yeast wall S. epidermidis, electron transport to mannan, 97 nitrate in, 343 Serine pathway of metabolism of Steady-state contents of adenosine reduced carbon compounds, 64 phosphates in growing cells, 265 Serine residues in yeast wall mannan Steady-state systems in relirtion to structure, 122 dissolved oxygen content, 246 Serratiu marinorubra, effect of pres- Sterilization by pressure, 188 sure on, 175, 185 Stimulation of respiration rate of effect of pressure on filament Klebsiella aerogenes by organic formation by, 229 compounds, 261 Serum albumin, effects of pressure on Streptococci, superoxide dismutase of, aggregation of, 202 44 Sexual agglutination factors of Sheptococcus faecalis, barophobic Hansenula wingei, location of, 95 nature of, 1 7 7 Sexual agglutination of yeast mannan barotolerance of, 180 mutants, 116 changes in volume accompanying Short-term survival and pressure, 185 glycolysis by, 1 7 1 Side chains of yeast wall mannans, and effect of pressure on biomass proimmunochemical determinants, 105 duction by, 1 7 9

414

SUBJECT INDEX

Streptococcus faecalis-continued effect of pressure on catabolism by, 217 effects of pressure on adenosine triphosphatase of, 208 Strep. n u t a n s , amino-acid composition of superoxide dismutase of, 47 Structural analysis of yeast wall mannans, 99 Structure of the mannan of the yeast re11 envelope, 93 Structures of specific yeast mannans, 107 Substrate control in bacterial respiration, 259 Subtilisin digestion of agglutination factors from Hansenula wingei, 13 I Subunit structure of bacterial nitrate reductases, 322 Subunits, ribosomal, effects of pressure on aggregation of, 203 Succinate, dehydrogenase, effect of pressure on activity of, 216 effect of, on respiration rate of Klebsiella aerogenes, 260 on synthesis of adenosine triphosphate by Klebsiella aerogenes, 270 Sugar degradation, effect of pressure on, 217 Sulphate-reducing bacteria, cytochromes of, 73 hydrogen metabolism by, 71 Sulphate reduction, by hydrogen in bacteria, 72 effect of pressure on, 220 Sulphisoxazole, effect of, on bacterial oxygen responses, 36 Sulphur wells, existence of high pressures in, 161 Superoxidase dismutase, assay of, 43 Superoxide dismutase, bacterial, amino-acid composition of, 47 in mammalian tissues, 4 3 in microbes, 29 Survival, long-term, under pressure, 174

Synchronization of growth, induction of, by pressure, 230 Synthesis of biopolymers, effect of pressure on, 222 Synthetases, mannan, intracellular location of, in yeast, 146

T Temperature, effect of, on Bacillus subtilis, 176 effect of, on growth of Escherichia coli, 181 on microbial pressure responses, 180 on pressure denaturation of proteins, 195 response of bacteria to, in relation to respiration rate, 256 Tension, dissolved oxygen, response of respiration rate to, 246 Terminal oxidases in micro-organisms, 294 Tetrahymena pyriformis, effect of pressure on biopolymer biosynthesis by, 229 effect of pressure on oxygen consumption by, 216 effects of pressure on, 206 Tetrathionate reductase, effect of chlorate resistance on, 361 Thio bacillus denitrificans, ribulose diphosphate carboxylase of, 63 Thiocynate, effect of, on nitrate reductase of bacteria, 329 Thiosulphate reductase, activity of Proteus mirabilis, 54 effect of chlorate resistance on, 361 Threonine residues in yeast mannan structure, 122 Tobacco mosaic virus, effects of pressure on, 205 Toxicity of oxygen for microorganisms, 35 Toxicity, oxygen, enzymic protection against, 40 Transforming deoxyribonucleic acid, effect of pressure on, 193

SUBJECT INDEX

41 5

Transient responses of bacteria to Vanadate, and nitrate reductase, 328 dissolved oxygen tension, 253 Veill0 n e lla a lca les c e n s , hydrogen proTransient states in relation to adenoduction by, 69 sine phosphate contents of cells, Veillonella alkalescens, cytochromes of, 350 269 Transition from helix to coil in Vesicles, intracellular, location of poiy -L -glutam ate, 2 00 mannan synthetases in, 146 Transition metals and gas metabolism Viability of yeast mannan mutants, by micro-organisms, 82 115 Transport, microbial, effect of pres- b'iiibrio marinus, effect of pressure on, sure on, 21 1 181 Tricarboxylic acid cycle, effect of effect of pressure on biopolymer pressure on activity of, 21 5 synthesis by, 225 Trichomonads, hydrogenase activity effect of pressure on, in relation to of, 69 medium composition, 185 Triton, use of, to solubilize bacterial V. succinogenes, cytochromes of, 350 nitrate reductases, 319 Vibrios, response of, to oxygen, 35 Tropocollagen, effects of pressure on Viruses, effect of pressure on, 186 aggregation of, 204 effects of pressure on, 205 Tryptophan permease, repression of Volatile amines, production of, 56 synthesis of, by ammonia, 25 Volatile sulphides, production of, by Tungstate, effect of, on formation of pseudomonads, 8 1 nitrate reductase, 325 Volume changes, associated with Turnover number of bacterial cytochemical bond breakage, 173 chrome oxidase, 294 associated with protein denaturTurnover of adenosine phosphates in ation, 201 bacteria, 263 during ionic bond formation, 183 Turnover of adenosine triphosphate in during polymeric interactions, 203 cells, 267 Volume decreases induced by pressure, Two-state hypo thesis for denaturation 168 of proteins, 196

U Uncoupling of growth and energyconserving mechanisms in bacteria, 255 Uptake of substrate, effect on, on rate of bacterial respiration, 262 Urate reduction by reduced ferredoxin, 69 Utilization Of gaseous carbon compounds, 56

Wall regeneration by yeast protoplasts, 145 Waste degradation, effect of pressure on, 221

X Xanthine reductase, as a molybdoprotein, 327 D-Xylose in yeast wall mannans, 97

v

Y

Vacuole fractions, presence of invertase in, 147

Yeast cell envelope, structure and biosynthesis of mannan of, 93

416

SUBJECT I N D E X

Yeast, effect of oxygen on nicotinamide nucleotides of, 37 glycolysis, effect of pressure on, 214 mannans, biosynthesis of, 138 properties of, 96 structures of, 107 mitochondria, biogenesis of, 30 effect of oxygen in composition of, 38 model for mannan biosynthesis in, 151 oscillations in contents of nicotinamide nucleotides in, 289 protoplasts, secretion of mannan by, 145 wall mannans, structural analysis of,

99

Yield, biomass, effect of pressure on, 177 coefficient, bacterial, response of, to temperature, 257 effect of methanol concentration on, with Pseudornonas extorquens, 262 'constant of Escherichia coli, effect of acidity on, 257 Yields, growth, effect of pressure on, 180

Z Zinc

76

in

bacterial

hydrogenases,