Advances in Microbial Physiology Volume 05

Advances in

MICROBIAL PHYSIOLOGY

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Advances in

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

and

J. F. WILKINSON Department of General Microbiology University of Edinburgh Scot land

VOLUME 5 1971

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111 FIFTH

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10003

0 1971 By ACADEMIC PRESS INC. (LONDON) LTD.

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111

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Contributors t o Volume 5 JOHN R. B E N E B I A N N , L)cpartmeiat of Biochemistry, University of California, Berkeley, California 94720, U.S.A. LV. W. FORREST*, C S I l i O , Division of Nutritional Biochemistry, Kintore d v t ~ t i ~ rB c ,d c b i d c , South Australia, Australia. JILLIAN C . GALBRAITH,Department of Biology, Paisley College of Technology, P(tislcy , ~ % o t h l d .

11.J. KLUG,Ikpartvnciit of Microbiology, Univcmity of Iowa, Iowa City, Iowa,

u.A!!f.L4 .

A. J. ~ L ~ ~ L I ~ O1)epartmmtt V E T Z , of Microbiology, University of Iowa,Iowa City, IO?UO, [ T AS.I PETER R. SIXPLAIR~, Biochemistry Ucpcwtment, University of Ktntucky Mc.dir.nl (’~ntcr,Lmi?igfo?i,Kentucky, 40506, l7.S.A . JoIm E. SMITH,1)cparfmcnt of d pplic d Microbiology, University of Strathclydp, (:kin!]oui, Scotki7ld.

D. J. W A L K E R , CI!?II?O i)ivisiotL of h ’ u t r i l i o n d Rioch~mistry,Kintore Avciaue, ddc [tridc. Soittli dirntrriliri, Aicstrnliri.

DAVIDC W m m , Biochemistry Bcpartmeiit, Uiiiversity of Kentucky Medical (’mt tc r , Le~iii!qfoia,l i c ri f ?icky, 40506, U .#.A. I t 4 Y B I O N D (>.

1)qxirtmc t i t of BiocI~c m i c i 94720, U.N.A.

ry. 1JnivPrsily of Cali-

* Present ciddrcss: Tho Australian Wine Research Inst.itiitt., Waite Road, Urrbrac,. Soutli Aiistralin, Australia.

t

Present address: Rockefcllcr University, New York, N. Y., U.S.A. V

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Contents

Contributors t o Volume 5

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.

v

Utilization of Aliphatic Hydrocarbons by Micro-organisms M. J. KLUG and A. J. MARKOVETZ

I. Intl.octuction

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11. Organisms . A. Ycasts . B. Filamentous Fungi . C. Bacteria 111. Ecological Studies

.

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IV. Growth as Indicator of Substrate Specificity A. Bacteria B. Yeasts . C. Filamentous Fungi . D. Comments .

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V. Induction and Repression of Hydrocarbon Oxidation VI. oxidation without Assimilation

VIII. Mechanisms of Oxidation A. 72-Alkanes . B. Alk-1-enes .

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IX. Occurrence and Biosynthesis of Aliphatic Hydrocarbons . X. Aclinowledgement References .

. vii

1

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2 2

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4

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5

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G

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7 7 9 13 14

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17

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17

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VII. I’athways of Hydrocarbon Oxidation A. Bacteria . 13. Yeasts . C. IMamentoiis Fungi .

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.

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18 19 24 29

30 30 37

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39

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39 39

...

Vlll

CONTENTS

Biochemical and Physiological Aspects of Differentiation i n t h e Fungi. J O H N E. SMITH and JILLIAN C. GALBRAITH I. Intl~otluctloll . IT Acrasiales . A. Life-Cycle . B. Cell Aggregation . C. Metabolism During Morphogenesis . 111. Division Mycota : Subdivision Myxomycotina : Class Myxomycetes . A. Life-Cycle . B. The Plasmodium . C. Sclerotium Formation . D. Sporulation . IV. Eumycotina . A. Cell Wall Construction and Morphogenesis . . B. Light-Induced Sporulation and Sporogenic Substances C. Biochemistry of Asexual Sporulation . D. Hormones and Sexual Reproduction E. Secondary Metabolites and Differentiation. . V. Acknov ledgemerits . References .

High-Energy Electrons i n Bacteria. RAYMOND C. VALENTINE

45 47 47

51 63

63 63 65

65 67 69 69 75 79 104 116 124 124

J O H N R. BENEMANN and

I. Introduction . . 11. High-Energy Electrons in Metabolizing Bacteria (in collaboration with P. F. Weaver) 111. I~erredoxin. The First High-Energy Electron Carrier IV. Flavodoxin V. Two New Carriers from Azotobacter . VI. Elcctron Chains in Anaerobic Bacteria . VII. High-Energy Electrons in Photosynthetic Bacteria . (in collaboration with P. F. Weaver) V I I I . Electron Flow in Aerobic Nitrogen Fixation by Azotobacter . I X . Nitrogcnase : A High-Energy Electron Acceptor X. Regulation and Genetics of Electron Chains . (in collaboration with C. W. Sheu) . X I . Concluding Remarks and Future Developments X I I . Acknowledgements . References .

135 137 140 144 147 150 1.72 154 157 160

.

163 169 169

ix

CONTENTS

Branched Electron-Transport Systems in Bacteria. DAVID C. WHITE and PETER R. SlNCLAlR

I. Introduction . 11. Methodology . A. Spectrophotometry . B. Oxygen electrodes . 111 Intcrprctation of the Data . IV. Branched Electron-Transport Systems . Al. Halophilic bacteria. B. Achromobacter . C. Azotobactrr . I). E'scherichin coli . E. Haemophilus parainjuenzae . . F. Bacteria containing cytochromes a3 and o G . Micrococcus clenitri$ca?c;s. . V. Aclrnowledgemc~iits . References .

173 174 174 181 182 183 183 186 188 192 198 207 207 208 208

.

The Generation and Utilization of Energy During Growth. W. W. FORREST and D. J. WALKER I. Introduction

.

213 214 214 217 22 1 223 227 227 236 249 249 264 267 269

11. The Requirement for Energy . A. Lithotrophic Carbon Dioxide Fixation H. Syntliesis of JIoiiomers . C. Polymeriz nt'1011 . . D. 'I'otd Synthesis of Bacterial Cells * 111. The Gcmeration of Energy A. Lithotrophic Metabolism *

B. Organotrophic Metabolism 1V. The Usage of Avnilable Encrgy A. Rlolnr Growth Yields . B. 'l'hrrmodpnnmic Assessments

V. Concliisions Rrfcreiices

Author Index

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Subject Index

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275

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289

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Utilization of Aliphatic Hydrocarbons by M icro-organ isms & tJ. I.KLUGand A. J. hrARKOVETZ

Depaif ment of Microbiology, University of Iowa, Iowa City, Iowa, U.S.A. I. Introduction T I . Organisms A. Yoasts

. .

1

2 2

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U. Filiimontous Fnrigi . C. Ihctrria . 111. Ecological Studics . IV. Growth as Iildicator of Substreto Specificity. A . Ihcteria . 13. Yeasts . C. Filamentous Fiiiigi . D. Comments . V. Induction and Repression of Hydrocarbon Oxidation VI. Oxidation without Assimilation . . VII. Pathways of Hytlroc:trl>ori Oxidation . A. Bacteria, . B. Ye:Lsts . C. Filameritoits Fungi . VIII. Mechanisms of Oxidation . A. n-Alkaiics u. A1B-l-etles . IX. Occurrence and Uiosynthesis of Aliphatic Hydrocarbons X. Acknowledgement . References .

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4 5

G 7 7 9 13 14 17 17 18 19 24 29 30 30 37 39 39 39

I. Introduction Biological interest in hydrocarbons has expanded t o such a degree in the past few years that it is no longer feasible t o attempt a review on all phases of microbial hydrocarbon oxidations. In the following pages certain aspects of the oxidation and assimilation of the simple aliphatic alkanes and alk-I-enes, for the most part microbial, will be discussed. The authors have attempted to extend and update those portions of the 1

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7

M. J. KLUG AND A. J . MARKOVETZ

cxccllcntl reviews by van der Linden and Thijsse ( 1963) and McKenna and Kallio (1965) concerned with alkanes and alk-1-enes. Since methane represents a somewhat specialized case, it will receive only limited comment in this article.

11. Organisms No attempt will be made to assemble a list of micro-organisms ~ ~ o s s ~ ~ s s ing the tLhility to oxidize aliphatic hydrocarbons. Information of this type has been tabulated by Beersteclier (1954) and Fuhs (1961). From these and other reviews cited previously, the iiuniber of bacteria recorded as being “hydrocarbon-oxidizers” far exceeded the number of yeasts and filamentous fungi. The greater propensity for oxidation of aliphatic hydrocarbons by bacteria was more apparent than real since it simply reflected the lack of investigations using yeasts and filamentous fungi. A cursory attempt will be made to review and update the information on the kinds of yeasts and molds implicated in aliphatic hydrocarbon oxidations. This is being done because later wc will be concerned with reviewing recent reports on the catabolism of hydrocarbons by these two groups. This information will be included, along with the more extensive investigations with bacterial systems, in a discussion of microbial oxidation of n-alkanes and alk-1-enes. A. YEASTS 1. n-Alkanes

Tausson (1939) first reported the assimilation of alkanes by members of the genera Debaryomyces, Endomyces, Hansenula, Torulopsis and Illonilia. Alkane assimilation by Candida lipolytica, Torulopsis colliculosu and Candida tropicalis was indicated by the work of Just et al. (1951). Markovctz and Kallio (1964) presented a hydrocarbon assimilation pattern demonstrating that species belonging to the genera Candida, Debaryoniyces, Hansenula, Rhodotorula and Trichosporon could grow a t the expense of certain n-alkanes of even-numbered carbon atoms, 10-18. Utilizationofn-alkanesofeven-numbered carbon atoms (10-16) by C. lipolytica was indicated by Azoulay et al. (1964). Miller et al. (1964) demonstrated high yields of cells when Candida intermedia was grown on alkanes of 12-1 8 carbons in mineral salts-hydrocarbon medium. Isolation and screening of 56 strains of yeasts capable of utilizing kerosene were described by Komogata et al. (1964): most of the organisms readily assimilated long-chain alkanes from 9-1 6 carbon atonis and, after taxonomic studies, most of the yeasts were classified as species of the genus Candida. A soil isolate, identified as being a species of Pichia, was reported by

UTILIZATION O F ALIPHATIC HYDROCARBONS BY MICRO-ORGANISMS

3

Ariiiia et al. (1965) to grow on a series of n-alkanes from CB to CI3. Candida rigida, Jfycotorula japonica, Candida utilis, Cryptococcus neoformans, Hansenula subpelliculosa, Rhodotorula glutinis and Saccharowyces chevalieri were observed to grow in a defined medium a t the expense
4

M. J. KLUG

AND A . J. MARKOVETZ

TABLE1. Genera of Yeasts Reported to Utilize Aliphatic Hydrocarbons for Growth n-Alkanes

Alk-1 -enes

Cnndida Cryptococcus Endomyces Hansenula Mycotorula Pichia Rhodotorula Torulopsis Trichosporon Saccharomyces

Candida Debaryomyces Hansenuln Rhodotorula

B. FILAMENTOUS FUNGI 1 . n-Alkanes

The observation by Miyoshi (1895) that Botrytis cinerea would attack paraffin presumably provided the beginning of hydrocarbon microbiology. Tausson (1925) showed that Aspergillus niger could utilize paraffin wax as the sole source of carbon for growth, and Hopkins and Chibnall (1932) found that Aspergillus versicolor grew on hydrocarbons from 23 through 34 carbons in length. It should be mentioned, simply because of the organisms concerned, that jet fuel has been reported to support growth of the filamentous fungi Cladosporium (Prince, 1961) and Hormodendron (Krynitsky and McClaren, 1962 ; Edmonds and Cooney, 1967). Fusarium moniliforme isolated from diesel fuel by Flippin et al. (1964) was found to grow on n-decane and n-dodecane. Koval et al. (1966) reported that n-alkanes of diesel fuel were utilized preferentially as the source of carbon for strains of Mucor, Cunninghamella, Penicillium, Trichoderma and Fusarium. A direct soil-baiting method utilizing a paraffin rod was used successfully by Rynearson and Peterson (1965) to isolate paraffinolytic fungi. Only 20 of the 31 cultures isolated from the rod grew when inoculated into a medium with paraffin as the sole carbon source. These fungi belonged to the genera Aspergillus, Chaetomium, Penicillium, Syncephalastrum and Cunninghamella. Paraffin utilization was also demonstrated with 3 out of 10 thermophilic fungi (Fergus, 1966). Krause and Lange (1965), studying the effect of addition of various water-insoluble fatty compounds to soil, showed that various n-alkanes (C11-C20, CZ2, CZ3 C32) would support vigorous growth of three species of Fusarium. Previously, soil strains of Fusarium, as well as Acremonium, had been

UTILIZATION O F ALIPHATIC HYDROCARBONS BY

MICRO-ORGANISMS

5

obtained in ethane an d propane enrichments (Dworkin a n d Foster, 1958; Kester, 1961). A number of molds were assayed b y Kester, as reported by Foster (1962), for their ability t o use n-tridecane as sole carbon source. The following organisms were found t o possess this capacity 1 Aspergillus alliaceus, Cephalosporium roseum, Colletotrichum rttramentarium , Acremonium, patronii, Fusarium bulbigenum and illonilia bonordenii. A species of Botrytis was able t o utilize n-nonane and n,-decane for growth (Yamada an d Torigoe, 1966). Tanaka et al. (1968) observed t h a t strains of Aspergillus, Penicillium, Fusarium and Clndosporium grew at the expense of a hydrocarbon mixture composed of n-undecane, wdodecane, n-tridecane an d n-tetradecane. A similar mixture of n-alkanes lacking n-undecane served as sole carbon source for species of Helicostylum, Rhizopus, Aspergillus, Penicillium a n d Fusarium (Ratledge, 1968). Lowery et al. (1968) surveyed a series of molds on nalkanes of C,-C,, plus C16. Genera able t o assimilate some members of the series were Aspergillus, Cephalosporium, Demutium, Epicoccum, Fusarium, Gliockdium, Graphium, Mucor, Paecilomyces, Penicillium and Trichoderma. A wide range of fungi were tested b y Nyns et al. (1968a, b) for their ability to assimilate a series of hydrocarbons which included n-alkanes, aromatic hydrocarbons a n d petroleum fractions. Three genera were particularly endowed with hydrocarbon-assimilating strains-Fusarium, Penicillium an d Aspergillus. Markovetz et al. (1968) checked 5 3 strains of filamentous fungi representing 32 species on five even-numbered n-alkanes of 1&18 carbon atoms. Species belonging to the genera Aspergillus, Cephalosporium, Fusarium, Helminthosporium, and Spicaria grew better th an the other organisms tested, with the exception of strains of Cunninghamella which exhibited profuse growth on all the substrates. 2. Alk-l-enes

Octadec-l-ene an d squalene have been reported to support the growth of three species of Fusarium (Krause an d Lange, 1965). I n a survey of filamentous fungi grown on even-numbered alk-l-enes (Clo-C18), Rlarkovetz et al. (1968) observed definite growth with the same species which utilized n-alkanes (see n-Alkanes, p. 3 ) . A tabulation of the genera of fungi reported t o utilize aliphat,ichydrocarbons is given in Table 2 .

C. BACTERIA Probably the only new “type” of bacterium not previously recorded in the annals of hydrocarbon-oxidizing bacteria is the thermophilic bacillus. Klug and Markovetz ( 1 9 6 7 ~ isolated ) a thermophilic bacillus which utilized n-tetradecane as its carbon source. Another thermophilic

M. J. KLUQ AND A . J . MARKOVETZ

TABLE 2. Genera of Filamentous Fungi Reported to Utilize Aliphatic Hydrocarbons for Growth n-Alkanes

Absidia Acremoniurn Aspergillus Botrytis Cepluclosporium Chaetomiurn Chloridium Cladosporiurn Colletotrichztm Cunninghamelkc Dematiuin Epicoccum Ftisarium Gliocladium Graphium Helicostylum Helminthosporiurn Monilia Mucor Oidiodendron Paecilornyces Penicillium Rhizopcs Scolecobusidiurn LYpicaria Syncep halastrwth Trichoderma

Alk- I-enes

Aspergillus Cephalosporium Cunninghamella Fuaarium Helminthosporium Spicaria

bacillus was isolated on long chain n-alkanes by Mateles et al. (1967), and was found to grow on n-alkanes from 12 through 20 carbon atoms.

111. Ecological Studies Ecological aspects were dealt with in a review by Fiihs (1961) and certain ecological principles with reference to micro-organisms utilizing hydrocarbons have been applied to the prospecting for petroleum. A review on this topic was compiled by Brisbane and Ladd (1 965). Jones and Edington (1968) conducted an ecological survey of the microflora of soil and underlying shale and, in all samples taken, hydrocarbon-oxidizing organisms were found. Perry and Scheld ( 1 968) isolated organisms of numerous genera from soil on non-hydrocarbon substrates and checked

UTILIZATIO?; O F ALIPHATIC HYDROCARBONS B Y MICRO-ORGANISMS

7

tli(’iii for their ability t o grow on hydrocarbon substrates. Generally, these studies tend t o verify t h a t the time-honored practice of using soil as a source of organisms capable of oxidizing hydrocarbons still has merit.

IV. Growth as Indicator of Substrate Specificity Attempts t o explain t h e significance of substrate specificity of nalkanes nnd ;ilk- 1 -cnm a s related t o growth studies may be a n exercise relegated t o futility. Nonetheless, certain growth studies will be mentioned togcthrr with possible explanations of the results.

A.

BA4CTERIA

Fostcr ( 1 %Z), geiicralizing on the ability of n-alkanes t o serve as groir t h substrates, indicated t h a t n-alkanes containing 10-1 8 carbons were attacked with the greatest frequency and rapidity (for older rcfcrenees S C P 13ecrstecher, 1966). Reference was made t o d a t a obtained by Lukins (1962) for 21 strains of Mycohacterium grown on n-alkanes of 1 through 18 (.arbom. Three strains utilized all t h e substrates from prop a n ’ throiiyli hcxadecane. Most of the cultures exhibited a preference for the lonq vhaiii c~onipoiindsconimericing with 9-1 1 carbons. n-Hexane u as found t o be toxic t o tlic growth which fWycobactPrium smeqmatis noriiinlly >l,ttiiill(d on other alkanes. This toxicity was proposed as a possiiblv (>xl)lil1iatiotlfor the refractoriness of intermediate chain length a1kaiic.s ((’-)-C0)t o su1)l)ort growth. However, it was noted t h a t n-hexane w a q utilized as il qrowth substrate by other niycobacteria used in the study. Sliort chiiiti ,/-i\lkn~~eq were not toxic t o a strain ofCorynebacterium which grew iLt t I I V vxpensc of R series of C3-- CIS, excluding C,, and (3,:. Ofthe alk-1-encs tcstcd, this Corynehactwium strain grew on dodec-1-ene, tetradcc- I -elic, Iicxadcc-1-ene a n d ortadec- 1 -ene. Olefins not supporting growth ~3 err 1.1 hylene, propylene, cis- and trans-but-2-ene (Kester and Foster, 1 9(i:r) Referring t o unpiiblished work of T. Ishikura, Foster (1962) rclmrtcd thnt intrrnicdiate chain length n-alkanes and alkcnes (C7)-(y07) u c r c inhihitory t o a number of bacteria, yeasts and fungi growing on non-hydrocarbon media 8 ~ v w a interesting 1 points appeared i n a 1)al)erby Finiierty cf nl. (1962) on alknnr-oxidizing inic,rococci. Growth rcspoiise~t o alkanes from rnetlian(\ tliroiiyh ~ ~ - c i c ~ o swere a u e checked, and generally growth T\ as absent when alknnw 5Iiorter t h a n the Clo-C12 range served as t h e carbon soiirccl. One strniii, X-12.2, isolated from dodec-1-ene, grew only on CR through Cl,. l < j r lovcririg the growth temperature from 25’ t o Z O O , oiie strain which utilized C,, a s the shortest n-alkane could now grow a t t h e expense of Clo, and the lower limit of growth response with another

8

M. J. KLUG AND

A.

J. MARKOVETZ

strain was extended from C12 down t o Cl0. The authors suggested t h a t lower vapor pressures of the n-alkanes a t the lower temperature indicated t h a t physical characteristics of the hydrocarbon as well as the metabolic potentials of the organism must be considered in assessing the utilizability of liydrocarbons as carbon sources for growth. Rased on these d a t a from Kallio’s laboratory, i t seems reasonable t o speculate that many micro-orgmisms which utilize the long-chain hydrocarbons t o t h e exclusion of t h e shorter members of a series find t h a t these shorter members are “toxic” because of their greater solubility and tliercfore their higher concentration. By lowering the temperature, and by extension the solubility of the hydrocarbon, t h e “toxicity” would bc lessened or eliminated. Indeed, Johnson (1964) stated t h a t the number of organisms growing on n-hexane increased if t h e hydrocarbon concentration in the medium was kept below the saturation level. Commenting on t h e micrococcus mentioned above, which did not grow on alkanes longer t h a n Cll, Johnson (1964) broached t h e subject of solubility. By extrapolation of the solubility d a t a of McAuliffe (1963) for short chain n-alkanes, Johnson suggested t h a t the concentration of n-decane and higher hydrocarbons in a n aqueous medium would be extremcly low. This could explain why some organisms do not grow on longer chain hydrocarbons. Another hypothesis t o account for growth on longer chain substrates suggested t h a t the micro-organism would attach t o a droplet of alkane with the long alkane chain becoming incorporated into the phospholipid micelle of the cell membrane, a n d t h a t a lyophobic pathway exists from outside t h e cell membrane t o the enzymic site responsible for initiating the attack on the substrate (Johnson, 1964). More recent data from McAuliffe (1969), Baker (1967), Peake a n d Hodgson (1966) a n d Franks (1966) indicate t h a t extrapolation of data from short chain hydrocarbons showing decreased solubility a s a function of increased chain length is not valid for longer chain n-alkanes (>Clo). Beginning with C, l-CIL, n-alkanes are “accommodated” in much higher concentration than anticipated from extrapolation of solubility measurements. Apparently the change from a state of true solubility (molecular dispersion) t o accommodation (aggregation)begins with CI1. XcAuliffe‘s plot, (1969) of his data along with the d a t a from t h e other workers listed above, indicated t h a t C12-C18 are “accommodated” in water a t approximately the same concentration. Mention of a paper b y Drost-Hansen (1965) dealing with the physical structure of water interfaces seems appropriate at this point. In coiisidering water-hydrocarbon interfaces, he proposes that a considerable “structuring” exists consisting of clusters or “cages” of water molecules which may serve a s “binding sites” for t h e molecules of hydrocarbon at

UTILIZATION O F ALIPHATIC HYDROCARBONS BY MICRO-ORGANISMS

9

the interface. Further, his data indicate that the interfacial tension of water and n-hexane show a complex behavior in the vicinity of 30”. Therefore it seems reasonable to assume that the inability of an organism to grow on a long-chain hydrocarbon is probably attributable to a metabolic deficiency and not t o the lack of “dissolved” or “accommodated” substrate. In the case of short-chain hydrocarbons the concentration of dissolved substrate may be high enough to be “toxic”, perhaps as suggested by others, by an effect on the cytoplasmic membrane. However, it seems strange that n-hexane, for example, would disrupt the integrity of the cytoplasmic membrane of one mycobacterium while serving as a growth substrate for another mycobacterium. presumably under the same experimental conditions. Two strains of micrococci were reported to grow at the expense of hexadec-1-ene (Stewart et al., 1960) and Makula and Finnerty (1968) grew .Micrococcus cerijicans on each of the following alk-1-enes as sole carbon source : dodec-1-ene, tetradec-1-ene, pentadec-1-ene, hexadec- 1ene and octadec-1-ene. A micrococcus strain (S-12.2),isolated by enrichment on dodec-1-ene by Finnerty et al. (1962),would not grow on the corresponding n-alkane, a point which will be discussed (p. 30). For older citations on alkene oxidation, refer to Beerstecher ( 1954). Before leaving the bacteria some mention should be made of the growth responses of pseudomonads to hydrocarbons since members of the genus Pseudomonas have been used extensively in studies concerned with the catabolism of aliphatic hydrocarbons. Konovaltschikoff-Mazoyer and Senez (1956) found 11 strains of Pseudomonas capable of growth a t the expense of n-alkanes (Ci-CI6). Thijsse and Zwilling-de Vries (1959) in a comparative study of branched and straight-chain alkanes reported that n-pentane through n-hexadecane were used for growth by a pseudomonad. Although no growth survey related to alk-1-enes has been published, Pseudomonas aeruginosa is known to utilize oct- 1-ene (Huybregtse and van der Linden, 1964) and tetradec-1-ene (Markovetz et al., 1967) as growth substrates.

B. YEASTS From statements in the literature one obtains the impression, which as it turns out may be correct, that yeasts and filamentous fungi more readily utilize long-chain rather than short-chain hydrocarbons. Only in the past several ycars have studies appeared on substrate specificity as related to growth, and only a few of these investigations employed a comprehensive series of substrates. I n an experiment initiated to select a yeast which would readily utilize long chain n-alkanes and alk- 1-enes, and thereby presumably be

10

M. J. KLUO AND A . J . MAEZKOVETZ

a good organism for a study of the catabolic degradation of these substrates, some 30 different yeasts were assayed to determine their ability to assimilate hydrocarbons (Markovetz and Kallio, 1964). Genera, from which representatives were found to assimilate some member of the series tested, are noted in the section on ORGANISMS (p. 4). Substrates used were eveii-numbered n-alkanes and alk- 1-ems of 10 through 18 carbons. The 14-CarboIl member of each series was utilized most frequently. As a group, the alk-1-enes were assimilated to a somewhat lesser degree. It was suggested that hydrocarbon assimilation tests may have potential value in delineation of species in certain genera. It was also noted that the hydrocarbon-air interface in agar slants frequently acted as a growth demarcator in that growth might occur above or below the surface of the hydrocarbons, sometimes depending on whether the substrate was an alkane or an alk-1-ene. The physicochemical and biochemical implications of cells growing essentially in an atmosphere containing substrate as opposed to cells actually immersed in the substrate were not pursued. Species of the genus Candida were used in a number of growth studies. C. lipolytica was unable to utilize shorter n-alkanes ((3,-C,) but it did assimilate n-dccane, n-dodecane, n-tetradecane and n-hexadecane. Cell yields increased with chain length (Azoulay et al., 1964). Of the Candida species checked on n-alkanes by Komogata et al. (1964), most of the organisms which grew utilized n-alkanes in the carbon range of 9 through 16, but not in the range of n-pentane through n-octane. n-Decane and n-tetradecane appeared to elicit the strongest assimilatory responses. Miller et al. (1964) demonstrated that the generation time for C. intermedia decreased as the chain length of the n-alkane increased from C1, through C18 (minus n-tridecane). Takahashi et al. (1965) checked C. tropicalis against a series of n-alkanes and alk-1-enes. The n-alkane series ranged from n-pentane through n-eicosane and growth was observed in the C12-C20 range with the best cell yields arising in the Cl5-Cls range. Even-numbered alk-1-enes from hex-1-ene through outadec- 1-ene and including liept- 1-ene were also employed. Alk-1-enes of 14, 16, and 18 carbons supported growth to approximately the same degree. Cell yields obtained from these alk- 1-enes were approximately the same as those obtained from the corresponding n-alkane of the same chain length. n-Nonane through n-octadecane were utilized by C. petrophillum with cell yields increasing with increased chain length to a maximum which leveled off in the range of CI4-Cl7, with a drop in yield occurring on n-octadecane (Mizuno et al., 1966). Ten species of Candida exhibited varying assimilation patterns on n-decane through n-hexadecane (even carbons only) as reported by Otsuka et al. (1966). I n regard to cell yield, three species gave the greatest response a t the expense of'

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11

wtlrwane, one on 11-undecanc,four on wtetradecanc and two on n-hcxadecane. An cxtcnsivc survey on malkanes (C9-C1,) and alk-1-encs (C,o-C,,, even numbers only) eniployed 55 strains representing 36 species of the genus Cawlidn (Klug and Markovetz, 1967s). Thc purpose of the survey was twofold: (i) to determine if the ability to assimilate hydrocarbons was limited to a few species of the genus, thereby being of possible taxonomic significance and, (ii) to select an organism for metabolic studies 15 Iiicli was a n active assimilator of these substratcs. A high percentage oftlre organisms exhibited an ability to assimilate some member of thc. series tested so it appeared that this capacity was not limited to a few isolated species. It was concluded that assimilatory patterns of this type were of marginal taxonomic value except 11 here they would perhaps

Fig. 1. Eelatiolisliip betwecxn the I ~ I I I I I ~ I Cof I qpceiei of the genus Caizdida which iitiliir hj diocaiboiis and t h e cham length. 0, utilization of >?-~1lIit3ileS; A , utilization of 1 d l w n r ~ .

complement some existing biochemical methods of classification within this genus. A plot of the number of species utilizing a particular chain length hydrocarbon against the chain length is shouw in Fig. 1. Only designations of “abundant” growth taken from the data in the published tables were enililoyed for the plot. The portion of the plots above CI3 with n-alkanes and above CI4 with alk-1-enes demonstrates a slight increase in assimilatory responses as a function of increased chain length. Tlie lower sections of the plots are interesting in that an increase is seen up to n-undecane followed by a decline to n-tridecane in the case of the n-alkanes. Approximately the same shaped curve is noted for the alk-1mes except that it is shifted to the right. The C,, and C,, n-alkanes are apparently assimilated with the greatest facility. It was also observed that greatest number of “maximum” growth responses was recorded for these chsin lengths. Tlie observation of a decided change in the assimilatory pattern of n-alkanes in the area of 11 carbons is reminiscent

1%

M. J. KLUG AND A. J. MARKOVETZ

of the data cited earlier on the physical properties of n-alkanes, i.e. in the area of this carbon number a change from molecular dispersion to accommodation occurs. The significance of such a correlation is moot. Another point on physical considerations may be noted. Canclicla lipoliticu (Grande) obtained from E. Azoulay was able to utilize n-nonane whereas Azoulay ef al. (1964) found that wdecane was the shortest n-alkane utilized by this organism. One may attribute this difference to temperature since in the survey of Klug and Markovetz the incubation temperature was lower than that used by the French group. This observation would be in accord with the temperature observations of Finnerty et al. (1962) cited on p. 7 . One generalization made from the data of Klug and Markovetz (1967a) was that n-alkanes, when compared with the corresponding chain length alk- 1-enes, are more readily utilized. This implied that the ability of a n individual species to utilize an n-alkane need not allow for the utilization of the corresponding alk-1-ene, and indeed this was found to be the case. This point will be mentioned again in the section on MECHANISMS OF OXIDATIOR. Tanalia and Fukui ( 1 96S), assessing the range of n-alkanes which C. aZbica?is could assimilate, observed growth on C,,) through C,, with n-decane and 71-dodecane yielding the greatest number of cells. u-Hexane and n-octane were the shortest substrates checked and neither supported growth. 111the survey of ?/-alkanesfrom CY1through C1,, (minus ?r-pent:~decane)by Lowery et ul. (1968),no growth was observed below <)-lo carbons in length for C'anditla species. The longer-chain members supported growth. n-Pentane through n-tridecane were assayed for their ability to support growth of a Pichia sp. n-Nonane supported limited growth with ndecane through n-tridecane being assimilated well, giving approximately equal quantitative yields (Arima et al., 1965). Aida and Yamaguchi (1966) found that all n-alkanes used in their study (n-octane through 9z-hexadecane) were assimilated by illycotorula japonica and they also observed that cell yield increased with chain length, attaining a maximum a t Cls. Two strains of Rhodotorula glutinis grew on n-alkanes from Clo-C16 in the survey of Lowery et aZ. (1968) mentioned previously. In summary, the bulk of investigations on determining substrate specificity by growth studies have employed yeasts of the genus Candida. With respect to these organisms, none utilized n-alkanes shorter than C, for growth. However, few attempts to determine utilization of shorter chain hydrocarbons have been published. It is difficult to generalize on the effect of chain length on amount of growth. Considering the carbon number range of 10 through 18, examples have been cited indicating that in some cases the shorter members of this series allowed for highest cell yields, in other cases it was the intermediate lengths, in others the longer members gave the highest response and in still other investigations

UTILIZATION OF ALIPHATIC HYDROCARBONS BY MICRO-ORGANISMS

13

vields iiicreascd with chain length or stayed relatively static from C14 through C , *. Apparently these differences are a reflection of t h e particular species or perhaps even the strain. Alk-1-enes serving as sole carbon murces show the same variations as well as can be determined by t h e Iiniited investigations with these compounds.

C. PILAMENTOUS FUNGI Unlike the yeasts, there is some evidence t h a t this group of organisms cmitains members which utilize short chitin hydrocarbons for growth. lndcrd, i ~ sincntioned previously, soil strains of Fusariurn a n d Acrru?oaium wwe isolated from ethane and propane enrichments (Uworlsin aiid Foster, 1!)58; Kester, 1961). It would be of interest t o know the range of n-alkanes which supports growth of these isolates. Yamada a n d Torigoe ( 1 966) isolated numerous strains of molds from samples of soils, water, fruits, etc., in a medium containing a 1 per cent mixture of nalkaiics (equal volumes of each alkane from n-nonane through n-octndccme). Ten of some 450 strains produced organic acids in a medium containing 3 ~ w cent r n-alkancs. By gas-liquid chromatographic analysis of t h c inedium i t n-as determined t h a t mnonane a n d n-decane were iitilizrd t o thc greatest extent, followed b y 77-undecane a n d 7b-dodecane. Alkanes n it11 greater t h a n 13 carboii atoms were not used. Apparently only one of thesc organisms was classified. being placed in the genus Roirytiq. In the survey of Lowery et ctl. (1968) employing alkalies of C1 through C,(j (minus CIS), growth with the different genera was most frequently foiind in the range of Cl0 through C16. One species of dsperqillrrs had a substrate assimilatory range from n-hexane through tz-hexadecane while Uliodrirlium crxteii uluturn initiated growth on wbutane aiid continued growth throughout the series. However, weak responses were recorded i n the Ci-Cll range. One Penicillium species utilized the whole series but growth on the shorter members was apparently sparse. Extensive screening for fungi able t o assimilate 72-alkanes was accomplished by Nyiis d al ( 1 9 G h ) with the following caonclusions. n-Alkanes with less thaii 10 carbon atoms supported the growth of very few Fusariurn, I’micillium and Aspergillus strains and, if growth occurred, i t was minimal. Rqn-esentatives of six othcr genera (Absidia, Yaecilomyces, Cu?in i n y h a m d n , Chloridium , Oidiodendron, a n d Scolacobasidium) did not assitnilate ra-alkanes shorter t h a n n-decane. However, the number of short chain n -allianes tested was limited. I n some studies n-heptane was apparently the only substrate tried while in others n-heptane, n-octane and n-nonaiic were employed. Marked differences between strains were noted on tz-decanc and n-undecane, and n-alkanes from C1.)through CIG were the best growth substrates. It was observed when growth occurred

14

M. J. KLUG AND A. J. MARKOVETZ

on this scries ofn-alkanes that one particular chain length usually gave a higher growth response, and that the particular n-alkane eliciting that response could be any member of the series. These investigators concluded that for the most part the property to assimilate hydrocarbons lacked taxonomic value. Aspergillus jlavzcs, A . niger, Penicillium notatzcin and Cladosporizim wsinae were tested on n-hexane, n-heptane, n-hexadecane and n-octadecaiie by Tanaka et al. (1968). The latter two n-alkanes supported good growth, whereas t h e two shorter substrates wcrc not assimilatcd. In the assimilation study employing eveii-numbered n-alkanes arid alk-I-cnes o f 10 through 18 carbon atoms, Alarkovetz et al. (1968) used rcpresentatives of 32 species. Generally, (i) the 14-carbon member of each series was utilized most frequently, (ii) the ability to grow a t the expense of aii n-alkane did not necessarily allow for thc assimilation of the corresponding alk- 1-ene and, (iii)the use of hydrocarbon assiniilatioii tests as a taxonomic tool was questioned.

1). COMMENTS It is difficult to translate the iiieaiiiiip of substrate specificity as determined by growth to specificity a t thc enzyme Icvcl. Tf an organism utilizes iL liydrocwhon for its sole sourrc of carbori arid cnc’rgy for growth, certain obvious generalizations may be made. If assimilation occiirs then the initial physical conditions were favorable, i.e., substrate miti oxygen availability, growth temperature, etc. Equdly obvious is the fact that either the substrate or an early oxidative intermediate was transported into the cell and subjected to the series of enzymic reactions which rclcased cticrpy and transformed substrate carbon into ccllnlar carbon. If any of thesc criteria arc not fulfilled then tlic organism will not grow. Certain physical considerations as related to the substrate have been mentioned prcviously. I t is difficult to cquate reports of growth on gaseous hydrocarbons, volatilc hydrocarbons o f interniediate chain length and long chain hydrocxrbons. In the few cases where growth has been reported on a series of n-alkanes from gaseous t o long chain, we may be wcing either an cxtremely versatile organism or, altrrnatively, a rnry fortuit o i i h expcrinient Based strictly on what has been cited above oii “accomn~odatioii”of long chain hydrocarbons in aqueous environments, together with solubility data on thc short chain gases, one can predict what may he metabolically significant differences in the amounts of substrate available to the organism in a growth survey on, for example, a series of n-alkanes from methane t o octadecane. Further, we may bc dealing with different types of transport pheiiom-

UTILIZATION O F ALIPHATIC HYDROCARBONS BY MICRO-ORGANISMS

15

ena when an organism “takes up’’ a soluble short chain substrate as (winpared to :L long cliain substrate. JYith respect to the long cliain 1 1 ) drocarbonx as L: groii~), it is conceivable that two typcs of transport ncwl to k w cwnsidcred, one by tlie aqueous route for “accomodated” substrate and one by direct transfer from tlie insoluble m a s s of substrate. Jolinsoii (1964) proposed n theory for the uptakc of insoluble hydrocarbons by an attacliment of the organism to a mass of insoluble hydrocarbon wliicli would become incorporated into the phospholipid niicelle of the cytoplasmic membrane. Whether or not one can visualize the polymeric structure of the cell walls of the various types of organisms which utilize long chain hydrocarbons to be amenable to a direct contact of the mcmbranc with the droplet poses an interesting problem. If this throry for transport of long chain hydrocarbons is correct thcn specificity of growth responses to different chain lengths cannot bc explained 011 thc basis of lack of specific transport mechanisms, i.e., “permeases”, since a11 these substrates should bc incorporated into the phospholipid mirclle and “transported” even though they may not be metabolized. Scverd experiments are interesting in this rcgard. Electronmicroyral)lis by L ~ d v i l r~t nl. (1968) indicated that hydrocarbons penetrated the cell n all of C c r ~ l i d ulipol?ylica and accumulated at the cytoplasmic meriibraiic wherc they evoked structural clianges. Especially noticeable was the increase in cytoplasmic membrane surface. Volfov&et nl. (1967) reported tliat protoplasts of this yeast lost the ability to oxidize nhexadecnne but retained the ability to oxidize glucose. They conjectured that this oxidative loss resulted from either a change in tlie surface of the cytoplasmic membrane with a destruction of the oxidative enzymes bound thereon, or a loss of ability to transport hydrocarbon into tlie cell. Apparently, the latter cxplanation with its inherent assumption that the cell w i l l played a role in hydrocarbon transport was preferred. Munk ef nl. (1969) followed the penetration of labelled hydrocarbon under anaerobic conditions and their results indicated that tlie hydrocarbons entered and left the cell in less than 1 min. Further, the maximum ainount of hydrocarbon accepted was directly proportional to tlie amount of cellular lipid, but apparently there was a spccific type transport since hydrocarbons not supporting growth did not penetrate. The role of cell wall in penetration and degradation of hydrocarbons was challenged by the findings from Azoulay’s laboratory (Lebeault et al., 1969, 1970%)whereby it was shown that protoplasts of C. t~opicalisdid not lose the ability to oxidize n-tetradecane and n-decane. Bos and de Boer (1968) observed that yeast cells growing on n-hexadecane were found clinging to droplets of the emulsified substrate, again suggesting tliat hydrocarbon transport is provided by direct contact of cell and substrate droplet. Electronmicrographs of hydrocarbon-grown cells

16

&I. J . KLUG AND A . J MARKOVETZ

slio~vcda dark edgy’, after appropriate fixing, which wits absent in glucosegrown cells. This was interpreted as evidence for a “lipophilic.” wall of cells grown on hydrocarbons. Such a wall would thereby facilitate contact with the droplet. Pictures of cellular sections appear very similar to the ones of Ludvik et al. (1968) which were interpreted as simply having a hydrocarbon coating which was absent in glucose-grown cells. However, Bos and de Boer (1968) took cells grown on glucose and shook them in tlie hydrocarbon medium for several hours and no dark edges were observed. This would tend to rule out normal adsorption of hydrocarbon to the surface layer as the cause of the “lipophilic” edge. It would have been interesting to know if, as the glucose-grown cells adapted to the oxidation of hexadecane, whether or not a concomitant increase in the “lipophobic” nature of the cell wall would have been observed. One final point should be made concerning the physical nature of the substrate as it applies to the numerous growth studies which have been conducted. Long chain hydrocarbons have been added as substrates by incorporating them with non-metabolizable carriers, by adding them as emulsions, or by simply adding the “straight” hydrocarbon. The work of Bakhuis and Bos (1969) sliowed a particularly striking effect on the growth rate of C. lipolyticn as a function of the size of the droplets in emulsioiis of tlie hydrocarbon substrates. When the diameter of the hydrocarbon droplet approximated the size of the cell, there was a decided drop in growth rate. Examining the data compiled on assirnilation of hydrocarbons one could presumably make some generalizations. However, when one considers simply the variables concerned with the physical state of the substrate, and then examines the variety of conditions under which these assimilation studies have been done, it seems that definitive statements on the physiology of any organism based on growth studies may not bc warranted. No one survey has been exhaustive enough to test for growth responses by, for example, varying the actual and not the apl~arentamount of substrate available to the organism, along with varying the aeration rate, the temperature, etc. Only when these have been acconiplishcd can it be said witli any degree of certainty that this group of organisms, or that species of a certain genus assimilates short cliain but not long chain hydrocarbons, or other statements of that nature. Indeed, this is not meant as a criticism of these experiments since they were not necessarily conceived with the thought in mind of solving the underlying physiological problems of transport, enzyme specificity, etc. Reasons for conducting these experiments have been (i) simply to obtain an organism which grew well enough on hydrocarbons t o be an adequate tool for studying the biochemistry of reactions involved in hydrocarbon oxidation; (ii) to determine under a specified set of

UTILIZATION OF ALIPHATIC HYDROCARBONS BY MICRO-ORGANISMS

17

conditions which hydrocarbon yielded the greatest amount of cells as a source of single cell protein, or (iii) the greatest yield of a certain byproduct, or (iv) oxidative intermediate ; (v) to ascertain under certain “standardized” conditions the merit of assimilation tests in microbial tnxonoiny ; etc. The intriguing questions of the physical interactions of hydrocarbon substrates, hydrocarbon transport and hydrocarbon enzymology are yet to be answered before broad statements on hydrocarbon assimilation have any real basis in fact concerning “hydrocarbon physiology .”

V. Induction and Repression of Hydrocarbon Oxidation Van der Linden and Thijsse (1965) discussed adaptive versus constitutive oxidation of hydrocarbons in their review. Generally the process has been shown to be inducible in the presence of hydrocarbon. Induction of hydrocarbon oxidation by non-hydrocarbon substrates has been reported. Davis et al. (1956) observed that Mycobacteriurn parafinicum did not lose the ability to oxidize ethane when subcultured on ethanol. Acetone was shown to induce the microbial oxidation of propane (Foster, 1962), and van der Linden and Huybregtse (1967) found that hexane- 1 ,B-diol induced alkane oxidation in Pseudomonas aeruginosa. Perry and Scheld (1968) were able to demonstrate that cells of an Arthrobacter adapted to o-phthalate oxidized propane without lag. Van Eyk and Bartels ( 1968) found that a variety of compounds induced alkane oxidation, such as cyclopropane and dimethoxypropane. It was concluded that molecular specificity involved in induction was not very restrictivc and the presence of a methyl group was apparently not needed. Van Eyk and Bartels ( 1 968) were also able to demonstrate repression of alkane oxidation. Glucose was found to be a strong repressor and induction mas completely repressed by malate. L-Alanine, L-arginine, L-proline and tyrosi sine gave rather low repressor values and malonate, a non-repressor, allowed gratuitous enzyme synthesis. It would appear that the biology of hydrocarbons is about to enter the age of molecular biology.

VI. Oxidation Without Assimilation Alkane oxidation which does not lead to cellular growth has boen reported. This oxidation occurs during the concomitant oxidation of an assiinilablc substrate which may, but need not be, another hydrocarbon. Early ex~)erinientsof this type have been discussed in the reviews by Poster (1962) and McKenna and Kallio (1965). Recently, Klein et al.

18

M. J . KLUC AND A . J . MARKOVETZ

(1968) reported on the production by an Arthrobacter species of 2 - , 3-, and 4-hexadecanone from n-hexadecane during growth at the expense of corn-steep liquor or yeast extract. Subsequently, the corresponding 2 - , 3-, and 4-alcohols were identified by Klein and Henning (1969). Glucose was observed to stimulate the production of these isomeric alcohols and ketones. Production of oxidation products via a n apparent “blind-alley” pathway suggests that these initial reactions result from non-specific enzymes whose primary functions are not rclated to hydrocnrhon oxidation ; alternatively them organisms arc in a stage which is cit81ierevolving towards or away from an assimilatory pathway for thcsc substrates. It appears from the studies from Klein‘s laboratory that this Arthrobncter lacks the capacity for producing the enzymes for the conversion of ketones to the next degradative products.

VII. Pathways of Hydrocarbon Oxidation Data used for the conceptualization of metabolic pathways for oxidation of aliphatic hydrocarbons have been obtained, for tlie most part, from investigations with whole cells. These studies have taken the form of ( i ) growing an organism a t the expense of the hydrocarbon substrate and then analyzing the growth-culture fluid, the cells or both, or (ii) using so-called “resting cells” plus substrate followed by analysis, as above. These analyses arc‘ concerned with finding oxidative products which rcflcct the carbon number of the parent substrate or which could logically have arisen from the degradation of such intermediates. Also, the problem of metabolic significance must be considered since it is difficult to ascertain which of tlie accumulated compounds represent the major p t h w a y ( s ) of catabolic significance to the organism. Products may bc directly derived from the substrate, but their subsequent accumulation may rcsnlt from the fact that they are either poorly metabolized or not rrictabolizcd a t all. Alternatively, many oxidation products may be of such a transient naturc that they iicver appear in detectable amounts during unimpaired oxidation of the substrate. With thesc considerations in mind, data obtained by whole-cell experiments permit the development of metabolic pathways involved in the microbial degradation of alkanes and alk- 1-enes. Confirmation of such pathways awaits enzymological verification, which a t the time of writing, has been obtaincd for several reactions. I n instances where dcgradation may follow alternate routes the rtietabolic significance of these must be determined by selective inhibition of each to determine its significance, and this apliroach is not without difficulties. M’e have chosen to separate the numerous reports concerniiig this subject according to the broad categories of niirro-organisms employed,

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i .c. bacteria, yeast and filamentous fungi. Pathways will be enumerated from these d a t a and subsequently many reports will be considered in inore detail in the section on MECHANISMS OF OXIDATION.

A. BACTERIA 1. n-Alkanes

Iktc~tcrialoxidation of n-alkanes was reviewed in detail by van dcr 1,incten ;111cl ‘I’hijssc (1965) so only limited space will be devoted t o a rcc:tpitulation of the subject matter prior t o the date of their review. Trccanni ( 1962) citing carlier work from his laboratory, demonstrated the formation of hexanoic acid and heptanoic acid from n-hexane and n-heptanc respectively, with different bacteria (Pseudomonas aeruginosa, Achiomobacter sp., ilrocardia sp. a n d Mycobacterium sp.). Senez a n d Konovalschikoff-Mazoyer ( 1956) also identified heptanoic acid in cultures of P. neruginosa grown on n-heptane. The isolation of cetyl palmitate from cultures of Jlicrococcus ceri$cans grown on n-hexadecane (Stcwnrt ef al., 1959) provided strong indication t h a t a methyl group was oxidized t o the primary alcohol followed b y subsequent oxidation and cstcrificntion yielding the Csz ester. Isolation of cetyl palmitate derived from l-14C-hexadecane in this system demonstrated t h a t t h e alcohol and acid of the ester arose directly from the hydrocarbon (Finnerty a n d Knllio, 1964). This work provided additional evidence for methyl group oxidation with t h e formation of a primary alcohol as t h e first stable intermediate. Hankin and Kolattukudy (1968) using 41. ceri$cans were ~inablct o detect waxy esters when n-nonacosane (CZ9)was thc growth substrate. However, in a cwoxidation experiment with 14C-n-iioii;t(’o~a~ie plus n-hcxadccane, radioactive esters with alcohol and arid fragments of primarily 16, 17 a n d 18 carbons were formed. These workers were inclined t o favor methyl group oxidation leading t o the CZ9primary alcohol which would be rapidly oxidized t o shorter fragments. These, in turn, could bc used b y the ester-synthesizing enzymes which appear t o prcfer medium-chain length fragments of approximately 16 carbons. Synthesis of primary alcohols from various n-alkanes was demonstrated in scvcral whole-cell systems (Proctor, 1960; Heydeman, 1960; Lcadbctter a n d Foster, 1960) a n d with cell-free extracts from pseudomoiinds (Baptist et al., 1963; Azoulay et al., 1963). Aldehyde dehydrogcnasc was also demonstrated in pseudomonad extracts b y Heydeman and Azoulay (1963) and Baptist et al. (1963). Recent work from Coon’s laboratory (Peterson et al., 1966) a n d from Kusunose et al. (1967b)using cell-free extracts of Pseudomonas oleovorans a n d P. desnzolytica, respectively, have shown t h a t t h e active enzyme system required three

20

M. J . KLUG AND A. J. MARKOVETZ

components for octane and decane hydroxylation. Cardini and Jurtshuk (1968) demonstrated that cell-free extracts of Corynebacterium sp. oxidized n-octane to octan-1-01. Resolution of the active system into two fractions was accomplished and the Corynebacterium system differed in certain aspects from the pseudomonad system of Coon's work. Mycobacterium rhodochrous terminally oxidized n-decane with the formation of the corresponding primary alcohol, aldehyde and acid (Fredricks, 1967), and Hart et al. (1968) indicated that n-tetradecane was oxidized t o tetradecan-1-01 and tetradecanoic acid by a thermophilic Bacillus sp. All of these reports verify that the C1 position is a susceptible target for alkane oxidation. The mechanism(s) whereby this alcohol intermediate could arise will be discussed later, but a t this point it should be mentioned that one such mechanism proposes that the first intermediate in alkane oxidation is the corresponding alk-1-ene. Evidence was prcsented by Chouteau et al. (1962) for the formation of hept-1-ene from n-heptane by whole cells of P. aeruginosa and subsequently, Wagner et al. (1967)indicated that hexadec-1-ene had been isolated and identified by several analytical tools after cultivation of the following organisms on n-hexadecane : iWicrococcus ceri$cans, Mycobacterium phlei, Nocardia sp. and a Pseudomonas sp. Abbott and Casida (1968) also reported on the formatioii of alkenes from alkanes by a resting cell suspension of a A'ocarclia sp. grown on glucose. However, in this case the alkenes were internally unsaturated. TVlien n-octaderane was the substrate the resulting mono-alkene mixture was prcdominantly octadec-9-ene) followed by decidedly decreasing amounts of the 8-, 7-, 6-, and .!-isomers. Whole cells would not produce the mono-alkenes under anaerobic conditions. These authors tended to rule out formation of these mono-alkenes by the aerobic mechanism for formation of unsaturated fatty acids. This was based on experiments in which cells were incubated with exogenously supplied fatty acids. Failure to detect unsaturated acids was taken as evidence for the lack of the aerobic desaturation mechanism. This may be tenuous reasoning since there is no assurance that the fatty acids entered the cell and, if indeed they did, there is no evidence to indicate that these long-chain free acids would be converted to the activated acyl derivatives. Therefore, it would seem that these mono-alkenes were formed non-specifically by the aerobic fatty-acid desaturation mechanism, and the significance of this to a mechanism for alkane degradation is questionable especially since no further oxygenated derivatives which could have been derived from the oxidation of an internal double bond were detected. A special type of terminal methyl-group oxidation was reported by Kester and Foster (1963) t o occur with an organism belonging to the genus Corynebacterium. This oxidation represented a diterminal attack

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21

on CtO-CI4 alkanes with the subsequent formation of w-hydroxy acids leading to the forniation of dicarboxylic acids. These authors proposed that this diterminal oxidation represented a minor pathway in the oxidative degradation of the carbon chain. The main pathway for alkane oxidation was thought to proceed to the monoic acid which would be subject to P-oxidation. Some of the monoic acid would undergo w-oxidation to the dioic acid which also would be oxidatively degraded via ,&oxidation. With the Corynebacterium sp. no dicarboxylic acids were detected from n-alkanes shorter than Clo. Ali Khan et al. (1963, 1964) obtained evidence for the conversion of n-octane to octanedioic acid mediated by a Pseudomonas sp. Coon has reported on a soluble enzyme system from Pseudomonas oleovorans which oxidizes octane (Baptist et al., 1963; Gholson et al., 1963) and w-oxidizes a series of fatty acids including octanoic (Kusunose et al., 1964a, b). Alkane oxidation by means other than through a terminal methyl group has been reported. Leadbetter and Foster (1959) observed that Pseudomonas methanica growing on methane could produce acetone and butaii-2-one by co-oxidation of propane and butane, respectively. Lukiris and Foster (1963) showed that acetone, butaii-2-one, pentan-2one and hexaii-%one were produced from the respective alkanes, i.e., propane, butane, pentane and hexane, by Mycobacterium smegmatis. Undecan-%one was shown by isotopic-dilution trapping to have arisen from n-undecane in cultures of M . rhodochrous. Fredricks (1967) did not detect methyl ketone formation from n-decane by M . rhodochrous. However, comparing this organism with P . aeruginosa grown on ndecane under identical conditions it was found that decan-2, 3-, 4-,and 5-ones were produced together with the corresponding secondary alcohols. Decan-%one was produced in highest concentration indicating that, in what appears to be a rather nonspecific attack on the substrate, the rnethylene group alpha to the methyl group was preferentially oxidized t o form the methyl ketone. Ketones were also formed from the transformation of r~-alkanesby an Arthrobacter sp. (see Section V I , p. 17). Vestal and Perry (1969), using a Brevibacterium sp., showed that pr0pane-2-~%in unlabeled competitor experiments was oxidized to acetone. Competitor experiments also indicated that isopropan01-2-~*C was oxidized to acetone and acetol. No acetol was detected from propane experiments. A pathway for propane utilization was visualized as : 0

0

I/

/I

CH3CHeCH3 + CHsCHOHCH3 + CH3CCHs + CH3CCH20H

4

+

CH3COOH

A pathway for oxidation of a long-chain methyl ketone, tridecan-2-one, has been established with cells and cell-free extracts of P. aeruginosa (Forney et al., 1967; Forney and Markovetz, 1968,1969).I n this pathway

22

M. J . KLUG AND A . J . MARKOVETZ

tridccan-2-one is oxidized via a Baeyer-Villigrr type rmctioii to uiidecyl acetate which is cleaved to undecan-1-01 and acetate. When the secondary alcohol (tridecan-2-01)was used as substrate it mas oxidized to the ketone, followed by the same products listed above. Recently, it was deiiionstrated that P. nemginosa growing on thc1 C I 3 alkane produced tridecan-2-01 and undecan-1-01 (Foriiey and Markovctz, 1 0 7 0 ) . On this basis the following pathway was formulated for thr oxidation of ntridecane via a pathway other than methyl group oxid at'ion:

CHa(CH*)&OOH

+

Further

oxidation

On the basis of types of compounds identified, Figure 2 depicts a skeletonized scheme summarizing alkane oxidation by bacteria. 2 . Alk-1-enes

Stewart P t al. (1960) suggested that bacteria attacked alk-1-enes by oxidation of the methyl group, leaving the double boitd intact. This was based on experiments with the ester-producing ilIicrococcus ceyi$cans (see previous section on n-Alkanes). Identification of hexadec- 15-enyl palmitate from cultures grown on hexadec-1-ene and octadec-17-enyl margarate, -palmitate, and -stearate from growth on octadec-1-ene verified that the double bond was preserved. In their review, van der Linden and Thijsse (1965) pointed out that the large concentration of octadec-17-enyl margarate was interesting and they questioned whether or not it could be taken as evidence that the double bond was oxidized, followed by decarboxylation to the saturated C1, acid which uould esterify with the w-unsaturated alcohol. It is wondered whether the absence of the doubly-unsaturated esters of twice the substrate carbon number could also be taken as evidence for an alternate mechanism through the double bond. Hept- 1-ene degradation by a heptane-grown pseudomonad in the presence of chloramphenicol was studied by Thijsse and van der Linden (1963). Accumulation of hept-6-enoic acid indicated that the methyl group was attacked. The study of alk-1-ene oxidation was extended by van der Linden (1963) and Huybregtse and van der Linden (1964) with the conclusion that minor reactions a t the double bond lead to the

UTILIZATION

OF ALIPHATIC HYDROCARBONS BY MICRO-ORGANISMS

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formation of an epoxide, a diol, an a-hydroxy acid and a saturated avid (possibly via the aldehyde). Oxidation of the methyl group leading to the formation of the w-unsaturated primary alcohol and acid was considered to be the main degradative pathway for alk- 1-enes. hlarkovetz 0H

0

II

c-c-c-c-c-

t

C-C-C-C-C-

t

c-c-c-c-

f--

c-c-c-

OH

0

II

c-c-c-c-

0

c-c-c-

II

I

I

OH

I

I - C---C,---C-COOH

HOC---Cn---C'

-

COOH

&/

Fig. 2. A schemo summarizing the reactions involved in alkane oxidation b y bacteria.

et al. (1967) studying tetradec-1-ene oxidation by P. aeruginosa corroborated methyl group oxidation of alk- 1-enes by isolating and identifying tetradec- 13-enoic acid. However, tentative identification of tetradecan-2-01 indicated that the terminal double bond was also subject to attack. The inability of octa-1,7-diene and tetradeca-l,l3-diene to support growth of this organism appeared to cast doubt on the alteration 2

24

M.

J. KLUG AND A . J. MARKOVETZ

of the double bond as an important step in alk-1-ene oxidation. However, there is insufficient knowledge concerning the solubility, toxicity, and transport of these dienes to make any definitive statements with wholecell experiments. Recently, Makula and Finnerty (1968) have indicated that M . cerijfcans may have two mechanisms for oxidation of hexadec-1-ene and octadec-1-ene, i.e., methyl group oxidation as shown by w-unsaturated OH C-C-Cn-C-C

I

t

c-c-cn-c-c

HOC-C-Cn--C=C

1

I

t

$.

HOOC-C-Cn-C=C

C--C-Cn-~-COOH I 0 H

Fig. 3. A scheme summarizing the reactions involved in alk-1 - e m oxidatioii by bacteria.

acids of substrate chain length, and double bond oxidation as indicated by the high percentage of saturated fatty acids one carbon shorter than the substrate. Figure 3 summarizes the oxidation of alk-1-enes by bacteria.

B. YEASTS 1. n-Alkanes

Dioic acids of 11, 9, 7, and 5 carbons were isolated by Ogina et al. (1965)from cultures of Pichia sp. growing a t the expense of n-undecane. This represented the first report of diterminal oxidation of hydrocarbons mediated by a yeast. Iizuka et al. (1966a, b) grew Candida rugosa on n-decane and recovered and identified the following compounds : decyl alcohol, decyl aldehyde, decanoic acid, decanedioic acid, octanedioic acid, hexanedioic acid and succinic acid. The obvious postulation was that n-decane was oxidized

UTILIZATION

OF ALIPHATIC

HYDROCARBONS BY MICRO-ORUANISMS

25

by a mono-terminal mechanism followed by diterminal oxidation prior to degradation of the carbon chain by /3-oxidation. Cell-free extracts of C. rugosa converted decane to decanoic acid and presumably there was some evidence of unsaturation in anaerobic experiments with decane, which suggested t o these workers that decane was initially dehydrogenated to dec-l-ene (Iizuka et al., 1968). Subsequently, Iizuka et al. (1969) identified dec-l-ene as its mercuric adduct by thin-layer chromatography from a resting-cell experiment. In a somewhat abbreviated notation it appeared that Kusunose et al. (1966)obtained oxidative activity on l-l%-octadecane using a cell-free extract of a Candida which had been grown on n-hexadecane. We assume that the radioactive product measured was either primary alcohol or monoic acid. Also included with the list of bacteria, mentioned in the previous section, which formed hexadec- 1-ene from n-hexadecane in whole-cell experiments was a yeast, Rhodotorula sp. (Wagner et al., 1967). Klug and Markovetz (1967b) working with Candida lipolytica reported that the presence of primary alcohols and monocarboxylic acids of the same chain length as the substrate in cultures grown on n-alkanes from 14 through 18 carbons was evidence for methyl group oxidation. However, secondary alcohols (2-hydroxy) of substrate chain length were also detected. The high specific activity of hexadecan-2-01 isolated from cultures grown on l-14C-hexadecane suggested that these compounds were synthesized directly from the n-alkane molecule (Klug, 1969). No methyl ketones were detected so the significance of a subterminal attack to the metabolism of these compounds is not clear. It may be that these alcohols are poorly metabolized after formation or not metabolized a t all. However, cells grown on n-hexadecane readily oxidized hexadecan-2-01 in manomctric experiments (M. J. Klug, unpublished results). Tulloch ~t al. (1962) found that Cl6-CZ4 alkanes and fatty esters were converted by Torulopsis magnoliae into w - and w - 1-hydroxy acids which separated from the culture fluid as sophorosides. Jones and Howe (1968a) using T.gropengiesseri also demonstrated a conversion of long chain alkanes (C1,-Ce4) into oxygenated derivatives which were incorporated into glycolipids. The yeast was grown on glucose for 24 hr. before addition of hydrocarbon. By that time the readily-available nitrogen in the medium had usually been assimilated. Acids of substrate chain length, hydroxylated in the w- and w - 1 positions, as well as the ccw-dicarboxylic acid, monocarboxylic acid and primary alcohol of substrate chain length were identified. It was proposed that the major pathway for alkane metabolism was formation of the primary alcohol, dehydrogenation to the corresponding acid, which subsequently was metabolized by p-oxidation or by hu\-droxylation to the w-hydroxy acid (and thence to the

26

B I . J . KLUG A N D A. J . MARKOVETZ

crw-dicarboxylic acid) or to the w-1-hydroxy acid. I n addition to these compounds, the lipids derived from alkane fermentation contained small amounts of alkan-2-01s of substrate chain length. This was considered to be a minor pathway since it was felt that as the alcohol was formed, it was protected against further oxidation by incorporation into waterinsoluble glycolipid. However, this raises the question of what would happen to the alkan-2-01s in the absence of glucose before nitrogen is H 0

I

t

T C4-Cn-COH

C-C-Cn-C,

40

H

I

0 H

J

HOC-C-Cn-COOH

\

/3-Oxidation

HOOC-C-Cn-COOH

Fig. 4.A scheine siiinrnm-izing the reactions iiivolvcd in alkane oxidation by yeasts.

limiting, which presumably would be before glycolipid formation. It is not, clear whether these alkan-2-01s were looked for in hydrocarbon growth experiments in the absence of glucose. Extracts prepared from protoplasts of Candida tropicalis were found to dehydrogenate n-decane. Attempts to separate the three dehydrogenase activities, i.e., alkane, alcohol and aldehyde dehydrogenases, were partially successful with the removal of the aldehyde dehydrogenase activity (Lebeault et al., 1969, 1970b). Oxidation of n-alkanes by yeasts is summarized in Fig. 4. 2. Alk-1-enes Bruyn (1954) provided the first evidence for biological oxidation of an alk-1-ene with the isolation of hexadecane-1,2-diol from cultures of

~JTILIZATIONOF ALIPHATIC HYDROCARBONS BY MICRO-ORGANISMS

27

Cnndida lipolytica growing at the expense of hexadec- 1-ene. This observation was confirmed by Stewart et al. (1960), and subsequently, atmospheric oxygen was implicated in the oxidation of the alk-1-ene to the diol (Ishikura and Foster, 1961). Klug and Jlarkovetz (1967b) reported that the 1,Z-diol and w-unwturated acid were produced which reflected the carbon number of the substrate when even carbon-numbered alk-1-enes from C14 through C,, were supplied as sole source of carbon for growth of C. lipolytica. These compounds clearly indicated that alk-1 -enes were oxidized at both the methyl end and the unsaturated end of the molecule. I n a more extcnsive study of the oxidative intermediates from hexadec-1-ene and lieptadec-1-ene, Klug and Markovetz (1968) isolated and identified the following compounds of substrate chain length : w-unsaturated acid, w-unsaturated primary and secondary (2-hydroxy) alcohols, 1,2epoxide, 1,2-diol and the 2-hydroxy acid. The identification of these compounds strengthened the hypothesis for methyl group and double bond oxidation and, the identification of the w-unsaturated alken-2-01 indicated a subterminal attack was also possible. Conccivabl-, the 1,Z-epoxide would be a logical intermediate in oxidation of the double bond to the 1,2-diol since these compounds may bc synthesized via epoxide formation a t the double bond. Foster (1962) reported on a mechanism proposed by Ishikura for the degradation of oc%adecanc-I ,Z-diol derived from octadec-1-ene which proceeds through the 2-hydroxy acid, followed by a decarboxylation to a fatty acid one carbon less than the original substrate. The results presented by us are consistent with Tshikura’s postulation since the 2-hydroxy acid was identified and the fatty wid one carbon shorter was present in a higher perccntag,lc than the 1)ercentagc of this same acid found when different alk- 1 -encs served as growth substrate. Identification of hexadecan- I and 2-01 from hexadrc- 1-enc cultures indicated that a mechanism other than 1,2-diol formation was operating on the double bond (Klug, 1969). Wc have assumed that methyl group oxidation of alk-1-enes is of ntetabolic significanw to C. Zipolytica as a catabolic pathway. However, no definitive evidence has been obtained for this. Only when octadec-1eiie served as substrate was any evidence obtained for presumptive P-oxidation, i.e., the w-unsaturated Cl8 acid and the C,, w-unsaturated acid were detected. Interestingly, the reverse situation was seen since when hexadcc- 1-ene was substratc the w-unsaturated acid was produced. Further, the CI8 dienoic acid, unsaturated in the w- and 9 positions, was also produced (Klug and Markovetz, 1968, 1969). This 2-carbon elongation and/or degradation may be occurring by reactions apart from the normal P-oxidative reactions. The fact that w-unsaturated acids, progressively shorter by 2-carbon increments, were not detected

28

M. J. KLUO AND A. J. MARKOVETZ

does not by itself mitigate against an operational P-oxidation since these intermediates need never be released from the “oxidative” complex. It would seem strange if a double bond in the w-position would render a long-chain fatty acid refractory to i3-oxidation in this organism. However, neither the w-unsaturated C1 primary alcohol nor the corresponding

/

C-C-Cn-C=C

I 0

H

\

\ C-C-Cn-C=C

HOC-C-Cn-C=C HOC-C-Cn-C=C

I .1

J1 -

C-C-Cn-C-C

I

0 H

C-C-Cn-C-C

I

H 0 H

\ C-C-Cn-C-C

‘d

HOOC-C-Cn-CEC

1

liZC \

c-c-cn-c-c

Chain elongation and desaturation

I

1

t

0 0

1 1”

H H

\

C--C-C,,--C~--COOH

\

I

0

C-C-Cn---COOH /3-Oxidation

i-2c

(‘IM ,iiid

Fig 5 . Al ~clic.inc*siiiniiiarimig thc rcactioiis in\ (JI\ c.d yeasts.

+ COz

it1

elongation dewturation

it1

allr- 1-ciic oxidation b y

acid werc oxidized to any appreciable extent in manometric cxperirnents and, neither supported appreciable growth when provided as sole carbon sources (Klug and Markovetz, 1968). Again, the shortcomings of growth experiments and manometric experiments with whole cells must be considered. Jones and Howe (l968a) employing alk-1-enes (CIG,C17, and CIS) in fermentation-type experiments like those discussed above with alkanes, obtained glycolipids which incorporated w- and w - 1-hydroxy acids and

UTILIZATION OF ALIPHATIC HYDROCARBONS BY MICRO-ORGANISMS

29

ctw-dicarboxylic acids of both substrate carbon number and carbon number one less than the substrate carbon number. The acids one carbon lcss than the substrate wcrc also suggested to have been formed by an over-all oxidative fission presumably of the type discussed above. :\nother mechanisrn of double-bond alteration was proposed t o account for acids of substrate chain length (see p. 30). Figure 5 illustrates alk-lm e oxidation by yeasts.

C. FILAMENTOUS FUNGI n-Alkanes and Alk-1-enes Yamada and Torigoe (1966) provided the initial evidence for oxidation of the methyl group of aliphatic hydrocarbons by filamentous fungi. Employing a mixture of n-alkanes (C9-CI8) as substrate they determined 2 C + HOC-C-Cn-

c -c--c-c-c /I 0

n-

1

I

c--c

I

0

H

'-1

or

c-c-cnI 0 0 H

H

71' or

2

Subterminal Oxidat ion

C=('

Rlethyl Group Oxidation

HOOC-C-C-C-Cn-C-C

p-Oxidation

+ C z Elongation

Fig. 6. A scheme showing the oxidative a t t a c k s on alkanes and alk-1-enes by fungi.

30

N . J. KLUG AND A. J . MARKOVETZ

that the main acids produced by a Botrytis sp. were the C, mono- and dioic acids, as wcll as the Clo mono- and dioic acids. Allen and Markovetz ( 1968) reported on the foi.ination by Cun?iii~yhamella blakesleeana of tetradecaiioic acid from n-tetradecane and tetradec-13-enoic acid from tetradec-1-ene, thus providing evidence for methyl group oxidation of an n-alkane and an alk-1-ene by this organism. Subsequently, Allen and Markovetz (1969) extended the study on oxidation of tetradcc-1-ene to include a Penicilliuwi sp. I n this case, no w-unsaturated C,, acid was detected but presumptive cvidcricc was presented on the identification of the CI4 w-unsaturated methyl ketone and the CI2 w-unsaturated primary alcohol with the obvious assumption being made that this organism was attacking the alk-1-erie by a subterminal rather than a terminal oxidative mechanism. Various secondary alcohols and ketones were tentatively identified from rultnes of Yrnicillium sp. grown on n-tetradecane and tetradec-lene (Allen and Markovctz, 1970). Tetradecaii-2-ol, dodecan-1-01, tetradecan-"one and tetradecan-4-one were formed from n-tetradecane. Tlie w-unsaturated ketones (tetradec-13-en-3-one,tetradec-13-en-4-one) and the corresponding alcohols were detected when tetradec- 1- m e served as substrate. Several other ketonic and secondary hydroxyl compounds were detected, but the theme that appears to emerge from this work is that Penicillium attacks these two types of aliphatic hydrocarbons via a subterrninal mechanism. This is in contrast to the terminal methyl group oxidation observed with C. blakesleeana. Figure 6 illustrates the oxidative attacks on aliphatic hydrocarbons by filamentous fungi.

VIII. Mechanisms of Oxidation A. n-ALKANES Terminal oxidation of n-alkanes involves the participation of molecular oxygen with tlic formation of a primary alcohol as the first stable intermediate. Mechanistically, the interesting problem becomes one of determining the genesis of this alcohol and, several mechanisnis have been proposed, i.e., hydroxylation, hydroprroxidation-reduction,and dehydrogenation-hydroxylation. Methyl group liydroxylation has been the subject of extensive work by Coon and coworkers (Baptist et al., 1963; Gholson et al., 1963; Peterson et al., 1966, 1967; Peterson and Coon, 1968). The picture which has evolved of this soluble system from Pseudomonas oleovorans is that 3 protein components participate in the initial w-hydroxylation of the methyl group of a fatty acid or the hydroxylation of an alkane methyl group. These protein components are, (i) rubredoxin, (ii) XADH-

UTILIZATION OF ALIPHATIC HYDROCARBONS BY MICRO-ORGANISMS

31

rubredoxin reductase and (iii) fatty acid w-hydroxylase or alkane 1-hydroxylase. It appears that a single hydroxylase system is active for both types of substrates. The specificity for chain length was interesting since it was established that C, was the optimal chain length for naIliit11es (Peterson et al., 1067) whereas C1., was found to be the optimal lcngth for :I series of fatty acids from 8-18 carbons (Kusunose et al., 1064b). l’hc following schcinc for methyl group hydroxylation was presented (Peterson et al., 1967) : NAAL)Hl [l
N \I)

ASP)

I
(Ihl)

Kuhredoun

l
H \ dro-,) litso -HzO

(F’P3+)

‘1

d 1,

HOCH2--R

A role for rubredoxin n s an electron carrier in a pathway involving molecular oxygen was therrby established in this aerobic bacterium. Kusunose P f nl. ( 1 067b) obtained similar results with P. desmolytica. I n a subsequent comini~nicationby Kusunose et al. (1967a) using this organism (reclassified as 1’. denitrzficans), a requirement of flavin adenine dinucleotide (FAD) was found for maximum hydroxylation of decane. It was suggested that fiavin participated as a cofactor of the NADH-rubredoxin reductase. Peterson et al. (1969) determined the stoiclziometry of the system and showed that the reaction could be classified as mixed function oxidation stoichiometrically dcscribed by : NADH

+ H’

1

0 2

t It-CHs

+ NAD’ t HzO

i

R-CHzOH

In the above-mentioned paper attention was called to the fact that whereas much information had been obtained concerning the soluble enzymes which transfer electrons from NADH t o the hydroxylase, little is known of the nature of the functional group in the essentially psrticulate hydroxylase. The partially purified hydroxylase does not contain detectable cytoclirorne P-450 (E. J. McKenna, personal communication). A4bsence of this cytochromc had been indicated previously by insensitivity to carbon monoxide (Peterson ef al., 1966). Cardini and Jurtshuk ( 1068) demonstrated the oxidation of n-octane by cell-free extracts of C’orynebactwium sp., strain 7ElC. A specific requirement for NADH and molecular O2 was shown, and the active system was resolved into two protein fractions, both of which were required for oxidation of n-octane. Spectral characteristics of one fraction wcrc consistent with cytochrome P-450. The presence of flavoprotein in the other fraction was also ascertained by spectral characteristics. These authors suggest that, although indirect, the evidence

32

M. J. KLUO AND A.

J. MARKOVETZ

indicates that cytochrome P-450 is involved in the hydroxylation of n-octane by this bacterium. The sensitivity of the reaction to carbon monoxide in the absence of other CO-binding components would tend to strengthen their case. These reports on methyl group hydroxylation point to alternate mcchanisms for n-alkane oxidation, ix., the CO sensitive-cytochrome P-450 containing system and the carbon monoxide insensitive system lacking cytocliromc P-450. The former system is similar to the microsoma1 hydroxylase from rabbit liver in that cytochrome P-450 is required (Lu and Coon, 1968; Wrada et ol., 1968; Lu et al., 1969). Cytorhromc P-450 has also been implicated in sterol liydroxylations (Omura et al., 1965) and in methyleue hydroxylation of camphor (Katagiri et al., 1968). ,4 hydroperoxidation-reduction mechanism could also account for the formation of the primary alcohol as the first, stable intermediate in methyl group oxidation. Leadbetter and Foster (1960) suggested thc participation of slkyl hydroperoxides arising via a free radical mcchanism i n the microbial oxidation of n-alkanes to account for the formation of methyl ketones and fatty acids of substrate chain length. A free radical equilibrium h t w e e n C1 and C 1 was visualized as leading to l-alkyland 2-alkyl hydroperoxides which presumably gave riw, rtqcctively, to the primary alcohol - + f a t t y acid and the sccondary alcohol --f methyl kctone. It was suggested by work from Kallio’s laboratory that rather than a direct hydroxylation of the alkane to form the alcohol, a l-alkyl hydroperoxide was formed which yielded the alcohol on reduction. This was based on the following: (i) 1-alkyl hydroperoxides were oxidized by alkane-grown cells (Stewart et al., 1959; Finnerty et al., 1962); (ii) the bacterium w1iic.h formed long chain esters from alkanes also produced thc same esters from the corresponding 1d k y l hydroperoxidcs (Finnerty et al., 1962); (iii) cells, as well as extracts of cells grown on n-hexadecane degraded 1-hexadecyl hydroperoxide (McKenna and Kallio, 1965); (iv) suggestive evidence for a hydroperoxide reductase in the presence of NADH was cited in the review article by McKenna and liallio (1 065). At any rate, the “0 incorporation data of Stewart et al. (1059) are consistent wit 11 either hydroxylation or the 1iydro~~‘~roxidation-reductioii mechanism. Hov ever, if R hydroperoxide reductase is postulated to function in the reactions for insertion of molecular oxygen into the substrate it is wondered whether the 1 : 1 :1 stoichiometry of substrate, O2 and KADH for w-oxidation of octanoic acid and alkane l-hydroxylation of octane as shown by Peterson et al. (1969) would hold since one could visualize two functions of NADH, i.e. in the supply of electrons for formation of “active oxygen” and in the reduction of the hydroperoxide t o the alcohol.

UTILIZATION O F ALIPHATIC HYDROCARBONS BY MICRO-ORGANISMS

33

Dehydrogenation as the initial reaction in n-alkane oxidation by a pseudomonad was proposed by Senez and Azoulay (1861) when it was demonstrated that pyocyanine or NAD was reduced in the presence of ?~lieptanc.Subsequently, an infrared spectrum was obtained of the purported alk-l-ene intermediate (Chouteau et al., 1962). Azoulay et al. ( 1963)after further work proposed that following dehydrogenation of thc illkane to thc alk-l-ene an oxygenation of the double bond would lead to n I ,2-epoxide which would be reduced to the primary alcohol. This proposal would not conflict with lsO-incorporation studies cited above. It has been pointed out that the energetics of NAD reduction by an alkane deliydrogcnase are unfavorable (Johnson, 1964 ; McKenna and Kallio, 1965).Further, Pcterson et al. (1967)found that enzyme fractions prepared from P. aeruginosa (used by the French group) and P.oleovorans (used by Coon’s group) were interchangeable in catalyzing the NADHdependent hydroxylation of the n-alkane. No NAD-dependent dehydrogenation of the hydrocarbon could be demonstratcd. Additional evidence against nlk- l-encs as free intermcdiates in alkane oxidation was provided by studies mhicli demonstrated that an n-alkanc and its corresponding alk-1-enc gave rise to different oxidative products (Huybrcgtse and van der Linden, 1964; Markovetz et al., 1967; Klug and Rlarkovetz, 1967b, 1968 ; Jones and Howe, 1968a).Assimilation studies of alkanes and the corresponding alk-l-enes have demonstrated that the ability of an organism to utilize a particular chain length n-alkane does not necessarily allow for the utilization of the alk-l-ene of the corresponding chain length (Klixg and Narkovetz, 1967a : Markovetz et al., 1968), thereby providing additional indications that alk- 1 -enes are not intermediates in alkane oxidation. I n only one case has the reverse situation been reported, i.e., growth on an alk-l-ene and not on the corresponding alkane (Finnerty et al., 1962). Howcver, reports of alk-l-ene formation from n-alkanes and NAIIdependent alkane dehydrogenations continue to appear and they cannot be ignored. n’ngner et al. (1967) identified hexadec-l-ene from n-hexadecane mediated by whole cells and a crude cell-free extract from 3ocardia. lizuka et al. (1968) claimed that a cell-free extract of Candida rugosa contained a NAD-dependent decane dehydrogenase, and subsequently dec-l-ene was identified (Iizuka et al., 1969). Interestingly enough, these authors felt they had presented evidence that the formation of the primary alcohol involved the addition of H,O to the double bond rather than molecular oxygen. Lebeault et al. (1969, 1970b) using mitochondria1 preparations from C. tropicalis grown on n-tetradecane, reported the reduction of NAD in the presence of decane. Again, the French group reported that this activity was measured in the absence of molecular oxygen. The dehydrogenase activity was proportional t o

34

31. J. KLUG AND A J. MARKOVETZ

enzyme concentration, except for concentrations higher than 0.5 mg protein a t which point a change in slope occurred. This change did not occur in the prcsence of ATP (lop341). Also, ATP was found to bc necessary for alkane dehydrogenation in the presence of oxygen. We have meiitioiied work which questions the importance of alk- 1-ene intermediates in alkane oxidatioii. Alternate explanations proposed for observed NAD reductiou have been, (i) traces of impurities in thc substrate which were present initially or which 1%ere formed by substrate emulsification procedures are being oxidized or, (ii) 0.)in thv anaerobic experiments, again possibly due to cniulsificutioii of the substrate, allowed for oxidation to a vompound which readily reduced NAD. The matter of energetics is the most questionable point concerning alkane dehydrogenation. If we accept that alkane dehydrogenation is coupled to NAD reduction then a most intriguing biochemical question remains, i.e. how is this accomplished? Perhaps the explanation put forth by Johnson (1964) needs careful evaluation in this system. Reference was made to a “reverse electron flow” situation demonstrated by the reaction shown by Chance and Hollinger (1961) and Sanadi et nl. (1964) NAD+ i succiriate

+ ATP

+ NADH

+ fumarate + ADP + PI + H+

An analogous reaction might be occurring with the 72-alkane, especially since ATP has been shown by the French group to exert some type of action on their system. Subterminal oxidation of the alkane chain leads to the formation of secondary alcohols and ketones. Free-radical equilibrium between the C-1 and C-2 positions, followed by formation of 1-alkyland 2-alkyl liydroperoxide was postulated to account for the formation of methyl ketones (Leadbetter and Foster, 1960).I n their scheme the 2-alkyl hydroperoxide was visualized as being reduced to the 2-OH alcohol prior to methyl ketone formation. If indeed the hydroperoxide is a transitory interniediate in ketone formation it could also be postulated that the alcohol need not be an intermediate since the ketone could be formed directly from the 2-alkyl hydroperoxide. For that matter, the aldehyde could also be formed directly from the 1-alkyl hydroperoxide thus bypassing the alcohol stage i n both cases. It is not clear whether %OH alcohols were searched for in work reported from Foster’s laboratory (Leadbetter and Foster, 1960; Lukins and Foster, 1963). Alcohols could be formed from the reduction of the aldehyde or ketone and thereby not be directly involved in the pathways under consideration. Forney and Markovetz (1968, 19G9)found that tridecan-2-one was not only oxidized by pseudomonad cells and cell-free extracts but also reduced to the corresponding secondary alcohol. A direct liydroxylation of methylene groups could also account for

UTILIZBTION O F ALIPHATIC HYDROCARBONS BY MICRO-ORGANISMS

38

secondary alcohols as the first stable intermediate in subterminal oxidation leading to ketone formation. At any rate, a free radical equilibrium among the various susceptible loci in the alkane chain may be an attractive hypothesis to account for the various isomeric alcohols and kctones which have been reported to occur in some microbial systems capable of subterminal alkane oxidation (Fredricks, 1967 ; Klein et al., 1968; Klein and Henning, 1969; Allen and Markovetz, 1970). Direct hydroxylation by a mixed-function oxidase of an internal methylene group in a hydrocarbon chain has been reported to occur in a plant system. Galliard and Stumpf (1966) demonstrated that a microsomal fraction from developing seeds of the castor bean hydroxylated oleyl CoA a t CI2 to form ricinoleic acid. At this point it is pertinent to mention a recent paper from the extensive work from the laboratory of Tullochand Spencer on the hydroxy acid glycosides produced by Torulopsis. Heinz et al. (1969) worked with a species of yeast of the genus Torulopsis which hydroxylated CIS fatty acids a t the w-1 position. It was determined that the oxygen introduced on hydroxylation was derived from molecular oxygen and not water. Three routes involving different intermediates were considered for hydroxylations at the penultimate carbon, i.e., (i) an interaction with molecular oxygen probably involving 1 proton, (ii) involvement of protons on adjacent carbon atoms, and (iii) a mechanism involving two protons of the w - 1 methylene group. Acids deuterated in the 18, the 16 and 18, and the 17 positions were incubated with whole cells. No deuterium atoms were lost from the 16 or 18 positions which would tend to rule out an unsaturated compound with a 17-18 or a 16-17 double bond as an intermediate (see (ii) above). Therefore the mechanism was confined to the w - 1 methylene group. To determine if postulation (iii) involving a methyl ketone intermediate was valid, stearic acid with two deuteriums a t the 17 position was employed, and it was found that most of the product recovered had lost only one deuterium atom. Finally, the hydroxylation occurred with retention of configuration since on incubation with N-L-and 17-~-deuterostearate it was determined that only the 17-~-deuteriumatom was lost in formation of the hydroxy acid of the 1,-configuration. Jones and Howe (1968a) using Torulopsis gropengiesseri found W and w - 1 hydroxylation of alkanoic acid. Acids of C16-Cls were near the optimum length for w - and w - 1 hydroxylation. An interesting proposal made by the authors is that both w - and w - 1 hydroxylation are catalyzed by a single enzyme on which the acid can adopt two alignments. One would expose the terminal methyl group and the other alignment would expose the w-1 methylene while shielding the methyl terminus from oxidation. The alignment favored would be the one in which distance

36

M. J. KLUQ

AND A . J. MARKOVETZ

between hydroxylation site and carboxyl end is closer to the optimum for efficient hydroxylation, in this case, a spacing of 14 methylene groups between carboxyl terminus and hydroxylation site. I n studying the mechanisms of hydroxylation Jones (1968) prepared methyl stearates stereospecifically labeled a t C17 with tritium and incubated each of these compounds mixed with uniformly 14C-labeled methyl stearate with the yeast. The 3H:14Cratio in the recovered methyl 17-~-hydroxystearate as compared to the ratio in the initial substrate mixture was lowered when the 1 7 - ~ - ~labeled H substrate was used. When the 1 7 - ~ - % labeled substrate was employed the ratio was only slightly changed. This indicated a stereospecific hydroxylation was taking place a t the W-1methylene group. The dimethyl octadecane-1,18-dioates arising as a result of w-oxidatmionactually were enriched in tritiated molecules. This was explained by an isotope effect due to a reaction involving a position other than the tritiated position, i.e., the w- rather than the tritiated W-1position. The conclusions reached were that W and w- 1 hydroxylations are independent reactions involving a direct substitution of a hydrogen by a hydroxyl group and furthermore, the w- 1 hydroxylation is a stereospecific substitution which takes place with retention of configuration. Jones and Howe (1968a) had observed, as had others, that alk-1-ene oxidation by this yeast gave products differing from those obtained when n-alkanes were oxidized, thereby indicating that alk-1-enes were not intermediates in alkane oxidation. However, the possibility of an enzyme-bound alk-1-ene intermediate could not be ruled out. Jones (1968) attempted to provide some evidence concerning this point. 1-Bromoheptadecane, tritiated a t CI6, was mixed with uniformly labeled 14C-heptadecaneand provided as substrate with the recovery of heptadecane-l,17-dioate. Jones and Howe (1968b) had determined that 1-bromoalkanes were oxidized to BrCH2(CHz),CHzOH -+ BrCHZ(CHZ),COOH -+ BrCHOH(CHz),COOH

+ OHC(CHz),COOH -+ HOOC(CHz),COOH

The 3H:14Cratio in the C17dicarboxylic acid showed no change from that of the initial substrate. This finding tended t o rule out an intermediate which would have been formed by abstraction of a tritium and hydrogen from the 16 and 17 carbons, i.e., an alk-1-ene intermediate. Retention of configuration in w-1 hydroxylation suggests an electrophilic displacement reaction. Jones (1968) pointed out that the retention of configuration noted in hydroxylation may occur because orientation of the substrate only allows exposure of one of the two methylene hydrogens. The breaking of the C-H bond and the subsequent formation of C-0 bond would probably occur on the same side of the carbon atom

UTILIZATION O F ALIPHATIC HYDROCARBONS BY MICRO-ORGANISMS

37

and therefore, Jones concludes-"the stereochemistry of the hydroxylation does not necessarily provide a distinction between reaction mechanisms involving nucleophilic substitution, electrophilic substitution, or a radical process."

B. ALK-~-ENES Oxidation products of alli-I-enes indicate that oxidative attack on these unsaturated substrates may occur a t several positions. Oxidations a t the methyl group and methylene CL to the methyl group in aliphatic chains havc been discussed above. The following will deal with oxidation at t,he terminal double bond. Ishikura and Foster (1961) demonstrated the involvement of molecular oxygen in terminal double bond oxidation by Candida lipolytica with lS0 experiments. Hexadecane-1 J-diol derived from hexadec-1-ene showed a 56.4% "0 enrichment while octadecane-l,2-diol from octadec-l-ene gave only a 32.2% enrichment. It was proposed that one atom of oxygen was incorporated in diol formation. However, the higher value seen with hexadec-1-ene could also suggest that two atoms of oxygen were incorporated. If one atom of oxygen is incorporated then an epoxide intermediate in the formation of the 1,2-diol would be an attractive consideration and, 1,2-epoxides have been isolated from oxidation of alk-1-enes (see PATHWAYS). Enzymatic hydroxylation of steroids are known whereby the hydroxylase capable of introducing a hydroxyl a t a given carbon atom is also capable of converting a double bond a t this same site into an epoxide. For a recent treatment of mechanisms of steroid hydroxylation the reader is referred to a review by Sih (1969). Van der Linden (1963) suggested a similar situation for alkanes and alk-I-enes, that is, a methyl group oxygenase when confronted with a terminal double bond would form an epoxide. If we consider the epoxide formed by the hydroxylase as an intermediate in double bond oxidation then one must conceive of several reactions to account for the products detected originating from double bond oxidation, i.e., 1,2-diol, saturated primary and secondary alcohols. Azoulay et al. (1963) proposed an epoxide reductase which presumably would cleave and reduce the oxirane ring between C-2 and oxygen forming a primary alcohol. Presumably one could as well postulate an epoxide reductase which cleaves and reduces on the C-1 side to account for alkan-2-01production. We are unaware of experimental evidence for such reductases. However, an epoxide has been shown as an intermediate

3x

31

J KLC(: AND A . J . MARKOVETZ

i l l ;I conil Jex single hydroxylation reaction involving an aliphatic polyolefin, squalene. The epoxide intermediate then rearranges and cyclizes into the steroidal configuration of lanosterol (van Tamelen et al., 1966; Corey et al., 1966). Huybregtse and van der Linden (1964) indicated that the epoxide formed from oct- 1-ene was hydrolyzed non-enzymatically to form the 1,a-diol. However, enzymic hydration of epoxy groups to form diols has been reported. I'ulloch (1963) and Hartman and Frear (1963) demonstrated t hc enzymic conversion of cis-9,IO-epoxyoctadecanoic acid to 9, I 0-diliydroxyoctadecanoic acid by plant rusts. Niehaus and Schroepfcr ( 1 907) demonstrated that the same soluble pseudomonad enzyme preparation which stereospecifically hydrates the double bond of oleic acid to form D- 10-hydroxystearic acid also catalyzes the hydration of cis- and trans-9,lO-epoxystearic acids yielding threo- and erythro9,IO-dihydroxystearic acids. The oxygen of water was found to be specifically incorporated a t C- 10 during hydrolysis of the epoxides. Jerina et al. (1968) found that benzene oxide was converted to the dihydroxy compound by an epoxide hydrase present in a rabbit liver microsomal fraction. 1,S-Diol formation from alk-1-enes could also occur via a cyclic peroxide which on cleavage would yield a 1,2-diol with both atoms of oxygen originating from molecular oxygen. Such a mechanism was proposed to account for the observation that both oxygens present in the hydroxyl groups of catechol, derived from oxidation of anthranilic acid, originated from molecular oxygen (Kobayashi et al., 1964). Gibson et al. (1968) indicated that cyclic peroxide rather than an epoxide may be an intermediate in catechol formation from benzene mediated by a pseudomonad cell-free extract. Hydroxylation of the double bond could lead directly to a methyl ketone or an aldehyde. Reduction of these compounds would account for the finding of saturated alkan-I- and 2-01s from alk-1-ene oxidation. However, aldehydes and ketones have been postulated intermediates of mixed-function oxidases and neither has been experimentally demonstrated as arising from double bond hydroxylation. I n concluding this section on mechanisms mention should be made of the work of Das et al. (1968) concerning a mixed-function oxidase system from rat liver microsomes. These workers concluded that hydroxylation of heptane, lauric acid and testosterone, as well as the oxidative demethylation of aminopyrine proceeded by way of a common microsomal hydroxylating system. I n light of this and other work cited above it will be interesting to see if one microbial system could account for most of the initial oxidation products arising from methyl and methylene groups and a terminal double bond of various aliphatic hydrocarbons.

UTILIZATIOS O F ALIPHATIC HYDROCARBONS B Y MICRO-ORGANISMS

39

IX. Occurrence and Biosynthesis of Aliphatic Hydrocarbons Hydrocarbons have been detected from diverse biological sources. Plants (see reviews by Eglinton and Hamilton, 1967 ; Kolattukudy, l9G8a), insects (see review by Gilby, 1965), human spleen (Liber and Rose, 1967), and human meninges and meningiomas (Cain et al., 1967)' to list only a few examples, have been found to contain various aliphatic hydrocarbons. Fungal spores (Or6 ef al., 1966), blue-green algae (Gelpi P f al., 1968; \\'inters P t al., 1969) and bacteria (Huston and Albro, 1964; Tornabene cf nl., 1967) also contain aliphatic paraffins and olefins. Information is becoming available concerning the biosynthesis of thcse compounds and it appears that several different mechanisms may be involved. Kolattukudy (19GSb)has provided evidence for a pathway to explain the synthesis of a CZD n-alkane in broccoli leaves. Palmitic acid is synthesized de novo from acetate and then elongated by another system to thc C3" acid which undergoes decarboxylation to the C,, hydrocarbon. However, Grice et al. (1968)have stated that Kolattukudy's mechanism would not account for the alkane pattern seen in heartwoods from the Guttiferae. By using acetate-l-"C, Kelson (1969) found that hydrocarbon synthesis occurred in the integument but not the fat body of thc American cockroach. The actual mechanism of synthesis was not ascertained. In a series of papers on hydrocarbon synthesis by Xarcina lutea, Albro and Dittmer (1969a, b, c, d ) have proposed that biosynthesis occurs by a condensation of two fatty acids to yield CO, and a monounsaturated hydrocarbon which is reduced to the saturated analogue. This represents a modification of the mechanism proposed by Chibnall and Piper (1934) for hydrocarbon synthesis via condensation of two fatty acids yielding an internal ketone plus CO,. The carbonyl function would be reduced to the secondary alcohol followed by dehydration to the unsaturated intermcdiate and finally reduction to the alkane. Further clarification of mechanisms involved in these proposed pathways on an enzyme level will be of considerable interest, as will be the reason for such a biosynthetic capability in micro-organisms.

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Biochemical and Physiological Aspects of Differentiation in the Fungi .JOHN E. SMITHand JILLIAN C. GALBRAITH Ihparlment of Applied illicrobiology, lJii iversity of Sfmthclyde, Qlasgow, Scotland, and Depwtment of Biology, Paisley College of Technolog!/, Paisley, Scotland 1. Iiitrodiiction . 11. Acrasiilles . . A4.Life-Cycle . 13. Ccll Aggrcfi"tliori . C. Metitt)olism Dnring Morphogoricsis . 111. Division Jlycota : Subdivision Rlyminycotiiia : C L r s Nyxoiiiycetc~s A . Llfc-Cyclt . . I < . Thc I'lasmodiiiin C. Sclcrotinm Formatioir . . D. Spornlation . I\-. Eiimycotiiia . A. Ccll Wall Construction and Morphogciicsis . 13. Light-Indiiced Sporiilation and Sporogrnic Srtbstanccs . C. Biochemistry of Asexual Spornlntion . D. Hormones and Sexiial Reproduction . E. Secondary Metabolitrs and Differentiatioii . V. Acknowledgements . References .

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45 47 47

51 53 63 03 65 65 67 69 69 75 79 104 116 124 124

I. Iiitroduction The pastj decade has seen extraordinary progress towards a fuller understanding of gene action. I n particular, the relationship between gene action and macromolecular synthesis has been intensively studied and it is now apparent that differentiation results from an interplay between an unchanging genome and a labile cytoplasm, the cytoplasm at any one period determining the sequence of gene expression while itself being modified as a consequence. This apparent paradox of cellular differentiation in the face of a constant genetic apparatus has prompted investigations into nuclear differentiation, extrachromosomal heredity and biochemical mechanisms of morphogenesis. The classical studies of Jacob and Monod (1961) on 45

46

JOHN E SMITH AND JILLIAN C. GALBRAITH

gene rcprcssion and induction in bacteria have provided a model of chemical differentiation without alteration of the primary genetic information, and it is now clear that differentiation results from changes i n the relative importance of biosyntlietic pathways as different products nccumulate scquentially. Thus, altered cell chemistry can be considered to be prerequisite to altered cell structure and behaviour. A completely satisfactory definition of differentiation is not yet possihle, but for the purposr of this rcvicn- differentiation 1% ill be considered as “thc slim of processes by which acquisition of specific metabolic ncw (or thc loss thereof) distinguishes daughter cells from each other or from thc parental cells” (Gross, 1968). For a compreliensive discussion on the biochemistry of differentiation in eukaryotic cells, see Gross (1968). Biochemical studies of differentiation aim to correlate chemical activities with morphological changes, either at the level of nucleic acids or enzymes. Currently most studies centre around the transition between primary gene action (transcription) and cell chemistry, and much real progress is being made. The second stage of transition between cell chemistry and changing structures and behaviour is less well understood. Huxlcy (1 924) drew a distinction between primary and sccondary phases of embryonic cliffcrcntiation. I n the primary phase the presumptive fatc of thc. cell is determined although there are no detectable morphological c,hanges, and in thc sec.ondary phase cytodifferentiation and tissue elaboration o(wu-. The grcntcst challenge is the cause of tlie chain of events lcading to thc visible effect. Such studies must involve the examination :uid separation of the primary from tlie secondary phase. Wright ( 1 966) lias pointed out the danger of searching for a single cause or trigger mechanism, I\ liich would oiily dclay the eventual understanding of a phenomenon often brought about by varied and independent forces. A study of niorphogcnesis poses the problem of distinguishing between tlie biocliemistry of cellular differentiation and the general biochemistry of growth, both of which are expressed through cell expansion and cell division. I n this respect, fungi are idcal subjects for such a study because differentiation is generally associated with the end of the period of active growth. Fungi are easily cultivated in the laboratory, the response of many is synchronous, and in most cases morphogenesis can be controlled by adjiistnient of certain physical or chemical factors in the environment. Once such a controlling factor has been found direct comparison of the biochemicd events in a differentiating and non-differentiating culture, which differ initially in only one factor, is possible. In recent years there have been several authorative reviews on various aspects of fungal morphogenesis and these should be consulted to obtain

ASPECTS OF DIFFERENTIATION IN THE FUNGI

47

n. completely comprehensive coverage (Nickerson and Bartnicki-Garcia,

1964 ; Baldwin and Rusch, 1965; Morton, 1 967 ; Gray and Alexopoulos, 1968 ; Sussniaii and Sussmuii, ll)(i!) ; Turian, 1960). ‘Vhroiighout this rctview the systc~mof classiiicatioii adopted is that of‘Xlexopoulos ( 1962).

11. Acrasiales Pew microbiologists can dispute the fact that the work of the past decade on the biochemistry of cellular slime mould differentiation represents one of the most exciting developments in contemporary cellular biology. Xot only have these studies set out valuable information concerning the initiation aiid control of the process of differentiation in slime moulds, but they have also significantly contributed to a clearer understanding of the evolution of morphogenesis in micro-organisms as well as higher organisms (Wright, 1970b). Several excellent reviews on various aspects of differentiation in tlie slime moulds have been published, aiid those by Gregg (1966), Wright (1964, 1966, 1970a, b), Bonner (1967) and Sussman arid Sussman (1969) are recommended to eiiibellish thc following brief account. The cellular slime moulds are eukaryotic soil protists which can be cultivated in association with bacteria. Whether or not these organisms are true fungi can still be debated though traditionally their study has very largely been tlie domain of the mycologist (Alexopoulos, 1966). One of the main advantages of the slime moulds is the ability to separate their life-cycle into a proliferating unicellular phase and a multicellular phase in which a series of morphogenetic events are carried out through extensive shifts in enzyme patterns and metabolite levels within the cells. Thus, unlike most higher plants and animals, the slime moulds undergo a transition from the unicellular, free-living conditions found in many micro-organisms to the multicellular integrated conditions associated with higher organisms. The component myxanioebae retain their individuality by not fusing yet co-operate a h members of a well organized community until sporulation occwrs. For this reason the Acrasiales have oftcn been termed communal slime moulds. Thus, a unique opportunity is available to determine the factors tliat permit independent cells to adapt to a multicellular existence and tlie subsequelit interactions that effect their course of development. A.

LIFE-CYCLE

The germinating spore releases a single uninucleated non-flagellated niyxamoeba. The myxamoebae typically feed on bacteria by phagovytosis and multiply exponentially by binary fission. Recently, axenic

48

J O H N E SMITH A N D J I L L I A N C. GALBRAITH

cdtures of Dictyostelium discoideum have been obtained on complex media rich in proteose peptone and yeast extract (Sussman, 1966; Sussman and Sussman, 1967); previously only Polysphondyliuw pallidium could be cultivated successfully in defined liquid medium (Hohl and Raper, 1963; Sussman, 1963). The importance of this devclopineiit in culture practice cannot be overstated as it will permit a detailed examination of tlie nutrient requirements of slime moulds. When the food source is becoming depleted and near exhaustion, the solitary vegetative myxarnoebae begin to aggregate and stream together to central collection points to form cell masses or communal pseudoplasmodia each containing about l o 5 cells. The component cells of the pseudoplasmodium are held together within a sheath of slime in which tlie cells move in a concerted and integrated manner. There is no fusion of the protoplasts and the uninucleate character of the myxamoebae cells is maintained throughout the various phases of development. Feeding stops sometime before aggregation and all future morphogenesis depends on the endogenous food materials in the myxamoebae to serve as the source of energy for completion of development (Gregg, 1966). However, n u r n ( ~ o ~low i s molecular weight organic or inorganic compounds can accelerate the rate of sorocarp formation without affecting tlie quality of morphogcnesis (Bradley et al., 1956; Iirichevsky and IYright, 1963; Iirichevsky et al., 1969). They appear to act by preventing leakage of RNA and protein from the cells and so promote an internal accumulation of rnononurleotidcs which accelerate niorphogenesis. For morphogenetic investigations by conventional biochemical procedures a degree of synchronous development is required and, to this end, the myxanioebae are harvested at the beginning of the stationary growth phase, washed, resuspended, and dispersed on non-nutrient solid substratum to begin their morphogenetic development. Several solid-surface techniques have been used including non-nutrient agar surfaces (Wright and Anderson, 1960; White and Sussman, 1961), buffered salts/strcptomycin solutions on 47 nim diameter Millipore filters or Whatman No. 50 filters (Sussman, 1966; Sussman and Sussman, 1969); or alternativcly, single aggregates may be examined by immunochemical and biochemical assays (Gregg, 1966). Each group of cells becomes polarized and forms a cartridge-shaped pseudoplasmodium which can migrate over the substratum along temperature gradients and towards light (Sussman and Sussman, 1969). The slug stage has been shown to contain two types of cells that differ in buoyant density, cytological appearance and enzymic composition (Miller et al., 1969). When the slug comes to rest, about one-third of the cells form a tapering stalk over which the remainder of the cells migrate and become encapsulated to form spores. Each spore is uninucleate and encapsulated in a hard

49

ASPECTS O F DIFFERENTIATION I N T H E F U N G I

cellulose spore case (Fig. 1). For further details of t h e life-cycle see Alcxopoulos (1966) a n d Bonner (1967). Studies with mutant, strains derived from haploid D.discoideum have shown t h a t genetic exchange can take place at a low frequency during

s

Pseudoplasmodium

t

\ Germinot ion

\

/

/

\

Amoebae

‘\

/

\

7 Meiosis 7 \

\ \

7

Plosmogamy

7

I

FIg. 1 . Life-cycle of Dictyostelium discoideum. Adapted from Alexopoulos (1962).

the life-cycle (Loomis a n d Ashworth, 1968; Sinha and Ashworth, 1969; Loomis, 196Ba). The participation of a parasexual cycle has been inferred by Sinha a n d Ashworth ( 1 969) who isolated a n d characterized heterozygous diploids from D.discoideum (Fig. 2 ) . These diploid cells were extremely unstable a n d api’ear t o lose individual chromosomes randomly over many generations forming aneuploid and haploid cells. The mean volumc of a diploid spore population is twice t h a t of a haploid spore

50

J O H N E. SMITH A N D J I L L I A N C . GALBRAITH

population and the distribution of volumes amongst a spore population can be analyzed in terms of the frequency of occurrence of haploids, diploids and aneuploids (Ashworth and Sackin, 1969). Haploid strain (XY

Haploid strain

1

(XY)

Heterozygotic spore (diploid)

Heterozygotic myxamoeba (diploid) X y XY

I

Aneuploid spore

Haploid spore

Haploidization

Aneuploid inyxamoeha

I

Haploidization

Haploid mpxamoeha Parental (xy) ( X Y ) Recombinant (XU) (Xy)

Frg. 3. The parasexual cyclc of Uictyostelizm discoideuna. x/X and y/Y represent two unlinked gencs, and the genotype of the various kinds of nuclei are enclosed i n parentheses (Sinha and Ashworth, 1969).

Treatment of myxamoebae with various mutagens has led to the isolation of strains with hereditable aberrations in the developmental sequence. Such mutants either stop at a stage prior to culmination or they mature with a fruit body of abnormal character. These mutants also display changes in the normal metabolic pattern consistent with the stages a t which the particular morphogenetic observation occurred

ASPECTS O P DIFFERENTIATION IN THE FUNGI

.5 1

(Sonneborn P t al., 1963; Sussman and Sussman, 1953; Yanagisawa et al., 1967). Mutants of D . discoideum, which have a temperature-sensitive step either in growth processes or in the development processes of aggregation and differentiation, have been isolated by Loomis ( 1 969a). Strains \vhose development is temperature sensitive probably carry mutations that affect the cells only during the phase of aggregation before the formation of the multicellular fruit body. Genetic studies suggest that several of these temperature-sensitive and morphological mutations are recessive and non-identical. Mutants which affect growth, but not development, have been described by Looniis arid Ashworth (1968). B. CELL AGGREGATION Cell aggregation comprises two distinct sets of activities, namely chemotaxis and adhesion. 1. Chemotaxis

The mechanism which enables cells of the slime mould t o make the transition from a unicellular state to the multicellular condition involves a chemotactic response. The aggregation process is mediated by a chemical substance, acrasin (Runyon, 1942; Bonner, 1947) which can be destroyed by an extracellular enzyme, acrasinase (Shaffer, 1956). Acrasin activity has been found in steroid animal hormones and A''stigmasten-3/3-01has been extracted from D. discoideum and shown to have weak acrasin activity (Heftman et al., 1960). I n Acytostelium leptosomum the alkaloid yohimbine tartarate was the most active acrasin-type compound amongst several alkaloids and steroids tested (Hostak and Raper, 1960). Many types of bacteria can support growth of D. discoideum and, furthermore, bacteria are not only the food source of the myxamoebae but also chemically attract the myxamoebae (Samuel, 1961). All Gram-positive and Gram-negative bacteria so far tested can attract myxamoebae and the attractant of Escherichia coli can activate several strains of Dictyostelium and Polysphondylium (Konijn, 1969). Myxamoebae close to their aggregation phase were most sensitive to the bacterial attractants, and high concentrations of bacterial attractant could disperse aggregates even when they were in an advanced stage of morphogenesis. It has recently been demonstrated that adenosine-3',5'-cyclic monophosphate attracts myxamoebae and can be considered to be an acrasin (Konijn et al., 1967, 1968, 1969). Acrasin may also be extracted from Escherichia coli (Bonner et al., 1969) as can cyclic AMP (Makman and Sutherland, 1965). Substantial quantities of cyclic AMP can be synthesized by D. discoideum and P. pallidium (Konijn et al., 1970; Barkley,

52

JOHN E. SMITH AND JILLIAN C. QALBRAITH

1969) and D. discoideum also produces a phosphodiesterase which specifically converts cyclic 3’,5’-AMP to 5’-AMP (Chang, 1968). Thus, for D.discoideum, cyclic AMP is clearly an acrasin and phosphodiesterase an acrasinane. Phosphodiesterase is secreted throughout the vegetative, aggregative and migrative steps of development. There is a hundred-fold increase in sensitivity t o cyclic AMP between the beginning of aggregation and late aggregation, falling to a low level during migration. The sensitivity t o phosphodiesterase also increases a hundred-fold during aggregation ; therefore the chemotactic system is at least l o 4 times more effective during aggregation than during the vegetative stage (Bonner et al., 1969). Thus, when a myxamoeba starves, it becomes highly sensitive to gradients of cyclic AMP and will move towards the source of secretion. If the source of cyclic AMP is a bacterial colony, feeding recommences; conversely, if the source is another myxamoeba of the same strain, aggregation will occur. Cyclic AMP is widely distributed in animal cells and is considered t o function as a second messenger or intermediate in certain hormone reactions (Sutherland et al., 1965). It has been considered that the hormone activates a cell-surface adeiiyl cyclase which converts ATP to cyclic AMP and this in turn triggers the hormone-induced reaction. The role of cyclic AMP in E. coli is not clearly understood though probably it is part of an internal biochemical regulatory system. I n cellular slime moulds cyclic AMP clearly can act as a hormone as well since it can convcy stimuli across large intercellular spaces (Bonner et al., 1969; Konijn, 1969). Besides the ability to attract myxamoebae, cyclic AMP can also stimulate: (a) rate of movement of the myxamoebae; (b)production of cell adhesiveness ; and (c) inhibition of centre formation (Bonner et al., 1969). It is of considerable interest to understand how 3’,5’ cyclic AMP promotes the orientation of myxamoebae. Many analogues of cyclic AMP have been tested on D.discoideum and only analogues having the 3 ‘ , 5’ ring structure were capable of causing some degrce of orientation (Konijn et al., 1969). It is not clear why only some cclls secrete cyclic AMP and thus initiate the process of aggregation. Early studies had suggested that a myxamoebae culture was not initially homogeneous but contained a few cells, called initiators or I-cells (Sussman and Noel, 1952), around which other cells will collect. It has been claimed that one I-cell per 2200 responder cells can be detected in populations of D.discoideum (Ennis and Sussman, 1958) and recently Ashworth and Sackin (1969) have considered aneuploid cells (inherent in the concept of a parasexual cycle) to be implicated in the initiation process.

ASPECTS OF DIFFERENTIATION I N T H E F U N G I

53

2 . Cell Adhesion

Cell adhesion is regarded as necessary in promoting morphogenetic interaction. The adhesion of the cells is specific and involves cell recognition and sorting out. There are changes in the cell surface such as the appearances of a new lipoprotein antigen in tlie cell membrane (Sonneborn et al., 1964). Only species whicli co-aggregate with D. discoideum contain cross-reacting antigens (Gregg, 1966). The adhesive material has yet to be idcntified although the evidence suggests substances like mucopolysaccliarides. A surface polysaccharide involved in speciesspecific adhesion contains a high concentration of N-acetylglucosamine (Gerisch et al., 1969) and it has been considered that an accumulation of the enzyme acetylglucosarniiiidase may be required for the cells to stick to each other to form a pseudoplasmodium. C. METABOLISMDURINGMORPHOGENESIS 1. Chanqes in Carbohydrates In slime moulds, morpliogeiiesis is initiated in part by some degree of nutritional deficiency. Furthermore, tlie availability of the appropriate energy source during the early stages of morphogenesis will inhibit or reverse morpliogeiiesis completely (Bonner, 1967). Since all nutrient uptake stops at the time of aggregation, endogenous substrates must provide the only source of energy for the process of fruit-body development and maturation. Such systems become self-sufficient and to a greater or lesser degree have cut themselves off from their environment (Wright, 1967). Extensive catabolic turnover of pre-existing endogenous constituents takes place and, a$ a result, cells lose a considerable fraction (approximately 30-50% of the initial weight of vegetative myxamoebnc) of their original dry weight (Liddel and Wright, 1961). It is considered that protein is the main energy source in this “closed system” and that the polysaccharide end-products of differentiation are derived largely from glycogen (Cleland and Coe, 1968; Baumann and Wright, 19G8). Calculated on the basis of the original dry weight, total carbohydrate does not vary greatly during development (Gregg and Bronsweig, 1956 ; White and Sussman, 1961 ; Ceccarini and Filosa, 1965).At aggregation 5% of the dry weight of the pseudoplasmodium is made u p of soluble glycogen and approximately I:/, by trehalose and glucose. At the terriiination of differentiation it is possible to account for roughly all of the total carbohydrate in the following identifiable compounds : the disaccharide trehalose (glucose 1,la-D-glucoside)which accumulates in the spore and apparently serves as a carbon and energy source during germination (Clegg and Filosa, 1961; Ceccarini and Filosa,

54

J O H N E. SMITH A N D J I L L I A N C. GALBRAITH

1965), 2.6% ; an acid inucopolysaccharide composed of galactose, Nacctylgalactosaniiiie and galacturonic acid and found associated exclusively with the spores in the mature fruit body, 0.7% (White and Sussman, 1963); soluble glycogen, 1.3% (Wright and Dahlberg, 1967); and the cellulose-glycogen cell-wall complex which in mature fruit bodies constitutes the integument of the stalk and is also present in the spore coats

Trehalose . 0

k' 0 0

---7iiov -0 10

20

-

Cellulose

1

30 40

50

Hours of development

FIG.3. Carbohydrate synthesis during morphogenesis in Dictyosteliurn discoideum (Sussman and Sussman, 1969). The experiments were performed with cells incubated 0 1 1 nori-nutrient agar.

(Gezelius and Ranby, 1957), 2.4%. Figure 3 summarizes the pattern of carbohydrate synthesis during morphogenesis 2 . Respiratory Metabolism

Vegetative myxamoebae are facultative anaerobes (Wright and Anderson, 1958, 1959) while all phases after aggregation are obligately aerobic (Bradley et al., 1956). Mitochondria have been isolated and characterized from D. discoideum (Erickson and Ashworth, 1969) and the electront
ASPECTS O F DIFFERENTIATION IN THE FUNGI

55

M'atson and Smith, 1967, 1968).Cytological evidence suggests that mitochondria (Duncan et al., 1970) and some mitochondria1 enzymes (Milne and Takeuchi, 1967) change during morphogenesis of D. discoideum. Isocitrate dehydrogenase, glucose 6-phosphate dehydrogenase, glutaiiiate dehydrogenase and glutamatelpyruvate aminotransferase do not vary significantly during development (Wright and Anderson, 1958). The activity of malate- and 6-phosphogluconate dehydrogenases increased during aggregation though the malic enzyme decreased in activity after the completion of culmination (Wright and Anderson, 1958). The anterior prestalk part of D. discoideum has the greatest alkaline phosphatase (Bonner et al., 1955; Krivanek, 1956) and 5'nucleotidase activity (Krivanek and Krivanek, 1958 ; Gezelius and Wright, 1065) as shown by histochemical analysis. High activity of cytochronic oxidase and succinate dehydrogenase was found in the posterior prespore area (Takeuchi, 1960). A complete glycolytic pathway via lactate dehydrogenase has been demonstrated in D.discoideum (Cleland and Coe, 1968).Alternate pathways of hexose metabolism diverge from all other hexose phosphates a t fructose l,B-diphosphate, and phosphofructokinase may be regarded as the first enzyme characteristic of the glycolytic sequence proper and therefore constitutes an important branch and control point subject t o strong metabolic regulation. This enzyme has been extensively studied in many organisms and it is implied that it plays an important role in energy-yielding metabolism, in particular, in regulating the balance between glycolysis and gluconeogenesis. Many observations suggest that, in the m yxamoebal stage, D. discoidpum grows mainly a t the expense of amino acids and proteins and glucose neither increases the total cell yield nor decreases the doubling time (Sussman and Bradley, 1954; Gezelius, 1962).It has been considered that, during growth of the myxamoebae, glucose is converted to glycogen which is subsequently used as a source of carbon precursors for synthesis of the various polysaccharides which accumulate during fruit-body maturation (Baumann and Wright, 1968). The low activity and weak regulation of phosphofructokinase in D. discoideum implies an altered physiological function for this enzyme (Baumann and Wright, 1968). Phosphofructokinase may serve primarily as a source of triose and acetate units for purely biosynthetic reactions or even for supplcmcntary energy-yielding metabolism in conditions where there is excess glucose in the medium (Baumann and Wright, 1968).Evideiice also exists for the presence of'a pcntose phosphate pathway in D. discoideum (Wright et al., 1964) and the metabolism of lirxose units through this system would give NADPH, ~ssentinl for 1l i t , 1)iosynthetic. re:i~tioii~assori:tted wit11 differentiation. 3

56

JOHX E. SMITH AND JILLIAN C. GALBRAITH

The extremely low activity of fructose 1,6-diphosphatase in D. discoideum (Cleland and Coe, 1968; Baumann and Wright, 1969) coupled with the observation that fructose 1,6-diphosphatase enzymes from D. discoideum (Baumaiin and Wright, 1969)and P . pallidiunz (Rosen, 1966) ‘we unaffected by AMP, ADP, or ATP, infers that glucoiieogenesis is not of major importance in slime mould development. Furthermore, 14C-glucoseis more readily incorporated into cell-wall material than is 14C-glutamate (Pannbacker and Wright, 1967) while conversely there is an increase in carbon dioxide evolution from 14C-glutamateas differentiation progresses (Wright, 1963). Thus, it becomes clear that proteins and amino acids serve as the main energy source in D.discoideum and that glucose is used primarily for glycogen and cell-wall synthesis. During differentiation when the organism is under starvation conditions there is a high rate of breakdown of macromolecular reserve materials. The high rate of protein turnover allows for extensive protein utilization as a n energy source, for maintaining the composition of critical proteins, and for synthesizing new enzymes. 3. Sequential Enzyme Development

Sussman and his coworkers have developed elegant replacement techniques for synchronous development of D. discoideurn in which differentiation can be completed in 24 hr. The reliable reproducibility of this technique has allowed accurate and detailed enzyinological investigations to be carried out a t various stages during the developmental cycle. The specific enzyme activities are assayed in crude extracts of cells harvested after different periods of incubation. At this stage it is still unclear whether the methods of measuring enzyme activity are satisfactory for the comparison of activities in crude extracts taken from organisms at different stages of growth, and care must be taken in interpreting such results. Specific activity of enzymes, under conditions of optimum substrate concentration, only measures changes in thc enzyme concentration. However, in vivo, the activity of a given enzyme concentration is dependent on substrate and cofactor availability, ionic environment, and the presence or absence of inhibitors ; failure to detect a change in enzyme concentration does not therefore imply that the activity is constant in vivo. I\?right (1963) has shown in the slime mould that an increase in the oxidative decarboxylation of glutamate occurs during development in response to the in vivo accumulation of glutamate, although the specific activity of glutamate dehydrogenase decreases during differentiation. It is generally recognized that the intracwllular concentrations of intcrmcdiary metabo1itc.s are mostly at suboptiinal levels which do iiot allow inaximal enzrme activity (Polakis

ASPECTS OF DIFFERENTIATION I N THE FUNQI

57

;iiid Bartley. 1966; Krebs, 1957). Therefore, caution must be taken in relating changing specific activities to the physiological situation during development. Also, in in vitro studies, the physiological system is incvitably distorted by the extraction procedure and in particular breakdown of oi~ganellcsmay bring enzymes and their inhibitors or inactivatiiig enzymes into contact,. Differential enzyme inactivation has been found to occur with six enzymes extracted at different stages of the life-cycle of D.discoideuw, namely isocitrate dehydrogenase, glucose 6-phosphate dehydrogenase (Wright, L960), pyruvate kinase (Cleland and Coe, 1968), cell-wall glycogen synthetase (Wright, 1965), cellulase (Rosness, 1968), and UDPG-pyropliosphorylase (Wright and Dahlberg, 1968). I n the case of UDPG-p?iroi)hosphorylase extracted at an early stage of development, the kinetics of inactivation mere lion-linear and it was not possible to determine the true specific enzyme activity. Although the enzyme from later stages of development was stable it could be inhibited by a heatlabile inhibitor present in extracts prepared a t an early stage. It is still not clear whether this type of inhibition is functioning in vivo or is purely an artifact of the extraction procedure. I n contrast the increase in the specific activity of ,tI-glucosidase during the first stages of development of D.discoideum may result from preferential protection of the enzyme present in vegetative cells from the fairly extensive protein turnover which occurs during pseudoplasriiodial formation (Coston and Looniis, 1970). Glutamate dehydrogenase, alanine-a-oxoglutarate transaminase, lactate dehydrogenase, an alkaline pliosphatase and glycogen synthetase do not, appear to be subject to differential inactivation in slime mould extracts (Wright, 1960, 1964 ; Wright and Dahlberg, 1967). A final point which must be taken into account is that the enzyme concentrations generally used in assay systems are lower than concentratioiis in the cell (Srere, 1967; Tomkins ct al., 1963). Different kinetic behaviour results with higher concentrations, such as glutamate dehydrogellase which exists as a polymer a t high concentrations and exhibits different kinetic and binding properties (Frieden and Colman, 1967). Nore expansive discussions on the topic of enzyme methodology in crude extracts of differentiating cells are given by Wright (1963), LTright and Dahlberg (1968), and Sussman and Sussman (1969). Sequential enzyme development has been studied in detail with particular eriiphasis on cell wall synthesis. a. Tyehalose &phosphate synthetase. This enzyme is concerned with the synthesis of trehitlose and is undetectable in vegetative niyxamoebae. It is first detected a t the primary stages of aggregation and there is a linear increase in activity u p t o 17 hr. of development followed by a

58

J O H N P. SMITH AND J I L L I A N C. QALBRAITH

decline prior to stalk formation (120th et al., 1968; Roth and Sussman, 1968). glucose &phosphate

+ UDPG

tiehalose 6 phosphatr

+ H20

+ trehalose 6-phosphate + UDI’ --f

trehalose

+ PI.

b. L’ricline cliphosphogalactose: polysacchaiide transfemse. This enzyme cxatnlyses the transfer of galactose from uridine diphosphate galactose to n polysaccharidc acceptor which is probably a precursor of a component of the slime sheath (Sussman and Osborn, 1964; Loomis and Sussman, 1966). Enzyme activity is first detected a t 12.5 hr. (advanwd pseudoplasmodial formation) ; it accumulates over an 8 hr. period and is then rapidly excreted into the external environment where the enzyiiie is destroyed. The product of the enzyme reaction, namely ~iiueopolysaccharid~, is retained within the organism. c. Uridine diphosphoglucose pyrophosphorylase. This eiizymc catalyses the formation of uridine diphosphate glucose, a major metabolite in carbohydrate-syiithesiziiig reactions including trehalose synthesis (Ashworth and Sussman, 1967; Newel1 and Sussman, 1969). glUC~030I -phosphate + UTP

+ UDPG + PPI

Low activity of this enzyme can be detected in vegetative myxamoebae. There is little increase up t o the end of cell aggregation when the activity increases linearly over a11 8 hr. period. Maximum activity coincides with the tcrmiiial stages of maturation. The activity associated with the stalk cells disappears while that associated with the spore is retained (Ashworth and Sussman, 1967). Other enzymes which show specific developmental patterns are trehalose (Ceccarini, 1967)) cellulase(s) (Rosness, 1968), P-glucosidase (Rosness, 1968; Coston and Loomis, 1970) and an enzyme that hydrolyses p-indopheiiyl-N-acetylglucosamine (Loomis, 1968).

4. Regulation of Enzyme Accumulation and Disappeurnnce A series of studies (see Sussman and Sussman, 1969) have been concerned with biochemical events and their causal and temporal interrelationships to RNA synthesis in slime moulds from the beginning of the stationary phase after proliferation to the completion of the mature fruit body. These studies have centred around the use of cycloheximide (actidione)which inhibits protein syiithesis reversibly, probably by iiiterfcring with the transfer of amino-acyl residues from t-RKA to the nascent polypeptide, and with actinomycin D which specifically inhibits DNAdependent RNA synthesis. Actiiiomycin D has become widely used for testing the relevance of transcription to a particular differentiation proccss. It is believed that the time at which actinomycin D treatment

59

ASPECTS O F DIFFERENTIATION I N T H E FUNGI

prevents subsequent appearance of the characteristic differentiated product of activity is in fact the time when the relevant transcription is read out from the genome (Gross, 1968).I n these studies, rates of enzyme synthesis were measured, and inhibitors were added to the cultures only after the proliferative stage had been completed and the residual bacterial food supply removed by centrifugation. The specific activity of a number of enzymes has been shown t o vary significantly during slime mould development. Experiments with either

Acetyl glucosominldase Trehalose phosphate synthetase Threonine dehydrose

UDP-Glucose pyrophosphory lase

-

c - i

-

Loomis (1969~) ~~

~

_

_

~-

Roth ef ol

W Allister ond W F Loomis (unpublished)

Roth el ol (1968)

UDP-Golactose polysacrhoride ironsferase

,3 -Glucosidose --

Time (hours)

FIG.4. Transcription and trailslation in development of Dictyostelium discoideum. A schematic summary of the periods of RNA synthesis (open bars) and protein synthcsis (filled bars) required for accumulation of seven developmentally controlled enzymes. Lag times were measured from the midpoint of the RNA synthetic period to the midpoint of the protein synthetic period for each developmentally controlled eiizyme (Loomis, 1909b).

actinomycin D or cyclohexiniide suggest that the increase in specific activity of seven enzymes, uridine diphosphoglucose pyrophosphorylase (Ashworth and Sussman, 1967), acetylglucosaminidase (Loomis, 1968), trehalose 6-phosphate synthetase (Roth and Sussman, 1968), uridine diphosphogalactose polysaccharide transferase (Sussman and Osborn, 1964), P-glucosidase (Coston and Loomis, 1970), alkaline phosphatase (Loomis, 1969b), threonine dehydrase (W. Allister and W. F. Loomis, unpublished observations) requires concomitant protein synthesis and prior ltNA synthesis (Fig. 4). There were significant differences between the time required for RNA synthesis and the period of accumulation among the various enzymes. It can be considered that the changes in specific enzyme activity probably represent alterations in specific gene activity. The transcriptive period of each enzyme was delineated by

~

-

fi0

.JOHK E. SMITH AND JILLTAN C. CALBRAITH

incorporat,iiig actinomycin D a t progressively later times during the developmental sequence, and the results imply that the transcriptive events and their duration are under developmenta.1 control. There exists some controversy concerning the extent of RNA metabolism during the period of programmed starvation. Pannbacker and Wright (1966) using 10 pg. actinomycin D/ml. found that the drug inhibited development when added up to the early stages of aggregation, but when added to aggregated cells and later stages of development it had no effect. They suggested that m-RNA synthesis may only occur during periods of growth and that the information for multicellular differentiation may be present in the myxamoebae prior to differentiation. Sussman and his coworkers, using concentrations of actinomycin D tenfold higher than Pannbacker and Wright, obtained gross and cytological derangement of development t.ogether with drastic depression of RNA synthesis. This inhibition of morphogenesis and cytodifferentiation coupled with the prevention of certain enzyme syntheses led them to conclude that RNA synthesis does play a major role in slime mould morphogenesis. More recent studies have confirmed the synthesis of new m-RNA during morphogenesis of slime moulds (Inselburg and Sussman, 1967; Sussman, R. R., 1967; Sussman and Sussman, 1969) although it has still to be shown conclusively that this m-RNA synthesis is intimately involved in differentiation and not merely serving for the maintenance of a specific protein in the different,iating system. It should also be noted that the concentrations of actinomycin D required to produce the disruption of differentiation are 2-3 orders of magnitude higher than those needed for inhibitors of RKA synthesis in ot>hersystems (Weinstein et al., 1965). In most studies using actinomycin D and/or cycloheximide, the inhibit'ors have normally been added only after the proliferative stage had been ronipleted and the residual bacterial food material removed by centrifugation. In contrast t o this, Hirschberg et al. (1968) have examined the effects of actinomycin I>, Miracil D (a bacteriost'atic and carcinostatic thiaxanthenone) and some of its derivatives, as well as cycloheximide, on myxamoebae during the logarithmic growth phase, in particular to assess the ability of the agents t o inhibit aggregation. Actinomycin D caused 50% inhibition of proliferation a t 2 x lo-* 1 11 whereas 2 x ilf was required to block aggregation (Table 1 ) . Miracil D was less effective in inhibiting proliferation than actinomycin D, while cycloheximide was also less active a t both stages. The inhibition of aggregation by these drugs was not related to cell death since myxamoebae exposed for up to 24 hr. t o concentrations ten times the minimum inhibitory concentration of actinomycin D or Miracil D aggregated normally when the drug was removed by repeated washing. Comprative

ASPECTS OF DIFFERENTIATION

61

IN THE FUNGI

TABLE1. Coniparative activity of inhibitors against proliferation arid aggregation of Dictyostelium discoideum

Compound Actinomycin D Miracil D Miracil D derivativosa AN 207 AN 216 AN 304 AN 305 AN 316 AN 317 Quinacrine Cycloheximide Puromycin

Concentration causing 50% inhibition of proliferation (M)

Minimum concentration preventing aggregation (M)

2 x 10-8 2 x 10-6

2 x 10-5 2 x 10-5

x 10-6

2 x 10-5 I x 10-5 7 x 10-5 >i x 10-4 >i x 10-4 1 x 10-5 6 x >i x 10-4 >i x 10-4

4 I 2 >5

x 10-6 x 10-5

10-5 10-5 10-6 10-5 10-5 Not examined x x 2 x >i x 4 x

>z

~~

a Miracil D = l-diethyleminoethylaniino-4-methyl-lO-thiaxanthen-9.one. The basic --NHCH~CHZN(C~H~)~ side chain of the parent compound is replaced by in AN 207, by -N(CH~)CH~CHZN(C~HR)~ in AN 216, and --N(CH3)CH2CH2N(C2H5)z by -N(CH3)2 in AN 317; AN 305 is Hz-NCHzCHzN(C2H5)z; AN 304 is 4-hydroxymethyl-Miracil D (Hycanthone); AN 316 is Miracil D sulphoxide (Hirschberg et d.,

1968).

studies on the rates of RNA synthesis in the middle and a t the end of the proliferative stage in the presence and absence of actinomycin D and Miracii D showed that : (a)the rate of RNA synthesis in rapidly growing and dividing cells was a t least twenty-five times that found in the stationary phase before aggregation ; and (b) concentrations of the drugs that effectively blocked proliferation or aggregation inhibited only a small portion of total RNA synthesis in cells harvested a t either phase. As a result of these studies, Hirschberg et al. (1968) have suggested that the various messenger-RNAs required for the normal sequence of morphogenetic development in D.discoideum may be formed during the logarithmic growth phase of the mould. Relatively stable and long-lived messenger-RNAs have been demonstrated in mammalian cells (see Gross, 1968) and it is suggested that the orderly sequence of morphogenesis in the slime mould may be reflected in the differential stability of the messenger-RNAs. Tn D.discoideum, the total transfer-RNA methylase capacity is decreased by 4096 eight hours after aggregation and drops still further in niature fruit bodies (Pillinger and Borek, 1969). This diminution is

62

J O H N E. SMITH AND JILLIAN C. GALBRAITH

considered to be due t o inhibitors that do not inhibit the base-specific enzyme to the same extent. Whether the altered capacity of the methylases is the result of the synthesis of new enzymes or of inhibition of previously existing ones remains t o be established. Clearly, there is much to be done in understanding the involvement of RNA synthesis and changing enzyme levels in differentiating systems. The patterns and levels of enzymes can be considered t o be the primary point of control during differentiation and any change in their concentration and activity may be reflected in an altered rate of synthesis of some material essential to differentiation. I n vivo, enzymes are generally in great excess compared to their substrates (Lowry and Passoneau, 1964; Srere, 1967) and undoubtedly many enzyme changes in differentiating systems will be of a quantitative rather than a qualitative nature (Wright, 1968). For these reasons perhaps too much attention is being given to a few enzymes which have been shown to have consistent temporal relationships and not enough attention to the essential metabolic pathways which function throughout the life cycle of the organism. Such pathways may not change qualitatively, but quantitative changes may regulate “catabolic competition” (Wright, 1966, 1970b). Wright considers that critical endogenous precursors may exist a t limiting concentrations in the cell and that differentiation may be intimately linked to the intracellular concentration of such metabolites. 5 . Kinetic Models of Differentiation

The extensive biochemical information now available on D . discoidewm has led Wright (1968,1970a)and Wright et al. (1968)t o attempt to devise a kinetic model of some aspects of metabolism essential to differentiation in this organism. By making use of the known accumulation patterns of UDPG, glucose 1-phosphate and uridine triphosphate (UTP) the K,,, values of UDPG synthetase for glucose I-phosphate and UTP have been calculatcd and a series of differential equations describing the synthesis and utilization of UDPG have been determined and used t o design a computer model for the conversion of glycogen through glucose I-phosphate and UDPG to the end products of differentiation. Analysis of this system suggests that an increase in UDPG pyrophosphorylase concentration in vivo cannot account for the enhanced rate of synthesis of UDPG nor for the accumulation patterns observed. The most important regulating factor in this system is the availability of glucose I-phosphate and this further emphasizes the importance of measuring concentrations of precursors in differentiating systems. Enzymes are normally in great excess in vivo compared to their substrates, and large fluctuations in enzyme concentrations may not be critical t o the rate of in vivo reaction. From such computer models, the analytical results

ASPECTS O F DIFFERENTIATION IN THE FUNGI

63

stress that, under the steady-state conditions of the living cell and over the time periods normally involved in differentiation processes, changes in the concentrations of some essential enzymes are not significant with respect to controlling the metabolic flux necessary to the accumulation of specializcd end-products. Thus it is clear that much more attention must be given to understanding the role of intermediary metabolites in regulating differentiation. The open discussion recorded in Wright's (1968) paper clearly shows that such computer studies on the regulation of flux in metabolic pathways will ultimately be of considerable value in explaining and predicting the changes that occur in differentiating systems.

111. Division Mycota: Subdivision Myxomycotina: Class Myxomycetes The Myxomycetes (the plasniodial, acellular, or true slinie moulds) differ froin most other organisms in that, during the growth phase of their life-cycle, they consist of a mass of protoplasm of indefinite shape containing up to several million nuclei. This mass of protoplasm, the plasmodium, in many ways resembles a large multinucleate amoeba mid biochemically may be considered as a single cell. Because of their size and synchronous development the Myxomycetes are being used to a greater and greater extent by cytologists, physiologists and biochemists to study many fundamental aspects of cellular metabolism not least being the elucidation of differentiation (Rusch, 1969; Sauer et al., 1969a). Taxonomically the Myxomycetes are considered t o show certain affinities to fungi (Alexopoulos, 1962) though others would consider them to be more closely related to protozoa (Kudo, 1954, Korn et al., 1965). Several excellent reviews summarizing the present knowledge of the biology of the Myxomycetes have been aritten by Hawker (1952), Alexopoulos (1963,1966),Gottsberger (1966),Gray and Alexopoulos (1968)and Rusch ( I 969). A. LIFE-CYCLE

Thc life-cycle of the Myxomycetes is initiated by the germination of haploid, uiiiiiucleate spores to give flagellated swarm cells or noiiflagellated niyx:hmoebac which ultimately can act as gametes (Fig. 5). In the presence of adequate nutrients and 011a solid surface, the myxamoebae will grow and multiply by binary fission, and if transferred t o a liquid medium will normally develop one or two flagella and become actively motile swarm-cells. Coiripatible c.clls fuse to form diploid zygotes. In heterotliallic Myxoiiiycetes the t M o gametes 1 1 1 i i h t of opposite mating-type and come from different spores. Dee (1966a, b) has

64

JOHN E. SMITH AND JILLIAN C. UALBRAITH

denionstrated that four matin! types can occur and she has also shown recombination between genetic markers. Whereas heterothallism is knowri t o occur in the Myxomycetes the existence of homothallism has

Germicnticm

\ \ \ A

/Fructification

Koryoqamy (zygote)

\

FIG.5. Life-cycle of P h y s c t r ~ ~polycephalzrm. ?)~ Adapted from A41csuporilos(1962).

not yet been proved and homothallic species may prove to be apogamic. Karyokinesis continues without cytoltinesis resulting in the formation of a macroscopic, multinncleate network of rhythmically streaming protoplasm. A plasniodiutn m a y be formed by the growth of a zygote, but it may also enlarge by successive coalesceiices with other zygotes.

ASPECTS OF DIFFERENTIATION I N THE FUNQI

65

Tlic plasniodial or somatic stage of the life-cycle is free-living, acellular. and mobile, feeding on bacteria. Under certain environmental conditions, such as limitations of food or dessication, the plasmodium can undergo extensive differentiation by sporulation, by encystment of the myxamoebae, or by sclerotial formation. During sporulation meiosis will occur in the large fruit bodies ultimately leading to the formation of haploid, uninucleate spores. Slthough there are over 400 known species of Nyxomycetes, the plasmodia1 stage of only approximately 30-40 have been grown in the laboratory (Henney and Henney, 1968), and in most cases this has been achieved only with media which contained living or dead bacteria. Although several species can now be grown on semi-defined or completely defined niedia (Daniel and Rusch, 1961, 1962a, b ; Daniel and Baldwin, 1964; Ross, 1964; Ross and Sunshiiie, 1965; Lucas P t al., 1968; Heiiney and Henney, 1968; Henney and Lynch, 1969) thew is still digculty in getting most Myxoinycetes to complete their life-cycle in pure culture in chemically defined media. Synchronous plasmodia1 cultures of Physarum polycephalum can be grown quite simply in petri dishes on filter papers or Milliporc membranes supported on the surface of nutrient medium by glass beads (Daniel and Baldwin, 1964; Guttes and Guttes, 1964; Guttes et al., 1961; Nygaard ct al., 1960). Illohberg and Rusch (1969) have recently developed a new technique that can produce plasmodia at least ten times larger than in the other methods. Microplasmodia can also be cultured in shaken flasks or large fermenters (Brewer, 1965).

B. THEPLASMODIUM This is the vegetative phase of the life-cycle and it is also the most characteristic. Since nuclear division can be precisely synchronized in P. polycephalum plasmodia it has become a popular organism for fundamental studies of mitosis. The brilliant studies of Rusch and his coworkers have been largely concerned with obtaining a better understsanding of the biochemical events leading to nuclear division and to what makes a plasmodium divide in a synchronous manner. A comprehensive review on the biochemical regulation of mitosis in P . polycephalzcin has rrceritly been published (Rusch, 1969).

c. SCLEROTIUM FORM-4TION Under certain adverse environmental conditions, Myxoniycetes can form sclerotia directly from the plasmodium. The sclerotium is composed of clusters of spherules each containing one or more diploid nuclei. Unlike sporulation, spherulation does not require niacin or light and can

66

JOHN E. 8R.IITII AND JILLIAN C. GALBRAITH

be induced simply by deprivation of nutrients (Guttes and Guttes, 1963) or by various chemical and physical methods (Jump, 1954). Recently spherulation has been obtained with a fully defined synthetic medium without involving starvation in a non-nutrient medium (Chet and Rusch, Uridine incorporation

1500r-

I

Jermindtion

f

n

i:

0)

E

cli

-./= -----.A-

500

a z

I'

n z O a n "-O :;t 0

ss

I--.--.

,

/

-.'.

u 2

Time (hr)

84 96

Time ( h r )

FIG.6. Synthesis of RNA, DNA, and protein during growth, spherulation and geririiiiation. Tho ciiltures of Physarurn polyceyhalum were transferred t o synthetic n i c d i i n n containing mannitol after 24 hr. of growth. Mannitol induces splwriilittion. The spheriilw were again transferred t o fresh medium 46 hr. after the. Iiegiiliiing of spherulation. The graphs on the right-hand side show incorporation of 3H-uridine into RNA during growth, spherulation, and germination ( 0 ) and the cffcct of nctinomycin D on this incorporation (0). Data from Chet and R~isch(19($9).

1969). Ultrastructural changes occurring during spherulatlion have been studied by Goodman and Rusch (1970). The total amounts of RNA, protein and DNA increased during growth, but decreased during spherulation (Chet and Rusch, 1969; Fig. 6). The rate of ItNA synthesis as measured by 3H-uridiiie incorporation varied during plasmodia1 growth, spherulation and germination of spherules and wa,s sensitive t o actinomycin D. Glycogen content increased during growth and germination, but decreased t o a low lcvel during spherule formation.

ASPECTS OF DIFFERENTIATION I N THE FUNGI

67

The amount and intracellular distribution of polyphosphate (a condensed polymer of inorganic orthophosphate) and other phosphoruscontaining compounds have been determined throughout plasmodia1 growth and spherule formation in P. polycephalum (Goodman et al., 1969).There mas a large difference in the concentration of polyphosphate during growth and spherulation. It was considered that, during growth and early spherule formation, polyphosphate was involved with energy relationships and with synthesis of nucleic acids. l n later phases of spherulation, the polyphosphates may be involved in maintaining osmotic balances by sequestering phosphate in an inactive form, and also as a storage product for use during germination. Further evidence for the role of polyphosphate as a storage product was obtained by Sauer d al. (1969~) who demonstrated transfer of 32Pfrom polyphosphate to RNA when starving microplasmodia were returned to a growth medium. Inhibition of RNA synthesis in plasmodia by actinomycin D resulted in a marked stimulation of "P incorporation into polyphosphate.

D. SPORULATION Many external factors have been linked with sporulation in the Myxomycetes, but the conditions which actually trigger the process are still unknown. Using replacement medium techniques, Daniel and Rusch (1962a, b) first discovered factors which induced sporulation in P. polycephalum in axenic cultures. As with most micro-organisms depletion of nutrients is one of the main conditions necessary to initiate the events leading to sporulation. The sporulating medium must contain niacin, niacinamide or tryptophan, and a period of illuniinstion following four days of starvation is essential. For a comprehensive summary of the environmental factors involved in the sporulation of P . polywphalum and other Myxomycetes, see Gray and Alexopoulos (1968) and Rusch (1969). During sporulation the entire plasmodium is converted into one or more fruit-bodies and, for this reason, the somatic and reproductive phases rarely occur simultaneously in the same individual. The process of differentiation can be reversed up to a certain critical period of development by the addition of nutrient, but after this critical point has been passed the plasmodium is reversibly committed to sporulate even if it is returned to a growth medium. Most of the information concerning the biochemical changes associated with sporulation have becn summarized by Daniel (1966), Gray and Alexopoulos (1968) and IZusch (1969). Light is necessary for induction of sporulation in yellow pigmented plasmodia, but not in non-pigmented species. The apparently non-piginented plasmodia which do require light may actually contain pigments

68

.JOHY E. SMITH AND JILLTAN C. QALBRAITH

in vcrx Ion- cvnecntratiou (Lieth, 1054). The metabolic changes accoinpanying the light effect include a decrease in respiration, fluctuation in ATP concentrations, inhibition of glucose uptake and an increase in noii-fwrous iron (Daniel, 1966). R a k o c q ( 1963) found that there was an inverse i~~lationship bet ween the length of the period of illumination for the initiation of sporulation and age of culture. He considered that a photochemically synthesized compound (substance 13) was the essential trigger of sporulation. This essential compound was believed to be formed from a precursor synthesized in thc vegetative plasmodium in either light or dark. light

Plnrniotliuin

__f

or darh

Substance A4+Substance H + Sporulation light

Gray and Alexopoulos (1908) have suggested that: (a) substance A may be produced from a metabolite of niacin or a metabolite synthesized through rtxnctioiis catalysed by niacin; (b) substance B is produced by a pliotochcmicd reaction iiivolviiig substance A and the photoreceptor ; aiid (c) substance B can cause an inactivation of sulphydryl groups whic~happear to inhibit sporulation (V’ard, 195Sa, b). It is also quite possiblc that, in the non-pigmcnted species, conversion of substance A t o substance 13 map also take place by another mechanism not directly involving light. 1)uriiig sporulation there are marked shifts in the activity of two separate oxidase systems. Plasmodia show six times as much ascorbic acid oxidase activity as spores while about three times as much cytochrome oxiduse activity is present in spores as in plasmodia (Ward, 1958a, b). Polysaccharidase B activity decreases in the presporangial stage (Zddriii aiid Ward, 1963a, b). In 1’. polywphnlum, starvation in itself is not sufficient to induce spriilation. Sporuhtion will only occur when starvation is followed by ;L pcriocl of illumiiiatioii. Studies using actinom ycin 1> and other DNA inhibitors have shown that there must be DNA synthesis late in starvation and prior to the period of illumiiiatioii (Sauer et d., 1969a). Protein synthesis owiirs tlirougliout the entire period of differentiation while RNA synthesis is essential until 3 hr. after the elid of the period of illumination. At this time, the organism is irreversibly committed to sporulatc. RNA from sporulating and starving non-sporulating plasmodia show several important differences, iiicludirig : (a) more rapid incorporationinto, and possibly higher turnover of, RNAprior to commitment t o sporulation ; (b) microsomal-RNA from sporulating cultures contains an extra peak in the radioactivity profiles not present in starving piasmodia ; (c) microsome-associated RNA from sporulating plasmodia incorporates more labelled uridine aiid contains relatively more large

ASPECTS OF DIFFERENTIATION I N THE FUNGI

60

RNA molecules than from starving plasmodia; (d) total RNA from sporulating cultures has a different pattern of hybridization after sucrosegradient fractionation than from non-sporulating cultures (Sauer et al., 1969b).These authors have also suggested that the essential role of light i ti P. polycephalum sporulation is to stimulate transcription and also t o align the protein-synthesizing systems in n manner conducive t o the ready translation of new informatJionfrom thc sporulating genome. Thus, during the transition to sporulation in both the Myxomycetes and Acrasiales, there is a n absence of growth, the process can be induced and is completely synchronous, and finally there is total conversion of the plasmodium or pseudoplasrnodium to sporing structures. Together these features allow for a much clearer interpretation of the biochemical events that accompany differentiation and make these organisms ideal for studying morphogenesis in eukaryotes.

IV. Eumycotina A. CELL WALLCONSTRUCTIONAND MORPHOGENESIS The presence of a rigid cell wall determines to a large extent the cellular form of fungi and, by its very nature, renders these organisms amenable to investigations of the molecular basis of their form. I n common with other microbial cell walls the fungal cell wall is a complex dynamic structure, the site of diverse enzymic activities and intimately involved in and responsible for cellular morpliogenesis. Studies on the chemical composition of cell walls have provided information on the nature of the macromolecular components of the wall fabric, while electron micrographs of wall material have revealed the spatial arrangement of some of the macromolecular aggregates. For more detailed studies of cell-wall chemistry in taxonomy (phylogeny) and morphogenesis (ontogeny), reference should be made to the recent review articles by Aronson (1965),Bartnicki-Garcia ( 1 963, 2968a, 1969), Nickerson (1963), Nickerson and Bartnicki-Garcia ( 1 064), and Villanueva (1966). 1.

Vegetative Differentiation and ljimorpkisni

Studies of fungi which exist in two vegetntivr fornis have provided a valuable approach to the biochemical basis of vegetative differentiation (Romano, 1966). This phenomenon has been known for more than a century and the special attention originally given to these fungi was due in part to the fact that many of them are pathogenic, causing deep mycoses i ti aiiirnals and mail. Fungi that exhibit diinorphisni can exist as filamentous mycelia (M form) or as spherical yeast-like cells (Y form)

70

JOHN E. SMITH AND JILLIAN C. GALBRAITH

wliich reproduce by budding. I n the yeast Candida albicans the Y form is a serious human pathogen whilst the M form grows saprophytically on plant residues or in soil and only becomes converted to the Y form when it invades the animal host. This duality of vegetative form in dimorphic fungi has been considered t o represent a plausible example of primitive morphogenesis (Haidle and Stork, 1966). Growth in tlic Ail form represents an interference with the mechanisms of cell division. Nickerson and Falcone (1966) considered that division in (2. albicans is a result of a chain of events that begins with the utilization of metabolically generated hydrogen by a cell-division enzyme, protein disulphide reductase, for the reduction of disulphide bonds in mannan-protein complexes of the cell wall. This reduction weakens the c d l wall making plastic deformation possible, and the subsequent bud formation is it purely physical consequence (Nickerson, 1963). M form differs from Y in that metabolically generated hydrogen is not coupled to disulphide rcduction so that M is characterized by an excess of reducing power (Nickerson and Falcone, 1956). Autoradiographs have revealed sulphydryl groups in Y form, but not in M (Nickerson and BartnickiGarcia, 1064). The division enzyme is of widesproad occurrence and is active with many proteins (Hatch and Turner, 1960). More recent comparisons of M and Y have shown that the Y form of Histoplasma capsulatu~nand Paracoccoides brasiliensis have a more active tricarboxylic cycle than the M form (Kanetsuma and Carbonell, J966), and that the change from Y t o M in Mucor rouxii is accompanied b y a shift from anaerobic to aerobic metabolism (Haidle and Storck, 1966).

Currently, studies on mould-yeast) dimorphism are concentrated itiainly on the non-pathogenic phycomycete $1.rouxii. Bartnicki-Garcia and Nickerson (1962a, b) first demonstrated that a mixture of carbon dioxidr and elemental nitrogen was necessary for production of the ycast 1)hase in several strains of J!!. rouxii. I n the absence of carbon dioxide, aerobically or anaerobically, development was typically as a branched coenocytic mycclium. From their studies they concluded that carbon dioxide plays a specific role in tho maintenance of yeast growth. 1)iffvrrnt morphology of growth was correlated with different cell-wall structure, tlie cell wall of the yeast phase containing six times more mannan than thc cell wall of the filamentous phase. Interestingly, mannan has been found to be abundantly present in different species of true ycast and abscnt in most filamentous fungi (Aronson, 1965). Little is known of' the mechanism by which tlie accumulation of mannan c.ould disrupt tlie cylindrical cell formation although the work of Robertso11 ( 1 Wi) is c~nitrihritingto ail urid(,rst~LricliriKof differcntintion iii ltyphal tips.

ASPECTS O F DIFFEREKTIATIOK I N T H E FUNGI

71

1)irnorl)hism is affected by environmental factors such as temperature, sulphydryl cotnpouiids, and aeration (Nickerson and Bartnicki-Garcia, 1 M 4 : Koninno, L 966). Bnrtnicki-Garcia (1963) regarded the crucial diff(wncc1 between the A1 and 1-forms t o be in tlie grom t h polarization. Thus, development of Y represented a selective inhibition or interference u it11 the morphological mechanisms which are indispensable for cylindricd (sell formation. Formation of Y was regarded as i t consequence of isotrol)ic 1)li: sicitl forces. Using J l . rozc.rii ( K liRL 1804) Haidle and Storck ( 1966) obtained yeast grot1 tli i n ;microbic conditions without carbon dioxide and concluded that other nutritional factors were involved in tlie control of dimorphism. Bartnic.ki-Garcia ( L 968b) using X.rozc.c:ii (IRZ-80)clcarly demoiistratcd that hoth Iicxoses nnd carbon dioxide are primary determinants of yeast clcvc~lopriicwtin J / u c o r spp., a n d t h a t their dimorphic effects are complcmeiitary; at i~ low p C 0 , a higli coilcentration of liexose is needed for c.oml)lete yeast-typc development and vice versa. If, however, t h e conccntrntion of hexose in the medium is high enough, carbon dioxide is not iwpiired. The effect of glucose concentration could not be attributed to iticwnscd production of carbon dioxide siiice maximal evolution of cwbon dioxido ~ v a sreached with only 0.lo, glucose. Hexose also influciices the aerobic yeast-like growth of M . rouxii (NRRL 1894) (‘I‘trcnzi ant1 Storvk, 1968) a n d C. nlhicans (Kickerson aiid Mmkowski, I953).

I’hcnc+hj 1 alcohol, n proven inhibitor of growth in bacterial, fungal and miim;il wlls (see ‘I’erenzi and Storck, 1960), caused spores of J I . rouzii (NICRI, 1894) t o form spherical budding cells instead of Iiypliw provided that tlir cnrbohj-drate source was a hexose a t 2-5:,. IVheii tllc cnrboliytlixtt, soiirce was xylose, maltose, s u ( ~ o s eor n mixture of ;miino acids thc morphology in the presence of plienetliyl alcohol was filamentous. I’hcnc~tlijI alcohol stimulated carbon dioxide niid ethyl alcohol I)t*odnctionand inliibitcd oxidative phosphorylation of extracted niitocliondria. It is intcrwtiiig t o note t h a t all of the factors n-liicli cause j-east-like tnorphology i n Jlzccor also favour fermentation. Furthermore, there arc‘ riimy other examplcs where inhibitors of respiration and consequent enhanwmc~iitof fermentation have led to a restriction of morphological differcwtiation in filamentousfungi (Schwalb and Miles, 1967; Kobrpt al., 1967 : Croc~lwnand ‘raturn, 1968). l’erciizi and Storck ( 1 969) havereceiitly considcred t h a t filamentous development in fungi can be regarded in ninny \I a ) s :LS :L morphogenetic expression of the Pasteur effect, a view that is in agrcenient with the concepts on oncogcncsis recently enunciated by \Vnrhitrg et (11. (1$)68).who states : “Respiration energy creates and m:iint;iins i i high differtntiation of body cells. Fermentation energy can 4

72

J O H Y E. SJlITH AXD J I L L I B S C . GALBRAITH

only tiiniiitain a lo\\. differentiation. It follows t h a t if respiration is replwcd by fermentation in body cells, high differentiation must disa1)pear." 2 . Dirrmyhism and Cell Tl'all C'onstruction

Some ycars ago Bartnicki-Garcia ( 1 963) proposed t h a t fiuigal diuiorl)liisiii could result from t v o d relit tiiodcs of cell-wall construction : ( i ) iiiiiforiiily t1isl)erscd in budding yeast cclls, mid (ii) apically localized in 11) pliw. This interl)rctation has been experimentally c.onfiniied by rccviit ;iiitot.ndioqral,liic studies of cell-a all formation in J I . i o z i ~ i i (B~~rtiiicki-C:~~rc.i:b aiid Lipl)tnaii, 1 %XI) in which tlic pattern of cell-wall coiistruction w a s cxainiiied in cylindrial and sl)lieric*alcells of X.r o z i ~ i i gro\v11 uiitler clt~finecl conditions (Barttiiclii-Garci:~ a n d Nicakerson, 1962a : I~;irttiicki-C:arcia, 19,t;Sb).Cell suspensions were exposed macrohicdly to trit intcd nT-iic.etylglucosamine, subsequently killed, and treatcd in siicli :I a s t o remove all cytoplasmic. radioactivity without destroying thc origin;il s l i a l ) ~of~ tlic cell. The resulting cell ghosts werc then staincd, fixed on a microscope slide, coated with nuclear eniulsioii and prowsseetl foi-aiitoradiogral)liy. \\'hen viewed under the light microscope tlw silver grains (*orrespondalmost entirely t o glucosamine a n d acetylgluc~osamincmolecnles incorporated into the cell-wall polymcrs (Fig. 7 ) . Ln hyl)liac., t h c c*cllwall appenrs t o be ~)rt~ferctitinllq synthcsized in the aI)iwl rcgion 11itli a sharply descending pradicnt of 11 all synthesis itig from the apcx. I n gerrninating spores aiid yeast cells of A!. , n-all formation occurred largely, if not entirely, in uiiiforriily dislwrseed fasliioii over the entire cell periphery. There was 110 evidence of 1)olarizntioii of wall synthesis in the yeast cclls. rl'lic~ difti~rentpatterns of cell-wall formation sliowii for cylindrical a n d s l h c r i c d cells of X.rouxii strongly imply t h a t the manlier of celliv:i]l construction is o f major importance in determining the shape of a fungal ccll. Tliesc studies together with the fluorescent antibody studies by Rlarc1i;~titaiid Smith ( 1 968) arc undoubtedly contributing t o a clearer undcrstanding of the biochemical a n d subcellular basis of apical growtli of fiingi and in t u r n t h e whole problem of fungal morphogeiiesis. It is iiow rlenr that there exists in the apical region of fungal liyphac :L (y)nsidernble degree of ititracellular differentiation. How far intracelluorg,ancllcs c w i be implicated in supplying the various cell-wall precUrs()rs together with synthetic a n d degradative enzymes for cell-wall g r o ~tih is still some.n.1iat unclear. Cytological studies have s1ioa.n t h e prc~s~~iice of certain organelles or vesicles unique t o the growing t i p in hot11 liiglicr aiid lower fuiigi (Girbardt, 1935 : Bracker, 1967; Bartnicki(;:arcia e/ ul , 1968; Bartnicki-Garcia, 1969; McClure et al., 1968; Grove ct nl., 1W:)). Clearly the mechanisms of cell-mall cotistructioii play a

ASPEC’TS O F U I P P E R E S T I A T I O S IS TILE PUl-Gl

73

tlvcisi, t x rolc in fiiiigal morphogeiiesis and a fuller understanding of tlie al)i(,altil) differentiation must certaiiily lead t o a better understanding of inow cotnples vegetative structures such a s rliizoniorl)hs, sclerotia :I i i d (’0reni i a .

FIG. 7. Photomicrographs showing patterns of cell-wall construction in M m o r rouxii. 1 shows germinated sporangiospore prior to germ tube emission with disperse pattern of wall synthesis. 2 shows a hypha with apical pattern. 3 depicts a yeast cell with three buds showing disperse patterns; one of the buds (arrow) also exhibits a band of basal wall synthesis probably related to septum formation. Cells were grown anaerobically under nitrogen (1 and 2 ) or 30% carbon dioxide (Bartnicki-Garcia, 1969).

3 r ,

(’4 Tlhll Composifioii ( i n d RPproclucfio,z

I li(> foregoing studies 1)resent a n d t o some extent prove tlie working liypotliesis t h a t a givcn cell morpholog- is deteimiined by, and is depeiid(~t~t u 1 ) o t i . tlir. c.hernicnl composition of t h e (.ell wall. 111 this respecst. it is siciiifiic.;riit t h a t differences exist in cell-u all structure of different ~iiorl)liologic.;tlstructures of one fungus (De Terra and ‘l’atntn, 19(i I : C’liin and Knight, 1963 : SeiitlieShaiimuqaiinthan aiid Nickerson, 1962 : JZc.\liirronch and Rose, I065 : \Vmg and Miles. lR(i6). A consideration of tht. factors involved in dimorphism could be profitable t o a study of sl)orul;rtioii since asexual sporulation resembles t h e change from a

74

JOIIh E . SIIJ'rH AND JILLIAN C. GALBRAITH

ni) c~41,il to a yeast form i l l that both represent a change from cylindrical to
ASPECTS O F DIFFERESTIATIOS I S THE FUJSGI

75

tire gr:~duallyuncovering some of the underlying biochemical basis of ontogenetic8 and phylogenetic development of the fungi.

13. LIGHT-INDT(TD SPORULAT~ON ASD SPOROGENIC SUBSTANCES The weiLlth of information on environmental factors which affect sporulatioii provides a good starting point t o biochemical a n d phj-siological studies. The cffwt of light, humidity, aeration, pH value, temperaturc, injury, mid nutrients on reproduction have been well summarized by Hawlwr ( 1 037, IUB6), Cochrane ( l 9 6 7 ) , aiid Turian (1969). There is 110 o h i o a s t hrinc undrrlying the complex effect of environmental factoi s. and thr, Klcbs generalization (Klebs. 1898).t h a t reproduction is initiatcd 1)) factors \\ liicli check growth, still stands today. The com1)lrx c>ffvctsof irradiation on sporulation (reviewedby Burnett. I !Hi8 ; H a \ \ kcr, 1966 : JIarsli et al., 1059 ; Carlile, 106.5) are little undcrstood in pl~ysiologicalor biochemical terms. To study the effect of light at this lcvel it is ncwssary t o identify the photoreceptors involved in tlie l)liotochemicd rcac+on. This problem niaj- be al)l)roachedin three iva> s. i i a i t i t ~ l , y by isolation aiid identific.ation of t h c pigments present, bjdetermining the action spectrum of the light-induced reaction, and by tlic e&ct of‘ mctabolic inhibitors of the pigments on photo-induction of t I i c rcac+ion. 1 . Yhotoreceptor.s

The I)roblt.ni of the 1)hotoreceptors has usually been iiivcstigated through t h c iwtion spectrum. Many morpliogcnctic responses are assoc*i;itedwith the blue eiid of the spectrum. and pliototropic ligltt-gro\vth cfticts oftcan share ii (witinion action spectrum \I itli mor1)hogenetic (>vents.C’arotcnoids and flavins are the popular contenders for the role of photo-inducers in fungi. Riboflavin seems likelj. where the action spectrum slion-s a peak in the ultraviolet region, sincc i t has a high al)sorl)tion pc.nk at 26.3 nni. The association of carotenoids with proteins or 1il)oljrotcins is theoretically necessary if they are t o function i n tiietabolism such a s t h e reception of light stimuli. However, such an atsociation has not yet been established (C‘ochrane, 1967). Carlile (1960) Iiiis suggrstcd a. pteridinc in relation to the indiiction effects due t o ultrtiviolet radiation a n d one' caurrent opinion is that caroteiioids are not the 1)rinciI):ilphotoreceptors in fungi, but protect tlie fungi from light damage (Carlil(~,1 !)M). Triii(*iand 13anbury ( I!lB9) were unable t o identify the photoreceptor involx cd i n the light stimulation of conidiopliore extension and carotenopwcsis in Aspergillus gignnfeus. They isolated ,&carotene and two unidentificd red and p l e orange carotenoids from the conidiophores,

it i

J O H S E . S\IITH A L D JILLIA?; C . GALBRAITH

aloiig 11 itli a y(4low mcthanol-soluble pigment wdiich might Imve been a n ~~trtlirac~riitione. Hov ever. this last pigment had no absorption peak i n t l r v visible region o f t h e sl)ectrum,whicli made it nii unlikely candidate for ~)liotor~cwptor unless t h e absorption spectrum was altcwit aftcr cxl~osiirct o light, or during extraction. I t also scenicti anlilicly t h a t cwotvnc was involved in its owii ~’lioto-induction.The work of 1,each is

lwgitiiiing t o exl)lain the 1)liysiology of tlie action of ultraviolet radiation. I t is eeiierallj assumed t h a t altraviolet radiation exerts its effect tlirougli nucl(.ica acid, most 1)robak)lj-through IIKA (hloselc>y,1968). Leach ( 1962) ol)s(mwl that sl)ornlatioii of 31 species of fungi w as more effectivcly incliicd hy tiear-ultraviolet radiation tlran by longer waveleiigtlis, and that long c.sposiires (w neither lethal nor inhibitory. Stimulation o c w t t . t d i~~gardlcss of iiiaiiy other environineiital factors. Leach ( I!)ci4) postiilatd t h a t ~)lioto-induccdasexual sporulation in many fungi iiir-olvw t h c sani(x tneclinnism. and t h a t radiant energy is ca1)tured by q-ntlresizrd by sonre fimgi in t h e dark 011 i t rich rnedium, but on a n incomplete nirdiiini :~n iml)ortant photochcmical reaction is nccessary t o induce its forniatioti. ‘I’he absorption ciirve of P3 IOC is similar t o t h a t of a thymine tlimc.i., 1)yriniidiiiv2 dirncrs, aiid an oxidation product of zeatin, altliorigli ot1ir.r 1)liysiwl and cliemical prol)erties differ. This iiidic~ites t h a t 1’3 1 0 niay btx similar t o somr of these compounds, but has different iiil)stitncnt giuii1)s (Trionc and Leach, 1969). Low doses of radiation a t uxvc~lcngtlisbelo\\. 300 nm. arc known t o cause t h e formation of p j ritnicliiicl dirntw in iiiicleic. w i d (Jagger, 1967). It is necessary t o I m t u l a t e thc ~ ) i * ( w i i of w p1iotorecc~l)tors suc~lias P310 becausc awiirate detcriiiinat ions of t lie wtion sl)ec+rum of Ascochyta pisi and other fungi (Trione ;ind I,t.ac.li, ICtO9) slron t h a t they do not ronform t o the usual ahsorption Y1)wtra of wt*oteiioids,flavol)rotein, or l~tcridiiies. I’no plij siologicd stages appear t o he involved in t h c sporiilatioii of S t ~ w p h y / i u~~~ T ! J O S Z L M(Leach, 1068). This fungus onl>- I)rodiic*cs I )rofust. conitliophores in alternating periods of light mid dark, suggesting t h a t tlic. first stel) is an iiiductive phase in whicli tlie formation of coiiidiol)lioim is stimiilated by ultrnviolct radiation. In the second terminal I)liasc. the folmiatioii of c.onidia is inhibited hy light. TT’avelengths which < L I T inliibitoq- during t h e teriiiiiial phase range from 240 t o 650 n n i , but their c4fec.t is clelwiident on teml)erature. Similar tw o-stel) proc(mes w e

ASPECTS O F DIFFEREXTIATION I S THE FUSGI

77

knov n in (‘honnrpliora(Barnett a n d Lilly, 1950), Thamnidium (Lythgoe, I !Ki 1. 1962) and PiZohoZus ( P a g e , t95li). Among the Basidioinycetes, light may br necessary for the initiation of the primordin, or inajr affect \iil)seqwiit stagcs of dcvelopiiieiit S L I C ~ I ~as stipe elongation. pileus tormation, or hyrnenium and sporc formation (Burnett, 1968). 2. In hih itor E z p r r i m PV ts

Attctril)tst o evnliiatcl the role of pigments asl)hotoreceptors have been inade by us(’ of diphenj lariiine, which decreases carotenoid synthesis, mid lysofliavin and inel)acrine t o inhibit riboflavin. A fluvin-mediated light nl)sorptioii is indicated in Piloholus, wlierc lyxoflaviii inhibits tro1)lioc~ st foiwiation. ail effect wverscd by additioiial exogenous riboflavin (Page. 1056). Ncithcxr cm-otogciicsis nor conidio1)hore exteiisioii of A . yiqnn/pzis v-as inliibitcd by tiiepncriiic or Iyxoflaviii. S o r did diphenylatiiiiie inhibit conidiopliore esteiisioii (Trinci aiid Banbury, 1 969), or show ;I c*lrnr effwt on f’ilobolics tropliocyst formation (Page, 1 956). 1 ) i ~ ~ l i ~ ~ i i ~tlcntnicut ~ I ~ ~ t t ~ iof i i eS.o~nssnresulted in repression of coilidid formation, along with inhibition of carotogencsis, but the two processes are not c;iusallj- relatcd because fully coiiidiated albino mutants are Imov 11 (Tutiaii, 1 9(i(ia). 5-Fluorouracil caii iiiliibit photo-induced sl)orul;ttion of Trichodwmcc. but this is a n effect on R S A (Gressel and (;ahin, 1 !)6’i) which will be discnsscd later. 3 . Jlrtnbolism of Photo-inductio7i

Sincc t h c tintiirc of tlie I)hotoreceptor niolceule (or molecules) is iiot ktion-n it i i not cnsy t o cwnsider subsequent reactions leading t o the wspoiisc’. Atlvocaates of riboflavin molecules a s I’liotoreceptors have relntcd its action t o tlie observations that it caii cntalyse the photooxidation of iiidolc-acetic acid (Galston, IU49), but thcre is no reason t o siipposc that this is an c ive growth regulator in fungi (Gruen, 1959). LAltei~natiwly. light may bring about the destruction of riboflavin, or may iiiitiatv activity i i i an clcctroii-transfer system containing t h e fl avo1)rotein corn1lo nent (Carli le. 1 Ni3). Thus, in Alter nu riu solnni, flav ins are ciscntial for conidial forrmtion, and are plroto-itiactivated (Lukcns, 1 !)(;:I) The furthest progress in undcrstandiii:: tlic biochemical vhaiiges itiducd by light 11;ive been made by Caiitiiio working on BZnstocZuciirZla. I lit, cliiuigcs ;rccotnpanyiiig t h e differeiitiatioii of B. rmrrsonii into thin\\ a l l r d ordinary colourlcss (OC) or resistant s1)orangia (KS), whicali can l)c conti*ollcclby I)icnrboiiate, liavc. beeii studied in considerable detail (Cantino, l!)(X).Theearly stagcs of ontogeiiy ofthe OC cell are accelerated by n-liitc liglit in tlic I)reseiicc of carbon dioxide, and this affects R number of parani(~trrs,sucali as tlie rate of nuclear reproduction aiid the rate of r 3

-78

J O I l h E. SJIITH AND JILLIAS C . GALBRAITH

glycitie u1)take. ‘I’hyniine synthesis may be a limiting barrier in tlie growth of’ B. enwrsonii because exogenous thymine can substitute for light ( T u r i m , I!Mh). The light receptor has not yet been identified, :Lltliough thcrc. iirc indications tliat it is a protein-bound porphyrin resembling cytoc*hronie (Cantino, l!l66). Blastocladiellu britunnica shows no response t o bicarbonate, hut develops into OC sporatigia in tlie light and Its in the dark. Like the response t o bicarbonate, this rvslwiise is reversible up t o two-thirds of the generation time (Horeiisteiii and Cantino, I Cf6.’). It is postulated that light-sensitive glucose uptake is it factor in determining morphology, since dark-grown cells have a fiLr grcatclr chapcity (Horeiisteiii a n d Cantino, 1964). Dry weight, soluble ])rotein, non-sc.dimentable iiucleic w i d and soluble polysacsc.haride iti(*reaseniorc, rnpidlj- when the organism is grown in white light t h a n in the dark. This conld i n e m thitt light inhibition inhibits tlie i)athn ay for glncosc degridntion. shunting t h e metabolism towards the ninnufacture of i)olys~Lc,c,liarid(,s. Such a hypothesis is consistent with the fact tliat the spwific artivity of glucose 6-phosphate dehydrogenase is at its highest in d:~rk-gro~\ ti c~lls from 5 5 ” , of the geiieratioii tiine but, in the light, eiizymc syiitliesis stops a t 80°, of the generation time (Goldstein a n d C‘;tntino, 1 ! ) W ) . The i i ~ f l n c n cof‘ ~ ~light in inducing carotogeiiesis and coiiitiiol)hore grou th in A . !ji!jantPus could not be trntisniitted from an illuiniiinted rcgion to ; L I ~ adjacwit region of tlie same mycelium in darkncss (l’rinri :tiid Banbur>.. I !f(i9). This would imply t h a t photo-inductioii does not involve tlw i)rodiivtioii of substances wliich are readily diffusible, niid tlie authors si1gg:cstc.d t h a t , in tlie case of conidiophore extension, t h e 1)lioto-iiiductivc response was closely associated with t h e cell wall. ‘I’lie photo-induction mas also dependent on tlie presence of free oxygen. The authors h l i e v e t h a t i t involves “low-energy” photoclic~rnical renctions in w.liic.li light wrves only as a trigger t o a chain of reactions \vliich niaiiitain grov th and carotogenesis. S o r is the light stimulus t o priinordia forrnatioii in the basidioniyretc ,lldniiotzts transmitted from illuminated to dark portions of the mycelium. lnductioii of primordin in Jfelanotus is a response t o light of waveIcngths 5 I ()-(; I() nni., somewhat higher t h a n is generally rcported, a n d is iiiorc clircc$ly ivliited t o the effects of light and temperature than t o the age' of tliv inyc*cliurii(Newman, 1968). Fungus photoscnsitivity has been linked t o an inhibitory effect of liplit on groutli (Riirnett. 1968). b u t substantiating evidence is lacking. X cwrrrlation Iwtn-cen light-induced sl)orulntioii and decreased grou t h is iiot apparent on IIelminthosporium stwzospilum (Freeman and Luke, 1960). The ol)I)osiiteeffects of light on asexual a n d sexual reproduction of f’hytophthom lias led both Lilly (1966) and Brasier (1969) to suggest

ASPECTS O F DIFFEREliTIATIOPi I S THE F U S G I

79

tlrat there is competition between the pathways leading to tlie production of the two types of spores. A simple action of light seems unlikely because its wtioii is affected by the composition of the medium (Lilly, 19G6).

C. BIOCHEMISTRY OF ASEXUAL SPORL-LATIOX 1. Blastocladiella

‘I’he miijority of the studies of Cantino and his coworkers have been the slwcics first isolated b y Cantino, Blastocladidla ~ r n ~ ~ s o nThc ii. triotile zoospores of the fungus settlc down, retract their flagellum and d(,vc,lol)ii uninucleate gcrn-tube which develo1)s into the rhizoidal system of the nnic*c~llular fungus. After a n exponential phase of invrease in d r y I\ (light. volunic and other features, tlie second developmental stage of (*(4I tliffercnti;Ltion is reached. Almost all of the thallus is converted into il sI)oraiigiurii in which the cytoplasm is cleaved u p into spores. The rc~ltaseand subsequent germination of these spores gives rise t o four different phenotypes. Between 99 a n d 100° of the population of sporcs v i l l form ordinary eolourless sporangia. (OC) or thick-wallcd pigmented resistant s1)ornngin (13s)depending on whether or not bicarbonate is pr(wnt. H o v cxver. depending on the growth medium selected. u p t o 0..5(’,, of tlic first generation thalli will form orange cells, due t o t h e l)ivwiicc> of y-carotenc, and another 0-0.5O{, of the population will t of “liltc rolourlrss (.ells” which differ frorn OC cells by their much longer yencriltion time (Fig. 8). When released from any of these types of sf)orangia,tlrc zoosporc~sinitiate a new cell generation (Cantino, 1967). Studies were conceiitruted on the RS and OC cells. This work was dealing with the differentiation of a single cell, since development is from a nniiiuc~leatespore t o a ~nultinucleate coenocyte. This fact, toq.ther u i t h the development of submerged, synchronized singleg(wcration cultures containing u p t o 10’- 109 individuals, provided an cllcgmt system for studying the relations between biochemicd and mor~)liological differentiation. The discovery t h a t the presence of bicarbonate during the c ~ x p o n ~ n t iphase al of growth led t o the development of ItS slwrangia, whereas essciitinllj- all spores developed along the OC pathway in the absciice of bicarbonate, provided a system in which t h e ”trigger” r(vwtion leading t o one type of differentiation rather t h a n another could be analyzed. The essential biochemical events of the bicarbonate trigger mechanism are associated with the tricarboxylic acid cycle (Fig. 9 ; Cantino, 1967). Cantino ( 1951) h a s concluded t h a t actively proliferating OC cells carry on a ])redominantlyhomofermentative type of metabolism, leading to tlie formation of lactic acid. The net outl’ut of carbon dioxide during active growth is detectable, but very low (Cantino, 1 % 1). Cell-free 011

YO

JOIIN E. SMITH A N D JILLIAhT C . GALBRAITH

reparations of OC cells exhibit most of the enzymic activities associated with the glycolytic I)athway leading from hexose phosphate, through exclusively SAl)P-specific reactions, t o pyruvie and lactic acids (Cantino, 1 ! ) . 3 ) . On tlw other hand, enzymic and chemical assays show t h a t the tricarbox) lir w i d cycle is a t least potentially operative in OC cells of various ages (Cnntino, 1933, 1959; Cantino and H y a t t , 193Ya, 1)) al-

A-

Orange plant (thin -waI led)

Ordinary :olourless plant (thin-walled)

7 5

‘2.5

I

t

990

0-5

I

+ 1

t

38

34

1

+

No

No

i

No

Yes

Flc.. 8 . The.

clrtdirlltr

f‘orii.

(I(.\

A v “gamma” particles per spore in plant

‘I’

AV percentage of less than

first 0.1 generatiov (usually zero) population

4

A v generation +Ime (hours) Melanin in wall 7

Carotene in pratap!ast 2

I08

i 1

Late colourless plant (thin-walled)

0.5

I

i 38

11

Yes

No

Yes

No

c’loptii(’tit;il p t h s n h i c h cmi bc takeii by sporcs of Hltrsto-

o / / c 1 : s o / t i i . iiti(1

< l l l o t h c ~ (l (~* < l l r t l t l o .

Resistant sparangial plant (brown thick-walled p i t t e d )

th(.

gas\

pavarnctcrs u hich cllstiiigiilsh thcrn fimni

otic

1961 ).

though it is coicaludcd t h a t it is a weakly functional system playing only a rniiior role in supl)lying energy. ll’hen bicarbonate is added t o a dcvelol)ing germling, it quickly induces a set of multiple enzymic lesions in the tricnrboxylic acid cycle. Hon ever, isocitrate dehydrogcnaxc specific for XADP rcm;iins functional, and begins t o operate in reverse, t n d i a t i n g rrdnctive carboxylation of a-oxoglutarate t o isocitrnte. At the snme time, bicarbonatc also indnces the formation of isoritrate Iynsc. which cleaves the isocitrate t o glyoxylate a n d succinate, and thus prevents its accurnulation. Finally, a constitutive glycine-alaninra transnminasc brings about the amination of glyoxylate t o glyvine a t t h e

ASPECTS O F DIFFEREYTI~4TION7I N T H E B U S G I

81

cxlwiisc’ of nlaiiine (McCiirdy and Cantino, 1960). Other d a t a are consistent with this liypothesis. Thus, mutants unable t o synthesize aoxoglutnrntr drhydrogennse are unable t o form RS cells in response t o bicarbotintr (Cantino, 1933: Cantino and Hyatt, 1953b, c ) . l i S cdls have a much lower oxygen consumption than OC cells (Cantino P t al., 1957) and i ~ almost n complete loss of tricarluoxylic acid cycle cwzyrnes (except NADP-dependent isocitrate dehydrogenase), a terminal c*ytoc~lii-omc oxidnse niid two ~pectropliotometricallyseparable

rS0 HC03-

OC P L A N T S

0 HC03-

--l R S PLANTS

Z-OXOGIdUTARATE

coz PIC..9. ‘1’11~1 t)ic*,wt)oriatcti ifig~xiinctchaiiimi I I L Hlrrstocltrdielln r~ttier.vo7iii(Caiitino, I96l). SOllCI IltlC% Itltilcatc ln
protein fi.;wtions which are found in OC cells (Cantino, L!).56, 1939; Cantino a n d Horenstein, 1065). Oxygccw c~onsnmptioni n liS cells (1r01)s t o about one-tenth of the original value mid this 1)mxllels a 90°, decrease in the intracellular a(wimul:it ion of cc-oxoglutarate drliydrogenase relative t o the isocitrate dehjdrogcnase in exponential growth (Cantino, 1967). ‘I’herefore, it appears that a-oxoglixtaratc dehydrogennsc causes a bottleneck in t h e tricnrboxylicb acid cycle, bringing the activity of t h e cycle t o n halt and initiating tliv R S pathway. KOsuch bottleneck occurs during exponential growth of OC cells because total activity of isocitrate dehydrogenase and cc-oxoglutnrate dehydrogenase is approximatel- the same ; there is no

82

JOHN E. SMITH A 4 D JILLIAN C . GALBRAITH

synthesis of isocitrate lyase and what little there was in the spore is diluted out. The supply routes of XADPH, and a-oxoglutarate necessary for the continuation of such a metabolic route in RS development are not yet fully docurnented. RS cells possess a polyphenol oxidase system whic~li medintcs electron transfer from tyrosine t o either oxygen or NADP (riot NAD). This system could be coupled in vitro with isocitrate dehydrogenasc t o drive reductive carboxylation of a-oxoglutarate t o isocitrate. Thus, a major source of reducing power lies in the path t o melanogenesis (tyrosine is converted through quinones t o melanin). rl'l~is1ij.pothcsis is supported by the fact t h a t bicarbonate induced CIE 7 7 0 ~ 0 synthesis of tyrosinase (Cantino and Horensteiii, 1$155, 1959 ; Caiitiiio and Lovett, 1964). A likely source of XADPH, is glucose fi-phosl h t e d c h y d t y p i a s e (Cantino and Lovett, 1964), which may itnpose a mechanism of self-regulation on differentiation through t h e changing I)alanw hctwcwi the soluble and bound forms of the enzyme (Pandhi atid Cantino, I%X). It becomes necessary t o consider compartmetitalizntioii within t h c cell t o explain the fact t h a t incubation with bicarbonate increases the pool of succinate. yet succinate relresses synthesis of isocitratc 1y;~sc(Cantino and Lovett, 1964). Glucose &phosphate dcliydrogennsc is present in both OC aiid RS cells, but bicnrbonate induction of morphog,c.ncsis is associated with a bic~~rbon~~tc-iiidu(~c[~ compartment nlizntion of glucose ti-phosphate dchydrogcnase in the cell. In OC cells intrawllular glucose 6-pliospliatc dehydrogenase is soluble, but during the exl)onential development of liS i t is localized in or near tlie cell wall or thc r n ~ r i i b i ~ i ~associated nc~ n i t h it. This is supported by in vivo ol)scrvations that the rate of synthesis of glucose Ci-l)hosphate dehydrogeiinsc in 11s cells reflects the exponential rate of deposition of the surface a r c ~ iof the cell rather than its weight or volume, whereas the opposite is true in developing OC cells. This is also confirmed b y enzyme extraction. 1)uring tlie early stages of ItS exponential growth most of the c~nzyriit~is insoluble, but the percentage which is soluble gradually incrcascxs until essentially all is soluble a t tlie point of no return (see b c a l o ~). I~~lcc~t~opliorcsis lias revealccl quite distinct isozymes appearing a s RS dcvc.lopnieiit proceeds, although t h e significance of this is not yet apparent (Cmitino, 1967). lluring ontopmy a point is reached beyond which the cell becomes irreversibly committed t o RS fortnatioii. This is the morphogenetic point of no rc,turti. Up t o this point. which is 437, of the generation time in the (widitions used, removal of bicarbonate from the medium causes reversion of the development path aiid maturation into a n OC cell. Beyond this point of no return, which coincides with a halt t o the increasc3 in cell size aiid the completion of the cross-wall formation, the 1weseric.c or absence of bicarbonate does not affect tlie ultimate nature of

ASPECTS O F DIFFERENTIATION I N THE FUNGI

83

the cell. nlariy of the events during RS development, both before and after thc point of no return have been traced. a n d are summarized in Fig. 10 (Cantino, 1967). Many components begin t o increase at an exponential rate, a n d (wise t o do so a t t h e point ofno return. Others begin much later, but still cnd a t this point. Yet other features continue t o increase to differcnt stagvs in ontogeny beyond the point of no return. Yet a fourth group comrn(~ncronly a t or beyond the point of no return. Of value t o a study of the causal events of morphogenesis is whether any of thc f m t urcs associated ith a n lZS cell during exponential cell development \\ onld wvert t o those more typical of OC development if bicarbonat(. I\ erc removtd bcfore tlie 1)oint of no return. n’hen this is done, some but not all of tlie RS cliaractr~risticsdo revert. In particular, the two kcy enzytncl systems thought t o be directly involved in K S formation do revert. Thus, the. total units 1)ercell of isocitrate dehydrogenase dropped s1i:wI)ly whcreas the total units per cell of woxoglutarate deliydrogciiase i~oscsliar1)ly (C:intino, I c f f i 1 : Cantino and Goldstein, 1962; Lovett and C ~ n t i i i o 196 , I ) . After tlie point of no return. removal of bicarbonate has 110 suc~li(.fie($ oti these enzymes. Cantino a n d Goldstein (196%)do not vicw the, clinnge frorn a plastic t o a unidirectional pathway of developincnt a s ciuc to any one single factor, but take the view t h a t i t is a cumulativcb wsult of many biochrmical results, each having their own point of no return tlispcrscd over a much broader range of time. Thc chcmicd ft.aturc,s of tlie last stages in differentiation have also bctw traced (Fig. 10; Cantino, 1967). In the final stages of development tov, izrds tlic thick-walled, pitted, pigmented RS. total synthesis of protcins and DXA stops, exogenous glucose is no longer utilized, and thew is ii dcc*rc.ascin oxygen ul’take and release of lactic acid into tlie mcdiuin : chitin, mc.lanin, lipid mid polysaccliaride syiitliesis increases several-fold. ‘I’rnnsformations occur in the RNA pool during RS maturation. R S A syntlic,sis occurs until the cell is nearly mature. with an iiicrwsc in the prol)ortion of sodium chloride-insoluble RNA t o about of the total. I t is bclievcd tliat insoluble RXA is laid down during diKwcwtintion in tlie form of protein-bound organelles which are finally vonvcrtcd t o tlic ~)lastoclndeaceous nuclear caps in the spores (Cantino, I S(i1). Hcrr thcn, w(~ have a proposal for tlie biochemistry of differentiation a t t h r lcvcl of t h e organelle. Energy for changes necessarj- for the formation of insoluble l i K A is derived frorn the polysaccharide pool, and fi-oin glucosc~fi-pliosphte dehydrogenase, which is the only enzyme nhoscb spc,c4ie aetivitj- is known t o rise during RS maturation (Lovett nnd Cniitino, 1 ! ) ( i l ) . In contrast, in sporogenesis of tlie OC cell, very little new R K A is sjwt hvsiztd although a considerable fraction of s1)orangial RNA is degradcd. Here ,\lurl)liy nnd Lovett ( 1 966) have shown t h a t the nuclcar cal) is formed by tzggrcgation of pre-existing ribosomes and not

Percentage Generation Time. KS Cell 0

Exponential Growth

>

Increase per Cell Volume, DNA, Soluble Protein, Glucosamine Synthetase, Glucose 6Phosphate Dehydrogenase, Glycine-Alanine Transaminase, Isocitratase, Isocitrate Dehydrogenase

1

Differentiation 100 hr. Change per Cell Tnrrease in Glucose Dehydrogenase, Lactir Acid : Decrease in Polysaccharides

[Glucose]-[Dry

Weight, Lipid, Total Nitrogen]

-[Chit

in, Polysaccharide]

-[Total

RNA] -[Melanin] Decrease in Organic Phosphate, Cytidylic Acid, Amino Acids: Increase in Organic Phosphate

Decrease in the ratio of a-0x0-Glutmate/ -1socitrate Dehydrogenase Activities 90% Decrease in Oxygen Consumption 9O0,

[Protein, RNA Turnover]-

FIG.10. A digest of some of the events which have been quantified during exponential growth and differentiation of an RS cell of Blastocladiella emersonii (Cantino, 1967).

ASPECTS O F DIFFERENTIATION I N TIlE FUNGI

85

from d p n o m synthesis. Details of the relation hetween structure aiid function in sporogenesis are emerging with the elcctron microscope work of Fuller ( 1 %Xi), Reichle a n d Fuller (1967), and Lessie and Lovett ( 1 968). Cantino‘steam hnvc extended their research into a study of the differeiitiatioii of thc Blastocladiella spore (Cantino, 1969),in particular the probtern of the trnnsformatioii of a snirnmiiig spore into a rounded cyst (Cantino P f al., 1968). Thc “rounding-up” process I\ 1iic.h precedes flagella retraction is closclj- linked on a temporal basis Lvith the release of a n uiiident ified fluorescent substance. genesis of flocculence coml)etence, formation of “vacuoles” which migrate t o the surface of the spore and al)parentlj-release their contents, n decrease in cell volume mid probable cli,tiiges i n sulphydryl coin poiients on thc spore surface. The fine htruc+nre and c1iatig:csduring zoospore differentiation have also rcccived attention from Puller (1066), Reichle and Fuller ( I U(i7), I m s i e and Lovett ( I $)(is)mid Rlurl)liy mid Lovett ( 1966). ‘l’hus, Cniitino aiid his coworkers are perhaps the first workers in fungal differentiation t o reach a stage when the? can break a w ~ yfrom the ‘.lincnr” ;tlqjroach (Cantino et nl., 1968) exprriinenting with (.rude honiogen:Ltcs of intricate systems interacting within a cell. and are able t o niove towards the study of smaller and smaller units of orgmizntion \I Iiich yield results with t h e minimum of approximations. Khnoa- and RlcCurdp (1R69) have obtained results which do iiot coiii1)letcly ngrce with those of Cantino a n d his coworkers. They have found t1i;rt all of the tricarboxylic-arid cyclc enzymes. t > x c y t woxoglutarate dchydrogeiiasc, are present in extracts from RS and OC cells and t h a t thcse c m z p i e s have a higher activity per plant in ItS cells (Fig. 1 1 ) . However, the liS cells are larger than the OC, aiid when specific activities \\ ere (misidered. these were slightly lower in RS cells for every enzyme cxceptiiip aconitate hydratase and the citrate synthase. Specific activities reached a maximum a t 24 hr., except for NAl) I-’-dependent isocitrate dchydrogenase, which iiicreasrd throughout the 3(i hr. period of growth of ItS cells. These results are in contradiction t o the findings of Cantino ( IO6,j) that tricarboxylic-acid cycle enzymes, excepting NAUP-clepeiident isocitrate dehydrogenase, are low or ahsent in 13s plants. They iiitlicate t h a t , in the coiiditions of these experiments. t h e metabolism of ()c(.ells is iiiaiiily oxidative. in contrast t o earlier reports (Cantino, 1951. l!ItjC).1 Cj(i1, 1966, 1OGT) t h a t OC cells (grown i n rnultigeiieration cultures \\-ith poor aeration) are homofermeiitatire. Neither do these rcsults confirm t h a t maturc R S cells are devoid of most tricarboxylic-acid c j d e enzymes. nor t h a t bicarbonate leads t o their loss or a significant decrease in activity during the period when its morphological effect is crucial. yet this is the basis of the hypothesis of Cantino (l!)BB) t o explain the morphogenetic effect of bicarbonate. Indeed Khnow and JIcCurdy ( 1069)

86

JOHK E. SMITH A S D JILLIAN C. GALBRAITH

lravc shown striking similarities between the tricarboxplic-acid cycle ciizyrne activities, and other characteristics, of RS and OC plants during their exponential growth. It is oiily when bicarbonate has become ineffec-

2

02

02

2

1

01

01

1

0 5

Hours

c

W

t

-0 0 Q

Hours

HoLrs

Hours

Hours

<m ?K

+

v l

:I

E, W C

I

Hours

Hours

Hours

c~lntioiiihipbvtn w i i rii~yirrc.activity dnd tlcvc,lopmeiit of UZostoclut//c.l/tc OC n ~ i t lRs cells. In dat,L for OC plants. c iridicates uiiiti of c.ilzyiiie ,irtirity pc’r m g protcLiii, aiid 0 miits pcxr plant x 106. I11 data for RS p L i i i t 5 , -ilitl~cntc~s I t l i l t 5 ~ X Y111gProt(’iI1, a11d 0-0 uliits p c plntit ~ x 1 0 6 (Kholl\I C ~ ~ i 111

McC‘l~~dy. lY(j9).

t l

ASPECTS OF DIFFERENTIATION I N THE FUNGI

87

tive in determining the path of development that significant changes occur in RS development. Khuow and McCurdy (1969) have made the valid criticism that Cantino and his coworkers have compared OC and RS cells on the basis of generation time, which assumes that their respective niaturation times reflect the achievements of analogous stages of growth and development. I n fact, exponential growth continues throughout the development of OC cells, whereas in RS cells it proceeds for a prolonged period of development in the absence of growth. Therefore, these workers (Khuow and McCurdy, 1969) consider comparisons to be most valid during the exponential growth phases. Since the results fail to the thesis that bicarbonate fixation via a-oxoglutarate leads to a loss in tricarboxylic-acid cycle activity, Khuow and McCurdy (1969) made 14C-bicarbonate fixation studies. The greatest amount of label appeared in aspartate, with lesser amounts in glycine and malate. Significant amounts were also present in other compounds, such as a-oxoglutarate and lactic acid. Since aspartic acid was the first compound t o be labelled, and it is rapidly formed from oxaloacetic acid, it is proposed that bicarbonate is fixed into oxaloacetate a t the site of carbon dioxide fixation into phosphoenolpyruvate or pyruvate. The metabolism of bicarbonate would then be dependent upon a functional tricarboxylic acid cycle, and on the relatively high levels of the corresponding enzymes existing before the point of no return. After this point, a decrease in the activities of these enzymes and a decreased rate of carbon dioxide fixation (Cantino, 1967) may be causally related to rnorphogenesis Other fungi in which the physiology or biochemistry of sporulation has been studied are inherently more difficult organisms than Blastocladiella from which t o obtain meaningful results, since they are multicellular. Within each hypha there are gradients of physiological age and differentiation, yet the majority of techniques have involved the examination of crude homogenates of the entire culture. This problem is multiplied in static cultures, in which variation of physiological status of the mycelium becomes magnified by additional variants of oxygen tension and nutrient concentration. Shake or continuous culture eliminate these complications only if the mycelium grows in a filamentous form rather than as pellets which are more typical of submerged fungal growth (Galbraitli and Smith, 1969a). Nutrients and oxygen are only freely available to the peripheral hyphae of a pellet, and autolysis sets in a t the centre while dense growth continues a t the edge (Clark, 1962; Camici et al., 1962). This is demonstrated in Sapromyces and Mindeniella, in which the amount of cytochrome is considerably less under decreased oxygen tension than with aeration, and in Aphanomyces astaci which shows fluctuations in QOn due to the age of the mycelium (Unestam and Gleason, 1968). 5

88

JOHN E. SMITH AND JILLIAN C. QALBRAITH

Although submerged growth is thus more desirable for biochemical studies of sporulation, it is beset with the hazard that sporulation is gencrally suppressed in submerged culture, even in those species which spore freely in static culture. I n some cases (reviewed by Galbraith and Smith, 1969b) sporulation or conidiation can be induced in submerged culture by manipulation of the culture medium, by the nature and concentration of the carbon and nitrogen sources, and by the presence of various trace elements. Yet an aerial stimulus remains the most potent effector of reproduction in mycelium grown in submerged conditions. This suggests that an oxidative metabolism is essential for sporulation. It is a common observation in static culture that only the uppermost layer of the mycelium bears the reproductive structures, which are orientated away from the medium. Morton (1961) has shown in Penicillium chrysogenum that conidiation occurs in subaerial conditions in media which will not stimulate reproduction in submerged conditions, and that the aerial stimulus can be reversed by re-sdbmergence. He postulated that a surface-active material, possibly protein, rapidly forms when the mycelium becomes aerial, and production of such a substance is reversed by re-submergence. There was also an increase in dehydrogeiiase activity on emergence, which decreased on re-wetting. He argued that the only essential difference between the aerial and submerged mycelium is the sudden formation of an airlwater interface, which possibly causes changes in the orientation of the polar molecules such as the unfolding of protein which would result in metabolic change. The effect of nutrients such as Ca2+on submerged conidiation could be correlated with such configurational changes. I n Neurospora, a change in the chemical nature of the surface of the conidiogenic hyphae is seen on emergence; there is an accumulation of lipoproteins which give the aerial hyphac their hydrophobic character (Turian, 1969). 2 . Neurosporu

The biochemical work of Turian on morphogenesis in Neurospora crassa emphasizes the importance of the aerial stimulus to conidiation. On solid medium, or in static culture, the asexual microconidia (more accurately termed arthrospores; Turian, 1969) form a t the tips of aerial conidiophores. Growth and conidiation are distinct processes (Turian, 1966s) and observations such as the formation of macroconidia in slope cultures, mainly a t the top where the agar medium is shallow, indicate that conidiation occurs characteristically in starvation conditions (Dicker et al., 1969). This illustrates the views of Wright (1966) that differentiation occurs in closed systems. Such observations lead t o the hypothesis that the utilization of the endogenous amino-acid pool,

ASPECTS OF DIFFERENTIATION I N THE FUNGI

89

expected to be maintained by proteolysis, is one of the major events during conidiation. Evidence was found in experiments in which normally aconidial mutants were induced to spore on media containing casamino acids. Addition of certain sugars negated partially or completely the effect of the amino acids, while other carbon sources such as pyruvate, acetate and tricarboxylic-acid cycle intermediates had no such effects (Dicker etal., 1969). DeBusk and DeBusk (1965) have in fact shown that glucose or sucrose considerably decrease amino acid transport and the size of the amino-acid pool in Neurospora. The negative effect of tricarboxylic-acid cycle intermediates further strengthens the argument that an oxidative metabolism is necessary for conidiogenesis. This was indicated by the observation that addition of tricarboxylic-acid cycle intermediates to a sucrose-nitrate medium greatly enhanced conidiation (Turian, 1964). Indeed conidiation is inhibited by preventing the formation of aerial hyphae with Tween 80 (Zalokar, 1954). However, malonate, an inhibitor of succinate dehydrogenase, favours conidial development (Turian, 1962). Conidiation on sucrose is affected by the temperature of incubation, being much more profuse a t 37" than 25". This rise in temperature was accompanied by an increase in the activities of enzymes associated with the succinic oxidase system in isolated mitochondria of young conidia (Ohja and Turian, 1968).

Investigation of the effect of acetate on conidiation revealed stimulation of the glyoxylate cycle as an alternative to the tricarboxylic-acid cycle in these conditions of culture. Isocitrate lyase was much more active in organisms grown on acetate than on sucrose (Turian, 1961 ; Turian and Kobr, 1965) ; it is also active in organisms grown on sucrose a t 37") but not a t 26" (Turian, 1961, 1963). Conidial differentiation could be induced on sucrose a t 25" in the presence of bicarbonate, and again isocitrate lyase was induced (Turian, 1963). Thus, three environmental variations inducing conidiation also induce activity of isocitrate lyase. The glyoxylate formed from isocitrate lyase, as in Blastocladiella, is transaminated with alanine to form glycine (Turian and Combepine, 1963). A source of glyoxylate rather than an active glyoxylate cycle appears to be vital for conidial development, since conidia are formed a t 25' under conditions of relatively low isocitrate lyase activity on the aconidial Westergaard-Mitchell medium supplemented with succinate or citrate. In this case, glyoxylate is supplied by splitting of pentose produced by the hexose monophosphate pathway (Turian, 1963). The nitrogen source in these experiments was nitrate, and its assimilation through nitrate reductase provides a mechanism of NADP regeneration essential for the continued function of the hexose monophosphate pathway. Addition of ammonium salts to the medium inhibited conidiation. It is preferentially

90

JOHN E. SMITH AND JILLIAN C. QALBRAITH

utilized, and probably uncouples nitrate reductase from the hexose monophosphate pathway and prevents conidiation (Turian, 1964). This hypothesis is supported by the absence of protoperithecial differentiation in the presence of nitrate (Hirsch, 1954)) the presence of NAD nucleotidase (Zalokar and Cochrane, 1966) and NADP-dependent glutamate dehydrogenase, but not the NAD-dependent enzyme, in conidia (Sanwal and Lata, 1961), and a low activity of cytochrome oxidase, indicating that the cytochrome system is dispensable (Turian, 1960). Direct evidence from nitrate reductase assays is not yet available. Glycine is the only amino acid which can overcome the inhibitory effect of ammonium ion. It prevents uptake of ammonium ions and heavy conidiation occurs (Oulevey-Matikian and Turian, 1968). Another line of evidence which rules out the glyoxylate cycle as a causal mechanism is that alteration in activities of enzymes such as isocitrate lyase and isocitrate dehydrogenase, by varying the carbon source, could not be correlated with any particular morphogenetic event (Kobr et al., 1965).

Active conidiation is accompanied by an increase in succinate dehydrogenase activity (Ohja and Turian, 1968) yet malonate which inhibits succinate dehydrogenase, stimulated conidiation (Turian, 1962). This paradox has been explained by Ohja et al. (1969), who found that, in vivo, malonate is metabolized by N . crassa and its stimulatory action seems to act through stimulation of succinate dehydrogenase. Metabolism of malonate could be through some decarboxylating acetate-producing reaction. Since the glyoxylate cycle is stimulated by acetate, malonate could, therefore, be acting indirectly through stimulation of the glyoxylate pathway. Alternatively, acetate may be acting as a lipogenic agent, since lipid synthesis is connected with conidiation (Bianchi and Turian, 1967). To return to the thesis that oxidative metabolism is essential to conidiogenesis, an absence of conidia would be expected in conditions favouring glycolysis. A four-day vegetative mycelium produced 80% more ethanol than a conidial mycelium; this was decreased by 90-96y0 on addition of p-chloromercuribenzoate, and the vegetative culture conidiated (Weiss and Turian, 1966).Conversely, blockage of the tricarboxylic-acid cycle with compounds such as fluoroacetate favoured glycolysis, and conidiating mycelia reverted to the vegetative condition. Differences in relative enzyme activities in conidial and Vegetative cultures were as expected ; thus, ethanol dehydrogenase and carboxylase activities were greater in vegetative mycelia. The occurrence of high glycolytic activity in filtrates of vegetative tissue was confirmed by manipulation of the environment in various ways, such as by the induction of conidiation in an amino acid/amnionium medium by in-

ASPECTS OF DIFFERENTIATION I N THE FUNGI

91

creased oxygen tension (Kobr et al., 1967), or the addition of glycine to an ammonium medium (Oulevey-Matikian and Turian, 1968). Therefore, conidiatioii is considered to be a morphogenetic expression of the Pasteur effect (Turian, 1969). It was suggested that conidiation is regulated by the balance between oxidative and glycolytic pathways, probably at the point of pyruvate. The relative concentrations of reduced and oxidized NAD may also play a regulatory role. An active hexose monophosphate pathway would be expected to decrease activity of tlie Embden-Meyerhof pathway through competition for glucose 6-phosphate (Turian, 1966b). A closer examination of alcohol dehydrogenase has shown the presence of two isozymes in conidial extracts, whereas there is predominantly one in mycelial extracts (Del Vecchio and Turian, 1968). Induction of oxidative metabolism is essential for the expression of the conidiation potential ; for example, it can be induced by nitrate which has tlie effect of re-oxidizing NADPH, which can be coupled with glucose 6-phosphate dehydrogenase. An effective oxidative metabolism is not necessarily dependent on the cytochrome system because variations in flavins are suggested by thc accumulation of riboflavins a t 37" (Ohja and Turian, 1968). When the mycelial felts break the surface of the culture solution and emerge into more aerobic conditions where conidia are formed, there is a predominance of the assimilatory type of nitrate reduction requiring only the functioning of flavin adenine dinucleotide and molybdenum to regenerate NADPH, (Walker and Nicholas, 1962). The dispensability of the cytochrome system in this process may explain the lower cytochrome oxidase activity measured in conidial homogenates compared with protoperithecial ones (Turian, 1960). I n this connexion, it is also important to note that a very active diphosphopyridine nucleotidasc has been detected in conidia of N . crasSa (Zalokar and Cochrane, 1956) and to remember that the NADP-dependent rather than NADdependent glutaniate dehydrogenase functions in these conidia (Sanwal and Latrc, 1961). Thus, it2 appears that a flavin type of metabolism coupled through NADPH2-NADP regeneration to the direct oxidation of sugars through the hexosc monophosphate pathway predominates during conidial differentiation in N . crassa. Table 2 and Fig. 12 summarize the differences between purely vegetative mycelium and conidial cultures, investigated for a number of biochemical characters. Preliminary investigations of nucleic acids have shown an average RNA:DNA ratio of 8 : 7 in the conidia (Owens et al., 1958); the base ratio of total RNA in hyphae and coriidia is the same (Minagawa et al., 1959). Ultrastructural studies of differentiation have accompanied these biochemical studies (Dicker et al., 1969 ; Ouleveyblatikian and Turian, 1968).

92

JOHN E. SMITH AND JILLIAN C. UALBRAITH Glucose 1 -phosphate

t

Glucose 6-phosphate

Iodoacetate Fluoride Quinone

Reserve polysaccharides

hl,,-C

,

KADPH2

6-Phosphogluconate

Pentoses

Shikimate

(protoperithecia microconidia)

Glycine

Nucleic acids

Conidiopenesis

FIG.12. Diagram of the principal metabolic pathways of morphogenesis of Neurosporacrassa: carotenoid-pigmented conidia, differentiation stimulated by extracellular acetate or glycine, increase in temperature ( 3 7 O ) , or suppression of glycolysis. FA = fluoroacetate, an inhibitor of conidiation. M. Mp and C indicate with fertile predominant pathways associated with undifferentiated mycelium (M), mycelium producing protoperithecial asci (Mp) and microconidia, and with the differentiation of macroconidia (C). Note the double role in conidiation (C) of gluconeogenesis from acetate, and the reducing power of NADPHz (favourable to lipid synthesis; Turian, 1969).

93

ASPECTS O F DIFFERENTIATION I N THE FUNGI

As in differentiation of Blastocladiella, a point of no return in the development of the pathway resulting in vegetative or conidial cultures can be detected. Using ammonium salts as a conidiatiori repressor in a nitrate-containing medium, it was seen that ammonium ion is only effective up t o 8 hr. after inoculation (Oulevey-Matikian and Turian, 1968; Turian, 1969). Similarly, trapping of acetaldehyde with bisulphite, TABLE2. Biochemical analysis a n d physiological differentiation of rnycelial a n d conidial cultures of Neurospora crassa (Turian, 1969)u Criterion

Mycelial cultures

Conidial cultures

Specific activity Enzymes : Glucose 6-phosphate dehydrogenase WADP nucleotidase NADPHpcytochrome c reductase Succinntc-cytochrome c reductasec Succinate dehydrogenasec Cytochrome oxidase Isocitrate lyaso Pyruvato carboxylase Ethanol dehydrogeriasn Cherriical Composition : Ethanold (mg./g. d r y wt.) Acetaldehyde (mg./g. d r y wt.) Carotenoids (pg./yod r y wt.) Physiological activity : Qo, C02/02

760 605* 309 12,618 205 743 648 771 92 109 8,720 8,070 45 96 14 7 1,659 125 Specific production I

896 2.1 290 12.8 4.2

118

0.3 12,000 18.9 1.2

Age of cultures 3-4 days; Enzymes = specific activity; chemical composition = specific production. a 3-4 day-old cultures were examined. * Value was underestimated by rapid destruction of NADP (see value for NADP nucleotidase). c I n isolated mitochondria instead of cell-free extracts. d Cultures were 4 days old.

which converts conidial t o mycelial cultures, is only effective during this period which must then be regarded as the critical one for conidial induction. Other independent lines of research on Neurospora differentiation include that of Zalokar (1959a, b) who studied the activity of succinate dehydrogenase, 8-galactosidase, aldolase and tryptophan synthetase, and concluded that an increase in succinate dehydrogenase activity is associated with conidial formation. He found that the growing end of

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JOHN E. SMITH AND JILLIAN C. OALBRAITH

the hyphae differed substantially from the rest of the young hyphae, the tips being slightly richer in proteins and significantly richer in protein-bound SH and RNA (Zalokar, 195913).Stiiie (1967)also confirmed the requirement for succinate dehydrogenase in differentiation of N . CPQSS(X.conidiophores. His later work (Stine, 1968)is a measurement of NAD- a i d NADP-dependent glutamate dehydrogenase and NAD nucleotidase throughout the asexual life cycle of X. crassa, from genninat ion to coizidiatioii. Activity of the NAD-dependent enzyme increased slowly from 7 hr. after germination to the time of conidiophore elongation, when it declined slightly. This contrasted with the behaviour in an aconidial mutant, in which activity of the NAD-dependent glutamate dehydrogenase increased four-fold a t the time of conidiophore formation, most of the enzyme being present in the conidiophore and remaining a t a constant level thereafter. Activity of the NADP-dependent enzyme declined overall from 24 hr. to conidial formation, except for an increase in activity associated with conidiophore formation. The drop in activity thereafter was considerably greater in the wild type than in the mutant. NAD iiucleotidase activity increased dramatically at the time of conidiophore differentiation in both mutant and wild type, and this enzyme accuinulated in the conidia of the latter. Thus, there is an apparent association between conidiophore differentiation and increased activity of NAD nucleotidase and NAD-dependent glutamate dehydrogenase in iV. crassa. Stirie (1968) favoured the opinion of Stachow and Sanwal (1964) that NAD-dependent glutamate dehydrogenase may be involved in glutamine synthesis, while the NADP-dependent enzyme is essential for normal metabolism during the period of logarithmic germination and mycelial growth. Zink and Shaw (1968) have noted three isozymes of the malic enzyme in N . crassa, whose distribution varies with the age of the mycelium. Isozymes 1 and 2 appeared during the early stages of growth and disappeared a t 24 hr., while isozyme 3 appeared at about 12 hr. and increased with age. Isozyme 3 is inhibited by aspartatc, which may be an important factor in the conservation oC C4 compounds during the later stages of growth when the concentration of exogenous carbon-containing compounds is low. 3. Aspergillus

The biochemistry of conidiation of Aspergillus niger has been studied by Galbraitlz and Smith (1969b, c). They worked with submerged shake cultures, comparing the characteristics of sterile and conidiating inycelia a t intervals throughout the growth cycle. This fungus is another example in which differentiation occurs after the rapid phase of growth, after the exhaustion of the limiting nutrient (Galbraith and Smith,

ASPECTS OF DIFFERENTIATION I N THE FUNGI

95

1969b). I n the systems used, conidiation followed exhaustion of either the carbon or nitrogen source. Where the carbon source (glucose) was limiting, spore induction was affected by the nature of the nitrogen source. Conidiation did not occur in the presence of ammonium ion in spite of glucose exhaustion, although nitrate had no such inhibitory effect. This recalls the inhibition of Neurospora conidiation by ammonium ion, mid as in iVeurospora there appeared to be a critical period for spore induction, since addition of ammonium ion to a conidiating medium up to 15 hr. after inoculation prevented normal development of vesicles and sports. Older cultures were unaffected (Galbraith, 1968). The majority of amino acids, acetate and pyruvate, tricarboxylic-acid cycle intermediates and glyoxylate were able t o overcome ammonium ion inhibition of conidiation. An attempt t o explain the action of the amino acids in terms of the wtivities of glutamate dehydrogenase, glutamate-oxaloacetate transaminase and glutamate-pyruvate transaminase activities did not show any clear correlation between the activity of these enzymes and morphogcnesis. A higher specific activity of NADPH,- and NADH,dependent glutamate dehydrogenases in pre-conidiating conditions indicated a more rapid rate of glutamate synthesis in certain media (Galbraith and Smith, 1969b).When used as an inducer of conidiation, a-oxoglutarate was a t least in part used in amino-acid synthesis, judging from the increased glutamate dehydrogenase activity. Investigation of the tricarboxylic acid and glyoxylate cycles gave results strongly reminiscent of enzyme activities associated with differentiation in Blastocladiella and Neurospora (Galbraith and Smith, 1(16!k).Using the absence or presence of a-oxoglutarate in an ammoniacontaining medium to control differentiation, NADPH,-dependent isocitrate dcliydrogenase (carboxylating) and isocitrate lyase showed much higher spccific activities at the critical period preceding conidiophore differentiation, tlian in sterile mycelia of the same age. I n the case of the NADPH,-dependent enzyme, activity was initially low in an ammonia-containing medium and dropped steadily to zero. I n ammoniacontaining medium supplemented with a-oxoglutarate, activity of the NADPH,-dependent isocitrate dehydrogenase WRS initially double that in sterile niycelia, and increased alniost threefold in the following 24 hr. ding the first morphological signs of conidiophore formation. On the other hand, activity of the NADP-dependent enzyme in both vegetative and conidiating mycelia was initially low, increasing during the last stages of the life-cycle. Malate dehydrogenase activity mas similar in vegetative and conidiating mycelia, being initially high and tlien dropping. Like NADPH,-dependent isocitrate dehydrogenase, isocitrate lyase in conidiating mycelia reached a peak during the preconidiating period, but remained low in activity in sterile mycelia.

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J O H N E. SMITH AND J I L L I A N C. GALBRAITH

The activity of the second enzyme of the glyoxylate cycle, malate synthetase, remained negligible throughout growth of both types of mycelium (Fig. 13). Glycine-alanine transaminase was detected qualitatively in pre-conidiating cultures, but not in sterile cultures. a-0x0Mc!ate dehyrogenase

Isocitrate dehydrogenase (NADP -dependent)

80 a x

c

40

20

'0

20

60 80

40

100

' 0

20

Hours

40

60 8 0 100

Hours lsocitrate lyase

lsocitrate dehydrogenase (NADPHZ-dependent)

40

005

20

rn

c

'0

20

40

60

80

100

'0

20

40

60

80 100

Hours

Hours Malate synthetase

z

x

c

'

5

'0

20

40

60

80

I' 3

Hours

FIG.13. Relationship botuern various enzyme activities and age in conidiating and vogetativr inycelium of Aspergillus niqer in shake culture (Galbraith and Smith, 1 9 6 9 ~ ) .

glutarate dehydrogenase was not detected at any stage in either type of mycelium. Thus, one can draw the somewhat tentabive conclusion (Galbraith, 1968) that the tricarboxylic-acid cycle is only ticking over slowly during thc rapid phase of growth due to catabolite repression; the absence of a-oxoglutarate dehydrogenase suggests that the tricarboxylic-

ASPECTS OF DIFFERENTIATION I N THE FUNGI

97

acid cycle may serve purely a synthetic function as previously suggested for A . niger (Kornberg et al., 1960). A major distinction between conidiating and sterile mycelia in the systems so far examined is the synthesis of glyoxylate, and possibly glycine, through NADP-dependent isocitrate dehydrogenase and isocitrate lyase. The source of a-oxoglutarate is possibly provided by the drop in glutamate dehydrogenase activity a t this time, that is exhaustion of glucose is accompanied by a decrease in synthetic activity of glutamate dehydrogenase, giving rise to an accumulation of a-oxoglutarate which is metabolized to isocitrate. Isocitrate lyase, freed from catabolite repression (Polakis and Bartley, 1965; Gollakota and Halvorson, 1959), catalyses cleavage of isocitrate to glyoxylate and succinate. Support for this hypothesis is found in the fact that there is an increase in the relative rate of conidiogenesis when A . niger is grown on a n acetate-containing medium, which induces the glyoxylate cycle as an alternative pathway to the tricarboxylic-acid cycle (Turian and Seydoux, 1961). The results of Galbraith and Smith (1969b, c) are insufficient to correlate with the conclusion of Behal and Eakin (1959a) that spore formation in A . nigrr is dependent on a functionally complete tricarboxylic-acid cycle. These workers interpreted inhibition of spore development, but not growth, by 6-ethylthiopurine as an interference with methionine metabolism, causing a decrease in the activity of the condensing enzyme (Behal, 1959). The increase in glucose uptake and carbon dioxide evolved after a period of inhibition by 6-ethylthiopurine, together with the activity of tricarboxylic-acid cycle intermediates and saccharides as counteractants of 6-ethylthiopurine, were regarded as indications of a requirement for a respiratory process providing energy for spore formation (Behal and Eakin, 195913). As yet, the respiratory process has not been firmly established as the tricarboxylic-acid cycle. Indeed, inhibition of the condensing enzyme would interfere with both tricarboxylic-acid and glyoxylate cycles, and does not exclude the possibility of the glyoxylate cycle functioning at the expense of the tricarboxylic-acid cycle. Considering the prime function of the glyoxylate cycle as an anaplerotic pathway, it would be expected to support, rather than exclude the tricarboxylic-acid cycle. I n fact, the results of oxygen uptake studies show that respiration is occurring throughout the life span of A . niger (J.E . Smith, unpublished results). Other studies of Aspergillus differentiation include that of Stokes and Gunness (1946), who found that A . niger mycelium before conidiation contained about 50% more protein made up of all of the amino acids except histidine, compared with conidiating mycelium. Conidiating mycelium and the spores differed by only 10% in their nitrogen content, but there was three times more histidine in the mycelium than in the

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JOHN E . SMITH AND JILLIAN C. GALBRAITH

spores. Yanagita and Kogane (1962, 1963) have made a cytocheniical study of A . niger colonies, and also of P. urticae which are essentially similar. The young vesicles and phialides were highly basophilic, but the intensity of the basophilia varied with the location of the conidiophores within t,he colony, being high in the middle zone where conidia were about to be formed, but lower in the central region where there were abundant conidia. This suggests that basophilic substances are translocated into the conidia as colony development proceeds. PhosphorusContaining compounds are also translocated from the mycelium into the conidia, and are converted into polyphosphates which accumulate as metachromatic granules. Such phosphorus-containing reserves are probably needed for the successive nuclear divisions which occur in the phialides. Sulphur protein was only demonstrated in the phialides and conidia, the conidia also containing large amounts of other sulphurcontaining compounds, especially choline sulphate. The changing pattern of enzymes of phosphorus metabolism, such as ribonuclease and deoxyribonuclease, in developing mycelia of A . niger has been described by Nagasaki (1968a; Fig. 14).Most enzymes showed two pH optima at acid and neutral values, and in general the enzymes showing the higher p H optima were active in younger mycelia, the others in older cultures. Drastic changes occurred around 4-5 days when the conidia formed, and on the 10th. day when the conidia matured. Cytochemical methods were used t o study the intracellular localization of phosphatases in the hyphae and conidiophores (Nagasaki, 196813). Alkaline phosphatase was located in the nuclei of young hyphae, its location becoming more diffuse in older hyphae. Acid phosphatase stained in small granules similar to mitochondria. The activity of both enzymes was greater in substrate hyphae than in aerial hyphae. I n both aerial and substrate hyphae, the activity of alkaline phosphatase exceeded that of acid phosphatase in young hyphae, and in older hyphae the situation became reversed. Each enzyme was composed of a t least two components which changed characteristically during the course of development. The activities of both enzymes were much higher in fruiting structures than in hyphae. I n the growing conidiophore, the apical regions were slightly less active, but activity became highly concentrated in the vesicles when their development began. Activity in the phialides was also high, although it failed to stain probably because of the less permeable nature of the conidial wall. As conidiation progressed, cytoplasmic materials containing phosphatases in the conidiophore shifted towards the vesicle, leaving the rest of the cell vacuolated. I n an autoradiographical study of A . oryxae hyphae which was not related t o the differentiation of reproductive structures, Nishi et al. (1968) found that the rate of the nuclear cycle of nuclei in the liyphal apex was almost

99

ASPECTS OF DIFFERENTIATION I N THE FUNGI

double that a t a distance of 600 pm distal to the apex. Activities of RNA and protein synthesis were apparently less in the apical regions than in the more distal regions, but it is argued that this is not necessarily

8

Deoxyribonuclease

x

pH 5.0

\

pH 7 8 W

0 x

Days Phosphomonoeslerase

c x

-

5

10

15

20

Days Phosphodieslerase

OI

0 ._

._ c

c

5-

3 4E

Days

x~o-4

Days Pyrophosphatase

7

. J

Days

FIG.14.Changes in various enzyme activities assayed at different periods during the course of cultural development in Aspergillus niger (Nagasaki, 1968a).

the real picture, since the presence of the nucleotide and amino-acid pool, highly concentrated in the apical region, possibly causes dilution of radioactive precursors taken up by hyphae. Growth of the aerial hyphae is probably dependent on the supply from dist.al cells, driven by an active cytoplasmic streaming and by vacuolation.

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JOHN E. SMITH AND JILLIAN C. QALBRAITH

4. Other Filamentous Fungi

There are many other less detailed studies of fungal sporulation. Stimulation of macroconidia formation occurs in Trichophyton mentagrophytes under increased carbon dioxide tensions, with simultaneous increase in various enzyme activities which may imply stimulation of the hexose monophosphate and/or Embden-Meyerhof pathways (Chin and Knight, 1963). There is a decrease in oxygen consumption on initiation of Penicillium conidiation (Jicinska, 1968); the decrease slows down a t the time of conidiophore differentiation to form a plateau. A transient rise in respiration half-way through the period between initiation and appearance of the conidiophores may mark the transition t o the oxidation of another substrate, or t o some type of oxidative metabolism other than the hexose monophosphate pathway which predominates in vegetative mycelium, for example the tricarboxylic-acid cycle. Evidence for a t least partial activity of the tricarboxylic-acid cycle during sporulation is seen in P. frequentans (Deshpande and Sarje, 1966). I n the glucosecontaining medium used, conidiation occurred on the second and third days, acetate being the product of glucose metabolism during the first four days. Acetate disappeared on the fifth day when citrate first appeared, presumably being formed from the condensation of oxaloacetate and acetate. I n X u c o r hiemalis, a study of enzyme localization using the nitro-blue tetrazolium staining technique showed the tricarboxylic-acid cycle to be active in young stages of differentiation (Gooday, 1968~). These experiments showed that the distribution of ethanol dehydrogenase and the tricarboxylic-acid cycle enzymes is complementary, as would be expected since respiration and fermentation compete for pyruvate (Holzer, 1961). Ethanol dehydrogenase was almost totally localized in the chlamydospores and columellae of the sporangiophores, whereas malate and isocitrate dehydrogenases were most active in vegetative hyphae, zygophores, gametangia, suspensor-cells and young sporangiophores. Glutamate dehydrogenase showed a similar distribution to the tricarboxylic-acid cycle enzymes, being most active in the gametangia and young sporangiophores, although only the NAD-dependent glutamate dehydrogenase and not the NADP-dependent enzyme was present in vegetative hyphae. Kuenzi and Fiechter (1969) found that an increase in turnover of carbon dioxide and oxygen coincided with the appearance of the first buds in Saccharomyces cerevisiae. I n the first stage of development of the individual yeast cell there was a steady increase in the content of reserve carbohydrates, glycogen and trehalose. When these reached a certain maximum, degradation occurred chiefly by the tricarboxylic acid cycle and glycolysis t o provide energy used in budding.

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Glucose is oxidized more rapidly by young mycelia of sporulating cultures of Endothia parasitica than comparable cultures which remain vegetative (McDowcll and DeHertogh, 1968). There is preferential stimulation of the hcxose monophosphate pathway in the sporulating culture, and also an indication of an increase in the activity of the glucuronic acid pathway. Acetate is incorporated into bianthraquinones and fatty acids during asexual reproduction. I n Gibberella xeae, the fact that certain carbon sources such as acetate and succinate support vigorous macroconidium production, while glucose and sucrose are poor sources (Cappellini and Peterson, 1969), is reminiscent of the metabolic mechanism of Bacillus sporulation. Bent aiid Morton have made a study of changes in the amino-acid and protein composition during development of several filamentous fungi (Bent and Morton, 1964a, b ; Bent, 1964, 1967). There are no differences in the pattern of free peptides or proteins of vegetative and conidiating mycelium of P . griseofulvum of the same physiological age, although the conidia have a smaller amino-acid pool with a higher level of glutamine and a lower level of alanine than the vegetative hyphae (Bent and Morton, 1964a). There was a striking change in the amino-acid pool during germination; the main constituents of the pool during the period of rapid growth were glutamate and alanine, and there was no substantial change apart from an increase in the content of y-aminobutyric acid, until nitrogen exhaustion. Then glutamate, ornithine and arginine were rapidly depleted, together with a more gradual fall in the proportions of glutamate and alanine. Changes in P. chrysogenum, P. expansum, A . niger and Trichoderma viride were essentially similar. Exhaustion of nitrogen by P . griseofulvum was accompanied by a rapid turnover of insoluble nitrogen, involving breakdown to amino acids (Bent, 1964). These studies were made on vegetative mycelium. Bent (1967) made a comparison of the changes in protein bands, shown by electrophoresis, of conidiating and non-conidiating mycelia of P. griseofulvum. The exhaustion of nitrogen a t 28-29 hr. in submerged vegetative cultures was accompanied by the appearance of new bands of proteins and a change in the intensity of others. There was a similar pattern a t 22 hr. in inycelia which had been induced t o spore by “crude” glucose. Thus, a “crude” glucose appears to induce some of the metabolic effects normally brought about by depletion of nitrogen. Jicinska (1968) has found that autolysis is a necessary condition for endotrophic conidiation of Penicillium. Conidiation was induced by transference of mycelial mats to medium lacking glucose. During induction, reserve substances were rapidly consumed and extensive autolysis was shown by an increase in the ammonia content of the mycelium. Ammonia, formed from oxidation of amino acids, was only

102

JOHN E. SMITH AND JILLIAN C. OALBRAITH

released under aerobic conditions ; under anaerobic conditions ammonia was not formed and differentiation stopped. The most marked changes in amino acids were for alanine, aspartate and glutamate which decreased, and leucine and isoleucine which increased in relation to the vegetative mycelia. Another manifestation of autolysis was the increased concentration of orthophosphates in the mycelium. Pitt (1969) has shown a high concentration of RNA and total protein in young phialides of P. notatum, with decreasing amounts in older structures. This is similar to the findings of Righelato et al. (1968) that no net synthesis of protein or RNA occurs in P. chrysogenum after the induction of conidiation. It seems that the induction mechanism alters the metabolism of the mycelium so that the existing protoplasmic components are translocated to the phialides where materials appropriate for conidial formation are produced. I n Sclerotinia sclerotiorum and S. trifoliorum there are three phenomena during the early phases of sclerotium development : the formation of liquid droplets containing soluble carbohydrates on the surfaces of young sclerotia, a decrease in the water content, and a decrease in endogenous mannitol and also of glucose and trehalose in S. trifoliorum. The third phenomenon probably represents the conversion of soluble carbohydrates t o storage and structural compounds, thus maintaining the movement of soluble carbohydrates into the developing sclerotia by creating a metabolic sink. Excretion of carbon compounds in droplets also has this effect (Cooke, 1969). An important paper has recently been published (Griffin and Breuker, 1969) in which a change in RNA synthesis associated with sporangial differentiation in Achlya is reported. Methods were developed for growing and treating large populations of mycelia so that the hyphal tips would differentiate into sporangia with considerable synchrony. Under the starvation conditions imposed for the differentiation of sporangia, net RNA, DNA, and protein syntheses ceased. However, incorporation of radioactive precursors into RNA continued a t a high rate throughout the period of differentiation, showing that the enzymic mechanism for RNA synthesis was still in an active state. Actinomycin D inhibited the differentiation of sporangia and the incorporation of labelled precursors into RNA. The concentration of actinomycin D used did not inhibit the normal outgrowth and branching of the mycelia that occurred during differentiation. Thus, DNA-dependent RNA synthesis was required for the differentiation of sporangia. Sucrose-gradient analysis of newly synthesized RNA showed that only the ribosomal and soluble fractions of RNA were labelled during vegetative growth. During differentiation of sporangia, ribosomal and soluble RNA fractions were also labelled, and, in addition, a heterodisperse fraction of labelled RNA which was heavier than ribosomal RNA appeared. The fact that this fraction was

ASPECTS OF DIFFERENTIATION I N THE FUNQI

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peculiar to the differentiation process suggested that they are not precursor ribosomal RNA. The patterns of incorporation showed that synthesis of ribosomal RNA continued up to the time of spore discharge, which differs from the situation in B. emersonii in which RNA synthesis c’casesduring differentiation of the sporangia (Murphy and Lovett, 1966). Gressel and Galun have also made a study of RNA changes in Trichoderma. They found that 5-fluorouracil inhibited photo-induced conidiation (Galun and Gressel, 1966), an effect overcome by the addition of uracil (Gressel and Galun, 1967). Chromatography of RNA from the fluorouracil-treated mycelium showed several differences from the control, the main difference being the appearance of a large peak between 4SRNA and 5SRNA. 8-Azaguanine was the only other RNA antimetabolite of a large group tested which acted like fluorouracil in suppressing conidiation while only slightly inhibiting growth, and it also induced production of the peculiar RNA. Fluorouracil and azaguanine arc incorporated into all types of RNA. It was concluded that photo-induction of conidiation requires RNA-mediated action, and that a feasible mode of action of fluorouracil in inhibiting photo-induced conidiation is the production of spurious proteins because of spuriously coded t-RNA. Suppression of normal ribosome production would not be a suficient explanation in itself because sufficient “old” ribosomes would be available for protein synthesis. Mention must bc made of the more novel possibilities of morphogenetic control mechanisms, such as specific sporulation factors and extrachromosomal heredity. Hadley and Harrold (1958a, b) found that inediuni from a mature mycelium promoted more rapid conidiation of vegetative mycelium of P. chrysogenum than did a fresh medium. Media which had supported growth of five other Penicillium spp. or A . niger had a similar effect. This was taken t o indicate the presence of sporulation factor(s) since it was not considered to be an effect of change in the concentration of nutrients or staling products. The accumulation in staling media of a fungal morphogen which has conccntration-dependent effect on fungal inorphogenesis, including the production of conidia, has been noted for Fusarium, Geotrichum and other fungi (Park, 1961, 1963; Park and Robinson, 1964, 1969; Robinson and Park, 1965). Stimulation of asexual reproduction of Pilobolus by Mucor plumbeus is d w to a volatile substance which may be ammonia (Page, 1959). It was proposed by Cantino and Hyatt (1953~)and Cantino and Horenstein (1954) that a cytoplasmic factor, designated gamma, controlled the incidence of development of orange, late colourless, and OC plants from RS plants. This was confirmed by Cantino and Horenstein (1956)by studying swarmers from these three types of cells, when it was found that there were three distribution patterns of mitochondria-like

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JOHN E . SMITH AND JILLIAN C. OALBRAITH

particles obtained from the three types of swarmers. A mutant developing only orange cells produced swarmers with distribution patterns like those of swarmers from orange plants, or when treated with diphenylamine t o suppress carotene formation, like swarmers from OC plants. Undoubtedly, extrachromosomal systems do participate in development and differentiation of fungi (Jinks, 1966).For instance, propagation of Aspwqillus glaucus for several generations by conidia causes a gradual loss in the capacity for sexual reproduction. If propagation is continually by hyphal tips, the ability to reproduce sexually is similarly lost, and later that for asexual reproduction (Mather and Jinks, 1958). Similar results have been obtained with N . crassa (Bertrand et al., 1968). Such work demonstrates the activity of cytoplasmic particles in determining the differentiation of reproductive structures, but rejuvenation experiments do not indicate that asexual and sexual differentiation is under the control of particles specific to each. The effect is more one of diluting out. Holliday (1969) has attributed clonal senescence in fungi to a loss in fidelity in protein synthesis, and has shown in Podospora that the development of senescence is strongly influenced by treatments affecting the accuracy of protein synthesis. I). HORMONES AND

SEXUAL REPRODUCTION

Sexual reproduction in fungi, as in other living organisms, involves the union of two compatible nuclei. Plasmogamy brings the two nuclei together in one cell and karyoganiy unites them into one diploid. I n higher fungi, in particular the Basidiomycetes, these two processes may be separated in time and space, and fusion may not take place until later in the life history of the fungus. This dikaryotic condition may be perpetuated from cell to cell by conjugate or simultaneous division of the two associated haploid nuclei. The ultimate stage of sexual reproduction involves meiosis which restores the haploid condition in the four nuclei which result from it. On a sexual basis fungi may be classified as hermaphroditic, dioecious, or sexually undifferentiated. I t is now generally considered that there are definite physiological mechanisms superimposed on the genetic genomes which govern sexuality. The secretion of sexual hormones (or gamones or sporogens) which control and direct the initiation of sexually active organs t o karyogamy is now well documented for several fungi. However, a n added complexity to the understanding of the sexual process in fungi is the presence in some fungi of a parasexual cycle. With limited exceptions t here is little known about the biochemical and physiological changes that occur within an organism during sexual morphogenesis.

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However, now that several hormones have been chemically characterized, such information may soon be forthcoming (Barksdale, 1969). A distinctive feature of sexual reproduction is the occurrence of meiotic nuclear division a t some point in the life-cycle of the organism. Thus, it becomes obvious that a fuller knowledge of the physiology of meiosis is necessary for understanding the mechanism of recombination in eukaryotic organisms. I n particular the transition from mitotic to meiotic division is of prime importance since it is considered that meiosis has evolved from a deviation from the pattern of normal mitotic division (Taylor, 1967). I n most fungi the occurrence of meiosis results from preceding events, such as fusion of nuclei or germination of the zygote, while in certain yeasts meiosis is directly induced by changes in the environmental conditions (Miller, 1959). For general background reading on sexuality in the fungi the reader is referred to the excellent review articles in “The Fungi”, volume 2 (1966; edited by G . C. Ainsworth and A. S. Sussman. Academic Press, N.Y .). 1. Sexual Reproduction in the Phycomycetes

a. Class Chytridiomycetes. This group is distinguished from other fungi mainly by the production of motile cells (zoospores or planogametes) each with a single, posterior, whiplash flagellum. I n recent times, species of Allomyccs and Blastocladiella have become valuable research tools in the study of the biochemical basis of morphogenesis (Cantino, 1966). I n species of Allomyces, in particular A . arbuscula and A . macrogynous, sexual reproduction is manifested by the copulation of free swimming anisogamous planogametes in an ambient liquid medium. The male gametes are orange due to the presence of y-carotene and are about half the size of the female gametes. The female gametes begin releasing a sperm-attractant, sirenin, prior to their release from the gametangium ; sirenin is active a t concentrations of M (Machlis, 1958a, b). Sirenin has been shown t o be an oxygenated sesquiterpene with four degrees of unsaturation and with both of its oxygen functions capable of forming esters (Fig. 15). The production, isolation, characterization and chemical structure of sirenin have recently been described (Machlis et al., 1966, 1968). Machlis (1968) has suggested that there may be several speciesspecific sirenins. Regulatory compounds such as sirenin have been termed hormones because the cells that respond t o the regulatory molecules are separated in space from the cells that secrete them (Barksdale, 1969). b. Class Oomycetes. Sexual reproduction is almost invariably heterogametangic and the formation of oospores is characteristic of all but the most primitive species. i. Order Saprolegniales. I n this group sexual reproduction involves the

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JOHN E. SMITH AND JILLIAN C. GALBRAITR

development either on the same thallus (homothallic) or on separate thalli (heterothallic) of oogonia with multinucleate eggs (oospheres) and antheridial hyphae. Fertilization is by means of gametangial contact, male gametes passing into the female gametangium through a fertilization tube. When potentially male and female thalli of Achlya occur in close proximity, a system involving several distinct hormones becomes operative and the sexual process is initiated. The full complexity of hormonal control in this type of sexual reproduction has been elegantly

A

HOCHz Trisporic acid

Sirenin

Antheridiol

FIG.15. Chemical structure of three compounds functioning in sexual reproduction in fungi. All three arc terpenoid in character.

demonstrated by Raper and his coworkers (see Barksdale, 1969). The basic investigations were carried out on two heterothallic species of Achlya, A . ambisexualis and A . bisexualis, though it has also been shown that the basic regulatory mechanisms prevail in homothallic forms (Barksdale, 1960). One of these specific hormonal substances, designated antheridiol, is secreted by the female plant and brings about the formation of antheridial hyphae on the male plant (Raper, 1957). Antheridiol has recently been isolated and characterized (McMorris and Barksdale, 1967 ; Arsenault et al., 1968) and if the proposed structure is correct it is the first steroidal sex hormone to be recognized in the plant kingdom (Fig. 15). In particular, it differs structurally from mammalian sex hormones in that is has a much longer side chain attached a t C- 17.

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ii. Order Peronosporales. Sexual reproduction is by means of well developed oogonia and antheridia borne on the same or different hyphae and represents the highest development of the class Oomycetes. Sterols have been found t o be necessary for sexual reproduction in ccrtain fungi in this Order, in particular Pythium and Phytophthora (Elliott etal., 1964; Haskinsetal., 1964; Hendrix, 1964; CheeandTurner, 1965). These fungi do not appear to synthesize sterols, and vegetative growth can occur in a sterol-free medium (Elliott et al., 1964; Hendrix, 1965; Schlosser and Gottlieb, 1966). Pythiaceous fungi are unusual in this respect since all other fungi investigated appear t o have a complete sterol-synthesizing system (Appleton et al., 1955; Cochrane, 1958). The growth of several Pythium species was increased between 65-100% if cholesterol was added to the growth medium and it was considered that optimum synthetic activity could only be achieved when sterols were present in the cellular membrane (Schlosser and Gottlieb, 1968). The stimulatory effect of sterols on sexual reproduction in Pythium may depend on the ion (Lenney and Klemner, 1966) and amino-acid (Sietsma and Haskins, 1967) composition of the medium. The connection between the sterol requirement and the formation of sexual organs is far from clear, but it is obvious that cholesterol (or closely related sterols) is essential for expression of sexuality in a t least t,his group of Oomycete fungi. c. Class Zygomycetes. Sexual reproduction in this group of fungi is by means of copulation of multinucleate gametangia which are in the main similar in structure but may differ in size. i. Order Mucorales. The classical experiments of Burgeff (1924) demonstrated that a diffusible substance(s) was probably responsible for the initiation of sexual reproduction in the Mucorales. This was the first demonstration of a hormonal sexual mechanism in the fungi. However, itt was many years later before Burgeff and Plempel(l956) demonstrated the presence of diffusible chemical substances in the culture medium of mated strains of Mucor mucedo which could induce zygophorc production in unmated cultures of the same fungus. Plempel (1963a, b) claimed that two hormones (gamones) were produced in mated cultures of M . mucedo and further that two substances (progamones) were present in the culture medium, one from each mating type in unmated cultures, that initiated production of these hormones in cultures of the opposite mating type. Ende (1967) obtained a substance from the culture filtrate of mated cultures of Blakeslea trispora that was active in cultures of M . mucedo. More recently Gooday (1968a, b) has extracted and characterized a compound from mated cultures of M . mucedo which is capable of inducing zygophores in unmated cultures of M . mucedo. Comparative studies showed that most of the hormone that

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could be extracted from a culture was in the mycelium and not in the medium. Furthermore, production of the hormone was controlled by the age of the culture and by the oxygen tension. There is a close similarity between the biological property of this hormone and of the trisporic acids isolated from culture filtrates of mated strains of B. tiispora (Caglioti et al., 1964; Cainelli et al., 1967; Sutter and Rafelson, 1968). The sexual factors obtained from culture filtrates of several members of the Mucorales have been identified with trisporic acids B and C (oxidized unsaturated derivatives of 1,1,3trimethyl-%(3-methylocty1)cyclohexane; Fig. 15; Ende, 1968; Austin et al., 1969b). Very little is known about the primary induction of sexual differentiation in these fungi. Physical contact between the mycelia of the two mating types is not necessary for maturation of sexuality. It has been suggested that a form of complementation may be involved in which each separate mating type may regularly produce a diffusible substance (progamone) that is converted into the hormone (gamone) by the other strain (Plempel, 1963a, b ; Banbury, 1964; Gooday, 1968a). An interesting aspect of sexuality in this group is the accumulation of carotenoids that accompanies sexual development (Burnett, 1965). Thomas and Goodwin (1967) have shown that there is an increase in carotene production in a culture of minus-mating type of B. trisporus on the addition of a crude extract of the medium of a mated culture. It has been suggested by Burnett (1965),Ende (1968)and Gooday (1968a) that the increase in /3-carotene content may be a secondary effect of the action of the hormone that induces production of zygophores. However, Austin et al. (1969a, b) prefer to consider the trisporic acids as degradation products of /3-carotene. This view is further strengthened by the fact that, in the car-mutant of Phycomyces (deficient in /3-carotene), the sexual process is also deficient (Heisenberg and Cerda-Olmedo, 1968). 2. Sexual Reproduction in the Ascomycetes a. Order Hemiascomycetidae. The Hemiascomycetidae are characterized by a simplicity of structure, often lacking true mycelium, and by the direct formation of asci without a concomitant development of ascogenous protective hyphae. Yeast-type members of this order have been intensively investigated, and sexual union can occur either between two somatic cells or between two ascospores. The product of this union becomes the diploid zygote cell, and in suitable environmental conditions will undergo meiosis and ascus formation. The duration of the haplophase and diplophase can vary considerably between organisms ; in

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Saccharomycodes the haploid condition occurs only in the ascospore and the vegetative budding yeast is diploid, while in Saccharomyces cerevisiae both haploid and diploid cells can proliferate vegetatively by budding. i. Agglutination. I n some heterothallic yeasts, in particular Hansenula wingei, opposite mating types will agglutinate when brought together in culture. It is considered that agglutination is due t o a specific substance, probably protein, present in the cell wall of one mating type combining with a specific substance, probably polysaccharide, present in the wall of the opposite mating type (Brock, 1959a, b). Both mating hypes must function and synthesize protein in order for conjugation to take place (Brock, 1961). A cell which has conjugated in one region of its cell wall is still able to conjugate with another cell in another region, so that triply and quadruply conjugated cells can be formed (Brock, 1965). There was no significant net increase in DNA, RNA, protein or carbohydrates that could be related to the conjugation process (Brock, 1965). ii. Sporogeriesis. Under appropriate conditions, sexual sporulation can be induced in nearly all of the members of a diploid yeast population. However, there is a t present little biochemical information on the differences between mitosis and meiosis and on the regulatory events which determine whether a diploid nucleus will undergo one or the other of these processes. I n practice meiosis is induced in a cell or culture on transfer from a rich growth medium to a sporulation medium containing acetate as carbon source (Miller, 1959). There are several indications of the actual onset of meiosis in an induced culture of yeast. These include : (a) enlargement of the nucleus (Nagel, 1946; Pontefract and Miller, 1962: Croes, 1967a); (b) vacuolar fragmentation (Miller P t al., 1963; Svihla et al., 1964; Croes, 1967a); (c) accumulation of glycogen and fat globules (Pontefract and Miller, 1962); and (d)an increase in cell volume (Deysson and Lan, 1963; Croes, 1967a). Nuclear enlargement coupled with vacuolar fragmentation are considered to be the most reliable indications of successful inducement (Croes, 1967a). Sporulation is dependent on acetate metabolism and also on the physiological age of the culture when transferred to the sporulat'ion medium (Crow, 1067a; Esposito et al., 1969).Although meiosis seems to be induced by contact with the sporulation medium, studies with ethioniiie have shown that, meiosis has been specifically prepared for before transfer of the cells t o the acetate-containing sporulation medium (Croes, 1967a). Thus, promotion of meiotic development has already started in the late phase of mitotic growth and acetate cannot be considered as the primary inducer of meiosis, but rather as a trigger allowing full expression of the meiotic proteins. Clearly much more attention

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must be given to analysing the premeiotic mitoses since it is a t this part in the growth cycle that the foundations of sporulation are being expressed. Extensive protein and RNA turnover and synthesis occur throughout sporulation (Miller, 1959; Croes, 196713; Esposito et al., 1969). Two maxima for protein synthesis occur, one before asci appear and a second during ascospore development (Esposito et al., 1969).The initial phase of intracellular differentiation during yeast sporulation is marked by protein degradation (Ramirez and Miller, 1964 ; Croes, 1967b) and active protcolytic activity has beeii reported (Chen and Miller, 1968). An important feature in the induction of meiosis is the change in carbohydrate metabolism from fernlentstion to intense oxidative respiration. During log-phase growth, cells are unable to oxidize acetate and thus cannot undergo sporulation. As the concentration of glucose decreases, the tricarboxylic acid cycle enzymes are derepressed and the cells progressively develop the high oxidative capacity essential for sporulation (Croes, 1967b). I n some cases, sporulation can occur utilizing only the end-product of fermentation, ethanol (Snider and Miller, 1966). The trigger action of acetate in sporulation appears to a c t by speeding up the encrgy-supplying processes in the cell, resulting in an insufficiency of the glyoxylate cycle (Croes, 1967b). The interrelationship of these pathways is shown in Fig. 16 (Croes, 1967b). Inhibition of mitosis a t T o(time of transfer to acetate medium) can be overcome by addition of a small amount of glyoxylate (Bettleheim and Gay, 1963). Thus, there appears to be a fundamental difference between the role of the glyoxylate cycle in yeast sexual sporulation and asexual sporulation in filamentous fungi (Galbraith and Smith, l969b, c). Under certain conditions, gibberellic acid and indole acetic acid have been shown to promote sporulation in Sacch. ellipsoideus. Both compounds were effective in promoting sporulation only when added before nuclear enlargcnicnt occurred in the sporulation culture (Kamisakaet al., 1967a). The promoting effect of gibberellic acid on sporulation was suppressed by inhibitors of RNA synthesis and protein synthesis (Kamisnka P t nl., 1967h). Hormone-like substances have recently been isolated from culture media of haploid strains of Sacch. cerevisiae. A substance ( C Y hormone) excreted by CY type cells made cells of the a type expand while correspondingly another substance ( a hormone) excreted by a cells caused cells of type to expand. The cell-expanding action of the a hormone was inhibited by actinomycin D, chloramphenicol and cycloheximide. The a hormone was heat-stable and dialysable (Yanagishima, 1969). b. Order Euascomycetidae. The basic pattern of sexual reproduction in the Euascornycetidae normally involves a coiled, septate hyphal struc-

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ture-the ascogonium or female reproductive organ. The male element, may be almost any kind of non-motile, unicellular body cut off from the parent plant such as oidia, spermatia or conidia. The union of two T4

TO

Late growth phase

phase growth ~Log _ _ _ _ _ _ _

Early

sporogenesis

i , f Y

Glutamic

acid

Amino

a

a

L

Proteins

a

FIG.16. Carbohydrat'e and protein metabolism during the two growth phases and early sporogenesis of Saccharonayces cereuisiae. The metabolic changes a t Gls and To are eonsidered to induce meiosis. Lines of different thickness mark the relative reaction rates of the processes shown (Croes, 1967b).

compatible haploid nuclei is brought about by one of several methods evolved within the Euascomycetidae (Alexopoulos, 1062), the most, common being gametangial copulation, gametangial contact, spermatization, and somatogamy. Plasmogamy is normally followed by a limited

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dikaryotic period of division, and nuclear fusion and subsequent meiosis eventually taking place in the ascus mother cell. A multihormonal system has been implicated in controlling and regulating sexual reproduction in Ascobolus stercorarius (for a review, see Machlis, 1966), and although this system has been extensively investigated the hormones have yet t o be extracted and characterized. Hormones have also been implicated in the sexual reproduction of several other Euascomycete fungi, but again little is known of their chemical and physical properties. Although a wealth of descriptive information has accumulated about the sexual development of many Euascomycete fungi (see Turian, 1966a, 1969) there is little information on the physiological and biochemical factors involved in these processes. The biochemistry of the male or conidial stage of Neurospora has been extensively studied by Turian and his coworkers (reviewed under Asexual Sporulation, p. 88) but surprisingly little is known about the ascogonial stage. 3. 8exual Reproduction in the Basidiomycetes

a. h'ub-Class Homobasidiomycetidae. Sexual reproduction in the liomobasidiomycetes is normally accomplished by somatic copulation by fusion of hyphae. The primary mycelium resulting from basidiospore gerniination is septate and uninucleate and the binucleate condition (plasmogamy) arises by the fusion of two compatible uninucleate protoplasts. A secondary mycelium develops from this fusion and it retains the biriucleate or dikaryotic condition during cellular division by means of clamp connections that ensure that sister nuclei arising from conjugate division of the dikaryotic cell become separated in the two daughter cells (for further details see Alexopoulos, 1962). The dikaryotic phase in the life-cycle can be of indefinite duration and only under defined conditions will the fruit body or sporophore be produced. I n the basidial cells of the sporophore, karyogamy and meiosis will occur resulting in the formation of uninucleate haploid basidiospores. Thus in the homobasidiomycetes there are no sex organs, the somatic hyphae taking over the sexual functions. Species may be homothallic, secondary homothallic or heterothallic. Sexual compatibility in heterothallic species may be bipolar or tetrapolar (Raper, 1966). i. Environmental control of sporophore formation. It is remarkable that so little attention has been given to understanding the mechanisms that regulate sporophore development in this group of fungi. This may be due in part to the mistaken belief that these large sporophores are only produced in natural environments. Indeed, more than 150 species of agarics, boletes and polypores have been successfully grown in the

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laboratory, although not all on synthetic media (Taber, 1966; Volz and Beneke, 1969). The initiation and subsequent development of Agaricus bisporus (the commercial mushroom) has long been a source of mystery to both mushroom growers and scientists. The pure-culture nutritional requirements for the riiycelial phase of growth were first fully documented by Treschow (1944), but he was unable to achieve sporophore development in culture. Changes in the concentration of sterols, sterol esters, and fatty acids have been associated with sporophore formation (Hughes, 1963) while the use of seed oils as a supplement in composts increased the crop yield (Schisler and Sinden, 1966). It has been suggested that in normal commercial practice and in natural environments successful sporophore formation is dependent on the presence of essential micro-organisms in the casing soil (Eger, 1963 ; Urayama, 1967). The stimulatory bacteria have been variously identified as Arthrobacter terregenes, Bacillus megaterium and Rhizobium meliloti by Park and Agnihotri (1969) and as Pseudomonas putida (whose multiplication was selectively favoured by metabolites of A . bisporus) by Hayes et al. (1969). I n each study, metabolites from the bacteria could serve as initiators of sporophore production. Further analysis of these metabolites may show a common initiating factor and the ability to complete the life-cycle of A. bisporus under defined conditions will greatly facilitate the understanding of the regulatory mechanisms of sporophore development. These experiments must also have a profound influence on commercial mushroom production in the future. Schizophyllum commune, a wood-rotting fungus, has been the subject of intensive genetical and nutritional investigations. This is due largely to the fact that the complete life-cycle can occur in a relatively simple medium under controlled laboratory conditions. Although formatmion of the sporophore normally occurs from the dikaryon, sporophores may develop from certain haploid mycelia supplied with exogenous chemical agents extracted from mycelial extracts of Hormodendrum cladosporoides, and sporophores of S . commune and A . bisporus (Leonard and Dick, 1968; Leonard and Raper, 1969). Comprehensive summaries of the environmental factors regulating morphogenesis in this fungus have been well documented (Wessels, 1965 ; Taber, 1966 ; Niederpruem and Wessels, 1969). Development of S. commune can be divided into four phases : (1) vegetative or non-differentiated growth ; ( 2 ) initiation of primordia and continued formation of non-differentiated hyphae ; ( 3 ) growth of primordia; and (4)formation of pilei or sporophores. The relationship between carbohydrate and nitrogen in mycelium and sporophores in a dikaryon of S. commune grown on synthetic medium is shown in Fig. 17. Using a replacement culture technique, Wessels (1965)

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has shown that : (a) initiation of sporophore primordia required external carbon and nitrogen sources; (b) growth of the primordia required a carbon source in the medium, but no exogenous nitrogen source; and

Time ( h r )

Myce Iium

300

Fruc t if icot ion

FIG. 17. Changes in total nitrogen and total carbohydrate of Schizophyllum conamune K.35 (cup mutant) and K.8 (wild-type stock) accompanying development. Part of the values for the whole organism is obtained by summation of the values for fructifications and mycelium. The schematic drawings a t the top of the graphs represent the various stages of development (Wessels, 1965).

(c)the synchronous formation of pilei proceeded in the absence of external carbon and nitrogen sources, drawing upon endogenous compounds and suggesting that a flow of materials occurs from stunted fructifications and mycelium to developing fruit-bodies.

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ii. Biochemical changes associated with sporophore development. The extensive literature on the cytodifferentiation and biochemical changes associated with morphogenesis in S. commune has recently been comprehensively reviewed by Niederpruem and Wessels (1969), and the following brief account will merely summarize the main biochemical changes that accompany morphogenesis. Since plasmogamy may occur shortly after germination of basidiospores, there has been extensive investigation of the metabolism of ungerminated and germinated basidiospores (Aitken and Niederpruem, 1968 ;Niederpruem, 1964; Niederpruem and Dennen, 1966; Niederpruem et al., 1065; \Vessels, 1965). The specific activities of two glutamate dehydrogenases varied during growth as a function of the stage of the life-cycle and the exogenous nitrogen source. During basidiospore germination the activity of the NADP-dependent glutamate dehydrogenase increased six to eight fold in specific activity whereas the activity of the NAD-dependent enzyme was depressed. I n dikaryotic mycelia activities of both glutamate dehydrogenase enzymes increased in a 1 :1 ratio, whereas during homokaryotic mycelial growth on glucoseammonium medium activity of the NADP-dependent enzyme was depressed and that of the NAD-dependent enzyme was increased six to eight fold (Dennen and Niederpruem, 1967). Simultaneously with the formation of sporophore primordia there was a drop in the ratio of RNAIprotein. During the next 24 hr., RNA synthesis was greater than protein synthesis and the RNA ratio increased (Wessels, 1965). Although sporophore primordia are formed in the presence of glucose, pileus formation will only occur a t glucose exhaustion. Associated with this development there is a decrease in cellular carbohydrate and a breakdown of cell-wall polysaccharides (Wessels, 1965). Regulation of a cell-wall glucan-degrading enzyme (R-glucanase) has been considered as an important feature of sexual rnorphogenesis in this fungus (Wessels, 1966; Wessels and Niederpruem, 1967). The enzyme acts on the /3-1+3, /3-1 -tB-linked R-glucans that constitutes, with chitin, the alkali-insoluble portion of the wall (Wessels, 1965) and may play an important role in initiating softening or lysis of insoluble fungal cell-wall /3-glucans which appears to be a requisite for pileus expansion (Wessels, 1969). The nature and distribution of certain enzymes of carbohydrate catabolism in vegetative monokaryotic mycelia and dikaryotic stalks and caps of Coprinus lagopus have been studied by Rao and Niederpruem (1969). Enzymes of hexose monophosphate catabolism, sugar alcohol dehydrogenase and trehalase were detectable throughout development although the ratio of xylitol dehydrogenase to sorbitol dehydrogenase varied with different growth phases.

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It has been implied that hormones or some form of endogenous growth regulation may be involved in the development of homobasidiomycete sporophores (Gruen, 1963). Some plant hormones such as gibberellic acid and indole acetic acid can have growth-promoting effects on agaric fungi (Volz and Beneke, 1969).

E. SECONDARY METABOLITES AND DIFFERENTIATION Microbial differentiation commonly occurs after the phase of rapid vegetative growth. Indeed, Schaeffer (1969) has pointed out that the diverse morphological processes, termed sporulation, in eubacteria, the actinomycetes, yeasts and the lower fungi, have in common the fact that they are all intracellular processes of differentiation in which the cell is subdivided by neomembrane formation ; this process remains unexpressed as long as growth is possible. Although growth and cell division on the one hand, and morphogenesis and differentiation on the other, are not necessarily completely incompatible, a certain antagonism is to be expected, if it is postulated that alternative metabolic pathways are involved in the processes. The incompatibility of growth and sporulation were observed by Klebs (1898) who made the generalization that reproduction is initiated by factors which check growth. This has been confirmed by the many studies of the effects of environmental factors on reproduction, and echoed in reviews such as that of Hawker (1957). Hawker concludes that conditions permitting spore formation are almost always of a narrower range than those permitting mycelial growth ;the requirements for sexual reproduction differ from those sufficient for growth far more than do the requirements for asexual reproduction in the same species. If growth and differentiation do not occur simultaneously, and are separated by a definite metabolic shift, the point of change is likely to be associated with limitation of vegetative growth due to nutrient exhaustion. There arc several examples among the fungi in which differentiation is associated with nutrient depletion. The most general condition for the induction of conidiation of P. griseofulvum, and several related fungi in submerged culture is the absence or exhaustion of assimilable nitrogen while carbohydrate is still present (Morton et al., 1958; Morton, 1961). Morton (1961) found that induction was completed three t o four hours after transfer of the mycelium to a nitrogen-free medium, although there were no morphological signs until seven hours. Addition of a nitrogen source before induction completely prevented reproduction, and addition during the period of induction delayed and decreased the number of sporogenous branches. Addition a t a still later stage increased the number of sporogenous and

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vegetative branches. Hadley and Harrold (1958b) also noted that conidiation of P.notatum was enhanced by a decrease of the concentration of nitrogen source, and likewise determined a critical phase for induction in which the presence of calcium ions was essential (Hadley and Harrold, 1958s). I n continuous-culture experiments with P.chrysogenum (Righelato et al., 1968) a decrease in the concentration of glucose to a very low “maintenance ration”, all other substrat)es being present in excess, induced conidiation. Conidiation did not occur in the complete absence of glucose, and the maintenance ration was thought to facilitate conidiation by preventing autolysis. There was a critical growth rate above which only vegetative growth occurred. The development of the sporocarp of Sordaria is dependent on only a limited supply of carbohydrate (Hawker and Chaudhuri, 1946). Amongst the basidiomycetes, development of the sporophore of S. commune is associated with successive limitations in the requirements for external nutrients (Wessels, 1965),and expansion of the pileus occurs only in the absence of a metabolizable carbon source (Wessels and Niederpruem, 1967). Morton (1967) has suggested that the occurrence of sporocarp initials only superficially or on aerial branches is related to limitation of nitrogen source or some other substrate. I n these studies, carbon and nitrogen are clearly implicated as the most important nutritional elements. Before considering how depletion of either of these elements essential for growth might direct cell metabolism towards sporulation, let us consider in general how exhaustion of the nutrient supply could cause a metabolic shift leading to morphogenesis. Differentiation occurs in essentially endogenous, self-sufficient systems which have t o a greater or lesser degree cut themselves off from the environment. I n the more primitive and easily influenced systems, morphogenesis is a t least in part initiated by some degree of nutritional deficiency, and is usually accompanied in the early stages by formation of permeability barriers (Wright, 1967, 1970b). Both efficient utilization and a high rate of breakdown of macromolecular components will be required by the endogenous metabolism in such a self-sufficient system. Precise regulation and the occurrence of events in a predictable sequence are consequences of the limited amount of compounds serving as energy source and precursors for rnorphogenesis. I n fact the changes associated with the depletion of endogenous reserves may actually trigger the final stages of morphogenesis (Wright, 1967, 1970b; Wright and Dahlberg, 1967), helping t o achieve a sequential order of events in differentiation. Normal morphogenesis is able to occur in spite of potentially hazardous variations in substrate levels because these compounds are a t a limiting concentration, and the complex interaction of the interacting pathways changes their operating

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range as opposed to rigidly controlling the end result. I n this way, premature or delayed accumulation of any one metabolite is unable either to trigger or prevent morphogenesis. We see experimental evidence for Wright’s predictions in the possible relationship between protease activity and fungal differentiation. A role for proteases in sporulation in providing the materials of protein iinabolisni from endogenous compounds is to be expected. Indeed, many moulds form appreciable amounts of proteolytic enzymes after a period of active growth, i.e. a t the time of sporulation initiation. The differences in the nature of proteins in conidiating and non-conidiating mycelia is illustrated in P . griseofulvum by Bent (1967). Other biochemical changes have been observed which provide a key to the way in which exhaustion of the nutrient supply could cause a metabolic shift leading to morphogenesis. Exhaustion of the nitrogen supply in the presence of glucose in the culture medium of Gibberella fujikuroi initiated a period offat storage, whereas fat breakdown occurred when glucose was the limiting nutrient (Borrow et al., 1961). I n Torulopsis utilis, ammonia assimilation in a carbon-free medium was accompanied by an increase in the rate of respiration, which was dependent on the levels of carbohydrate reserves. The assimilated nitrogen appeared mainly as glutamate, glutaniine, alanine, and later as protein (Yemm and Folkes, 1954). However, ammonia assimilation by Scopulariopsis brevicaulis continued for only two hours after depletion of the glucose supply, and changes in the amino-acid pool of nitrogen-starved mycelium on addition of ammonium salts were similar with or without glucose (MacMillan, 1956). This difference from T. utilis (Yemm and Folkes, 1954) may be due to the spatial separation of the endogenous carbon source and the nitrogen assimilatory enzymes in S . brevicaulis. When vegetative growth of fungi ends, secondary metabolites frequently accumulate and many are made use of industrially. These secondary metabolites are derived from primary metabolites which accumulate from primary synthetic processes ; they are often highly specific and confined to one or a few fungal species. When microbial growth stops due to a deficiency in such nutrients as carbohydrate, nitrogen, phosphates and trace elements, the intracellular level of primary metabolites will be high. Repression of enzyme synthesis will lower the rate of synthesis of metabolic pool components, and feedback inhibition can close off biochemical pathways from appropriate branch points. But, even though rates of synthesis are low, those intermediates prior to the branch points will attain exceedingly high levels in the cell (Woodruff, 1966). The result of “normal” enzymes acting on abnormal accumulations of pool substrates is the accumulation of products of no direct value to the cell. This line of thought arose in the study

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of antibiotic synthesis, and the term “shunt metabolism” was coined by Foster (1947) to describe the modification of normal pool metabolites resulting in antibiotic formation. Secondary metabolites such as antikliotics are eliminated from the cell. Woodruff (1966) belives this to be undesirable, and that cells have solved such a situation by evolving into iiiore differentiated cells, with a capacity for precise control of metabolic processes which makes antibiotic production no longer possible. In an intermediate position between these two extremes are certain microbes which have learned to use their shunt metabolites to some advantage. These are microbes which form specialized bodies such as spores which frequently contain molecules closely related in structure to the antibiotics. Thus, Woodruff considers that this is one branch of evolutionary development in which selective advantage is derived from secondary metabolites, but the main stream of development has led to a cellular specialization and elimination of shunt metabolism and of the accumulation of secondary metabolites. Bu’Lock (1961) has put forward the hypothesis that “the selective advantage of secondary metabolism is that it serves to maintain mechanisms essential to cell multiplication in operative order when multiplication in that cell is no longer possible”. This is not the place for a discussion of what Burnett (1968) considers to be an unlikely hypothesis, but of interest to this review are the connections being recognized between typical examples of secondary metabolite formation and the development of specialized structures, such as spores, conidia, and sclerotia, which prolong survival under adverse conditions. The correlation between bacterial sporulation and the production of antibiotics, exoenzymes and exotoxins is well known (Schaeffer, 1969). Some examples of ail association between fungal sporulation and secondary metabolism are discussed below. 1. Isoprenoids a. Carotenoids. There have been several attempts to relate causally

sporulation and the development of pigments in the reproductive structures. It is more likely t h a t pigmentation is a consequence of lightinduced metabolic changes. On the other hand, pigmentation is not obligatorily associated with light. The conidial carotenoids of N . crmsa develop in the dark (Zalokar, 1954, 1955), and carotogenesis in many other fungi does not require light, although it has a stirnulatory effect (Chichester et al., 1954; Garton et al., 1961). The sexual activity of fungi in the Mucorales is often accompanied by an increased accumulation of carotenoids (reviewed by van den Ende, 1068). For example, during sexual reproduction of Blakrslea twkpoya, the formation of hormones runs p~~rallel with the accumulation of 6

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carotenoids (Plenipel, 1965). As already mentioned, the existence of albino fully conidiated mutants of N . crassa dispels any causal relation in this fungus (Hungate, 1945; Sheng and Slieng, 1952) although Turian ( 1 966a) does report partial repression of conidiation in wild-type 1' . crassa grown in the presence of diphenylamine which represses carotene form;ttion. Albino mutants in Pyronema (Carlile and Friend, 1956; Bean and Brooks, 1932) capable of normal reproduction again show otdy a casual relation between development of a pigment and formation of npothecia. In l'hycomyces blakesleeanus, the time course for carotene formation indicates that the bulk of the carotene is formed after growth has been completed (Goodwin and Willmcr, 1952). In the gametophyte of Allornyces javanicus, the carotenoid pigments and colourless polyenes are restricted to the male gametangia (Emerson and Fox, 1940 ; Turian and Haxo, 1954).Differentiation on one myceliuni of orange male and colourless female gametangia represents a pheiiotypic sex determination, since the nuclei of both are derived by mitosis from the haploid nuclei of the parent mycelium. The formation of orange pigment and concomitant production of the orange sex organs on tlie sporophyte can be induced by cycloheximide (Whiffcw, 1951). Although inhibiting carotene synthesis, diplieti~-laminedid not irii ])air sexual reproduction (Turian, 1952), thereby denying a causal relation. Heterothallic Blnstocladida also produces orange or colourless niul tinucleate cells which, although sexually non-functional due to the inability of the swarmers t o retract the flagellum, are apparently similar to Allom?yces. Their formation can be similarly manipulated by inhibitors and stimulators of carotogenesis (Cantino, 1966). Investigation of the enzyme activity of Blastocladiella (Cantino and Hyatt, 1953a, b) furthcr dispelled the notion of a causal relation between differentiation and carotogenesis. The colourless cells havo a fully functional tricarboxylic-acid cycle, but in orange cells the activities of furnarate hydratase and malate dehydrogennse arc lower by 50% or more and aconitate hydrstase and CC-0x0qlutarate dehydrogenase are almost inactive. This lesion in the tricarboxylir-acid cycle causes a lack of reducing pressure power, which leads to an accumulation of carotene. The colour ofthe sex organs is also correlated with the distribution of a Nadi-positive, cytoplasmic gamma particle which ma,v be a lysosome (Cantino and Horenstcin, 1954, 1956; Cantino et a1 , 1963). There are relatively few gamma particles in orange cells, and their number responds in tlie same way as carotogenesis to diphenylainine and cycloheximide. A metabolic lesion in the tricarboxylic-acid cycle of orange cells was also found in Allomyces. Like Rlastocladiella, this species also lacks ~ - o x o g l ~ i t ~ ~dehydrogenase, rate but the second missing enzyme is succinatc dchjdrogenase (Turian, 1960). The lesion is compensated for

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~J,I- i i i i iiicwasc i i i tlic activity in the inale of the first vnzynie of thr gly oxylate cycle, isocitrat e lyase, and a n increase in glycine-alanine transaminasc (Turian, 106 I ) . The glyoxylate cycle is strongly functional on an acetate carbon source. Strong evidence for the causal relation between the glyoxylate cycle and differentiation of male cells is found in the observation that acetate induced increased maleness in Allomyces, along with an increase in isocitrate lyase activity (Turian, 1961). A link between carotogenesis and the lesion in the tricarboxylic acid cycle in these two water moulds has still to be proved, but tho primary observation of the relation between sexual differentiation and pigmentation has led to one of the few understandings of the biochemical processes preceding differentiation. b. Sterols. Sterols have already been shown to be required for sexual reproduction in the Oomycetes and they are also required for the production of normal sporangia and zoospores (Chee and Turner, 1965; Hendrix, 1965). Thus Pythium does not synthesize or require sterols for normal vegetative growth. If these compounds are supplied in the medium they are incorporated into the cell membrane, which shows changes in temperature sensitivity and cell-wall permeability. Cholesterol, or closely related sterols, appear to be essential for sexual reproduction although this effect is nullified by the presence of acidic amino acids (Sietsma and Haskins, 1967; Haskins et al., 1964). Sordavia Jimicola is able to synthesize sterols. However, the presence of additional sterols, particularly cholesterol, in tlie growth medium increased perithecial production. Hypocholesteremic compounds, which inhibit sterol synthesis, also inhibited growth, so this species a t least appears to require sterols for vegetative growth as well as reproduction. Two growth phases were induced by sub-inhibitory concentrations of the hypocliolesteremic compounds, the first rapid phase of growth being followed by a slower phase. A ring of perithecia was formed a t the hyphal front when the growth rate changed, and subsequently rings of perithecia wcre formcd during the second growth phase ; these numbered a t least twice those formed in tlie drug-free controls (Elliott, 1969).

2 . Anaino Acid and Peptide Derivatives This group of conipounds includes a number of antibiotics. Antibiotics are not to be regarded as a unique class of compounds, but as secondary metabolites derived from an abnormal coilcentration of cellular constituents resulting from the growth-limited state of the culture (Bu’Lock, 1961; IYoodruff, 1966). They have become distinguished as a special physiological class due to their frequency of observation, rather than their frequency of occurrence. Peptide antibiotics are produced a t sporulation by certain fungi.

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Conidia are produced in static cultures of Pithomgces chartarum only when the medium is depleted of its nitrogen source and there is a drop in growth rate. Sporidesmolides appear in the culture in the third day, the concentration being proportional to the number of conidia. There are no sporidesmolides present in submerged, non-sporulating cultures, yet a change of conditions from submerged to surface culture is accompanied by the formation of conidia and of sporodesmolides. There is no such association between sporulation and production of sporidesmin (Dingley et al., 1962). A related non-toxic metabolite, sporidesmolic acid B, can be isolated from felts of sporing cultures of Sporidesmium bakeri, and it is suggested that this compound functions as a water-insoluble coat on spores (Russel and Brown, 1960). 3. Processes Involving Phenol Oxidases

Bu’Lock (1961) pointed out that phenol oxidases seem t o be involved mainly in secondary metabolic change. There is a correlation between laccase formation and morphogenesis of the perithecia of Podospora anserina. This is shown in mutants with developmental defects and decreased melanin formation. The alteration in pigmentation is dependent on the formation of a qualitatively altered laccase, the expression of the mutant being abolished by a suppressor which also restores the enzyme level. Hirsch (1954) has shown the specific appearance of tyrosinase metabolism leading t o melanin production, with protoperithecial differentiation in N . crassa. However, the relationship is not causal because female sterile mutants which have lost their ability to form tyrosinase do not form protoperithecia even when tyrosinase formation is triggered by induction (Horowitz et al., 1961). The presence of yolyphenol oxidasc, specific for polyphenols, in Penicillium was associated with the differentiation of conidiophores and with sporulation. No polyphenol oxidase was present in vegetative mycelium, but synthesis of the enzyme started in the second half of the spore-induction phase a short time before differentiation began. Maximum activity was reached during spore production. The appearance of enzyme activity was preceded by the formation of polyphenols (Jicinska, 1968). Other cases in which phenol oxidases are implicated are the development of phenolic pigments in a number of wood-rotting polypores (quoted in Bu’Lock, 1961); polymeric pigments which invest the cell walls of the conidia and sporophores of Daldinia concentrica (Allport and Bu’Lock, 1960) ; and the strong correlation between melanin synthesis and microsclerotia development in Verticillium (Brandt, 1964). Indeed, Bu’Lock (1961) has shown that a wide variety of secondary metabolites seem to be related to structural elements in cell walls, and that spore formation

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clearly involves drastic changes in cell walls (Salton and Marshall, 1959). The possibility of an association between protease activity and sporulation induction in P. griseofulvum was investigated by Morton et al. (1960)who studied a group of intracellular proteolytic enzymes they termed “proteinase”. Conidiation was induced by exhaustion of the nitrogen supply, and a t the same time there was an eight-fold increase in proteinase activity. Prevention of conidiation by addition of a nitrogen source was accompanied by a sharp drop in proteinase. Aspergillus niger showed a similar increase in proteinase activity on transference t o medium lacking a nitrogen source. However, this relationship is not strictly causal since further experiments revealed conditions which prevented conidiation, but the usual increase in proteinase occurred. The authors suggested that such results are strong evidence that protein turnover is a basic feature of fungal metabolism and that increased proteinase activity is essentially connected with conidiation in some conditions, such as nitrogen-source depletion. No direct correlation between conidiation and peptidase A could be established in P. janthinellum (Thangamani and Hofman, 1966). Peptidase A is an extracellular enzyme which activates trypsinogen, which in turn appears at the time of the onset of sporulation in certain media. However, suppression of the enzyme with acridine orange did not completely prevent conidiation, whereas 6-ethylthiopurine decreased spore formation, but stimulated production of peptidase A. Spore formation was unaffected by transfer of log-phase cultures t o medium lacking a nitrogen source although high levels of peptidase were induced. Other studies of proteases (assayed using casein as substrate) have shown that, in P. cyanofulvum, the p H value of the medium regulated proteuse activity (Hill and Martin, 1966); in A . niger proteinase production occurs biphasically, and the two proteinases have been termed A and B. It is thought that A is functional close t o the hyphal tip and B in older hyphae, and that the sites shift as hyphae elongate (Ohama et al., 1966). The shift of the protease-forming region with hyphal growth has been confirmed by Yanagita and Nomachi (1967). The initial phase of intracellular differentiation during yeast sporulation is marked by protein degradation (Ramirez and Miller, 1964; Croes, 1967a). Chen and Miller (1968) have noted that, when vegetative cells of Sacch. cerevisiae were induced to spore, intact cells showed a great increase in capacity to degrade haemoglobin. The maximum capacity was a t the time when the vacuolar membranes break down as the spores begin to appear. This may release proteinase A into the cell wall, where it can hydrolyse exogenous protein a t the cell surface. Thus, so far it has not been possible

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to observe direct connect ion lwtu w n prote;tsc for’m;Ltion and spolulat ion as found in Ucccillus (Bernlohr, 1964 ; Kernlolir aiid Novelli, 1963).

V. Acknowledgements We are indebted to Drs. J. 31. Ashworth, T. W.Konijn, 117. F. Loomis Jr., H. P. Rusch, M. Sussman and B. E. Wright for making available to us manuscripts in advance of publication, and to Miss Isla Baker, Miss Anne Stuart and Dr. M. F. McCallum for technical assistance in preparing the manuscript. The work from the laboratory of J.E.S. reported here was supported by grants from A.R.C.. S.R.C. and

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High-Energy Electrons in Bacteria ,JOHN R. BENEMANN and RAYMOND C. VALENTINH Department of Biochemistry, (Jniversity of California, Bwkeley, California 94720, 1J.AS.A. I. Introduction . 11. High-Energy Electrons in Metabolizing Bacteria . (in collaboration with P. F. Weaver) . 111. Ferredoxiri : The First High-Energy Electron Carrier IV. Flavodoxin . V. Two New Carriers from Azotobactcr . VI. Electron Chains in Anaerobic Bacteria. . VII. High-Energy Electrons in Photosynthetic 13act,eria (in collaboration with P. F. Weaver) VIII. Electron Flow in Aerobic Nitrogen Fixation by Azotobackr IX. Nitrogenase : A High-Energy Electron Acceptor . X. Regulation and Genetics of Electron Chains . (in Collaboration with C. W. Sheu) XI. Concluding Remarks and Future Developments . XII. Acknowledgements . References .

135 137 140 144 147 150 152

.

154 157 160 165 169 169

I. Introduction All species of bacteria carry out oxidation-reduction reactions and possess a complement of electron carriers to fit their specific needs. These redox reactions play a vital role in many phases of bacterial metabolism, including energy conversion and biosyntheses. During the last ten years two new classes of electron carriers have been discovered in bacteria, the ferredoxins and the flavodoxins. They are high-energy (low redox potential) electron carriers and represent the strongest reducing agents isolated from bacterial cells with redox potentials comparable to that of molecular hydrogen. These electron-transport proteins have now been found in anaerobic and aerobic nitrogen-fixing bacteria, green and red photosynthetic bacteria, rumen bacteria and many others. Until 135 c

136

J O I I K R. HF:?;ER.IAUN 4 U D RAYMOKD C . VALEKTTKE

recently they had b c ~ foiuid i onlj- in anaerohic bacteria, hut with t h . isolation of s r i c s l i carriers froin a strict :icrol)c ( A l z o / o h a c ~ i err i ) / d u ~ / i itlw ), concept of high-energy electron carriers has bcen extended t o the aerobic bactciia. A s the list of ferredoxin- and flavodoxin-containing organisms grows it is bccwrning clear that these carriers rank in importance with cytoc.hrotnw mid flavoproteins iii thc clec+tron chains of tlic bacterial cell. \Yc hope that thc idra of “higli-mcrgy electrons” is not (.oafused with thv well esta1)lished usage of t h e term ‘‘high cnergy” in reference t o the 1)yropliospliate or anhydride bonds of ATI’ and other energy-rich c o n 1)outids. \\‘c refer here t o those electrons with a redox potential more High Volts

Light -activated electrons 7

-0 8

:I: 7.

Y

W

0

Nitrogenase 7

Ferredoxin 2‘ H 2 Flavodoxin Nicotinamide nucleotides F lovoproteins Cytochromes

+O 8

LOW

ncgativc than t h a t of the nicwtitiatnide iiucleotides ( - 0 . 3 2 V ) as liiglienergy electrons and their 1)rotciii c-twrirrs, such as ferredoxin, a s Iiiglicnergy clcctron carriers (sec, Fig. 1). The high-energy electron is indeed rich in cliernic:d cncrg.v, a n d in rnany cases the bacterial cell t a p s this energy su1)pIy directly for cxryiiig out dif’ficult chemical reductive reactions such as nitrogen fixation. In one sense then t h e high-energy electron is a prirnary energy source for the bacterial cell. The generation, transport and utilization of high-energy electrons in the bacterial cell are t h c topics developed below, with examples froin different bacterial systems. A number of r nt reviews are available for the reader who would prefer n morc extensive review of a given area (Buchatian and Arnoti, 1970: Hardy and Knight. 1068; Malkiii arid Kabinowitz, 1967; Hal1 nnd 15viLlis, 1969; \‘iLlentitie, 1 !#W).

I ~ I ~ ~ I I - E ~ EELECTKOKS I~GY I&* BACTERIL4

137

11. High-Energy Electrons in Metabolizing Bacteria (in collaboration with T’. F . CVPavw) Perhaps i i o hiologist I ~ R Sever h ~ i its i deel’ly fimcinated with cellular oxiditt ioii-reduction r(’itctions a s the renowiled Dutch microbiologist, A. ,J. Kluyvcr. His cliissic formulation of the “Unity Principle of Riocliemistry”, a cornerstone of modern biology, w a s in large 1 w - t derived from his work on the trensliytlrogenation reactions in bacteria (Klujwer, I!)%). Kluyver wits il master in the art of discovcriiig unity in the microbial world, and saw in the chains of catalytic oxido-reduction reactions a lic1.y principle of cellular activity. One favet of his work was coiiceriied with the redox potentials established in metabolizing cultures of various micro-organisms. I n Kluyver‘s words “ a closer study of the connection between tlie rcdox potentials established iii microbial cultures and t h e nature of tlie metabolic processcxs . . . w a s a logical consequence of the attempt t o rcduce the totality of biochemical events t o (*hains of cil,titll.ti(s oxido-reduction reactions” (ElcInil et al., 1934). Klnyvcr Iioped t o tag quantitatively it given metabolic process with a redox nurnbcr, whit-11 lie suspected would be diff’ercnt a n d I)erliaps specific for each organism nud process. I n other words, each different biochemical yroc~ssmight need a special reducing agent. Kluyver and his coworkers made a number of attempts t o measure and cvaluate t h e redox values of nietaholizing (.ells but met with only limited success. Kluyver did, however, establish n set of organic redox dyes which he used a s “bioindicators’‘ t o probe the redox reactions of the cell. His “universal indicator mixt,ure” contained a wide variety of dyes covering t h e known spectrum of biological potentials. Using these indicators, for example, i t W;LS found t h a t the potentials of fermenting yeast susp i s i o n s wcrc much lower t h a n previously thought, a n d fell in the range of-0.3 to -0.4 V (Hoogerheide a n d Kluyver, 1936). This valuc is in good ageerneiit with the rcdox I)otclitiill assigned today t o the nicotinamidt iiuc.leotid(,~triosr phosphate deliydrogcnnse cou1)le which is known t o be a key rcdox re:wtion of glucose breitkdown in yeast cells. Similar studies wcrc cnrrird out wit 11 bacterial cultures. These studies iicvcr led Kluyver t o tlie hopcd for qumititative theory of biocutalysis ; however, many later workers were t o come t o al)precitLte arid build on Kluyver’s investigations. This is particwlnrly true of workers wlio specialize in the highenergy (low redox) biologicd chains so important in photosynthesis and nitrogen fixation. It is doubtful whether Kluyver would have been very snrpriscd a t tlir findings t h a t some metabolic reactions in bacteria producc and require rxtrernely strong, biological reducing agents, coilsiderably strongcr thnri thoscl lie measured for fermenting yeast. Iluriiig the design of his 111iiv(>rsiilhio-indicator set of rcdox dyes, Kluyver must, have asked himself iit some point : what is tlie strongest reducing

13s

.JOIlY R

R E X E S I A Y S *4T1) HAYMOXD C . VAILEYTIKF:

sril)st;inw(s)I)rodiiccd by microbes and with what metabolic process is t1ii.c iwlric.iiirz cwmporiiid(s) associated? T o t r j t o ; ~ i i s w this t ~ question, a series of reclox dyes with potentials I ciiiziilip h.lo\+ th(L iiicwtinamidc niicleotitle Icvrl were tcstctl in oiir hh)ixtoi*>. ‘ l ’ h ~ j w(wL di1)yridyI dyes simihr t o methyl- and briizyl viologen : u I i o p d to use tlicsr, clyes as indicators of high-energy ria,. ‘I’hcse dyes were originally developed bccaausc of tlicir hc>rl)icidnlnc*tivity (Homer ef al.. 1960) which depends on the ease with \vhich they : ~ r creduced. Reduction of the dyes produces stable watc.i.-solri\)le fie(. rndicals posscisiing one added electron whicah are coiiwrtccl t o tlic, original ions i n tlic presence of oxygen (Boon, 1961). ‘ I ’ l i ( w dycs mx~retluc.cd b y chloroplasts in the light (Kok ef nl., 1965) exl)laiiiing \vhy tlic4r Iwrbicidnl activity is dependent on sunlight. The rcdricwl tlj-cxs in their frcc-radical state arc strong reductants and could s ( m ;IS~ tioii-~)liysiologic~~l reductants for many cellular rrdox rractions, \vliic*li I\ oiiltl rcsnlt in the “short-circuiting” of the metabolic pathways. ‘l’li(~availal)l(~cvitlenw therefore points t o the free radical (or rediiwd tlycl) t i 4 tlic toxic species t o the plant On(, \vould vxpect, therefort., t h a t growth iiihibitioii of bacterial rultrnw 1,) tlicw dyes would occur by a similar meclianism. It has been knon 11 for 21 long time t h a t methyl viologen a n d henzyl viologen can friiiction a s aitificinl non-sl)ecific electron carriers in low-redox reactions. It is ;I 1)owil)ilitjrt h a t such lack of redox specificity could have stronglj inliihitorj. c.ffvcts for the c ~ l l This . “short-c>ircuiting” of electron chains is mc,ntioiicd a s a possil)le mode of action of these drugs. However, it should bc 1)oitited out t h a t the precise mode of action is not definitely c~st;il)lish(d In one instance in n bacterial system, the dye methyl vio1ogc.n \\ a< foruid to shuttle electrons non-specifically between hydro~ ? c i iant1 SXI)I’ or ?;All. n-licrcas the natural system was specific for i\’AI )I’ (\‘alciit in(.. I 964). ‘l’hv .lon,tli-inliil)itory propertics of the dipyridyls has been studied ;inti thv cx\rl)(,i*iiiients reported in this section were done by P. M’eaver mid (’ I\’. Slicii of this 1)epartmciit. In one of t h e experiments with t l i c v c l j ~ ~ it s . was found t h a t a nitrogen-fixing species of Klehsiella p,icnnioriino ( D K - 4 0 ) (Pengrn and \\‘ilson, 1938) grew aerobically on l)lntc,sc.ontniiiiiig I)cnzyl viologen and reduced t h e dye witliiri the colony [Fig ? ( a )I. ‘I’his is surprising sirire dippridyliunl dyes are strongly aiitoxit1iz~~l)lr. KOother organism since tested h w exhibited the maintcxiianrc. of dycl rcduc~tionin a n aerobic cultnrc. Mutnnt strains were later isolated I\ I i i c B h failed t o rcdncc the d j ~ e[Fig. .‘(a)]. These m u t m t s also la(~lwdthe ability t o fix atmosplicric nitrogen. Preliminary experiments n 3 h c~cll-fiwc.xti-acts of one of these mutants indicates :m altc~rcd form iLt (> rnct abolism . (1

15'3

I I I G H - E N ER(;Y ELECTRONS I N BAC'TERIA

In many vases, however, the dipyridyls were found t o be extremely toxic for harteria. For example, as little as 1-2 pg. of methyl viologen per millilitre strongly inhibited the growth of K . pneumoniae. Methyl viologen was also a potent inhibitor of a number of other bacterial strains. The dyes served as "bio-indicators" for the intracellular redox potential, since the redox potential of the dyes could be correlated with their

Volts Redox dye -~

Organism

(-0 7 4 ) 1.1'-Trimethylene4,4'-dimethyl-

It

Rhodospfnllum

Azorobocter nnelondrf Klebsrello

2,2'- dipyridyl (-0 6 4 ) 1,l'-Trlmethylene2.2'- dipyridyl

(-0 56) 1,l'-Trimethylene2,2'- di pyridyl

DK-40

L (-0315) Benzyl viologen

(4

(b)

FIG.2. ( a ) Photograph of a plittc citltrire showing reduction of beiizyl viologcti by Klabsicllii pnrumonine (DK-40). This nitrogen-fixing species of Klebsiellu was naturally wsist.ant to benzyl viologen and readily reduced t h e dye at the colony level (dark colotiicxs).A rnitt,atit,form (colo~crless)was isolated which tiid tiot reducc: the tlyr ; t>hisstrait1 was unitblc t,o fix Iiitjrogeri,indicating a lesion in the riitrogc?tiasc: chitin. ( b ) Rctfox scale for several bacteria as measured by t,oxicity pattcriis tJowards the dipyridyl dyes. See text for details. The bacterial strains are listed alongside the name of the dipyridyl dye of lowest potential which showed growth inhibitmionof t h c bacterium. In most, instances, inhibition was observed also with all dyes more electropositive on the scale. The dipyridyl dyes were kindly supplied by Dr.C. A.Calderbank. Jealotts Hill Research Station, Bracknell, Berks., England.

toxicity. An example might help clarify this point. Let us assume t h a t all of the dyes down t o a redox potential of -0.64 V were toxic for a red photosynthetic bacterium. We would conclude t h a t a reductant powerful enough t o reduce these dyes t o the toxic-free radical was generated b y the red cell, thereby leading t o growth inhibition. Information about the redox potential of cellular reductants was thus obtained. The dyes with

I40

.JOllh I<

l%lC\l~Chl4VYA \ L ) K A Y A l O N I )

(’

VALEhTI5E

c~xtrc.ni(~ iiogativc~potentid w u l d not be rctiuc.cld. and werv tlicw,fore not toxic. 1)yc.s in thc i d o x range of‘ -0.:3 t o -0.8 V were tested on several I)nc$c+id sl)wirs. I n I)ril(’ticcthis “voltage” measurement consisted of following growth iiiliibition by the dyes and d rmining tlic lowest redox potential whirh ~ ) r o d i i c :Ld dctectable inhibition of the cultnrr. The ha(.terial (wltiiw wiis assigned this voltage as shown in Fig. Z(b).T h e greatest suscq)tihilit>.t o the lower potential dyes occurred with a red photorium cultured on a glutamate-containing medium. arc knou ii t o induce the nitrogenase pathway and plnotolij t l r o ~ i wevolution in this organism (Kamen a n d Gest, 1949). In a iiumbw of c.xI)erimcnts with the red bacteria, it was observed t h a t d j e it111il)itionwas strongly d(.piideiit on the composition of t h e growth mrdiuun For c,xaml)le, i n cwtaiii complex media 110 strong photorcduc.tant ;~1)1)ears t o he 1)roduced as measured b y dye inhibition. The tivxt oi~gmiisrnion tht. 1)iological redox scale W ~ L Ssomething of n surprise A . v i t / d u t / d i i .i ~ nci-ol)ic n nitrogen fixer which has a highly oxidative mttal)olisni ( I n d i u m of‘N~t\iton et nl., 1953). Other nitrogen fixers rated high : it should be mcntioncd t h a t i t was difficult t o obtain consistent wsults 11 itlr c*lostridial caltiires. perhaps because of hydrogeiiase which maj- have. detoxified the dj-cs. and for this reason this important group is not listctl The caontrol organism for tlic work was m i Eschwichin coli K 12 strain grown on iiutricnt broth. Kli~yvcrhiis discussed the many technicd problems associated with su(xli ti?(’iLsii1.(~nicints s u ( b h a s dye permeability. possible breakdown, a n d oxygc’n (4Twts ( Hoogerhcidc and Kluyver, 1 W i ) . The results arc, a t best, only quditative. Still, the findings with the bio-indicators fit in well with t h c knowti biochemical rtnctioiis of the various organisms. As expected, the. tiitt,og(,tr-fixitng spwivs \+ere sensitive t o the high-energy dyes, indic.ating tliat thcsc organisms 1)rodixc.rverjr strong reducing agents. The norin;il (~(~lluliir function of these strong tduc-iiig agents was presumcd to I)(, iiivolvctl i n reductioii of molecular nitrogen. This introductory swtion was intcnded t o give, some historical I)erspective t o this area as cvcll i i s t o foc.iis attcntion on the nature of the high-energy clcc*troiicaliaiiis iis they ol)(’ixtei n living k)wteria. The remainder of this review mill deal with JZ ork done at thr 1)iochemical level. ~

111. Ferredoxin: The First High-Energy Electron Carrier ‘I’h(. ( ~ ) m n i o no c c i i r i ~ ~ n of w molecular hydrogen as a fermentation prodiirt hiis long been a c*luet o t h e operation of low-redox electrontixns1)ort c*liiiinsi n 1)actc~ria(Graj and s t . I O(i.5) It is then not sitq)ri\iiig t l i t i t I)ac+cw;bl fcvrdoxiii ( P d ) M discovered diiritig studies 0 1 1 t Iiv riiwhiinistn of‘ formiLtion of molccii1:Lr hydrogen by extracts of

HIGH-ESERGY ETJECTRON? TU BACTERTA

141

Clostr’irliw~n p a s t e u k n u r n , an nnncroI)ic*, nitrogen-fixiiig l)nc*tc.i*ium (Mortenson et al., 1962). It was already recognized by early workers in the field that synthesis of molecular hydrogen from pyruvs
site; perhaps the electron migrates from F e t o Fe within the matrix of the prosthetic group, like a valency electron in a n Fe-S “basket”. The chemical rcactivity of Fe in the ferredoxins is clearly of great interest, but the cahcmistr\- of the electron-cmryiiig “basket” has thus far proved difficult t o unravel. Ya11g ( 1 969) has recently prepared a model ironsulphur cmtii1)lex hy mixing ferric or ferrous iron with sodium sulphidc aiid mt~rc.aptoethanol.The absorption spectrum of the complex w a s chartwterized hy maxima a t 318 and 412 nm., a spectrum similar t o ferredoxiii mid other Fe-S proteins. This complex may serve as n model coni])ound for the active group of ferredoxin. Several models for Fe-S linkage bonding in Fd have been I)rol)ose“d. In the model proposed by Phillips P t nl. (1965) and Tanalra ef al. ( 1966) the iron atoms are arranged in a linear manner and are each bound t o two cysteine molec.ules by sulphur bridges and t o two inorganic sulphidc atoms. excel’t for the two end iron atoms [Fig. 3(11)1.As described helow, tliv timillo w i d sc’cliiviiws of‘ t\ro c.lostridial fwrcdoxiiis Iiilve cliscaloscd D regular arrangemcnt of the c*ystciiieresidues in t h c peptide chain and

144

.1Oll\

I< l % K N K M A 3 1 AN11 RAYXlOhII)

(I.

VALEhTIhE

Ii;ivc. lctl to tlic. sl)cculation thnt the polypeptide chain m a y fold like a hnir1)in t o ~\c~c,omiiiodiite its Fe-8 core. One of the most interesting recent findings of fcrredoxiii chemistry n i ~ sthe complete rec~onstitutionof fiwwloxin by acidition of S“ and F e salts t o the apoprotein (Jlalkin
IV. Flavodoxin St~v(~i-;il j C;II’S after thc discovery of fcrredoxin, another high-energy carrier a s isolntcd from C. pastpuritcnum a t the Dul’ont Laboratories. rI’Iiis ( w ~ i c i .whicli . c~)titaiiiedno mrtal or sulphide, was called flavodoxin 1966; h c n u s c ~ of its fiavin (I’hlN) prosthetic group (Knight et d., Kiiiglit ~ ~ Hardy, n d 1966). A similar compound has recently been isolated from PPpto.\trPptoroc.c.11.s plsdpnii (Fig. 3 ) . Flavodoxin replaced bacterial f i ~ ~ * ~ d oi t ixsc~ei.al in rcac.tions including evolution of n i o l e d n r hydrogen, 1)yruv:itc. oxidation, nitrog:cn fixution. and reduction of NAIIP in rxtriL(*tso f (‘. pnstpurinnum. Flavodoxin u a s obtained from cells of C. pustPurinnzcrn qrourn in an iron-defivient medium atid appears t o be indiic*cdundcr these conditions. Since the iron-deficient cell contains very little fiwetloxitr, it sCerns likely t h a t flnvodoxin fuiic.tionnlly rel)lac*es fi~ri~cdoxin in c ~ l l i i l a imctal)olism ~ in the iron-deficient cell. \l’hen cornl);ir(yi 0 1 1 a n tqiiirnoli~i~ basis t o ferredoxin, its cffec.tiveness ranged from :joO,, i n tiiti.og:cii fixation to 7 0 in hydrogen evolution (Knight a n d H;irtlj . I W(i) This tliffi.rc~nc.ccoulcl he due t o a difTerencae iti the specificit01’i t 1 tlrv iwIo\ I)otc.titi;il ot’t Iic. t’riwtloxiti atid fiiivotloxiti Plavotloxin (witailis one nioleculc of PMN us its (presumed) uctivc

RTOH-ENERGY ELECTRONS TN RACTERTA

145

site. It is clear that PMN in this instance behaves as a high-energy electron carrier. Flavins have long been known t o be extremely versatile carriers, but this is a new role. The nature of FMN binding to the apoprotein is not known, but the displacement of the FMN by sodium mersalyl suggested that the flavin

FIG.4. Crystals of flavodoxin produced by the rumen microbe Peptostreptococc~~ elsdenii. The micrograph was kindly supplied by S. G. Mayhew and V. Massey.

146

JOTTN R . BBNEMANN AND RAYMOND

('. VAT,ENTTNR

was bound, a t least in part, to the protein through the cysteine sulphhydryl group (Hardy and Knight, 1967). This interpretation was complicated since the FMN was released only slowly on treatment with sodium mersalyl and only when high ratios of mersalyl to flavodoxin were used. The increase in free sulphhydryl on removal of the FMN from flavodoxin also implicated the one cysteine residue of flavodoxin in the binding of the flavin. It has not been possible to reconstitute flavodoxin from FMN and apoflavodoxin. Flavodoxin apoprotein of C. pasteurianum contained 149 amino acid residues with a preponderance of acidic amino acids (Knight and Hardy, 1966). The dissimilarities between ferredoxin and flavodoxin were striking. The two most prominent differences were in the cysteine and basic amino acid content. Ferredoxin contained eight cysteine residues per 6000 molecular weight, whereas flavodoxin contained only one cysteine residue per 14,600 molecular weight. Flavodoxin contained 10 lysine and two arginine residues, whereas ,ferredoxin contained only one lysine residue. Flavodoxin from P. elsdenii had no histidine (Mayhew and Massey, 1969).The results of Mayhew et al. (1969)showed that flavodoxin from P. elsdenii accepted two electrons, a t different oxidation-reduction potentials. Addition of one reducing equivalent of sodium dithionite (or NADPH, in the presence of NADP-ferredoxin reductase) generates a new species of flavoprotein, the flavin semiquinone. This reduction is almost instantaneous and can be easily observed since the colour of the flavodoxin changes from yellow to blue. This step in the reduction of flavodoxin has a potential of -0.115 V a t p H 7. Further reduction with two reducing equivalents of dithionite gave the colourless fully reduced semiquinone. This second step in the reduction of flavodoxin is probably the more important in reactions in which this protein substitutes for bacterial ferredoxin because the oxidation-reduction potential for this second step was -0.373 V a t p H 7, somewhat above that of ferredoxin (-0.42 V) but below that of the NADP/NADPH, couple (-0.32 V). This could account for the lower reactivity of flavodoxin when substituted for ferredoxin as electron carrier. But differences in specificity can of course not be excluded. AS expected, from the redox potentials, flavodoxin could not be completely reduced by NADPH, and ferredoxinNADP reductase, even in the presence of a large excess of NADPH,. The importance of the apoprotein in influencing the redox and catalytic properties of the flavin prosthetic group can not be overstated : the oxidation-reduction potential of free FMN is -0-2. A number of studies with flavodoxin and other flavoproteins show that the redox properties of the flavin group are highly dependent upon the structural integrity of the protein. I n other words, the protein groups surrounding the PMN moiety strongly influence its redox potential.

HTGH-ENERGY RTAECTRONSTN BACTERTA

147

V. Two New Carriers from Azotobacter The high-energy electron carriers discussed in the last two sections were all from anaerobic bacteria. Two electron carriers coupling to nitrogenase in the strict aerobe A . vinelandii have now been isolated: azotoflavin and azotobacter ferredoxin (Benemann et al., 1969; Yoch et al., 1970). They are the first known examples of high-energy electron carriers in aerobic bacteria. The carriers were discovered due to their activity in the coupled chloroplast-nitrogenase assay developed by Yoch and Arnon (1969) [Fig. 5(a)].I n this assay illuminated chloroplasts serve as an artificial reductant to nitrogenase. A high-energy electron carrier such as ferredoxin is required to transport the high-energy electrons produced by the illuminated chloroplasts to the nitrogenase. Using this assay the Azobacter carriers were discovered in the clear supernatant, of an A . vinelandii cell extract which had been centrifuged at high speed to sediment the nitrogenase [Fig. 5(b)].There were two different electron carriers present which were later purified from a butanol extract of Azotobacter cells by chromatography on DEAE-cellulose. The electroncarrier activity was separated into two components [Fig. 5(c)], one of which was a yellow flavoprotein called azotoflavin, the other a non-haem iron sulphur protein named Azotobacter ferredoxin. Azotoflavin appeared to be identical t o the flavoprotein from A . vinelandii isolated by Shethna et al. (1966) and Hinckson and Bulen (1967).Azotoflavin was only very slightly active in the phosphoroclastic reaction of clostridia or in the chloroplast photo-NADP reaction. It was thus a highly specific reductant for Azotobacter nitrogenase. The most unusual property of azotoflavin was its oxidation-reduction behaviour. It was reduced only slowly by excess dithionite to give a blue coloured protein, the semiquinone flavoprotein. A very large excess (100-fold) of dithionite for several hours was required for reduction to the colourless fully reduced form. Re-oxidation in air was also very slow; the semiquinone form was extremely resistant taking over 12 hr. for complete oxidation. These properties are distinctly different from the behaviour of the flavodoxins, which are easily reduced and oxidized. The flavin prosthetic group appears to be complexed in this protein in such a way that the reduced species are not accessible to oxygen. The resistance of reduced azotoflavin to oxidation by air may be a distinctive adaptive advantage for transporting high-energy electrons to nitrogenase in the highly aerobic cellular environment of Azotobacter. Illuminated chloroplasts reduce azotoflavin to the semiquinone form. It remains to be definitely established if this species is the actual electron donor t o nitrogenase. Unlike flavodoxin, azotoflavin is not a product of growth on low concentrations of iron but was found under all growth conditions

14s

.lOll\

It

I%ICr\ IC.11.2VY AUI) I
(’

VALICV‘l’TVTi:

studicd iiic.Iuditig gi.owtli 011 1)reformed amtnonia Azotoflavin, nlthough ( [ t i it(. di.;titic*tft*otnflnvodoxiii iii hot 11 clieniicd and biological prol)wtics, ( x i i hc, gt~)u1)ecl with t 111. flnvodoxins ; ~ n dI)hytoflavin, a fliavodoxiii-lik(~ carrier froin i~1g:i~c (Sttrillic, I !)(i3. 1965), i n tlic ~ 1 of flavoproteiii ~ s higlic’ticrgy clertron carricm. Azotobac$cr f i w d o x i i i , t h e second 1iigh-r.iic.rgg carrier of Azotobacter, w i ~n s brown non-linetn iron protein caoiitaiiiing approximately six atoms of iron atid six atoms of sulphide per riiolccsulnr weight of 20,000 ( c d cwlated from t h e amino twid comlmsition). Unlike azotoflavin, Azoto1)ac.tr.r ferredoxiii is t ~ b l et o mediate tlicl I)liotoreduction of NADP by s1)iiiacli c~hloroplasts.‘l’liis nhility t o trai1sf:c.r electrons t o fcrrcdoxinSAT) P rcduct;wc cliniw4erized all ferredoxins ; ~ t i dindicates a low-rcdox 1)otctitial siniiltw to tliiLt of other ferredoxiiis. In the clostridial ~ h o s I)lioroc.lnstic.rrtwtion it cxliibits low activity, about the simie as t h a t

Chloroplast

r--- - - - - - I I I

Carrier

N=N

I

hv

4

I I I I

I I

L --------- J

Itl(:~I-E\ ER(:Y

ELE('1'KOSS I N SAC

I49

of s1)itiacIi ferrdoxin. Azotobacter ferredoxiii undergoes reversible reduc,tion~oxid~Ltioii. I t s spectrum is similar t o t h a t of other ferredoxin except tliiit it has only broad shoulders instead of defined peaks. Iteductioti w a s ~tc.c.ortiI)lislirdanaerobically with a small excess of sodium tlrtliioiiitv aiid \ \ i t s coniplete a t five minutes. The decrease in the nbsorptioti in thc visiblc rcgion n-as considerably less tliaii t h a t observcd with ot1ic.r fmwdoxiiis. Re-oxidation of the reduced Azotobacter ferredoxin in air swincd t o owur in two steps. About 309, re-oxidation oc*curred in lcss thaii it riiiiiutc, while further re-oxidation proceedcd only slowly nnd took about :in Iio~ir.I n its sluggish re-oxidation by oxygen, Azotobactcr fwrcdoxin resembled azotoflavin. which n-as even more resistant t o I-c-oxidation by oxygen. The resistaiice of these electron carriers from A . viridnndii t o re-oxidation b y oxygeii inay reflect an adal)tatioii t o (*onscrve,iii ail aerobic environment, the strong reducing power needed for iiitrogw fixation. From the above lwoperties it is reasonable t o call this c*nrritlra ferredoxin. But its I'roperties are neither t h a t of c-lostridinl

2 .o

1.5

~

\

F

\hF

L

1.0

Q

PIG.5. The high-energy electron carriers of Azotob(rcter ,&wltsndii. (a) Diagram showing the basis of the coupled chloroplast-riitrogeiiaseassay for the low-redox carriers of A . vinelnndii. (b) Separat,ion of thr nitrogenase enzymes into a soluble titid pellet fraction by centrifiigattion a,t 145.000 I/ for 6 hr. (c) Srparation of two high-energy carriers by DEAE-chromatography.

I .yo

.ioit\

12

R m m r A N x AND RAYMOXD

c-. VALENTTUE

nor of‘phiit ty1w ferredoxin ; therefore it is best considered a new type of‘ fwrcdoxiii. It sliould be recalled t h a t 110 ferredoxin had been found l,re\,ionsl\ in a i i ! strict11 aerobic bacterium, including Azotobacter. ‘I’Iiiy ty1w of f‘eiwcloxin may then be rcpresentative of a new class of ‘‘a(qy)I)i fiwcdoxins. (8’’

VI. Electron Chains in Anaerobic Bacteria A, f’t.15 c x n i n ~ ) l e sof high-energy (low redox) electron-transport chains of ;n\wrohi(~bacteria mill be discussed in this section, stressing the iiiil)oi*taiiwof tlic orgaiiization and close coupling between individual

units of the ch:iin. Elcctron-traiisl)ort chains in this respect probably ar(\ similar t o other biochemical l ) a t h w a ~ ~ either s, biosynthetic or ckgrndat ive, in kvhich n number of different enzymes function as a n

otyinizc4 unit, Ivith each enzyme member of the pathway tightly rcgiulnted i n its activity and/or strategically located in a large enzyme c.oml)lex. H igli-eiicrgy electrons are, of course, never free in t h e cyto1)lastii: they exist a s valency electrons of their high-energy carriers. I n tliis r(qard%all 1)roteiiis either generatirig, transporting or accepting tltcw vlc.ctroiis i ~ r chigh-energy clectron carriers. Since these carriers are I)rotciiis, tliis t i i c n i i s tlint electron transport can orcur only if the carriers interact with c ~ ~ cotlirlr li a t least t o the extent t h a t their electron-carrying g i ~ ) ~ i lsii(2Ii ) s :IS tnrt:&i and flavins are close enough together t o permit tliv c~lwti~oii t o rnigmtc from one carrier t o the next. In other words, tlic (yii.ri(w may need t o touch for electron exchanges t o take place. This Icads us aqain t o the concept of specificity. These interactions must be very sl)wific t o insure that these very reducing electrons only migrate in tlirl tlesird 1)atIiw , otherwise we would have the same problem tliat ilic wII en(miiiters when reducing the dyes, namely a general short cin*iuitiiigof tlicl ccllular rcdox processes. It is not surprising then t h a t tlic, c ~ l l sbuild up their electron cliaiiis into Iiiglily organized and structurnll? intc,ractiiig systems. In these complexes the carriers may be I)Iiysic*allyliiihed by 1)acking onc protein carrier adjacent t o another in tlic. c.oriil)lcx. The prosthetic groups of the carriers may be even more strategicdly located t o allow smooth transfer of electrons t o take place. ‘I’li(~ rc;rc.tion between soluble electron-transport carriers and the other I)i’otc.iiii i n thr. c*liain must occur in tlic formation of a complex. For ( ~ ~ a i t i l ) lin ( ~ tlie . sl)inac*hcahloroplast NADP-reductase system the NAUPfiywclosin rednc*tasc.,a flavoprotein looscly associated with the rhloro1)l;ists. fotwi.; stak)le protein complexes with spinach ferredoxin iii an c~strc~tiicl> fitst i.cwc.tion ( IToust rt al., I 969). T h c ~el~c>troii-carryiiig ( Y ) I I I ~ ) I C S ( ~ S I)c+\vcwi two soluble carriers \vcre coniplctely k)roken down

I51

lIT(~FI-F,?;RRGY ELECTRONS I N RAC’TElZlA

at, high ionic strength suggesting protein-protein bonding wits primarily electrostatic in this case. Salts a t concentrations which broke up t h e complcx also inhibited photoreduction of NADP, indicating t h a t these coni1)lcxes are irn1)ortmt in catalysis. Although such complexes have not \-ct bccn studied i n bacteria, similar reactions must occur between t h e clc>mcntsof bactcrial electron carrier chains. Fei.rdoxin is not t h e only protein t h a t possesses a iioii-Ii;wm ironsulphur prosthetic. group. There are several bacterial enzymes containing iron n t i t l sulpliitle. such a s nitrogenase mid urate reductase. The irortsull)liiclc.groul)is tightly k)ouiidand intcgrated t o Inrge protein complexes i n thew w s r s It is not 1)ossible t o rt’move it without destroying thr enzymic activity. This brings us t o t h e idea t h a t high-energy chains might I)(, hnilt up of scvernl repeating ferredoxin-like iron-sulphide prostlictic groul’s t o form a kind of biological “metal wire”. An interesting clecti.on (,hain in the urate-fermenting bacterium, C. a c i d i - u ~ i c imay , be mndc uI) just this way with several Fe-S groups found in different proteins of the (*hain,as illustrated in Fig. 6. In this chain, electrons frorn pyruvate, I)rodwed during t h e metabolism of t h e uric acid, are ed t o ferredoxin and filially t o urate in a n important reductive reaction for t h e (TII (Valentine, 1964). Pyruvate is it strong reducing agent ; ~ t i dis oftcn uscd in t h e cell t o donate electrons t o ferredoxin. Active aldehyde (electron-donor) Urate (electron acceptor)

I

TPP - C - OH

dehydrogenase Acetyl- PJ

+ co,

\

.

Fd(red.)

reductase

1

Xanthine

FIG.6. A model of’ the pyruvate-urate clectroii-trailsport chain of Clostridiutn midi-urici, illustrating the “iron-wire’’ model of electron transport. See text for details.

Actually, a pyruvate derivative, active aldehyde, or Iiydroxyethylthiamine pyrophosphate, formed during the pyruvate dehydrogeriase

I -52

JOli\

I<. H I C ? E h l A N L ‘ A N D ttAYMO>D

(*.

VA1,I‘:NTINIE

rtlaction, is thought t o he the more dirrct electron donor t o ferredoxin in these reactions. As shown in the scheme, active aldehyde may first pass its electrons t o a n electron-carrying group bound tightly t o t h e tleliydrogcliilse eti zyin e cwmplex.This group presuni ably in close proximity t o the aldehydtl moietj niay tlicn i n t u r n redu oluble ferredoxin. In hiil)l)ort of this scheme, Kaeburn and Rabinowitz ( 1965) have found t h a t 1)yruwrte dcliydrogenase of C. cxcidi-urici contained the Fe-S group typical of‘ noii-harm iron electron carriers. ‘l’hc last reaction in the series involved t lit, transfer of electrons from reduced ferredoxin t o urate via urate rcdirctasc This is a key reaction for this unusua1 organism which must reduct urnte t o xnntliine in the first reaction of its major fermentative pathway (Barker, 1956). Studies with bacterial iirate rednctase s1iowt-c.dthat this enzyme was an oxidation-reduction catalyst of the 11oii-liaetii iroti tylw (Bradslinw a n d Barker, 1960). I t also contained flxvin. Oiic of its unusual features was its molybdenum content. The I)oint t o Iw strcwied ahout this particular sequence was the tandem use by the w I I of a scrirs of noii-hacm groups. In this it is thought t h a t as many a s 2 0 or so l+ atoms may take part in this relatively short transport chain from pyruvate t o urate. These electrowtransfer proteins arc indeed rich in iron.

VII. High-Energy Electrons in Photosynthetic Bacteria (it! collaboration with f’. F . Il‘ecxver) A f v of ~ tliv r e w i t developments in bacterial photos.vnthesis will be revicwed h i e f l y in this section wit11 the major emphasis being on the low-rcdox cliains of these organisms. It is clear t h a t both red a n d green bacteria possess low-redox cliains a s indicated by the presence of pathways for 1)Iiotoliydrogen cvolution a n d photonitrogen fixation b y many species. Fcrredoxins have also been isolated from many strains. For example. thr ~)liotos\~iitIictic~ bacterium Rhocloqpirilluin rubruwL produces molecwlnr 11) dropen diiring anacrobic photosynthetic growth on organic cornpoutids siich as tnalatc, when the combined nitrogen source is growth limiting. Under these conditions. hydrogen is continuously a n d rapidly evolved xvhilc the cells are illuminated (Gest and Karnen, 1949). Hydrogen cvolution a 1 q ) t ” ~ t o b r a manifestation of the iiitrogcnase system in these (.ells u h i c l i in the presence of ATP and reducing energj-, but in the ; ~ l ) s e nof c ~tiitrogw, will evolve hydrogen (see Section IX. p. 159). Thc nitrogeii;rw path\vay niay servc as a convcnient sink for t h c organism t o get rid of r x i w s rediictaiit and XTP produced b y photochemical or nictabolic. pathn a . This can be easily observed with resting, nongt’i)\ziiig c ~ l lof s li’ i r r b i U H I which photometaholize ncetatc and dicarboxyIic ;ic.ids fotmiing 11) tlrog!eii aiicl carl)oti dioxide in qriantities closely

(TitOlS I\

n \(’TlSlITA

1.73

a ~ ~ ~ ~ r o x i r n attIiitoi g s~ c’xpected from coni1)lrtc decomposition of substrate ( ( k s t P / (11.. I!)(i2). ‘ i ’ h c w remarkable mctabolic umversiotis ikl)fle>irt o occur by tlie c,xtt,iisivc.olwr:Ltion of an anacrobic citric acid chycble cvupled I\ itli ~ ~ l i o t o ~ ~ l ~ o s ~ ~ l ~which o t ~ y l brings ~ i t i o nabout the re-oxidation of I cduced nicotii~atiiidt~ nucleotides (produrcd by the oxidative steps of the citric. acid c j c . l r x ) through liberation o f hydrogen. This is. of course, a tentative s ( h t n r , in \vhich the role of light is limited t o photophos-

I)liorylatioii. ‘ 1 ’ 1 ~ Iwssihlc involvemcnt o f fc~twdoxitrhas not I r ) c ~ ~ t(i. s t a b lislicd 01’ruled orit. Evans (Jt (11. ( I !)ti(i) clwcribed n tie\\ ft.rrctloxiti-litiked 1)i~occssfor the iniilation o f carbon dioxide by the green bwterium, (’hZo~obiurtithiosu~;ztopiLiZurtr.One complcte t u r n of the cycle, wliicli they d l c d the reductivc c a r h x y l i c acid cyc~lc,inc*orporated four niolecules of carbon dioxide itlid resulted in t h e net synthesis of oxaloacetate. r l I Iius, beginning with onc molecule of oxaloacetate, one complete turn of t h e reductive. carbox) lic acid cycle rcgenerated the starting molecule i ~ n dyicllded, i n iddition, a second molecule of oxaloacetate forrned by the reductive fixation of four molecules of cwbon dioxide ‘I’lie four (‘ilrboxylibtiotlsteps in the complete reductive carboxylic acid cycle inclndtd t w o 11ca rcwkioiis for carbon dioxide fixation t h a t a e r e dcpcndent on ~ d u c w ftwedoxin l : the I’yruvate s p t h a s c (Biic~h~~nan Pt az., I $ W ) ( I ) and the. cc-ke~to#lutRratesynt1l;Lse (Rnc+h;Ln>Lnand I~:v2LIIs, I !Hi$)) ( 2 ) .

The reductive wlboxylic acid cycle appears t o functioti a s ii biosynthetic I)tLtIrway that is particularly suited to provide thc carbon skeletons for tlic tniLit1 Iwoducts of bacterial pliotosyiithesis, which are ~)rcdoniiiiantlyiunino acids. Thus, the rclactions of the cycle supply a-ketoglutarnte for the. synthesis of glutarnute, oxaloacetnte for iLspartat(., m i d pyruvatc, for nlanine. Other ferredoxiii-linked reatctions have I ) c ~ wrelwrtctl Iricli m a y bc iinlmrtant t o the ~ ~ h o t o s j ~ n t h eI)avtcria tic LI c~nvironrriental conditions For c~xt~niple, i t H ,-b’d-XAL> rc.nction was rr1)ortcd in the red and grren bacteria which might alloathese bacteria t o us(’ tnolecular hydrogen RS a source of high-energy clcctrons (Wcavcr rt ul., 1966; Buchanan mid Evans, 1 96!)). The E’d-

I .i4

.JOITV I<. i i E K m ) t n - A U r) I ~ A Y V O W L (1)

VAI.IC;LTIVR

NAI) iwwtiou may also play an important role in t h e ~)Iiotorediiction of‘ 1)yridiiio iiric.lcotides. I h c ~ cc a i i be no argunient t h a t the energy for driving these low-redox 1)roccsst’s, suc*h i ~ scarbon dioxide fixation, photolijdrogei~production ~ 1 ~)hotonitrogen d fixation, comes from light itself‘. The present controin this area is coiiceriied with t h e photochernical riiecliariism of synthesis of tlic high-energy electrons i i i these specks. One group of workcw ftLvoiirs what might be called the “chloroplast ”-type mecliaiiism which features ferredoxiri as electron a c c q t o r for electrons activated during thr I)hotoclieniical act (Kozaki P t al., 1 U(i5 : Buchnnan and Evans, l!N!), sce Fig. 7). A second group (Gest, 1966; Keister aiid J7ike, 1 9 6 7 ; J o t i c ~a i i d \7ci*tion,1969) has suggcstcd t h a t thc higli-c,nergj electrons may sytithcsized in these bacteria by an ATI’-driven rcvcrsal of c.lc~troii fiou t o the iiicotiiiamide iiucleotide lcvcl (Fig. 7 ) . ‘I’lie reader , I

/ / Electron

I<’I(.. 7. A l t c ~ r t I ~ l t~ol l o d ~for l s rlrctrolr f l o ~111 p h o t o ~ y ~ ~btahc ~ t t t~~, ~~ .

is rt~ftrredt o review articles b y Gest ( 1 966) a n d Vernon ( L 968) for a (.omplctc (lis(vssion of this argument. We have 110 iiew information t o add but \vonld suggest t h a t in the final analysis both groups may be correct with diff(wnt species of photosynthetic bacteria using different pathways dr.l)(~tidingoii the energy of light availablc mid otlier eiiviroiimental cwiiditions. I t is true t h a t until this argument is closed it will be difficult to work out many of the interesting low-redox renctions of these bacteria Still, some progress has been made and regardless of the outcome of’ the controversy i t is now clear t h a t ferredoxin plays a kejr role in I)liotosyntlietic bacteria

VIII. Electron Flow in Aerobic Nitrogen Fixation by Azotobacter Ihc liigli-eiiergy electroti-transl,ort chains supplying reducing power

r ,

for iiitiwgcn fixation are among the most important examples of higlienergy chains in bacteria. These ehair~sC O L I ~ ~ the C reducing power

TTT(ITT-RNERC,Y ET,ECTRONS TU BAC‘TERTA

1 5.;

geiieratcd in the metabolizing cell t o nitrogenase (nitrogen reductase). Indccct, cvcti iiitrogenase itself can bc reg:arded as link in this chain, receiving higli-energy electrons and passing them on t o molecular iiitrogcn. This rduc*tioiiof the nitrogen molecule t o ammonia represents :I difficwlt uiidc.rt&ing because of the high activation energy of thc inert nitrogen inolccde ; therefore strong reductants are required. The electroil cliain t h a t sul)l)licsthis strong reductnnt is c*lenrlya vital part of the tiitrogcii-fixtLtioii 1)athway. hi tlic C I L S ~of the anaerobic nitrogen fixer, Clostridiurr~paskurianurn, reduced fiwcdoxin (or flavodoxin), generated b y the pho q h r o c l a s t i c reaction, 1)rovidcd the high-energy electrons needed for nitrogen fixatioii (I)’Ihstnc*hioiuid Hardy, 196-1; Mortensoii, I9M; Knight and H:~rdy, 1 $Mi).It is suspected t h a t a similar pathway may fuiic$ion in other bacterial systclins where 1)j.ruvate snl)ports nitrogen fixation (Grau and l\’ilsoii, l!W: Hamilton et al., 1904). Tn t h e photosynthetic bacteria nitrogen fixkbtion (and hydrogen evolution) is a light-dependent process. But, as discussed in tlie last section, thc actual electron chain is not yet clenrly rl;tnblished for these bacteria. Some strictly aerobic bacteria such as A . / i t i e l a d i i are also able t o fix nitrogen. ‘I’licy must therefore have high-energy electron-gerieratiiig enzymes and carriers. Since Azotobacter has no clastic enzyme system, this would suggest t h a t another pathway must be operative. With the isolation of two clec%ron carriers from Xzotobacter involved in nitrogen tixation (see Section V, p. 1-17), the way opened for the education of the electron-transport pathway in this organism (TI. C. Yoch and J. R . Benernanii, unpublished observation). X tentative scheme based on this worli for thc nitrogciiase electron-transport chain of Xzotobar.ter is + H20

Glucose

ATP

I

NADH2

Oxidative phosphorylation

f

Pyruvate

T

\

Transhydrogenare

&N=N Azotoflavin

dehydrogenase

Ferredoxin reductase

Nitrogenase

Biosynthetic reactions l thc c l c c t ~ o r i - t ~ ~ i i i ~ cliniti ~ ~ ) o litrkiiig i~t iiit~rogcirnsci t 1 F’ic:. 8. \Vot.liitig t ~ i o d ( ~for d zotohrrctcj.r c~itrcltrrttlii.Svt. t,oxt, for tt tlisc~tssioiiof ttic pathway.

I .3i

.TOlTN It. TI E K IGX’IANU AND R A T M O N D C’. TAT,KVTTVR

s h o t~i i n Fig. 8 . In the. proposed pathway, NXT)PH generated from isocitrate rcduccs nitrog(~tim(~ through the elec-troii curriers azotoflaviii

and/or Xzotobactcr ferrcdoxin. The available evidence supporting this be sammarizcd in this section. T l i citric ~ wid c~yclcWLS indirectly implicated in nitrogen fixation by this orgaiiisrn (Mumford P t al., 19.59) because scveral citric acid-cycle intcrmcdiates (for exattiplc, isocitrate) were found t o support nitrogen fixittion i n a tnutant uiiablr t o fix nitrogen from glucose. U’c observed lov but cwnsistcnt lcvels of iiitrogenasc activities i n crude extracts of Azotohactc,r using isocitrate (or related substrates) a s reductaiit. Excess ;wototlnvin ;LSn.c.11 n s NI’I’wcre wquired for this rewtion. Recently, the isoc*itratt, de1iydrog~cn;~sc of this organism wits purified (Cliung and ~ h i i z ( ~ n I !)(j!l), , atid it wits found t h a t AzotobacUtci* is i i rich soiirw of S X I)l’-linlted (~nzynic~, a h i v h makes u p about I O(, of thc total solublc I)tvtcin. Other c e l l u l ~ rSADP-specific deliydrogenascs c i ~ nd s o supply NAI)I’H, t o the electron (.hitin. Lt was shown t h a t NAl)L’H, played a central role i n this pathway. 1)inlysis oftlie Aeotobacter extract resulted in cotnplete loss of nitrogcnfixittion w t i v i t y from isocitrate. Addition of NAUP resulted in rcstorcd itctivity while NAD had no effect. Substratr levels of NADPH, or a n S A I)I’H ,-grnerating system (glucose 6-phosphate dchydrogennse) replitcwl isocitrate as reductant. Azotoflavin was required for activity ; Azotobacter ferredoxin stimulatcd the reaction in extracts treated with DEAE cellulose. Thus the two Alzotobac*tcr carriers were implicated in this reaction. Excess Azotobacter fiwtdoxin did not replace tlic azotoflavin requirement, sitggcstiiig that I)oth carriers may be nreded in this pathway. I t is not get defiiiitcly wtublislicd whether azotoflavin and Azotobactcr ferrtdoxiii reduce i i itrogcxnase. I t e c ~ c n t cxperinients Iiavc indicated t h a t other cnzyrnes rnay be rc.qiiircd in this reaction. These factors are probably traiishydrogeti,ztioii cwz.ynics for t h e various carriers and are currently being studied At first glancc it is difficult t o understand how NADP could function in the nitrogenasc Imthway (Fig. 8 ) , because the reaction is thrrmodynamically uphill from NADPH t o ferredoxin. This energy barrier might be over(wine by tlic cell in two \vnys. First, tlic unfavourahle NAl)PH,-ferredoxin reaction could be couplcd with the extremely active isocitrate dctiydrogcwasc system, maintaining a high NAUI’H (and low NADP) c.onc.c.iitr;ttioti in tlie cell. ‘I’lius almost all the XAUI’ in the cell would in tlic rcduccd fortn, tlicreby helping push tlic rcvwtion. Secondly, thr NAI)l’H,-ferredoxin rcv~c*tionis coupled in the cell with the A‘I’Ptlrivc.ii nitrogcnasc rrac.tion which is highly irreversiblc. thus p l l i n g t h c N A 1)I’H ,-ftwcdoxin reawtion t o completion. Sinw t h e thermodynamic h c * l i ( . n i cw ill

iii(;ii-Eh E K G Y H r m q m o N s ~h R A C ~ T K R I A

I.i7

Iwxtiwters have not yet been worked out for the reaction involving azotoflavin or Azotobacter ferredoxin i t has not been possible t o assign frcc-cncrgyvalues t o the reactions. However, coupled reactions represent i i basic and well-known method t h a t cells use for overcoming individual ivactioii barriws. In the prc’sence of such “piish niid pull” reactions, the tiic.otitinmidc niiclcotides become n sourre for high-energy electrons. This energy harrier is not I)rcsent in clostridia, which utilize t h e energetically inore f i i v o i ~ ~ ~pyruvate ~ ~ b t e ~~Iios~~lioroclastic reaction for generation of i w l i i c w l ferrdoxiti. H owcvw, it hits been reported t h a t reduced KADP (mi also fiinc~tion :LS reductant in the clostridial nitrogenase system ( I)‘l~~iistac*hio and Hardy. I 964) raising the possibility t h a t a similar rc>actionoperates in this organism a s a n alternative t o the better known 1)yritvate reaction. A s discussed in the last section, a iiicotinamide niiclcotide t o nitrogcnasc system seems t o operate in the photosynthetic h;wteria and (as will be discussed a t the cnd of this review) in soybean i-oot notlalts. It, is therefore possible t h a t the pathway discovered in Azotobnctcr has a miicli wider applicability and t h a t iiicotinamidc iiucleotidrs art’ the reductants for nitrogen fixation in a large variety of bacteria.

IX. Nitrogenase: A High-Energy Electron Acceptor I n thc last section we discussed the pathway which feeds electrons t o 1iitivg:ciiasc.. \Ye arc now going t o discuss nitrogenase itself, as a n examl)lc of a high-encrgj- electron acceptor. One of t h e most interesting nspects of t h c rc.action catal,vsed bq this enzyme is the low redox natiire of the clcctrons handlcd during nitrogen reduction. Biological nitrogen fixation is t h c conversion of molecular nitrogen t o ammonia b y the iiitrogcnase c’iizymc in a six-electron reduction reaction. The list of 1)ncteri;iI species ablt t o fix nitrogen includes examples from many b;ic.tcrinl geiicrit such as red and green bacteria, clostridia, Azotobacter, roids (rhizobia), Klebsiella and bacillus. Using cell-free extracts of nitrogen-fixing cultures i t was found t h a t nitrogen fixation r q u i r e s hoth a stroiigrrdiictant and ATP (Mortenson, 1964 ; D’Eustachio and H a i d j - . I !)64: Dilworth rt nl., 1965). In clostridia reduced ferrctloxin (or flavodoxin) ~ v a sthe electron donor t o nitrogenase. In Xzotobavter. clostritlial ferrcdoxin could also act as electron donor (Bulen ( ~ f ((1.. I!)(i4). I)ut thv endogenous electron carriers are the recently isolattd ;izotoflavin and Aeotohacter ferredoxin. A number of strong inorgniiic*rct1rict;iiits sric~hiis ditliioiiitr atid dyes (I3rtl(~tiet a / . . I !Mi;

I5X

.JOTTY 1% RENEMANU AND RAYMOND C . VALENTINE

l)’KiistacIiio and Hardy, 1 !)64) can also donate electrons t o nitrogeiiasc. This cnzymcx is therefore a high-energy electron-acceptor which will iise electrons from a variety of low-potential electron donors. R u t nitrogen fixation (nnd oxidation of the strong reductant) proceeds only in the presmce of X‘I’P during which ATP is hydrolysed (Hardy and Iiniglit, I!)(;(;). Nitrogenase is thus a n elaborate redox catalyst consuming h t h A‘I’P and higli-energy electrons during the reduction o f molecular tiitrogcn. ‘I’hcl prolwrties of nitrogcnase, a s a. low potential redox catalyst?, and tliv nic~i~lianistn of nitrogen fixation. n ill be discussed in this section. 1)vspitc t h e f w t that they ctttiilysed t h e s t m e reactions, the uitrogt1n;tse.s of Azotohwtrr and calostridia seemed a t first t o be very d i f f e r f ~ t . \\’hiwm thc clostridial nitrogenase activity was soluble a n d 11011scditncntal)lc, Azotohnctcr nitrogenase could be sediniented b y high sl)ccd centrifiigation ( 144,000 9 for 5 hr.) indicating a high molccnlarivcight conil)lex (Bulen P f nl.. 1964). Also, the clostridial nitrogenase u’as very quicltly irrevcmibly inactivated by oxygen, whereas t h e Azotobnctcr cnzymc was stable in air for long periods of time. Purification of Azotobacter nitrogennsc (Bulen et nl., 1966) revealed t h a t i t was made up of t\vo oxygeii-labile proteins (1a n d 11) both of which are needed togvtlier for tiitrogcnilse activity. Both proteins contain Fe-S groups : the sinnllcr l)i*otcin ( I I ) has n molecular weight of about 40,000 while the larger (1)h i ~ s;L molccular weight of about 100,000 a n d contains molybdcnum. 7’u o very similar proteins were found t o make u p clostridiibl nitrogcnnsc~(Mortenson, 1 066). Recently such proteins have been found in several other nitrogen-fixing organisms (Klucas et al., 1968; Detroy P t ~ 1 . .1 96s). CoinI’lenieiitations of t h e two proteins obtained from diffwcnt org:misms are possible in many cases (Detroy P f al., 1968). The big differcnw between the Azotobscter a n d clostridial nitrogenase is not tlic cnzynie itsclf or, iis we shall scc later, the mechanism ofnitrogen fixiLtioii, but tlie organization of the protein components of nitrogenase. I he oxygen resistance of Azotobncter nitrogenase is due t o the organization of tlic tiitrogcnase into a high molecixlar-weight complex. Purificibtion of Azotobactcr nitrogenase involved breaking u p the complex by I)rotninine siilphatc precipitation followed by separation of the two proteins by 1) EAEhellulosc column chromatography. n’hen the complex of Azotobwtcr nit rogellitsr \ w s broken, the nitrogenase activity becairn(> \el.! svnsiti\v t o oxygen. It is evident that the organization of the tu.0 niti.ogl;t~iiawI)roteins into tlie native enzyme complex in t h r cell pro the cnzynic. from ox>-gen inhibition or denaturation. Clostridia, living in i t n ~uiwt*ol)ic cnvironmcnt, do not l i e d t o have a n oxygen-resistant nitrogcnnsc~.0x~rg:c.nresistance is only one of the possible advantages tliat ;\I] oreiitiizrtl nitrogentise coni~)Icxcwnld have for the Azotobactcr , I

TITC: FT-EKEROY ET,EC’TRON4 I N BACTERTA

159

( * ( > I 1 .\aotol)~tc*tc~t* tlitrog:c.tiilht.is :\Is0 c.xt~~\tiic.I~ Ilt,itt htill)l(’ ((;Om. I O i i j i t i . ) . but agaiti oiily in t h r cotnl)lex. Blthougli the Azotobactcr nitrogrnase is stable in air, recent experiments have shown t h a t tlic nitro~ ( ’ i i i ~ srciwtion e in Azotobacter is sensitive t o oxygen (1)ulton and I’ostgate, 1968). How then does Azotobacter fix nitrogen in an aerobic (.tiviroiitncnt I A1)1);wcntlythis is accomplished I)y using u p t h e oxyg:cw insidta thc rell, ~)roducainglocalized ttnaerobiosis around the nitrogenaw. (‘oiisitlering t h a t Azotobacter is tlie most oxygeti-consuming organism ktto\\ ti. this seems quite I)ossihle. There might thcrefore be other protc.ins and c’iizj.nicls iLssociatcd with the nitrogennsr cmii1)lex involvrcl i n tlicw r(vwtioiis. I k t t i c ~ worlc r in tltv area of the t r i ~ ~ c r o t n o l e r i organimtioii tl~~i~ of this i n t ~ ~ r ~ ~ sc+omI)lex t i n g should be very fruitful. Kitrog:c.nasc was found t o catttlyse a number of other reac.tiotis besides nitrogen reduction. Many small triple-bonded corn pounds such u s acctylen(. (LIilworth, 1966). nitrous oxide, cyanide a n d azide (Hardy and Knight, l!)C,8) can be rcduced by nitrogennsc, both in cell extracts and, t o sonie extent, in whole cells. Reduction of acetylene t o ethylene by tiitrogcnasc. as detected by gas chromatography, is used a s a most versatilc and sensitivc assay for nitrogen fixation (Hardy aiid Knight, 1Hfi(ia : S(~hollhoriiaiid Hurris. 1!)137). I n the absence of a suitable substrate 01‘ in the pi’esence of a wrnpctitive inhibitor such as carbon monoxide. tiitrogcnase evolves hydrogen in a n irreversible reaction dependent on both ATP mid reductant. During these reductions or hydrogen evolution, Al’t’ is liydrolysed t o A LIP, this reductant-deI)endeiit ATPase activity brling indc~pendtwtof t h e 1)reserice of substrates or inhibitors of nitrogenase (Hnrdy atid Knight, I966b). The uriiouiit of A‘I’P used is very Inrge, in the neiglibourhood of 15 Al’P for each nitrogen reducwl (Bulen et al., lU(i(i; ]\‘inter and Burris. 1068). Since all t h e nitrogen-fixing enzymes isolated from a wide variety of very differcnt micro-organisms catalyse the same reactions arid are composed of similar proteins, t h e same riiechanisms of nitrogen fixation would be expected t o olwrate in all C I L S C S . Any 1)roposed mechunisni of nitrogen fixation must take into iu.c.ount a l l thc iwwtions catalysed by thc nitrogenasc proteins i i t l d inforinat ion ncciitnult~tedabout nitrogen fixation. ,, I he c~lcc+roiisfeeding the iiitrogenase coml’lex have among tlie liiglirst c n c ~ g yknown i n thc ccll ; still, as seen from t h e A’L‘P rccluiremcnt, they arc not sufficient t o reduce t h e inert nitrogen molerule. ISlectrons of e v ~ n Iiiglier eiicrgy may be needed. Several mechanisnis for nitrogeii fixation have been proposed involving electron activation b y ATP (Hardy P t al., 1963 : Hardy and Knight, 1968). I n one model, high-energy electrons from the clectroii carrier reduce a n iron-sulphide group a t the electroiipting site on t h e nitropenase. The electrons then move t o the c~l(~c.ti~oti-;ic~ti\.nti~ig site \vlierc they itre activ;ited t o RII ev(111 higher 1evc.l

160

JOTTY R

nmmr,\uv

AYI)

n4rarosn c. I-ALEYTTYE

(Io\\c’r I)otvnti;il)i n i i i i ATP-reqitirilig renctioii ‘I’his stcl) iiivol\ w tlrc. of fivc A W niolecules for thc uctivation o f t u o clcctrons. ‘l’lic~ iiivolvenic~ntof molybdenum at this site lras been sugqcsted (Hardy and 13nrns, l!NS). One interesting idea is t h a t A T P is used t o phospliorylate i i chivntivc of rnolybdeiiuni giving 310 P. Part of t h e chemical energy of the Alo 1’ I)ond might then be converted into redox energy witlr t IIC subseqiwnt ~ ~ l c a sofe iiiorgaiiic phosphate. After activation, thch to the substrate-comI)lexiiig site \ v h c i ~nitrogen, or its :ui:dogiics, ar(1I)ound nnd reduced. ltcdurtion of nitrogcw is iwcom plislied by t lire(. c*onscc*ut ivrl two-electroil transfer steps with thc intermediates strongly cfiorn~)lcxetl at t h e site. For differcnt analogues of nitrogcn, thc tiutiiI)er of rcdurtion steps varies from one for acetylenc u ~ t )o six for isocynnidc. The afinity of the intermediates for the substr~ite-~~itidiiig site probably determines the extent of reduction. As pointed out b , ~ Hard? and Burns (1968) the coupling between the electron-activation site and subst,riltr-bindill~ site is not perfect with a, leak of sonic elrrtrons t o thc solvent yiclding hydrogen gas as a by-product. There are many uiiaiis~ver~d questions about t h e rnechniiism of nitrogen fixation : the ro1r.s o f t h c t u o 1)roteiiisa n d molybdenurn, the localization o f t h e various x t i v e sitcs mtl how A T P is bound m d liydrolysed. 7‘hc most exciting aspect of this 1)roblem is the electron-activatiori mechaiiisni atid t h e iwtiial Iwtentid of the electrons in t h c nitrogenase reaction.

-

-

X. Regulation and Genetics of Electron Chains (i7~ collaboration with C . Ti’. Xheu) r , I he rcgu1;ition and control ofthe synthesis a n d function of high-energy electron chnins, such as the nitrogenase p t h w a y , is a n important subject that has so ftw rcccived relatively little attention. ,Just like any other nictabolic pathway, the high-energy electron chain must be under tight regulatory control, both in biosj-nthesis a n d activity. That low iron wnc.entrat ions r c ’ p s s ferredoxiii synthesis and induce flavodoxin has alrtwly been mentioncd (see Section TV, p. 144). How this control tncc*linnisin o1)eratc.s is not knowii. In photosynthetic. bavteria, t h e ~ ~ l i o t o s ~ n t l i c:LIq):wittus tic~ (cliromatopliores) is iiidnccd when t h e cells w c shifted frorn i ~ i iaerobic t o an anaerobic environment. Light lins no tlirwt c+Tec*t0 1 1 this induction (Cohcn-Razire and Kunisawa, 1960 : Schtin and l)rc~ws,I !)66). A regulatory iiiechanistn for cliromato~)hore involving the redox state of some cell component has been I)roI)osed (Cohcm-Bazire et nl., 1957). The nitrogeii-fixation pathway provides a useful system in which t o study regulatory mechanisms of high-energy electron transport chains. r 1 I he iiitrogcnnsc system represents a considerable proportioil of t h e cell i)i-ott.iti ; ~ n dit is probable t h a t the supporting pathways which sul)ply

ti I ( ; H - EX EH(: Y ELE:(’TROU

Ifi I

A‘l’l’ and rcdiic*tunt must bc especially active in the nitrogen-fixing ~ ~ 1 1 . It is not a11 overstatement t o say t h a t a large part of the cellular economy is engaged in the maintenance of this process. Therefore one would sushe cell might have control mechanisms which regnlatr md function of the nitrogenase pathway. It has bcwi obsc.rvrd since the earliest work with nitrogen-fixing ct.11~t h a t . in the pr(www of ammonia or other metabolizable fixed nitrogen sources, bacteria (lo not fix nitrogen. For example, ammonia or urea a t a coticetitration of 10 p.p.m. inhibited nitrogen fixation in intact cells of A . t*i)idudiicompletely, while aspartate or glutamate were without specific on nitrogen fixation even a t high concentrations (Burris and M’ilson. l!)46). (irowth on limiting ammonia resulted in a lag of about 30 min. between exhairstion of combined nitrogen soiirces aiid growth on molecular iiitrogcn (Strundbcrg mid Wilson, 1968) showing t h e diauxie phenommon first rel)ortcd hy Moiiod. Cell-free extracts of ammonia-grown c ~ l l c~xhil)itetl s no iiitrogeiiase activity aiid had no nitrogeiiase enzymes. It w a s therefore suggested t h a t ammonia, or a cell metabolite drrived from it, rcl)r(~sss“d syiitlicsis of tlie tiitrogeriase enzymes. It is of somr interest t h a t iioiie of t h e common amino acids were repressors of nitrogen fixation in wholc cells of Azotobarter (J. R . Benemanii, uiipublished ol)scrv:itions). Sirnilnr findings werc reported by Yoch a n d Pengra (1966) for 1ilel)siclla. This might be due t o t h e fact t h a t t h e cell cannot metabolize the amino arids a t a rate siiffivient t o use them as sole sourres of nitrogen. Xitrogenuse activity (as measured b y the acetylene assay) in whole c ~ l l sof A . vinclniidii was inhibited hj- ammonia in a manner which suggestd fredbark inhibit ion (Strandberg a n d Wilson, I968 : Benemann, 1 ! ) 7 0 ) ‘I’hc . inhihition of acctylenc reduction b y adding ammonia t o whole c ~ l l sv, a s not immediate, indicating t h a t some metabolitc derived from ninnionin v a s tlic feedback inhibitor of nitrogenase. However, neither :\mtrioiiiit 11oranj. oftlie (wriimoii amino acids or other metabolites testtd inhibited nitrogmase in cell-free extracts, indicating t h a t feedback inhibition may not occur a t t h e level of nitrogenase. It is possible t h a t f d b n c k control is elsewhere d o n g t h e electroii-transi)ort chain linking nitrog:cnmc~.Altcmintively, some untested cell metabolites derived from ammonia might inhibit nitrogenase. It slioiild be pointed out t h a t i i o feedl)acl
Lfi2

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H&K”:MANN A N D ItAYMONI) (2

VALEhTIXE

activity irr I-itro. Since largc amounts of ATP are caonsumcd during nitrogen reduction (15 per molerule of molccwlar nitrogen). it, ma?’ be 1)eneficiiilfor the nitrogen-fixing organism t o rcpnlate closel~7the activity of n it roget ~ ~ sw( i1t h the avai lnblc energy supply. Alt Iiough nitrogennse biosynthesis in Azotobncter is rc>l)ressed bj, growth on amtnoiiin, t h r Azotobarter electron carriers seem t o be cwnstitutivc. compoiients of t h e cells (U. C . Yoch a n d J. K. Benemann, iinpul)lishcd results). This might indicate t h a t these clertron carriers play a role in some unknown reactions other t h a n nitrogen fixation. I t is ( h l i ~ ~from r the above discussion t h a t t h e area of regidation of the nitrogcnnse pathway is wide open for future research. Studies on the genetic rc,gulation and expression of nitrogeiiase might have important practicd c.onscc~uenc.cs,such as the development of more active nitro~Icv-fixiiigstrains of bnctcria. Nitrogen-fixing organisms which are excrctcw of large q m n t itivs of ammonia aiid are not rcprmsed or inhibited i n their nitrogcnme by availablc fixed nitrogen, c~oultlfind gwat u s e i n agricultureas chcnp and non-polluting fertilizer. Construction of such m u t m t s is well within the theoretical framework of biochemical genct irs. Biochemical mutants blocked in different steps of the nitrogen fixation pathway or, a s mentioned above. in the regulatory mechanisms, might be inilmrt;mt tools for studying some of the unsolved features of biological nitrogcn fixation. The organism which seems t o hold out the most l)romisc. for gmetic studies of nitrogen fixation and its high-energy electroii-traiisl,ort chain is again Azotobacter. The reasons for choosing this organism were purcly practical considerations, such as ease of h ~ ~ i i ~ l lwrobic ing growth conditions and rapid growth rate. The general ~)rocedurcfor obta,ining mutants of Azotobacter a n d preliminary results achieved xvith thein arc detailed below. Brfore this field ran realizc its full I)c)t(ttitiiLl,it will br ini1)ortant t o develop suitable methods of gem. trmsfclr i n Azotobacter, so t h a t a genetic map of the genes involved in nitrogen fixation eiin be made. Techniques for isolation of nitrogeriase mutants of Azotobacter were developed b j ~Dr. Cliingju Sheu aiid t h r ant hors a t 12(~kcley. Tlicy were modified from procedures used h j 7 various nut Iiors who have isolated tiitrogenase-deficiciit mutants in t h e past (Karlsson atid Barker, 1948: Alumford etal.. 1959; M‘y-ss and \\-yss, 1950; Green P t nl.. I9.73). The rnnin emphasis of this work was on the isolation of niutnnts which fix nitrogcw a t 30” but not at 39”. B u t the procedures develo1)eti during the isolation of these temperature-sensitive (ts) mutants can I)(, applied t o t h v isolation of other interesting bioc.hemica1 mutants. ‘I’hc isolation of thcse mutants will lw described i n some detail as n guitlv for those workers interested i n this tit‘eil.

llI(~H-lCNER(~Y ELECTROKS 1U BACTERIA

1 (iX

.-Izotohncttr vinelrindii strain 0 was grown on a modified Burk’s nitrogenfrw media and plated on the same media. Ion agar was used in the plates. s i n w normal agar is not nitrogen free Mutants w r e prepared as follous .I w I I cwlturc for miitagenesis was prepared by inoculation of a young c~oloii,~ iiito 20 in1 of nitrogen-free media follo\+cd by incubation to a cell dciisit>. of about 5 x lo8 cells/ml. Thc cells \\ere centrifuged at I0,OOO rc~v./miti for 5 min. and \\aslied ~ i t Tris-maleic h (TM) buffer (04.5dl-Tris ;und 0.05 M-maleic acid; pH 6.1) to remove cell nutrients. For mutagenesis thc wlls w e r e suspended in 10 ml. of TM buffer and an equal volume of a fresh solution ofnitrosoguanidine (NTG; 200 mg./ml.) in0.05 M-Tris buffer. Tlic culturc was incubated with the mutagen for 30 min. a t 30”.About 99% of the cells \I ere killed by this procedure. The mutagenized cclls 15 ere waslieti conseciit ircly I\ itli TM buffer and then nitrogen-free media and finally suspeii(kd in about 40 ml. nitrogen-free media at a final cell density of I - 2 x I ( ) * cc~lls/nil.The cells \\ere incubated with shaking for 12-18 111. t o ;I final cell density of about 5 x 1 0 ’ cells/ml This procedure allowed i~uclcat~ wgiqqitioti to takc place and diluted out slowgrowing and \\ cak miitants. Thc rclls \I c w next I\ aslieti once with fresh nitrogen-free media t o t’cmovc any fixed nitrogen and resuspended in the same volume of nitrogen-free media. The washcd cells were incubated for 1-2 hr. a t 3 9 ‘ to adapt the cells to the higher temperature and exhaust nutrient supplies in the /s mutants Penicillin-Cr (300 units/ml.) u as added and incubation continued for a further 5 hr. About 99% of the viable cell population was killrd by this procedui*c.The penicilliri-treated cells were washed twice u it11 .50 ml of distilled water to lyse the penicillin-affected cells, and once 11 itlr nitrogen-free media. The remaining cells were then suspended in nitrogcnfrce mctlia to a cell density of 5 x 10’ cells/ml. and incubated for 15 min to allov all I,vscd cclls to settle. Scrial dilutions were made and plated on lon nttrogtm platcs containing 25 units of chloramphenicol/ml. The p r c w i i r ’ o f this antibiotic inhibited most contaminants acquired (luring the mutagcticsis and selection procedure About 100 plates ere usually platcd to gtvc about 300 colonies per platc. The platvs \\ ere i n c u h t e d a t the non-permissive temperaturcx (39 ) for about three days until the colonies \yere fully grown. The plates \\ere screened for “t illy” colonies [any colony considerably smaller than normal ; scc Fig 9(a)]I\ liich indicated a temperature-sensitive lesion. These strains \I pickrtl \\ itli stwile toothpicks and streaked on t n o nitrogen-frec plates and tn o 1iitt.ogcn-containiiig plates (about 40 “tinj-” colonies streaked per platc). Identical plates ere incubated a t 30” (permissive) and a t 39” (non-permissire). After about 2-3 days the plates were compared and screcncd for strains that gre\\ at 30” on nitrogen-free media but a t 30” only on the plates I\ itli pre-fixed ammonia. Samples \\ere inoculated from the nitrogen-frec platc into a small volume (5 ml ) of nitrogen-free medium. Stains that did not multiply a t 39” within 24 hr. \\crt discarded. Stocks \\ c w prq)ar(d from mutants \vith good thci.niosensitive properties 111 a typical mutagencsis cxperiment about 1 lo(), of the colonies on thc. original plates \+crc‘ “tiny”. Of these, about 10-30% showed a deficiency (

~

1

~

I64

J O H N H . . B E N E M A N N AND RAYMOND C . V A L E N T I N E

30"

39" N2

FIG.9. Temperatmc-serisitive nitrogellase mutarits of Azotobacter 7iineZandii. (a) Photograph of a plate culture showing the small size of colonies of mutant strains. (b) Demonstration of temperature-sensitive lesions in the mutants.

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opc~iitixitioil but only i i srnall percentage of t l i c w h t i a i i i s scotwl as Prii~~lic~~~rnor~c. 0 1 1 1 ~ . n l ) o i t t I O 0 , ) oftlic, total /.s s t i i i i t i s \\(~w sriitablc

/A ~ i i i t t i i t i t ~ .

biochmic*alniiitarit s Hevernl hundred nitrogcnuse mutants were picked 111) by this proc d i i r c . Most of the ts mutants were unsuitable for l)ioc*lieniicalstudies I)cc*auscof rclatively poor growtlr rutc a t 90 and i*itl)idrcvc~sionof stocks to a tyiw iiidistiiiguisliabI(~from the parent strlLin. ‘I’crnperaturc shift-III)cxlwiriients with the nintnnts indicated t h a t sonic c~)mponent(s) of tlic nitrogc.nnsc pathway was heat labile. Studies i ~ I)resently r ~ under \\ ay t o drtc~tm~ine the nainre of the biochemical block i n these mutants; prcsiiniahIjr sonic’ of these mutants may be blocked itt tlie Irvel of the nitrogc~nasc.I)athwny. Following a similar procedure, IGslier and Brill ( I !16$))liavc recently isolated several mutants in iiitrogenase itself. These uorliers selected several strains unablc t o fix nitrogen for biochcmical studies. Extracts prepared froni difkrent mutitnts were tested for corn~~lcriient~ition with tlic two sel)arated nitrogenasc proteins I and 11isolated from the parent wild-tj1w strititi. Tliese coin~~lernetitntioti tests revealed t h a t one of the mutants \vas lacking nitrogenuse 1)rotein 1, while the b1oc.k in the sec*ond strain wits tnorc cwinl)lex with both cornponei~ts1 ttiitl I1 a,l)sent from tlie extract,. l n suin rnary, it is now cleur thiit a mutant approwli t o tlie low-redox pathways su(*Ii a s the nitrogen-fixation system is a workable way of attacking the problem and may a d d a new dimension t o our understanding of tlicscl pathways. It is easy t o visualize ii 1);itIiway divided and furtliw subdivided by genetic blocks so t h a t e\-t.ii t h r . finest details of t h e rwction s c ~ c ~ u ecome n ~ ~t~o light.

XI. Concluding Remarks and Future Developments High-energy cllcctrons arc of fund~tnientalimportance t o the bacterial cell. They play important roles in rnnny of the basic pro^ metabolisni bot Ii i t r aerobic ant1 ;uiacrobic cells (for ;I s i i i n n i a ~ rof ~ these reactions s w Fig. 10). HigIi-energy (xlectroiis produced i n one biochemical rraction are linked virc specific electron carriers t o tduc.tive cellulttr pi’o(wscs. 1 1 1 the sitriplest instanc*cs ferrcdoxin works with a deli ydrogeiinse and a rtductnse linking tliese two enzymes together. I n some cases the ~ ~ I e c t r o n - t r ~ ~ nchain s ~ ~ oofr t such 1”tthways is more coiriplex, as in Azobacter, where additional carriers are involved. As shown in Fig. 10, the electrons tli2Lt feed into these chains are supplied from donors such as a-keto acids. formate. molecular hydrogen, liypox~inthine, acetaldehyde, “exc4ed” chlorophyll and reduced nicotinarnidc nucleotides. dl of wliic~li WY’ strong c ~ e l l u l ;reductants. ~~~ Sirnil;u*ly, a varictj. of

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for higll-mcrgy clccti*oiis, soill(' ituportaiit c . x a i n l ) l c h Iwiiig niwtiiiatnide nucleotides (KAI) aiitl NADt'). carbon dioxide. m d molecular nitrogen. I Y P would like t o single out one of thc rclactions, the rcduced nicotinamide nucleotide t o ferredoxiii cmuplc, for more discwssion because this reaction aplicars t o have been Ii~rgclyovcrlookcd in the past a s a11 important source of high-energy elcc.trons mid may bc found to 1)hy a key role in several organisms. The nic*otinntnidc nuclcotide-Fd reaction as written is tlierinodyiiarnically uiif;ivoura~)le.St i l l , t l ~ c r eitre several cascs now known in which this rcvic*tioii 1)hys ;L c*ruc*ialrolc for t he cell. I n tlie case of the ethaunolfitr m v i i t i tig org;i I I isi n (' h s t r d i urr! kluyveri t he 11icot iiiamidc nuchleotide-Fd rw(*tioilis intitn;rtcly cwnnec~tcdt o tlic energy metabolisrn of tlie c ~ l l .

v) v)

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111 this anaerobic organism, oxidation of ethanol is accompanied by ;wctyl-CoA formation mid ATl' synthesis oiily if excess electrons derived fi-om thc oxidative prowss (wi be discarded as molecular hydrogen (Barker, 1 !)56).I n other words, the fermentation of ethanol yielding net ,4TP t o the cell can only proceed if t h e electrons from its nicotinamide nuc~leotidr~ canrriers are constaiitly removed from t h e cell as molecular Irydrog:rn freeing thrse ciwricrs for further catalytic action. Because of t h c ( ~ n c ~ g y - p onature or of the ethnnol fermentation it is uiilikelj- that addit ioiial cwcrgy ~ L HA'I'P can be exlxiided t o drive the reduced nicotiiimiidc nnc*lcotidr to tnolccwliir reaction. 111 support of this argumeiit, ATl' 11 a s not required for the iiicotinsmide nucleotide-H, reaction using extracts of C. klzcyveri (Jungermann et al., 1969; Predericks and Stadtinaii, 1965).This reaction was apparently readily reversible depeiiding on tlie partial pressure of hydrogen gas, a high hydrogen pressure reversing the clcc+xon flow from H,-nicotinamide nucleotide, whereas a low concentration of hydrogen gas resulted in evolution of molecular 11) tlrogctl (,Jungertiimii ef /iZ , 1969) Ferredoxin a n d the same enzyme

167

HIGH-ENERGY ELECTRONS IN BACTERIA

catalysts are believed t o be required in both directions indicating that the nicotinamide nucleotide-Fd reaction is readily reversible. The NADPH,-H, reaction of C.pasteurianum (D'Eustachio and Hardy, 1964) may occur by it similar mechanism and may be important to the organism when NADP-linked substrates other than pyruvate are available for nitrogen fixation. As discussed in this review, this nicotinamide nucleotidc-Fd reaction also appears t o be vital for the nitrogen-fixation pathway of Azotobacter and photosynthetic bacteria. Thus the reaction plays a key role in nitrogen fixation. Since the source of the reductant of nitrogenase remains a mystery in many bacteria, it is entirely possible that the reaction will be found to operate in other organisms. One important group of bacteria comes immediately to mind, the nodule bacteroids (rhizobia) of leguminous plants. These bacteria are semi-aerobic nitrogen-fixing organisms and might be expected to have biochemical pathways for nitrogen

0-Hydroxybutyrate

0-Hydroxybutyrate

r"--., NADHz C 4 3 Fd

c

3 N2

FIG.1 1 . Hypothetical scheme for the nitrogenase pathway of soybean nodule bacteria.

fixation similar to Azotobacter. Indeed, preliminary experiments with nitrogen-fixing extracts of soybean root nodule bacteroids have shown that these organisms have an active high-energy electron carrier, with ferredoxin activity which links t o the nitrogenase of this organism (Yoch et al., 1970). Although the rest of the electron chain has not yet been worked out it appears that reduced nicotinamide nucleotides serve as reductants for nitrogenase in bacteroids as reported by Klucas and Evans (1968). A tentative scheme for nitrogen fixation in leguminous plants modified from Klucas and Evans (1968) is given in Fig. 11. This scheme is intended as a working model only, since a number of reactions of the pathway have not yet been studied in detail. I n the bacteroid system the bacterial storage product, polyhydroxybutyrate, is thought to play an important role as a reservoir of available reducing power for nitrogen fixation. The possible linkage of the @-hydroxybutyratesystem to the general metabolism of the bacteroid is not known. but this storage 8

1G8

J O H N R . BENEMANN AND RAYMOND C . VALENTINE

polymer may accumulate in the bacterium during active periods of synthesis by the plant host. Note the proposed role of the NAD-Fd reaction for linking the reducing power produced from /3-hydroxybutyrate to iiitrogenase in bacteroids. One of the most interesting aspects of the low-redox chains discussed above is the high degree of specificity of the redox reactions. It seems clcar that the high-energy carriers must possess a complex series of active sites on their surfaces to allow this very selective coupling to other carriers. This specificity of action is, of course, a common feature of enzyme catalysts. Still, the specificity of high-energy carriers has some special meaning, since it means the individual electrons themselves must be tucked away, or perhaps buried, in the interior of the protein body, thus protecting them from seizure by surrounding electron-deficient compounds. The Specificity of these powerful cellular reductants clearly separates them from many classes of chemical reductants, which often sliow poor specificity and give up their electrons to most substances lying below thein on tlic energy scale. The protection of high-energy electrons on the carrier molecules may be more important in some environments than others. For example, strictly anaerobic bacteria normally do not face problems of autoxidation of their carriers by molecular oxygen, a strong oxidant. The strictly aerobic bacteria, in order to run high-energy chains, must avoid this problem. Azotobacter represents the case of a n organism in a highly oxidative environment ; yet Azotobacter maintains a crucially important high-energy chain for its nitrogen fixation pathway. As discussed above, the electron carriers and the nitrogenasc of Azotobacter have unique properties which allow them t o function in an aerobic environment. If the electron-carrying prosthetic group is buried in the structure of its protein, then another question immediately arises. How do other carriers pass electrons to and from this site? Perhaps the prosthetic group is strategically located t o accommodate linking carriers. Electron flow from one carrier t o the next might also be regulated by allosteric effector molecules which bind to the carrier. Indeed, some recent studies indicate that this may be the case for the NADPH,-Fd-H, reaction of C. kluyueri, a reaction discussed earlier in this section. Jungermann et al. (1969) have reported that this pathway is effectively regulated by the NAD/NADH, redox couple. Nicotinamide adenine dinucleotide strongly activated cvolution of molecular hydrogen, while NADH, inhibited the pathway ; NAD therefore behaved as a positive effector for the system, while NADH, was a negative effector. This regulatory reaction was at some level prior to hydrogenase, the terminal enzyme along the chain. A second interesting regulatory reaction has also been reported by the same workers (Thauer et al., 1969) for a related low-redox chain in C. kluyveri.

HIGH-ENERGY ELECTRONS I N BACTERIA

169

The NADH,-Fd-H, chain of C. Eluyveri was found t o have a strict requirement for acetyl-CoA which presumably acted as a positive effector on one of the enzymes of the pathway. Aeetyl-CoA had no stirnulatory effect on the NADPH,-Fd-H, reaction above nor did NAD stimulate the NADH,-Fd-H, reaction, indicating that these effector molecules were specific for each pathway. It is interesting to speculate that both acetyl-CoA and NAD function as allosteric effectors perhaps a t the level of the nicotinamide nucleotide-Fd reductase, which links electron flow from the reduced nicotinamide nucleotides to ferredoxin thereby shutting off or turning on electron flow from nicotinamide nucleotides to ferredoxin. These and other regulatory switches which govern the low-redox reactions in bacteria represent still another interesting area for future work.

XII. Acknowledgements We are indebted to Drs. D. C. Yoch and D. I. Arnon for many stimulating discussions during the course of this work. The studies on the Azotobactcr nitrogcriase pathway were carried out in collaboration with Drs. Yoch and Arrion a t the Department of Cell Physiology, University of California, Berkeley. Original research was supported by grants from the National Science Foundation. REFERENCES Barker, H. A . (1956). I n “Bacterial Fermentations”, p. 1-28. John Wiley and Sons, Inc., New York. Benemniin. J . R., Y‘och, D. C., Valentine, R. C. and Amon, D. I. (1969), Proc. natn. Acnd. S c i . I;.S.A. 64, 1079. Brnemanii, ,J. R. (1970). Ph.D. Thesis: University of California, Berkeley, Callfonlla.

Boon, W. R. (19G4).Outlook on Agricul. 4. 163. Bradshaw, W. H. mid Ihrker, H. A. (1960).J . biol. Chem. 235, 3620. Bucharian, 13. 13. arid ICvans, M. C. W. (1969). Riochim. biophys. Act0 180, 123. Huchanitn, I%. 1%. nitd Rabinowitz, J. C. (1964).J. Buct. 88, 806. 13uchanaii, 13. I<., h c h o f e n , R. and Amon, D. I. (1964). Proc. n a t n . A c a d . S c i . 7J.S.A. 52, 839. Brilcn. W. ‘I., ~ ~ l U . l i 5R. , C. and Le Conte, J. R. (1964). Biochcwa. biophys. Res. Goii&?iaun.17. 265. Buleii, W. A., 13iiriis, R. C. and Le Conte, J. R. (1966).Proc. n a t n . A c a d . Sci. U.S.A. 56, 979. Biirris. R. H. :tiid Wilson, P. W. (1946).J. Bact. 52, 506. Chiuig, A. E. t i l i d Franzeii, J. S. (1969). Biochemistry, N.Y. 8, 3173. Cohen-Bazlrc., C. a r i d Iiunisawa, R. (1960). Proc.natn. A c n d . S c i . U.S.A. 46, 1343. Cohen-Rwire. (i.. Sistroin, W. R. and Stanier, R. Y. (1957).J. cell. comp. Physaol. 49. 2 5 .

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Dalton, H. and Postgate, J. R. (1968).J . gen. Microbiol. 54, 463. Detroy, R. W., Witz, D. F., Parejko, R. A. and Wilson, P. W. (1968). Proc. natn. Acad. S C ~U.. S . A . 61, 537. D’Eristachio, A. J. and Hardy, R. W. F. (1964). Biochem. biophys. Res. Commun. 15, 319. Devanathan, T., Akagi, J. M.,Hersh, R. T. and Himes, R. H. (1969). J . biol. Chem 244, 2846. Dilworth, R l . J . (1966). B~ochZ‘n~. biophys. Acta 127, 283. Dilworth, 31.J.,Subramanian, D., Rlunson, T. 0. and Burris, R. H. (1965). Biochim. biophys. d c t n 99, 486. Elemn, B., \’an Dalfsen, J. W. and Iihiyver, A. J. (1934). Biochem. 2. 270, 317. Evans, M. C. W., Huchanan, B. B. and Arnon, D. I. (1966). Proc. natn. Acad. S%a. [J.S.A. 55, 928. Fisher, R. J. and Brill, W. J. (1969). Biochzna. bzophys. Acta 184, 99. Foust. G. P., Mayhew, S. (4. and Massey, V. (1969).J . biol. C h n z . 244, 964. Frcdriclts, W. W. and Stadtnian, E. R. (1965).J . biol. Chem. 240, 4065. Gest, H. (1966). Nature, L o w L 209, 879. Gcst, H. and Kamen. 31.U. (1949).Science, N . Y . 109, 558. Gest, H.. Ormerod, C:. and Ormerod, I<. S. (1962).drchs Biochem. Biophys. 97, 21. Grau, E’. H. and Wilson, P. W.(1963).J . Bact. 85, 446. Gray, C. T. and Gest, H. (1963).Sczence, N . Y . 148, 186. Green, &I.,Alexander, Af. and Wilson, P. W. (1953).J . Bact. 66, 623. Hall, D. 0. and Evans, IT. C . W. (1969). Nature, Lond. 223, 1342. Hamilton, I. R., Burris, It. H. and Wilson, P. W. (1964). Proc. natn. Acad. Sci. U.S.A. 52, 637. Hardy, R. W.F. and Burns, R. C. (1968). A . Rev. Biochem. 37, 331. Hardy, R. W. F. and Knight, E. J. (1966a).BiociLem. biopiiys. Res. Commun. 23,409. Hardy, R. W.F. aiid Knight, E. J. (196613). Biochim. biophys. Acta 122, 520. Hardy, R. W. F. and Knight, E. J. (1967).J . biol. Chem. 242, 1370. Hardy, R. W. F. and Knight, E. J. (1968). I n “Progress in Phytochomistry”, (L. Reiiihold, ed.), p. 387, John Wiley and Sons, Inc., New York. Hardy, R. W. F., Knight, E. J . arid D’Eustachio, A. J. (1965). Biochem. biophys. Res. Commun. 20, 539. Hincksori, J . W. and Bulen, W. A . (1967).J . biol. Chem. 242, 3345. Homer, R . F.,Aloes, G. G. and Tomhnson, T. E. (1960).J . Sci. Fd Agric. 11, 309. Hoogerheide, J. C. aiid Kluyver, A. .J. (1936). Enzymologia, 1, 1 . Jones, C. W.and Vernon, L. P. (1969). Biochim. biophys. Acta 180, 149. Jungcrmaiiri, K., Thaner, R., Rupprecht, E., Ohrloff, C. and Decker, K. (1969). PEBS Letters, 3, 144. Kamen, RI. D. and Gest, H. (1949).Science, N . Y . 109, 560. Karlsson, J . L. and Barker, 1%.A. (1948).J . Bact. 56, 671. Keister, 11. and 1-ike, N. J. (1967). Archs Baochem. Biophys. 121, 415. Klucas. H. lr.and Evans, H. J. (1968). PI. Physiol., Luncaster 43, 1458. Klucas. R . V., Koch, R., Riissel, S. A. and Evans. H. J. (1968). PI. Physiol., Lrtncaster 43, 1906. KlIiI ver, A. J . (1936). I n “The Jlicrobc’s Contribution to Biology”, (by A. J . Iiluyver and C. B. Van h-iel). Harvard University Press, ?vlassachusetts. Knight, E. J. arid Hardy, R. W. F. (1966).J . biol. C?iem. 241, 2732. Iiiiipht, E. J., D’Eustachio, A. J. arid Hardy, R. W. F. (1966). Biochiin. biophys. 113, 626. Tiocpscll, J . H. and Johnson, M. J. (1942).J . biol. Chem. 145, 189.

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Kok, U., Rnrainski, H. J. and Owens, 0. V. H. (1965). Biochirn. biophys. Acta 109, 347. Lipmann, F. and Tuttle, I,. C. (1945).J . b i d . Chem. 159, 21. Lovenbcrg, W., Buchanan, B. R. arid Rabinowitz, J. C. (1963).J . biol. Chem. 238, 3899. U:Llkiti, R. n t i d Rabinowit/. ,J. C. (1966). Bzochem. biophys. Res. Commctn. 23, 822. 12dki11,R. a l l d Ritbiriowitz, J. C. (1967). A . Rev. Biochem. 36, 113. hlntsubnra, H., Jukes, T. H. and Cantor, C. R. (1969). Brookhavan National Laboratory Syinposium in Biology, “Structure, Function aiid Evolution in Protcins”. p. 201. Mayhew, S. G., Foust, G. E’. aiid Massey, V. (1969). J . biol. Chem. 244, 803. Nayhew, S. G . and Massey, V. (1969).J . b i d . Chern. 244, 769. Morteiison, L. E. (1964). Proc. nntn. Acad.Sci. U.S.A. 52, 272. hlortensoii, L. E. (1966). Bzochim. Biophys. Actn 127, 18. Illortenson, L. E., Valentinc, R . C. and Carnahan, J. E. (1962). Bzochem. biophys. Res. Commun. 7, 448. blortlock, R. I)., Valentine, R. C. and Wolfe, R . S. (1959).J . biol. Chem. 234, 1653. Mumford. F. E.. Carnahan, J. E. and Castle, J. E. (1969).J . Bact. 77, 86. Ne\\toii, J. W.. Wilson, P. W.and Burris, R. H. (1953). J . bioZ. Chem. 204, 445. Nozaki, 11.. ‘I‘agawa, K. and Arnoii, D. I. (1965).I n “Bacterial Photosynthesis”, (H. Cest. A. & i i i Piotro arid I,. P. Vernon, cds.), p. 175. Antioch Press, Yellow Springs, Ohio. Pcrigra, R. M. mid Wilson, P. IV. (1958).J . Bact. 75, 21. I’hillipS, llr.J., Knight, E. J r . and Blomstrom, P. C. (1965).I n “Non-Heme Iron Protrins: Role 111 Energy Conversion”, (A. San Pietro, ed.), Antioch Press, Yellow Springs, Ohio. Racburii, S. atid Rabinowitz, J . C. (1965). I n “Non-Heme Iron Proteins: Role in Energy Conversion”, (A. Sail Pietro, ed.), Antioch Press, Yellow Springs, Ohio. Schon, G. and Drevs, G. (1966). Arch. Mikrobiol. 54, 199. Scholhorn, K . itrid Burris, R. H. (1967). Proc. natn. Acad. SCL.U.S.A. 58, 213. Shethna, I-.I., Wilson, P. W. mid Beinrrt, H. (1966). Biochim. biophys. Acta 113, 225. Sinlllie. R. JI. (1963). PI. PhysioZ., Lancmter 38, 28. Smilhe. It.hl. (1963). Biochem. bzophys. Res. Commun. 20, 621. Stmndbrrg, C. W. and Wilson, P. W. (1968). Can. J . Microbiol. 14, 25. Tagawa,,K. arid Amon, 11. I. (1962). Nature, Lond. 195, 537. T:tiiaka, M.. Nakashima, T., Bciison. A., Mower, H. F. and Yusuriobn, K. T. (1966). Rzochemistry, N . Y . 5, 1666. Thaiirr, R. l i , Jringcwnrtnn, I<., Rupprrcht, E. and Decker, K. (1969). F E B S Letters, 4, 108. \’aleritinc, R. C. (1964). Bnct. REV.28, 497. \.’aleritine, R. C., Jackson, R. L. and Wolfe, R. 8. (1962). Biochem. biophys. Res. Commztn. 7, 453. Vcriiori, L. P. (1968). Bact. Rev. 32, 243. Weavcr, P. F., Tiiikrr, I<. and Valentine, R. C. (1965). Biochem. biophys. Res. Coi)imzcn. 21, 9.5. Winter, H. C. arid Burris, R. H. (1968).J . biol. Chem. 243, 942. Wolfe, R . S. and O’ICane, D. J. (1953).J . b i d . Chem. 205, 755. \Vyss, D. and Wyss. M. B. (1950).J . Bact. 50, 287. C. S. (1969). Fedn Proc. F d n Am. Socs. exp. Biol. 28, 529. Yoch. TI. C. arid Anion, D. I. (1970). Biochzm. Bzophys. Actn 197, 180. Yoch, D. C. and Pengra, R. M. (1966).J . Bact. 92, 618.

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Toch, D. C., Benemann, J. R., Amon, D. I. and Valentine, R. C. (1970). Biochem. bio phys . 12es. Commun. 38, 83 8. Yoch, D. C., Benernann, J. R., Valentine, It. C. and Amon, D. I. (1969). Proc. nntn. Acad. Sci. U.S.A. 64, 1404.

Branched Electron-Transport Systems in Bacteria* [)AVID

c. ITHITF: Rlld P E T E R R. S I N C LA I R t

l3iocJicniistl.y Department, University of Kentucky Medical Center, Lexingtoti, Kentucky, 40506, U . S . A . I. Introdactiou . 11. Mrthodology . A. Spectrophotometry . . 13. Oxygell clcctrodes . . 111. Zuterpwtation of the Data . IV. I
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173 174 174 181 182 183 183 186 188 192 198 207 207 208 208

I. Introduction In the last few ycars the electron-transport systems of bacteria have received a great deal of study. Much of this work has been coniprehensively reviewed in an excellent book (Gel’man et al., 1967) and in several recent reviews (Aleern, 1970; Bartsch, 1968; Chance et al., 1968; Smith, 1961, 1968). In this review, an aspect of the electron-transport system in bacteria that has not been extensively discussed will be emphasized. Some bacteria can form a membrane-bound electron transport system that is considerably more complex than the system found in the mitochondria of eukaryotic cells. These systems are more complex, as they contain maiiy membrane-bound dehydrogenases which are the donors of electrons to the respiratory system and the many reductases by which

* This review was completed in March

1970. Present address : Rockefeller University, New York, N.Y., U.S.A. 173

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electrons reduce oxygen and alternative terminal electron acceptors. The ruultiplc primary dehydrogenases and oxidases are linked by pathways which overlap and interconnect to varying degrees. These interactions result in “branching” in the electron-transport systems. Electrons can enter the system through numerous dehydrogenases and leave by way of various oxidases. The major portion of this review will discuss some microbial electrontransport systems for which there are sufficient data for obtaining an insight into the fuiictioning of these systems. It is hoped that detailed understanding of the function of components of the electron-transport system citii provide somc insight into the processes by which the complex membrane might be synthesized and organized into a functional unit. At the risk of being teleological, it appears that bacteria gain evolutionary efficiency in having the capacity to synthesize branched electrontransport systems. For example, Haemophilus parainjluenzae produces a more extensive and complex electron-transport system as the oxygen tension in the growth medium is lowered (White, 1962). By means of this process, a respiratory system with a progressively greater affinity for oxygcii and the ability to utilize alternative electron acceptors enables the organism to maintain a rapid growth-rate (White, 1963b). This capacity for modification of the composition of the electron-transport system is unnecessary in the carefully controlled intracellular environment in which the mitochondria of eukaryotic cells must function.

11. Methodology I n studies of the electron-transport system, the investigator is somewhat more at the mercy of “black boxes” than in many other types of research. The “black boxes” may add difficulties themselves but an equally hazardous trap lies in the interpretation of the data. Therefore any discussion of the intricacies of branching of the electron-transport system innst become essentially a discussion of methodology. Rather than discussing the methodology of each specific study we have attempted a t this point to discuss in detail both the incthodology and the interpretation of the data. A. SPECTROPHOTOMETRY 1. Hand Spectroscopes

The simplest, and often the most useful, instrument for observing changes in the electron-transport system is the hand spectroscope. This inexpensive device can be used with a microscope or licld directly in the

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hand. The pioneering, and possibly the most elegant, studies of the electron-transport system involved the use of this apparatus (Keilin, 1966). If bacteria have a high endogenous rate of respiration it can be very difficult,to oxidize the respiratory pigments. Oxidized respiratory pigments are necessary for difference spectroscopy (see p. 177). The major cytochromes can be identified rapidly in a thick suspension of cells with the hand spectroscope. Mutants in which the cytochromes are not reduced c ~ i nhe detected using the hand spect,roscope if cells contain sufficient amounts of cytochromes. The hand spectroscope is extraordinarily useful during purifications of haemoproteins. 2 . Spectrophotometers

With the introduction of photomultiplier tubes it became possible t o assess quantitatively the absorbance changes in the electron-transport chain. Two types of iiistruments have been especially useful. The first type, the split-beam spectrophotorneter, contains a single monochromator and the beam is alternatively switched bctween two suspensions and the difference in absorbance between the two suspensions is recorded (Yang and Legallais, 1954).I n the second type, the double-beam spectrophotometer, light from two monochromators is alternately beamed through the same solution and the difference in absorbance is recorded (Chance, 1951b). The first type of apparatus is used primarily to scan the spectrum, and the second type to follow the kinetics, of various respiratory components. I n the first type of instrument there is compensation for the increase in energy of the light with decrease in wavelength, either by narrowing the slit width (Cary models 14 or 15) or by changing the photomultiplier voltage (Aminco-Chance or Phoenix dual wavelength spectrophotometers). The geometry of the sample compartment is important, as maximum possible transmitted light must fall on the surface of the photomultiplier and the path length of each beam must be carefully balanced. I n these instruments the small differences in absorbance in very dense turbid suspensions are amplified differentially and (%an be displayed so that one-tenth of an absorbancy unit can he spread over 10 in. of chart paper without excessive noise. I n general these instruments have a relatively slow response time, and the scanning speed should be adjusted in accordance with the manufacturer’s recommendations. It is important t o note the slit width when using instruments like the Cary spectrophotometer, as the wider the slit width the less precisc the wavelength interval and hence the resolution. The slit width can be decreased by intensifying the light source and making the greatest usc of the transmitted light. An end-on photomultiplier directly behind the sample or matched integrating spheres mill assist in collecting the

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transmitted and scattered light. The scattered transmission accessory for the Cary model 14 with a controllable light source has proved satisfactory for a number of years in our laboratory. Attention to the sample itself can save grief. The resolution can often be improved by suspending the bacteria in a glycerol-water solution. Glycerol lowers the difference in refractive index between the medium and the bacteria which increases the transmission of the sample. It is important t o cstablish that glycerol is not readily utilized by the cells or it will be impossible to maintain cells in which the respiratory pigments are oxidized. Glycerol also helps keep larger cells from settling. Aggregates of cells should be dispersed by gentle homogenization if possible. The cytochromcs can often be conccritrated by rupture of the cells and recovery of thc membrane fragments containing the cytochrornes. Cytochrome absorption may be changed and some components of the electrontransport system can be lost during preparation of fragments (Smith and ilihite, 1962).For quantitative spectroscopy it is assumed that the optical puth length is known accurately. I n turbid suspensions of bacteria there is much scattering of light with an increase in the true path length. If suspensions of cells containing 10-15 mg. dry weight of Micrococcus Zysodeikticus per millilitrc are lysed with lysozyme, the difference spectrum before and after treatment is not significantly different, although lysis of the cells results in a considerable decrease in turbidity (L. Smith, unpublished observations). This indicates that there is not much error due to turbidity changing the path length in the quantitative determination of bacterial suspensions. The methodology of difference spectroscopy has been discussed extensively (Chance, 1957%).The absorbance difference between cells with respiratory pigments reduced minus cells with respiratory pigments oxidized between 400 and 700 nni. is most useful. This is known as the “reduced-minus-oxidized difference spectrum” A problem in the examination of wliole cells is the tendency of samples with oxidized respiratory pigments to reduce these pigments as a result of metabolism of endogenous substrates. It is important to rule out endogenous reduction of the oxidized sample each time a spectrum is determined. Often the endogenous respiration can be minimized by washing the cells (White and Smith, 1962)or by exhausting the nutrients in the cells by prolonged incubation with aeration. A combination of vigorous mixing with a vortex mixer of suspensions chilled in an ice bath has proved adequate for oxidizing Xtaphylococcus aurezcs (Frerman and White, 1967). A direct spectrum of a limited portion of the spectrum can be obtained by using ground glass of the appropriate light-scattering properties as the reference (White, 196%). I n most cases the cells must be ruptured and the membranes containing the electron-transport pigments washed carefully

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to stop reduction o f the pigments by endogenous substrates. However, some of the primary dehydrogenases or cytochromes may be lost after breakage and washing o f the fragments (Smith and White, 1962; White, 1964).

It is also important t o be sure that the respiratory pigments are completely reduced. Some substrates react slowly with the cells and cause incomplete reduction of the cytochromes (White and Smith, 1964). It is convenient to monitor reduction of the cytochromes with an oxygen electrode (White, 1966).A small amount of fresh, solid sodium dithionite may be added to reduce the pigments. The dithionite probably acts by rendering the solution anaerobic (Chance, 1961a). Excessive dithionite can damage the haem prosthetic groups of the cytochromes (Falk, 1964). Once the initial oxidized-minus-reduced difference spectrum has been determined, the difference in adsorption between cells reduced and saturated with carbon monoxide minus cells with the respiratory pigments reduccd (reduced carbon monoxide-saturated minus reduced difference-spectrum) should be determined. This is the classic test for an oxidase (Warburg, 1926; Keiliii and Hartree, 1939). It is important to be sure that tlie suspension is completely saturated with carbon monoxide and that the cell suspension has been sparged for sufficient time so that all of the components will have reacted (Broberg and Smith, 1967). Complete combination with carbon monoxide is easily checked by repeating the spectral scan until there is no change in absorbance. The more definitive determination of an oxidase is the identity of the photochemical action spectrum, for the relief of carbon monoxide inhibition of respiration, with the reduced carbon monoxide-saturated minus reduced difference-spectrum (Castor and Chance, 1955). I n this determination tlie change in oxygen utilization of carbon monoxideinhibited preparations is measured in the presence of light of different wavelengths. Most of the published photochemical action spectra have such poor resolution that it is difficult to make definitive determinations of the oxidase functions of the carbon monoxide-combining components. Even though the component combines with carbon monoxide, and the photochemical action spectrum matches the absorption spectrum, this is still presumptive evidence that the component is an oxidase as there may be transfer of energy from one component t o another (Castor and Chance, 1955; Jones and Redfearn, 1967b). In H . parainfluenme cytochromes a*, a2 and o all form recognizable carbon monoxide complexes (White, 1962).However, the photochemical action spectrum corresponds to cytochromes a2 and o (D. C. White and P. R. Sinclair, unpublished results). The obligate anaerobe Bacteroides ruminicola contains an enzymically reducible carbon monoxide-combining pigment (White et ab., 1962). This organism is killed by the slightest traces of oxygen so it is

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difficult to conceive of a role for an oxidase in growth of this organism. Certain cytochromes combine with carbon monoxide after denatur at’1011. Cytoclirome c from mammals combines with carbon monoxide only when it is dcnatiired (Butt and Keilin, 1962). Peroxidase which can be demonstrated in bacteria under the proper conditions (Barrett and Sinclair, 1967) may combine with carbon monoxide after reduction by NADH, (Yokota and Yamazaki, 1965). Addition of carbon monoxide may cause a shift in the steady-state reduction levels of some components (Appleby, 19691)). The resolution and intensity of the absorption of the cytochromes can be increased in the alpha region in liquid nitrogen (Keilin and Hartree, 1949; Estabrook, 1966). This treatment apparently results in a 25-fold increase in the optical path length (Keilin and Hartree, 1949). If diluted samples are plunged into liquid nitrogen a t time intervals after addition of substrate, “frozen” steady-state reduction levels of the respiratory pigments can be recorded (Chance and Spencer, 1959), thereby permitting identification of the cytochromes reduced during the steady state. The intensity of room-temperature spectra can be increased by adding Kaolin or calcium carbonate to increase the turbidity and thereby the optical path length (Butler, 1962). The light source must be intcnsificd t o compelisate for the decreased transmittance. Reccntly, commercial reflectance spectrophotonieters have becorne available. These devices are particularly useful where the endogenous metabolism makes it impossible t o measure the respiratory pignients from reduced-minus-oxidized difference spectra or where the material is too dense for transmittance spectroscopy as in bacterial mats. These spectra are difficult to quantitate (Itickard et al., 1967). Once thc eytochrome spectra are a t hand, it is helpful if the recoinmendations of the I.U.B. Subcommittee on Cytochrome Nomenclature are followed (Florkin and Stotz, 1965). It is extremely helpful to know the nature of the prosthetic group of the cytochrome. The non-covalently bound Eiaeins (protohaem, the haem a and the chlorin haem d ) can be identified by their reduced pyridine haemochromes after extractioii (Falk, 1964).A very good discussion of the chemistry of the cytochromes is to be found in a recent review (Bartsch, 1968). The double-beam spectrophotometer is particularly useful in examining the kinetics of oxidation or reduction of the electron-transport chain (Chance, 1951b, 1954). I n this instrument, light beams of two different wavelengths are alternately passed through the sample. The wavelength of the sample beam is set a t the absorbance maximum of the particular component under study and the wavelength of the reference beam is set ideally a t a nearby isosbestic point. At a n isosbestic point, the absorbance does not change during reduction or oxidation. Light froin a single

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source traverses both monochromators to the photomultiplier so variations in intensity of the light source will be nullified. Turbidity changes can result from changes in the shape of bacteria that occur during activity of the electron-transport system (Bovell et al., 1963). Turbidity (*analso change when solutions containing substrates or acceptors are mixed with the cells or when some organisms show phototactic responses. These changes in turbidity are essentially equal a t wavelengths sufficiently close togcther. Consequently the use of the double-beam technique nullifies these turbidity effects. The apparatus can be used with pulsed-flow or stopped-flow instruments in which reactions in the 10millisecond range can be studied (Chance, 1951a). The major pitfall in the use of the double-beam apparatus lies in choosing the proper wavelength pairs and then interpreting the data. Uiifortunatcly cytochroine absorbancies even in the alpha region overlap. For example the absorbance maxima of cytochromes b and c in H . parainjiuenxac differ by only 8 nni. We initially determined the contribution of cytochrome c to the absorbance at 560 nm. (the maximum for cytochrome b ) and the contribution of cytochrome b to the absorbance a t 552 nm. (the maximum for cytochrome c) from a comparison of the spectra of the isolated H . parainjhenzne cytochrome c with that of cells grown so that they contained very little cytochrome c (TVhite and Smith, 1964). The expression for the correction of overlap was very useful for cells containing high or low proportions of cytochrome c relative to the cytochrome b. A second expression, calculated after determining a different contribution of cytochrome b to the total absorption a t 552 nm. in which the cytoclirome c absorption increment (A)= 1.37 A,(;, nm, - 0.62 A,,, nm, and cytochrome b absorption increment (A)= 1.07 - 0.15 A552n,,l., gives values which correlate with the total extractible protohaem (cytochrome b ) and the covalently bound haem c (cytochrome c) over a 20: 1 and 1:20 range (Sinclair and White, 1970). The cytochrome b absorbance a t 560 nm. is also complicated by the absorption of cytochrome oxidase o. Cytochrome o can be roughly estimated from the reduced-plus-carbon-monoxide-minus-reduced difference spectra, and accounts for less than 25% of the absorbance at 560 am. (Sinclair and '15;hite, 1970). Consequently the changes in absorbance measured a t 560 nm., which has classically been taken to represent cytochrome b, contains a possible 25% contribution from cytochrome 0.The problem is perhaps not so important in studies with mitochondria, as the proportions of cytochromes are relatively constant. But in bacteria, where the cytochrome composition can vary widely in response to different growth conditions (White, 1962), the overlap can be considerable. The validity of selected wavelength pairs is rarely considered in the published material currently available. Errors introduced by over-

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SINCLAIR

lapping absorbances can be corrected. The components absorbing at thc selected wavelengths can be identified in several ways. If the reaction is slow enough, or can be made slow enough by dropping the temperature or using inhibitors, difference spectra can be run over the 540-570 nm. range a t different times during the reaction (Ruiz-Herrera and DeMoss, 1969; Sinclair et al., 1970). As discussed previously, the steady-state spectra of samples can be “trapped” by freezing in liquid nitrogen. The cytochrome components reduced at each steady state can be identified from the dual wavelength tracing by measuring the absorbance difference a t the steady states between the reference wavelength and the wavelength a t 1-iim. intervals across the appropriate spectral region. When the spectra of the steady states are available, then the equations such as those developed above for H . parainfluenzae can be applied for calculation of the true contributions of each cytochrome to the steady state. Using wavelength pairs that are widely separated increases the risk of complicating the kinetics by involving multiple components. A convenient check of the validity of the wavelength pairs is t o compare a referelice wavelength at an isosbestic point using a .u\avelength greater than the sample wavelength, with a second referelice wavelength below the maximum. There should be little differciice in the kinetics. 111H . parainjuenzae we have found little difference whether 538 or 573 nm. was used as the reference when observing cytochrome c a t 352 nm. or cytochrome b a t 560 iim. (Sinclair et al., 1970). Microbiology’s greatest gift to biochcmists interested in cytochromes is cytochrome oxidase a 2 , whose spectral maximum at 631 iim. is uncomplicated by other pigments. If there is overlap in the alpha region of the cytochromes then there is much greater overlap for wavelength pairs in the Soret (390-450 nm.) region, because the absorption maxima are much wider. The gain in sensitivity from using the higher extinction coefficient is lost because of the increased overlapping of respiratory pigments in the Soret region. A particular note of caution should be exercized in using 470-505 rim. to measure flavoproteins. Cytochromes, non-haem iron proteins as well as flavoproteins absorb in this region (Klingeiibcrg aiid Bucher, 1959 ; Pullman and Schatz, 1967; Nicholls and Malviya, 1968). It should be obvious that the advantages of the double-beam spectrophotometer are lost when each wavelength is used to measure a different component, i.e. 340 nni. for reduced nicotinamide adenine nucleotides and 550 nm. for cytochroine c. It would be desirable to run separate experiments using small wavelength intervals for each component. Besides the trap of the cytochrome overlap, the investigator should be aware of the response time of the recorders, electronic components aiid the beain-switching frequency of his system. The same considera-

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tions of slit width and spectral dispersion should be made, as with the split-beam spectrophotometers.

B. OXYGENELECTRODES The ratc a t which the slowest reactant in the respiratory system is reduced can be measured polarographically with an oxygen electrode (Estabrook, 1967; Scliindler, 1967). The electrodes can be made more stable h j covering the surface with a thin plastic film (Clark et al., 1953) but this increases the response time to several seconds. Small electrodes with rxlwscd platinuni surfaces can be used to monitor oxygen reduction with a. sufficiently rapid response time to be used simultaneously with s~)cc~tro~)liotonictcrs. Since the oxygen electrode is a polarograph which reacts with the oxygen in solution, it must continually be exposed t o new solution. This is done by stirring the suspension with a magnetic stirring bar or by vibrating the electrode. It should be emphasized that the initial rate of oxygen uptake measures only the rate of the ratelimiting step of the electron-transport system. This rate-limiting step is usually at the primary dehydrogenase in microbial systems (White, 18G-i). The initial rate of oxygen utilization does not define the characteristics of the terminal oxidase reactions. A very useful but perhaps confusing parameter is the critical oxygen concentration. This is the oxygen concentration at which the rate of oxygen utilization shifts from a pseudo-zero order reaction to a pseudofirst order reaction (with respect to oxygen concentration ; Chance, 1957b). Usually the measurement requires very sensitive apparatus as the critical oxygen concentration is less than 1 0 p&l. Apparently one of the factors that determine the critical oxygen concentration is the flux of electrons to the cytochrome oxidases, as decreasing the flux to the oxidascs increases the critical oxygen concentration (White, 1963b). When the electron-transport system becomes branched, the critical oxygen concentration is often raised (Sinclair et al., 1970). I n Staph. nureus, when the electron-transport system is induced by aeration, the cytochrome concentration increases faster than the cell inass and the critical oxygen concentration decreases progressively (Frerman and White, 1867). Systems with rapid rates of oxygen utilization and high critical oxygen concentrations are often branched. When measuring the critical oxygen concentration, at least three determinations should be made on each sample and there should be no dependence on the cell concentration (Sinclair and White, 1970). Simultaneous recording of oxygen concentration and absorbance change in the double-beam spectrophotometer permits ready identification of the aerobic and anaerobic steady states (Chance and Williams,

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1955a). I n the commercially available Aminco-Chance spectrophotometer, the authors found it necessary t o construct a rectangular cell so that the vibrating electrode could be out of the optical path of the spcctrophotometer. Simultaneous measurement of the rate of reduction of oxygen and of the alternative electron acceptor shows a clear branching of the electron-transport system. I n many bacteria, nitrate is not reduced until the oxygen is exhausted (Skerman and Macrae, 1957; Pinchinoty and D’Ornano, 1961; Lindeberg et al., 1963).

111. Interpretation of the Data Several steady-state reduction levels for the various components can be determined a t different stages of the reduction of the electrontransport system. Initially there is the steady state of the respiratory carriers during the time interval that the rate of oxygen uptakeis pseudozero order. At this time, the steady-state levels depend on the ratelimiting primary dehydrogenase. Up t o now it has been difficult to demonstrate respiratory control in bacteria. Some substrates reduce only a portion of the electron-transport system and the addition of multiple substrates can show whether the pathways overlap (White and Smith, 1964). A new steady state is usually assumed at, or following, anaerobiosis, although not all of the cytochromes may be reduced a t this time (Jones and Redfearn, 1967b; Sinclair et al., 1970). It has proved difficult to add sufficient oxygen t o study the rapid kinetics of cytochrome oxidation. Adding hydrogen peroxide and catalase or oxygen-saturated buffer sometimes is effective (Smith et al., 1970). The sequence of reduction or oxidation can often be determined by comparing the initial rates of oxidation and reduction with the overall rate of respiration. I n general, slower electron donors produce lower steady-state reduction levels than faster donors, and blocking the terminal step of the chain with carbon monoxide or cyanide greatly increases the redox level of reduction (Kroger and Klingenberg, 1967). A very useful technique involving a pulsed-flow apparatus (Chance, 1951a) and the double-beam spectrophotometer allows direct calculation of the rate constants of each of the elements of the respiratory chain for which wavelength pairs are sufficiently unique (Chance and Williams, 1955b). Inhibitors have been used to help determine the sequence in the respiratory chain. It is usually assumed that inhibitors have a similar locus of inhibition in mitochondria and in bacteria, although this has been clearly shown not to be true in some cases (Jurtshuk et al., 1969b). It is also assumed these inhibitors do not have multiple sites of action, an assumption that must be established in each instance. The same caution should be observed in the interpretation o f data when artificial

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electron donors or acceptors are used. By-pass reactions are common and donors must be shown not to have multiple sites of reaction. TT'hen a review such as this is written, the fragmentation of much of the work reported on bacterial electron-transport systems becomes obvious. Perhaps the following criteria could make such studies much easier to correlate : ( 1 ) Identification of the respiratory pigments should be made using reduced-minus-oxidized and reduced-plus carbon monoxide-minusreduced differcncc spectra a t room temperature and at liquid nitrogen temperature. Diffcrent substrates and dithionite should be used. (2) Spectra should be correlated with the chemical identification of the extractable and covalently bound haems by their pyridine haemochromes. The amount of each cytochrome should correspond t o the amount of its haem. (3) Determinations should be made of the rate-limiting step in oxygen utilization and of the critical oxygen concentration. (4) Dctermination should be made of the sequence of reaction with substrate or oxygen either with a pulsed-flow apparatus or with specific inhibitors if they can be found. ( 5 ) The kinetics should be examined for discontinuities that are suggestive of branching, particularly in relation to the reduction of alternative terminal electron acceptors. Particular attention to overlapping cytochromes should be paid when choosing wavelength pairs and in the interpretation of the data.

IV. Branched Electron-Transport Systems A. HALOPHILIC BACTERIA The lialophilic strict aerobes require 4-5 M-sodium chloride for optimum growth and contain a complex electron-transport system. Halobacterium cutirubrunz contains two b-type cytochromes, possibly two c-type cytochromes and an a-type cytochrome (Lanyi, 1968). These cytochrornes were detected at -196'. The presence of haem a, protohaem and haein c was verified with pyridine haemochromes. Broken-cell preparations rapidly oxidize NADH2, L-a-glycerophosphate and succinate. Oxidation of NADH, involves cytochrome b,5, if measured a t pH 7.2 and cytochromes b,,, and b,,, if measured at pH 9-4. Further work (Lanyi, 1969) has indicated that reduction of the two cytochromes b show different salt dependence. The rates of reduction of the two cytochromes b are very much slower at both pH 7.4 and 9.4 than the overall rate of NADH, oxidase activity, although the rate of cytochrome

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oxitlascb (asvorbate-tetramethyl-p-phenylenediamine as donor and oxygen as acceptor), equine cytochrome c reductase or menadioiie rcductase (NADH, a s donor) and NADH, reductase (2,G-dichloroindophenol as acceptor) are all equal or greater t h a n the overall NADH, oxidase rate. Lanyi ( I 969) concludes t h a t the cytochromes b arc not on the main pathways of electron transport. However, he notes t h a t t h e rates of oxidation of tlic cytochromes ?I are too rapid t o measure acciilatvlj~ Ratcls of i d u c ~ t i o i arc. i not always valid criteria for inclusion i t i t l i t , direct c1ec.t ron-t I-nnsport lmthn-ay between KAD H and oxygen. Sii1c.c iinc*oupled SJ qteins llilve the primary dehydrogenase as tlie ratelimiting step in all the systrms t h a t have been carefully studied, rates of oxidation are n much more valid criteria for assessing the participation of ;L component iii a given electron-transport chain. Laiiyi ( 1969) carefully tried t o assess the rates of the primary dehydrogenases with artificiid donors or acceptors, but i t is still possible t h a t these agents mrasure shunts or bypasses rather t h a n the direct pathways. The c011cc11trittioiI for 507{, inhibition b y HOQNO (2-n-hepty1-4-hpdroxyquinoline N-osidc) for c;i(shof tlie cytochronies 11 differ b y a t least 40-fold. This compound inhil)its clcc%ronflohv to oxygen, cytochromc c, FRIP; aiid cytovhrome 135(i:3 h u t iiot t o q.toc~hroineh,,, using NADH, as electron d 0 1 1 0 1 - . Siicac*in;Lt(x oxidilsc is not inhibited by HOQNO. This result sugi t sc.i)watc>pathway through cytovhrome b,,, t o oxygen. Lanyi caonsidcr the I)lil(’(’ of‘ cytoclirome o in his formulation, ruin tyl)ical of cytovhrome o was detcvted (Lanyi, 1968). Tlie possibility of damngc t o the delicate membrane by incubation at, 1)H 9.4 wuld also csornplicatt. the analysis. The time-course of the a l ) ~ ~ of ~ cytoclirome i i ~ i ~ h,,,, iit p H 9.4 would be useful in determining whether incubation nt the alkalinp pH value is introducing an artifact. Clicnli ( 1 969) liils also studied the electroii-transport system of H . cicii~icb~i~n7 and found cytorliromcs of t h e a, b and c types (as well as tlic appropriate hacms). Some of t h e cytochrome a combines with carbon monoxide, and h r refers t o the carbon monoxide-combining portion a s c~ytoc~hi*onic~ a3. Cytochronit. o is also present and may account for about linlf t he 560 n m . (c*ytoc~liroine 13)absor1)tion. Both L-cr-glyceropliosphate and ascwbate r r d u c ~the c~ytochromeb and a. The alpha maximum of cytochromt. 1) is at 557 n m .a t 2 2 - after reduction of membrane fragments with ~ l ~ - c r ~ r o ~ ~ l i o swhereas p l i a t e the alpha maximum is a t 560 iim. after rctluc*tion 1% itli ascorbate. The two maxima suggest t h a t a t least two cytocsliromes 11 aiid possib1.v also cytochromc o are present. Oxidation of reduced niemlxtne fragments in the presence of 74 &-HOQNO rwcnls the prcsriicc of reduced cytochrome cjj3. 2-n-Heptyl-4-hydroxyquiiiolinc N-oxide inhibits cytochromc reduetion between cytocliromes 1) and c.

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At the temperature of liquid nitrogen, cytochromes a , b and c are reduced by dithionite but only cytochromes a and b (plus cytochronie 0) are reduced by ascorbate. Ascorbate in the presence of tetramcthylp-phenylene diamine reduces cytochromes a , b and c. Apparently this reductant is necessary for electron donation t o cytochrorne c. A soluble haemoprotein with protohaem as the prosthetic group and with the spectral characteristics of cytochrome o (maxima at 41 8, 338 and 575 nm. ; minima at 430 and 558 nm. in the dithionite-reduced-pluscarbon monoxide-minus-dithionite reduced difference spectrum)has been isolated. This 1)igment is recovered from particles after treatment with buffers of low ionic strength. This haemoprotein is not reduced by ascorbate and could not oxidize reduced equine cytochrome c. It is not possible to conclude whether this haemoprotein is denatured cytochrome oxidase o or sonic other protohaem-containing protein. Further work has shown that 12.halobium, H . salinarium as well as H . cutirubrum contain cytochrome oxidase o (Cheah, 1970a). Addition of 2 mM-cyanide eliminates the Soret absorption of cytochrome a3 from the reduced-ph~s-carboiimonoxide-minus-reduced difference spectrum of cytochronie oxidase 0. The carbon monoxide complex of reduced cytochrome oxidase a , can be recorded separately from cytochrorne o if the spectrum is recorded in the first few minutes of sparging with carbon monoxide. As sparging continues the cytochrome a3 (absorption maximum 431 nm.)disappears in thelarge cytochromeo-carbon monoxide complex with its maximum a t 416 nm. These techniques suggest very strongly that cytochrome oxidase a3 ( H . cutirubrum, H . salinarium) or cytochrome oxidase a , (EI. halobium) have a much higher atfinity for carbon monoxide and cyanide than cytochrome 0. A differential rate of reaction of cytochromes a3 and o with carbon monoxide has also been reported in Bacillus megaterium (Broberg and Smith, 1967, 1968) and in the cestode Moniexia expansa (Cheah, 1968). Using the hazardous technique of choosing wavelength pairs in the Soret region of the spectrum of H . halobium, Cheah (1970b) has found some strange results during reduction of the cytochromes by ascorbatetetramethyl-p-phenylenediamine. The aerobic steady state lasts 8 sec. for cytochrome a , (measured as 442-465 nni.) and 1 7 see. for cytochrome b (430-410 nm.). In the aerobic steady state, 337; of the enzymically reducible cytochrome b and 29% of the enzymically reducible cytochrome a , are reduced. Addition of 1 mM-cyanide has no effect on the cytochronie al but increased the reduction level of cytochrome b. The kinetics of reduction of particles from H . salinarium have also been studied (Cheah, 1 9 7 0 ~ )The . author used wavelength pairs of 606-630 nm. for cytochrome a , 444-4636 nm. for cytochrome oxidase a , and 560-575 nm. for the cytochrome b complex. The cytochroine b absorption

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includes components with maxima a t 555, 557 and 562 nm. when the spectra are examined a t -196’. The time between substrate addition and the aerobic steady state using ascorbate as the electron donor is 2 . 5 sec. for both cytochrome b and a3 but 14 sec. for cytochrome a, suggesting that cytochrome a is on a branch of the main electron-transport system. Both ascorbate or ascorbate plus tetramethyl-p-phenylenediamine produced rates of electron transport 25-fold greater than succinate or L-a-glycerophosphate with these particles. The “branch” involving cytochrome a could be the result of the artificial electron donor, ascorbate. Acceptors other than oxygen have not been examined, but careful experiments with these organisms show that a t least two cytochrome oxidases function simultaneously and that one has a higher affinity for cyanide and carbon monoxide than the other. B. ACHROMOBACTER

Achromobacter strain D contains cytochromes a l , a , and b which are reduced by primary dehydrogenases reacting with succinate and NADH, (Mizushima and Arima, 1960). The cytochromes can be isolated in membrane fragments. The reduced pyridine haemochromes of protohaem and haem a, were prepared from the acid-acetone extracts of the membrane fragments. When these cells are grown in the presence of high concentrations of oxygen, respiration (measured as the rate of oxygen uptake in the presence of substrate) is inhibited 50% by 10 pill-cyanide. Cells grown with 1 mM-cyanide or with low oxygen concentrations form cytochromes al and a2. I n the cells containing cytochrome a2, the respiration is 50% inhibited by 1 miM-cyanide, and is also more resistant to azide and 2,3-dimercapto-l-propanolthan the cells grown without cyanide. Cytochrome b measured with wavelength pairs 561-545 nm. is reduced more slowly than cytochrome a2 (measured a t 630-650 nm.). These organisms contain large amounts of cytochrome o which probably contributes significantly to the absorbance a t 561 nm. Reduction of cytochrome a2 by succinate is markedly inhibited by 1 mN-cyanide. When a suspension of membrane fragments, reduced in the presence of succinate, is aerated both cytochromes b and a2 oxidize “instantaneously” and then reduce a t the same rate as before aeration. However, addition of 1 mM-cyanide slows oxidation of cytochrome b and prevents oxidation of cytochrome a2. I n a later study (Arima and Oka, 1965) cytochrome oxidase o was shown to be the oxidase in cells grown with high aeration. I n cells grown with 1 mM-cyanide twice the cytochrome b and a,, three times the cytochrome a , and half the cytochrome oxidase o are formed. Cells grown with low aeration form a similar pattern of cytochromes to that

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of cells grown with high aeration in the presence of cyanide. If the extinction coefficient for cytochrome o a t 416 nm. is assumed to be 80,000 (Chance, 196lb), approximately half the absorbance a t 560 nni. is cytochrome oxidase o. This probably accounts for the slow reduction at 560 nm. reported as “cytochrome b”. Achromobacter will also grow mitli nitrate as the terminal electron acceptor. After growth with nitrate the cells contained three times the aerobic level of cytochrome b, half the aerobic level of cytochrome a , and o and about the same level of cytochroine a,. Tlie cyanide concentration for 50% inhibition of the respiration of nitratc-grown cells is intermediate between that of the resistant and higlily aerobically grown cells. No c-type cytochroine could be detected under any of the growth conditions. Tlie authors have attempted t o account for the changes in the resistance to cyanide by showing that the bacteria synthesize a cytochrome which progressively increases the resistance to cyanide (Oka and Arima, 1965). Accompanying the increase in cyanide resistance, a cytochrome oxidase is synthesized whose carbon monoxide complex is dissociated by red light. Dissociation of the carbon monoxide complex is measured by the relief of inhibition of oxygen utilization. The red light supposedly dissociates thc carbon monoxide complex of cytochrome oxidase a , (absorption maximum at 637 nni.). The carbon monoxide complex of the oxidase of the cyanide-sensitive cells is dissociated by light passed through a blue filter which transmits light in the Soret region. Red light does not relieve the inhibition in the cyanide-sensitive cells. The interpretation of this experiment assumes that there is insignificant absorption for cytochrome a , in the Soret region. Using bacteria that contain both cytochromes o and a,,the blue light becomes less and less effective in relieving the inhibition of the carbon monoxide as the cyanide concentration is raised. This indicates that cytochrome o is inhibited by cyanide. Thc release of inhibition by red light is not affected by an increase in cyanide concentration. The cytochrome a , is the cyanideresistant oxidase. This organism modifies the composition of its electron-transport system when the external environment is changed. These cells evidently contain at least two cytochrome oxidases that function simultaneously. These oxidases are cytochrome oxidase a,, which is less sensitive to cyanide and whose carbon monoxide complex is photodissociated by red light, and the cytochrome oxidase o which is more sensitive to inhibition by cyanide and which has a carbon monoxide complex that is photodissociated by blue light. Nitrate can serve as an alternative terminal electron acceptor for growth and cytochrome oxidation in these cells (Arima and Oka, 1965). Addition of 50 pH-nitrate to a suspension of cells inhibits the rate of

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oxygcn utilization in the presence of succiiiate by 2.5-folcl. This indicates that these cells can simultaneously reduce both oxygen and nitrate, which is unusual in bacteria, and indicates the presence of a further branching of the electron-transport system that is similar t o that found in H . parainjluenzae. Nitrite is formed from nitrate by the Achromobactcr nitrate reductase, but is not reduced by this strain of Achromob a c k . Also nitrite has no efTect on the initial rate of oxygen utilization, wlicreas it increases the critical oxygen concentration. Comparison of the rates of oxygen utilization in the presence of higher conceiitratioiis of nitrite indicates that nitrite is a competitive inhibitor with oxygen for cytochrome oxidase. Nitrate inhibits oxygen utilization by diverting the flow of electrons from the oxidases to the nitrate reductase. Nitrite inhibits oxygen utilization by competing with oxygen for the cytochromc oxidases. The cytochrome a , of Escheiichia coli, Aerobacter aerogenes and Pseudomonas pseudomallei is the most resistant to cyanide of the oxidases found in these organisms (T. Oka and K. Arima, unpublished results).

C. AZOTOBACTER Azotobactvr has a complex electron-transport system. Membranebound primary dehpdrogenases liavc bceii demonstrated for succinate, D-lactate and NADH, (Jones and Redfearn, 1966; Jurtshuk and Harper, 1968; Jurtshuk et al., 1969a). The activity of these dehydrogenases is not dependent on NAD. Numerous artificial electron donors and acceptors will react with the primary dehydrogenases. Rates of oxygen uptake i n the presence of several substrates added simultaneously have not be011 reported. The fact that the total rate of oxygen utilization for several substrates is less than the sum of the rates for individual substrates indicates that the pathways overlap aiid electrons competr for a ratc-limiting electron carrier other than the primary dehydrogenase. Coenzyme Qy (about 2 pmoles/g. dry weight) is found in the membrane fraction (Lester and Crane, 1959; Jones and Redfearn, 1966). No trace of menaquiiiorie was found by the latter workers. Cytochronies a,, a,, 0,b aiid c can be detected in the difference spectra (Temperli and Wilson, 1960; Jones and Redfearn, 1966). Examination of the difference spectrum at -196" did not show additional components (Jones and Redfearn, 1066). There is no splitting of the alpha maxima of either cytoclirome Z, or cytochrome c a t -196" although the maxima shifted from 560 nm. at 25" to 557 nm. a t -196" and from 561 to 549 nm. The cytochrome c actually consists of two cytochrornes which have been purified; cytochrome c1 (alpha maximum 551 mi.)aiid cytochrome c5 (alpha maximum a t 5.55 nm.; Swank and Burris, 1969). I n the strain used by Jones and

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Redfcarn, there was very little cytochrome c5. The redixced-plus-carbon monoxide-minus-reduced difference spectrum reveals t h e presence of cytochrome a L ,cytochrome a , and cytoclirome o (Jones and Redfearii, I!)67a). From the absorbaiice difference between 416 iiin. a n d 435 iim. i n the carbon monoxide-reduced-plus-carbon monoxide-minus-reduced spectrum, and the extinction coefficient of 80,000 for cytochrome 0, the contribution of cytochrome o t o the absorption a t 560 iim. (cytochrome h ) at room temperature is calculated t o he about 5%. The photochemical avtion spcctriim for tlie re1ic.f of carbon iiioiioxidr inhibition of respirat ion shows tnasima at 638 nni. (cytochrome a,), 590 11111. R I I 4%) ~ iim4:lfi mi (possibly c.ytoclirorne a,) a n d in tlw 5 2 0 ntii -540 nm. raiigc (cytoclirome 0 ) (Jones : ~ n dIicdfearn, 1 9 6 7 ~ ~The ) . relief of inhibition due t o light! a t 530-540 i i i n . was difficult to measure reproducibly and l ) ( ~ h a l )refhats s t h c lov affinity of cytochrome o for c~arbonmonoxide. 1)uriiig most growth conditions there is remarkably little variation in the 1)roportions of the cytochrornes (Knowles a n d Redfearii, 1968). In cells qronn on urea-containing medium tliere is a 5 - t o %fold decrease in cytoclirornc a , and poqsibly a small but defiiiitc inci.ease in the ratio of cytoclirome c :cytochrome 6. During all growth conditions, the mol:ir ratio of coenzyme Q8 t o cytochrorne b is constant, very much like the mtio of 2-detnetliylvit:~tnin K, :cytochrome h in H . parainjzienane (IVliite, 1965;~). The kinetics of reduction of tlic cytoclironies of Azotobacter have bceti s t ndied with the double-beam spectropliotonietcr. Cytochrome c 1%as c4m;Ltcd TI ith the 551-544 iim. wavelength pairs, cytochrome b from the 560-568 11111. wavelength pairs, and cytoclirome a , from the 630-619 iiin. wnvclengtli pairs (Jones niid Redfearn, 196Ta). The absorbancies a t 560 and 551 nm. are nearly equal in these cells, which indicates at Icxnst a 3O0$ coiitribution by cytoclirome b t o the cytochrome c adsorption if the cytochromes have spectra similar t o those in H . parainJCuenzae. No attempt was made t o correct for tlie overlap of cytochrome c a t 560 nm. or cytoclirome b a t 551 nm., although the kinetic differences reported below indicate overlap may not have been serious. The cytochrorne o in tliese cells makes a very small contribution t o the 560 nm. absorption of cytochrome b , as discussed previously. In the aerobic steady state. the reduction level of cytochrome c (30-407/,) is greater t h a n the cytocliroine b (20-%?/,), which in t u r n is greater than the cytochroine a , (07;) when L-malate, succiiiate or NADH, are used as substrates with pnrticulate preparations. In the anaerobic steady state, 80-100~oof the cytocxliroincs are reduced. The cytochrome b shows slow reduction after anaerobiosis similar t o t h a t seen in H . parainfluenme (Sinclair et al., 1970). Jones a n d Rcdfcarri (1967a) conclude from these d a t a t h a t a typical linear arrangement of substrate, cytochrome b , cytochrome c.

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cytochromc a3, oxygen, does not exist in Azotobacter. In 12. parainjizcenzac the slow reduction of a cytochrome after anaerobiosis represents the activity of a cytochrome oxidase (cytochromc oxidase o) with a low affinityfor oxygen. Jones and Redfearn (1967a) show that ascorbate plus 2,6-dichlorophenolindoplienol(DCIP) maintained a high aerobic steady-state reduction level of cytochrome c without having much effect on the aerobic steady-state reduction level of cytochrome b , and suggcsted that thc ascorbat,e-DCIP mixture donated electrons directly t o the rytoclirome c. Jurtshuk ~t al. (196913) found that ascorbate alone reduccd cytorhronic c, but not cytochromes 6 , a , and a,, suggesting again that there are two pathways, one involving cytochromes c and o and the other cytochromes b, a , and a,. Jones and Redfearn (1967a) used inhibitors to explain the kinetic data. Low concentrations of cyanide (40 p N ) or azide (4 mM) completely inhibited oxygen utilization with a mixture of ascorbate and DCIP as electron donor and had little effect on oxygen utilization with either succinate or NADH, as electron donors. High concentrations of cyanide ( 2)m & ! or azide (100 mM) are necessary for inhibition of succinate or NADH, oxidase. Since the ascorbate-DCIP mixture results in higher (79%) reduction of cytochrome c than succinate (4196) in the aerobic steady state, Jones and Redfearn (1967a) proposed that succinate oxidase and ascorbate-DCIP oxidase involve different pathways. I n this experiment both the succinate oxidase and ascorbateDCIP oxidase have the same specific activity ; the differences in steady states are not due to different rates of electron flux. Inhibitors were used to support the suggestion that electron flow is branched to two oxidases, one pathway containing cytochromes b and a, (insensitive to low concentrations of cyanide and azide) and another containing cytochromes c and 0 (sensitive to low concentrations of cyanide and azide). Low concentrations of azide ( 2 ma!) increase the reduction level of cytochrome c and are without effect on the reduction level of cytochrome b in the aerobic steady state. High concentrations of azide (200 mM) substantially increase the aerobic steady-state reduction level of cytochrome b when the particles are reduced with NADH,. The aerobic steady-state reduction of cytochrome b is not much affected by high concentrations of azide when succinate is used as donor, but the clcctron flux from succinate is four times less than from NADH,. If succinate dehydrogenase is inhibited by malonate, the aerobic reduction of cytochrome b is decreased more than that of cytochrome c. This suggests that electrons from succinate preferentially reduce cytochrome c. I n the presence of malonate, high Concentrations of cyanide ( 5 m N )which inhibit both cytochrome oxidases raise the aerobic steadystate of sytochrome c considerably and produce much less than the expected increase in the aerobic steady-state of cytochrome b. Thus, a t

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thc. low dehydrogenase activity, the steady state of cytochronic 6 cannot be iiicreascd by oxidase inhibitors, although the steady statc of cytocbhrome c is increased considerably. This suggests that cytochrornes c and h inay be attached to separate oxidases of different activities and wnsitivities to inhibitors, and that most of the electron flux involves cptochrome c. From the inhibitor data, it appears that NADH, oxidasc involves c,ytochromes b and a,. NADH, oxidase is strongly inhibited by carbon monoxide. Inhibition of NADH, oxidase by carbon nionoxidc is reversed by strong white light as well as by red (610 nm.) light. Blue light (385-490 nni.) is effective in the relief of carbon monoxide inhibition only a t very low concentrations of oxygen. Addition of 50 piW-cyanide has no effect oil tlie pattern of relief of inhibition of carbon monoxide by light or on the rate of respiration. Cytochrome oxidase a , is an oxidase which has i~ high affinity for oxygen and carbon monoxide, a low affinity for cyanide and azide, and a carbon monoxide complex that is photodissociated by red light. The ascorbate-DCIP (cytochromes c and 0) oxidase activity is less sensitive to carbon monoxide inhibition than NADH, oxidase, but the carbon monoxide inhibition that is present is relieved by both blue and red light. Cyanide (50 pLM) inhibits both respiration and relief of carbon monoxide inhibition by blue light. These data implicate cytochrome oxidase o as the oxidase of ascorbate-DCIP oxidase activity. Cytochrome o is an oxidase with a low affinity for both oxygen and carbon monoxide, a high affinity for cymide and azide, and a carbon monoxide complex that is photodissociated by blue light. The photodissociation of the cytochrome o-carbon monoxide complex by red light is unexpected. Jones and Redfearn (1967a) suggest that a charge-transfer reaction causes the dissociation. Evidence thus far indicates the existence of two pathways : succinate or NADH, to cytochrome b to cytochrome oxidase a , to oxygen, and ascorbate-DCIP to cytochrome c to cytochrome oxidase o to oxygen ; both are functional in Azotobacter. There is an overlap of these pathways as parts of each can be involved in both reactions. Jones and Redfearn (1967b) were able to fractionate membrane preparations into red and green particles by treating the membrane with deoxycholate and 660 mM-potassium chloride. The particles were fractionated on sucrose density-gradients and further purified. About 20yo (10% of each type) of the protein in the original membrane preparations was recovered in the red and green particles. NADH, and malate oxidase activities were destroyed The green particles contained predominantly cytochromes b , u 1 and a,. No succinate oxidase or succinate diaphorase activity could be detected in the green particles, but a “considerably lower” rate of

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asc~orb~~tc~-diclilorol,henoli~ido~~lie~iol, ascorbatc-tetramethyl-~-~,l?eng.lencdianiine and ascorbate-cytochrome c4 plus c5 oxidase activities could be detected. As expected for cytochrorne oxidase a,, tlie oxidasc activity was rcmarkably insensitive to inhibition by 50 pM-cyanide and 2 mMnzidc. The rcd particles retained 5076 of the succinate oxidase activitj and tt.n times the ascorbate-DCI P activity of tlie original niembrane 1)repwations. The green particles contained primarily cytochromes c aiid cytocliromc oxidase 0. As expected, the oxidase activity was markedly inhibited by 50 pJl-cyanide and the inhibition by carbon nionoxide was rclieved b) blue but not by red light. Separation of the red and green particles is a significant advance in the understanding of the branching in Azotobacter. Some caution is necessary in interpreting the separation of functional complexes. The re-assembly of complexes into a partially functional system has been used as an argutnent for sequential linear arrangements in mitochondria1 studies (Yamashita and Racker, 1969). Despite the untimely death of Eric Redfearn, definitive experiments on this system have been initiated by Colin Jones. Jones is attempting to establisli that two pathways for ATP synthesis exist in Azotobacter. Acccptablc P : O ratios for NADH, ( 1 - l ) ,succinate and lactate (0.8), inalate (0.6) and ascorbate-DC1P (0.2) have been reported for carefully prepared incinbranch fragments from Azotobacter (Ackrell and Jones, 1970). Swank and Burris (1969) have shown that oxidation of reduced purified cytochromes c 4 and c5 by particles is inhibited 50% by 10 pLM-cyanide.Tlicy suggest that P :0 ratio measurements in the presence or absence of low concentrations of cyanide would establish the activity of tlic two pathways in ATP synthesis. This work with Azotobacter represents one of the most carefully studied bacterial electron-transport systems. L).

Esche~ichiacoli

Microbial electron-transport systems contain either the benzoquinonc, coenzyme-& or the naphthoquinone, vitamin K,. Escherichia coli coiltains both, and the studies of the interaction of pathways containing these two quinones can provide insight into the function of the electrontransport system. Branching in the electroii-trailsport system of E . coli involving the (pinones was first studied by Kasliket and Brodie (1963a). Large and small particles from cells may be prepared which have a small but significant capacity for oxidative phosphorylation. The particles differ in their ability t o oxidize malatc and succinate. The large particles contain cytocliromes a g ,b , 0, and coenzyme Q8 but no vitamin Kg(45) and oxidize succinate ten times more rapidly than inalate. Small particles

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cvxitain the same pigments, but 0.14 pmoles vitamin K,(45) per milligram protein. The small particles oxidize malate as rapidly as succinate. If the particles are irradiated a t 360 nm., succinate oxidase activity is lost and a “decrease in quinone concentration can be demonstrated” (Brodie, 1962). Addition of coenzyme Qlo sometimes restored succinate oxidase activity to the large particles and vitamin K,( 35) occasionally restored malate oxidase activity t o small particles if it was added with phospholipid. Some caution is necessary in the interpretation of the efTects of the 360 nm. irradiation which might not be specific in inactivating quinones (Itagaki, 1964). Irradiation a t 350 nm., 370 nm. and 254 nm. of particulate preparations from E . coli inactivates formate dehydrogenase, nitrate reductase and NADH, oxidase, and formatenitrate reductase almost to the same extent (Itagaki, 1964). Itagaki confirmed that vitamin K, in hexane is more sensitive to light a t 360 nm. than coenzyme Qs in ethanol. Bragg and Hou (1967a,b)found E . coli grown with high aeration does not contain vitamin K2. NADH, oxidase activity is located in a particulate preparation which also contains coenzyme QR.Irradiation with light a t 360 nm. depresses the activity which can not be restored by adding back coenzyme Qs. The proportions of coenzyme Qs and vitamin K, in E. coli have been shown to change with the culture conditions. Aerobic growth favours coenzyme Q formation and anaerobic conditions favour vitamin K, synthesis (Polglase et al., 1966). I n further studies Whistance and his colleagues (Whistance and Threlfall, 1968 ; Whistaiice et nl., 1969) confirm that in many species of enteric bacteria coenzyme Q is formcd during aerobic growth as well as 2-demethylvitamin K,. During anaerobic growth the content of 2-demethylvitamin K, decreases and that of vitamin I<, increases. Further studies of the effects of growth conditions and of the absence of alternative terminal electron acceptors should be very useful in studies of the role of the two quinones in electroiitransport pathways t o both oxygen and other terminal electron acceptors. Mutants unable to synthesize either coenzyme Q or vitamin K, may offer insight into the function of the quinones in electron transport. Multiple aromatic suxotrophs of E . coli are unable to synthesize coenzyme Q in the absence of 4-hydroxybenzoic acid. Rapid aerobic growth of this mutant requires 4-hydroxybenzoic acid (Jones and Lascelles, 1967) whereas anaerobic growth does not. The requirement for 4-hydroxybenzoic acid for aerobic growth can be spared by succinate and amino acids that spare the succinyl-CoA requirement. I n the absence of 4-hydroxyybenzoir acid, alternative mechanisms for the oxidation of NADH, appear (lactateand ethanol dehydrogenases). The mutant forms coenzyme Q only when 4-hydroxybenzoate is present (Jones, 1967).

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Little or no vitamin K, is formed. Cells grown without 4-hydroxybenzoate have no detectible coenzyme Q and have very low particulate NADH, oxidase and NADH,-cytochrome hl reductase activities. Preincubation of the particles with coenzyme QBrestores activity. Vitamin K, was ineffective. The primary NADH, dehydrogenase measured by various diaphorase activities is not affected by lack of coenzyme Q. Succinate oxidase is much less affected by the lack of coenzyme Q than NADH, oxidase activity. The involvement of coenzyme Q in NADH, oxidase activities indicates that this strain of E . coli is not a t all like the strain studied by Kashket and Brodie who found NADH,-linked substrates utilized a vitamin K2-linked pathway exclusively. The use of quinone mutants has been exploited by Gibson’s group and may finally lead to definitive answers to the role of the quinones in electron transport. Mutants unable to form coenzyme Q and which contain five times the normal amount of vitamin K,, or mutants forming no vitamin K, but three times the normal amount of coenzyme Q have been isolated (Cox et al., 1968a)b).Malate oxidase activity in particles from the coenzyme Q nuxotroph is stimulated 150% by addition of coenzyme Q versus a 30% stimulation of the wild-type activity. Menadione stimulates the malate oxidase activity of this mutant 10yo a t five times the concentration of coenzyme Q. This suggests that, in the coenzyme Q mutant, lack of coenzyme Q is the rate-limiting step in malate oxidation. Coenzyme Q in the particles of the wild type is fully oxidized and becomes 35% reduced within 2 min. after addition of malate. It seems clear that, in this strain of E . coli, the vitamin K, is not involved in the malate oxidase pathway. Dicoumarol is supposedly a specific inhibitor of vitamin K,-mediated electron transport. Dicoumarol unexpectedly inhibits malate oxidase in the low vitamin K mutant to a much greater extent than it does in the low coenzyme Q mutant. Piericidin A, an insecticide, inhibits the coenzyme &-dependent NADH, oxidase, malate oxidase and lactate oxidasc The inhibition is reversed by the addition of coenzyme Q. Piericidin A also inhibits the a-glycerophosphate and dihydro-orotate oxidases but this inhibition is not reversed by coenzyme Q (Snoswell and Cox, 1968). Further development of strains using cotransduction systems, and their characterization both biochemically and genetically (Cox et al., 1969)has allowed more intensive studies on the involvement of coenzyme Q in aerobic respiration (Cox et al., 1970). The coenzyme Q mutant when grown under aerobic conditions gave an anaerobic type of growth yield, and accumulated lactate. There was no difference in aerobic growth yields or in anaerobic growth yields in the presence of nitrate between the mutant and the wild type. Itagaki (1964) however concluded that

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coenzyme Q is involved in the NADH,-cytochrome b, nitrate reductase complex. The oxidase activities for NADH,, lactate and malate are markedly decreased, the concentration of cytochrome o is a little lower and the concentrations of the flavins and cytochrome a , are somewhat higher in the mutant. The concentration of cytochrome b is the same in the mutant and wild type. I n the wild type the molar ratio of coenzyme Q : cytochromc: b , is 25 : 1 and in the mutant < 0.2 : 1. The mutant contains a vitamin K,:cytochrome b , molar ratio of 14: 1, and the wild type a molar ratio of 3.5: 1. Using a double-beam spectrophotometer and the wavelength pairs for cytochrome b (560-570 nm.), cytochrome a, (630615 nm.) and “flavoprotein” (475-495 nm.) (see p. 179), the aerobic steady-state reduction was determined in the wild type and mutant using NADH, and lactate as substrates. I n the mutant, the lack of coenzyme Q causes an increase in the aerobic steady-statereduction of the “flavins” and cytochrome b, without affecting cytochrome a,. This effect was similar to that of 130 p M piericidin A or 2-n-heptyl-4-hydroxyquinoline N-oxide on the wild type. From further work with electron spin resonance, these authors have concluded that coenzyme Q is not on the main pathway of the electrontransport system (Hamilton et al., 1970) functioning in a t least two positions. The effects of piericidin A are explained by its separating ubiquinone from the remainder of the chain. Birdsell and Cota-Robles (1970) attempted to prepare particles from E . coli with the properties of the Kashket and Brodie preparation. No difference in the composition between large and small particles was detected. I n summary, it appears that the evidence places coenzyme Q in the E. coli electron pathway between the flavin-linked dehydrogenases and the cytochrome oxidases with a vital but possibly indirect role i n electron transport. The relationship between pathways containing vitamin K, and coenzyme Q remains for future studies. Evidence for branching in the electron-transport system in membrane fragments derived from E. coli has been presented by Birdsell and Cota-Robles (1070). Cyanide (1 mM) inhibits formate oxidase by 98% but inhibits NADH, oxidase by 39% and succinate oxidase by only 13%. Azide (20 mM) inhibits formate oxidase but had very little effect on either NADH, oxidase or succinate oxidase. These results may indicate separate electron pathways from formate to oxygen and from NADH, and succinate to oxygen. Cyanide and azide may have inhibited the formate dehydrogenase rather than the terminal oxidase in which case the cytochromes b would not be more reduced in the presence of the inhibitor during the aerobic steady-state than in the absence of the inhibitor. Azide does not increase the aerobic steady-state of cytochrome b but inhibits oxygen uptake by 98:4 indicating that the inhibitory

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effect is probably not a t the oxidase. Pairs of substrates or substrates added in sequence reduce a t anaerobiosis less cytochrome b than the total reduced with each substrate added individually. This may suggest that there are overlapping electron pathways, although the rates of oxygen utilization and the aerobic steady-state reductions of the cytochromes would have been a more definitive measurement. The inhibitor 2-nheptyl-4-hydroxyquinolineN-oxide does not increase the aerobic steadystate reduction of cytochrome b with NADH, or succinate as substrates, even though the initial rate of respiration is inhibited by up to 82%. Perhaps part of the cytochrome b is not on the direct pathway t o oxygen, or 2-n-heptyl-4-hydroxyquinoline N-oxide is inhibiting after the cytochrome b as suggested by Cox et al. (1970). Escherichia coli reduces both nitrate and nitrite. The electron-transport systems involved in their reduction have been studied intensively and suggest further branching of the systems. An NADH2-nitrite reductase system is formed in E . coli, but this system apparently does not interact significantly with the rest of the electron-transport system. The system involves a NADH, dehydrogenase, cytochrome c552and nitrite reductase (Fujita and Sato, 1966; Cole, 1968). Addition of nitrite t o growing anaerobic cultures stimulates a rapid synthesis of cytochrome cjl,?, about 89yoof which seeins to be non-functional and can be released on rupture of thc cells (Cole, 19G8). The cytochrome is present in much greater amounts than is necessary for optimum activity of the NADH2-nitrite reductase system. Escherichia coli evidently can synthesize excess cytochrome c under conditions when Haemophilus parai??,fluenzaealso produces excess cytochrome c. The exact function of the E . coli cytochrome cjg2 in the KADH,-nitrite reductase system is not completely settled as yet. Wimpeniiy ( 1 969) summarized the effects of the presence of different electron acceptors on the formation of the cytochromes, nitrate reductase and nitrite reductase activities in E . coli. With an anaerobic chemostat, Cole and Wimpcnny (1968) found that cytochrome c552,cytochrome a2 and nitrite reductase are formed maximally when the nitrate in the feed solution is a t 20 mM. Above a concentration of 20 m M nitrate, nitrate reductase and cytochrome b activities increase. Nitrate oxidizes endogenously reduced cytochromes a*, a2, b and c, but only cytochrome c is oxidized hy nitrite. Formate does not reduce cytochrome c. From the growth experiments and the specific oxidation and reduction of the cytochromes, it is concluded that there is an NADH,-nitrite reductase pathway which includes cytochrome c and a formate-nitrate reductase pathway that includes cytochrome b. The location of the nitrate reductase system in the meinbrane has been confirmed by Showe and DeMoss (1968). Mutants lacking the ability to

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reduce nitrate call be selected (Ruiz-Herrera et al., 1969). A total of 38 isolates were selected, all of which have less than 7% of the nitrate reductase activity of the wild type. The mutants have decreased activity of the formate deliydrogenase, cytochrome b,,, or nitrate reductase, 01' combinations of two lesions. All cytoelirome b mutants have between 10 and 4Oq/, of the wild-type level of cytoclironie b and no mutants lack all tliiw activities. Revertants a t rates of' lo-.' indicated that many of thc mutants were point mutations. The three proteins evidently are essential for nitrate reductase activity and i t is unlikely that other proteins arc involved as no mutants were detected which retained all three activities. No nieasurenients of coenzyme Q or vitamin K, were reported. I t a p k i ( 1964) showed conclusively that coenzyme Q was functional in a t least one type of nitrate reductase pathway. Since three mutants lacked only formate dehydrogenase activity, formate is probably the only donor for the nitrate reductase system. Therefore the nitrate reductase system cannot be an alternative mechanism for oxidation of rcduced nicotinamide nucleotide. This is the role usually assumed for the elcctroii-transport system with alternative electron acceptors. The intcraction of the nitrate reductase system with other portions of tlic E . coli elcctroii-transport system has been examined (RuizHerrcra and DcJIoss, 1969). Mutants defective in formate dehydrogenasc are of two types. Some are unable to reduce methylene blue but still reduce bcnzyl viologcn and produce gas anaerobically in the absence of nitrate. These mutants are classified as retaining the formate dehydrogenase necessary €or formate hydrogrnylase activity. Escheiichia coli thus has a t least two formate dehydrogenase activities. Since these mutants revert at a high rate, it seems that the two enzymes share some common subunit. The authors also detected two types of cytochrome b . If the cells are grown anaerobically in the absence of nitrate they form cytocliroine hb5". When grown with nitrate, cytochrome b,,, is formed (examined i i i difYerencc spcctra measured at -196"). Mutants lacking cytocliromc b j5j but containing hs5*, and mutants with less of both types of cytochromes 11, have been found. The authors claim to have detected two cytocliroinc b,>, components which have identical spectra but have different kinetics of oxidation of the reduced cytochrome by the anaerobic addition of nitrate. The two components were identified by the lag in oxidation by nitrate, by the reduction of only one of them by ascorbate, tmd bjr the inhibition of the oxidation of one of them by 2-ii-heptyl-4-liydrox~c.juiiioliiieN-oxide. These data were obtained by dual wavelength spectrophotometry which were verified by reduced minus oxidized difference spectra. The cytoclirome b,,, seems to be involved in the NADH, oxidase pathway. Tlic authors feel that the formate-nitrate reductase pathway

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is distinct from tho rest of the electron-transport system in cdls in thc. log phase of growth although they note that the NADH,-nitrate reductase activity found in stationary-phase cells may be a part of the complete electron-transport system. Different types of nitrate reductase mutants have been intensively studied by Picliinoty's group. These mutants are resistant to chlorate, and map in a different part of the chroinosome from the formate-nitrate reductase mutants studied by DeMoss and his coworkers. TWOmutants whose extracts can complement each other when combined have been distinguished (Azoulay et al., 1969).These mutants are chlorate-resistant, lack nitrate reductase activity, hydrogenlyase activity, chloratc reductasc activity, and tetrathionate and thiosulphatc reductase activities. Cytochrome b is involved in this system, and the mutants have lost significant NADH,-oxidase activity, pyruvate dehydrogenase activity, and NADH,-ferricyanide reductase activity. These mutants offer the exciting possibility that they are defective in the organization of the membrane (perhaps by having a defective structural protein). The use of genetic analysis is increasing for the dissection of bacterial electrontransport systems, and it is likely that future advances will stem from the use of this powerful tool.

E. Haemophilus parainjluenxae The attention of our laboratory for the past several years has focussed on the formation and function of the electron-transport system of H . parainjuenxae. This organism has some remarkable characteristics that make it particularly useful in studies of the respiratory system. If the cells are washed in 50 mM-phosphate buffer (White and Smith, 1962), endogenous respiration stops. If the cells are returned to growth medium within ten minutes from the dilute buffer, the cells continue t o grow a t the pretreatment rate. The intact cells are remarkably permeable, and many substrates stimulate oxygen utilization (White, 1966). If NADH, is added to the washed intact cells, there is an immediate and rapid oxygen uptake with no measurable lag (R7hiteand Smith, 1962). When nicotinamide nuclcotide-linked substrates (malate, glucose, gluconate) are added to cells suspended in dilute buffer, nicotinamide nucleotide is reduced (100-200 nm./g. dry weight) after the oxygen in the cuvette is utilized. This can be detected as an increase in the absorbance a t 340 nm. after reduced nicotinamide nucleotide reduction is complete (White and Smith, 1964). Adding other metabolites, NADP or NAD, does not result in further increases in reduced nicotinamide nucleotides. If the cells are held a t 0" for several hours, the rate of oxygen utilization with many substrates can be increased by adding NAD (White and Smith,

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1964). These aged cells generate 100-times the NADHz found in freshly isolated cells. Reduced NAD generated from the added NAD accumuIates after the cells become anaerobic in the presence of a nicotinamide-linked substrate like citrate. When the suspension is rapidly aerated, the rate of oxygen utilization from the endogenously generated NADH, is equal to the rate at which added NADH, is oxidized (White and Smith, 1964). These experiments are possible as H . parainjuenzae has remarkable permeability, and apparently cannot oxidize reduced nicotinamide nucleotide in the absence of terminal acceptors for the electron-transport system (White, 1966). Haemophilus parainjhenxae is incapable of classical glycolytic growth. Growth, glucose catabolism and oxidation of reduced nicotinamide nucleotide occurs only in the presence of the terminal electron acceptors oxygen, nitrate, fumarate or pyruvate (White, 1966). Inhibitors of the electron-transport system like 2-nheptyl-4-hydroxyquinoline N-oxide or cyanide stop glucose catabolism, oxidation of reduced nicotinamide nucleotide and growth in the presence of terminal electron acceptors (White, 1966). It is possible t o achieve “pseudo” glycolytic growth by adding either 10 mM-NAD or a mixture containing 0-5 pN-NAD, 50 mM-glucose and beef heart lactate dehydrogenase to the anaerobic culture vessel (White, 1966; Sinclair and White, 1970). The haemin-independent Haemophilus species can grow glycolytically and do not form a detectable electron-transport system (White, 1963b). The ease with which the endogenous respiratory activity can be eliminated has greatly facilitated spectral studies of the respiratory pigments. Cytochromes a,, a2, 0, b and cb5, have been detected in difference spectra a t room temperature (White and Smith, 1962).A small amount of cytochrome c550can be detected in difference spectra a t -196” (White and Smith, 1962). Carbon monoxide combines with reduced cytochromes al, a2 and o (White and Smith, 1962), although the photodissociation action spectrum of the carbon monoxide-inhibited respiration corresponds only t o the absorption spectrum of cytochromes a2 and o (P. R. Sinclair and D. C. White, unpublished data). The cytochromes are reversibly reduced and oxidized by substrates and air. The anaerobic addition of nitrate, pyruvate, NAD or fumarate to cells reduced with formate results in oxidation of cytochrome c. A larger proportion of the cytochrome a , and the flavoprotein seems to be oxidized by nitrate than the other cytochromes (White and Smith, 1962). The small amount of cytochrome a I present in the cells has prevented further understanding of its role in electron transport. Protohaem and haem a2 ( d ) (identified from the spectrum of the reduced pyridine haemochrome) can be extracted from the cells with acid-acetone. The haem c remains in the residue. The amount of haems c and protohaem correspond to the 9

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amounts of cytochrome c and cytochromes b + o (Sinclair and White, 1970).

One of the most useful characteristics of H . parainfluenme is its ability t o modify the composition of the electron-transport system in response to changes in the external environment. The variations in the types and concentrations of the primary membrane-bound dehydrogenases are primarily effected by the nature of the major catabolites supplied in the medium (White, 1963a, 1964, 1967). The proportions of the cytochromes and of quinone are dependent on the nature and concentration of the terminal electron acceptor (White, 1963, 1965a; Sinclair and White, 1970) and are relatively independent of the major catabolite supplied to the medium (White, 1967). Haemophilus parainjluenzae contains the unusual quinone 2-demethylvitamin K, (Lester et al., 1964). Isoprenologues of 2-demethylvitamin K, with side chains of 0, 1, 2, 3, 4, 5, 6, 7, 8 and 9 isoprenoid units have been detected (Hammond and White, 1969). The cytochrome b and 2-demethylvitamin K, maintain a 1 : 1 5 molar ratio even though the cytochrome b content can vary eight-fold (White, 1965a). The other respiratory pigments apparently can be present in proportions that are independent of each other. High oxygen concentrations in the growth environment depress synthesis of cytochrome b and little or no cytochrome c, a,,or a2 is formed. Synthesis of cytochromes a , and c, and t o a lesser extent cytochromes b and 0, is stimulated by lowering oxygen tension or during anaerobic growth with alternative electron acceptors (White, 1963a; Sinclair and White, 1970). Under the conditions of extensive cytochrome formation, large amounts of cytochrome c are produced. Part of the cytochrome c is neither membrane-bound nor enzymically reducible, and can be recovered quantitatively in the supernatant after rupture of the cells (Smith and White, 1962; White, 1963a). The changes in the proportions of the respiratory pigments involve protein synthesis as they are inhibited in the presence of 0.15 pM-chloramphenicol (Sinclair and White, 1970), and these respiratory pigment changes coincide with transient changes in the proportions, rate of synthesis and turnover of the membrane phospholipids (White and Tucker, 1969). All of the combinations of respiratory pigments that have been examined possess the capacity for rapid oxygen utilization and all of the cytochromes that are membrane-bound are enzymically reducible. It is clear from these studies that the functional electron-transport system of H . parainjluenzae cannot be made up of simple structures consisting of subunits in which the components are present in stoichiometric amounts, as has been widely publicized as the basic structure for electron-transport systems (Green, 1964). Clearly, a large network with multiple “slots)’ is more likely to explain the variations in this organism.

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The remarkable permeability of the membrane of H . parainjluenzae allows a comparison to be made of the activities of the intact cells and various membrane components. Membrane fragments prepared by grinding with alumina, or rupture with the French pressure cell or Hughes press, have the same rates of oxygen utilization with NADH,, formate, succinate or D- and L-lactate per unit of cytochrome b as the intact cells (White and Smith, 1964).The substrate concentration giving half maximal rates of oxygen utilization are the same in the membrane particles and in the intact cells (White and Smith, 1964). The membrane particles contain all of the enzymically reducible cytochromes, the protohaem and haem d , the ferricyanide reductase activities of the primary dehydrogenases, the 2-demethylvitamin K2isoprenologues, the phospholipids and the extractable fatty acids found in the intact cells (White, 1964, 1965b; White and Smith, 1964; White and Cox, 1967). Rupture of the cells by mechanical means or by osmotic lysis of penicillininduced sphaeroplasts is complete as the membrane preparations completely lose nicotinamide-linked dehydrogenase activities for malate and glucose. Enzymic activities of the whole cells, such as aldolase and glyceraldehyde 3-phosphate dehydrogenase, are quantitatively recovered in the supernatant fractions after centrifugation (White and Smith, 1964; Wright and White, 1966). Prolonged sonication removes some of the primary dehydrogenase activities (White, 1964). Freezethawing, followed by washing in dilute buffer, removes cytochrome c (Smith and White, 1962) and aqueous acetone extraction removes 2-demethylvitamin K2 from the membrane fragments (White, 1965b). The sequence of the respiratory pigments of H . ParainJluenzae has been established in a number of ways. Cytochrome c can be removed by washing frozen-thawed membrane fragments with diluted buffer (Smith and White, 1962). Removing the cytochrome c decreases the rate of oxygen utilization in the presence of substrate. If the washing is continued, oxygen utilization in the presence of substrate eventually stops. Although the overall rate of electron transport is stopped, the ferricyanide reductase activity in the presence of substrate and the oxygen utilization in the presence of silicomolybdate are unaffected by removal of the cytochrome c. As will be shown below, ferricyanide reduction in the presence of substrate is a good measure of the activity of the primary dehydrogenases. I n the presence of 2-n-heptyl-4-hydroxyquinoline N-oxide, which blocks reduction of the electron-transport system between cytochromes b and c (White and Smith, 1962))the high-potential electron donor, silicomolybdate, can stimulate oxygen utilization. This places the cytochrome c between the primary dehydrogenases and the oxidases (Smith and White, 1962). Unfortunately, adding purified

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cytochrome c552 from H . parainjluenzae or equine cytochrome c does not restore activity. 2-Demethylvitamin K2can be removed from membrane fragments by extraction with aqueous acetone. After removal, oxygen utilization in the presence of substrate is abolished. Neotetrazolium is an artificial electron acceptor that is about 10% as efficient as oxygen, and neotetrazolium reduction in thc presence of substrate is inhibited by 2-nheptyl-4-hydroxyquinolinc N-oxide. The inhibitor-sensitive neotetrazolium reduotase activity can be restored to the extracted fragments by addition of the amount of 2-demethylvitamin K, that was removed by the acetone extraction (White, 196513). Activity is restored by 2demethylvitamin K, but not by vitamin K, or coenzyme Qlo. The 2-demethylvitamin K, added back is reduced by all of the substrates that reduce the respiratory pigments. This places 2-demethylvitamin K, between the primary dehydrogenases and the site of inhibition by 2-n-heptyl-4-hydroxyquinoline N-oxide. The inhibitors malonate, secobarbital, 2-n-heptyl-4-hydroxyquinoline N-oxide and cyanide inhibit reduction of the cytochromes by succinate. If the respiratory pigments are reduced by substrate, the inhibitors added and the membrane fragments oxidized by shaking in air, all of the respiratory carriers between the point of inhibition and the oxidase should be oxidized and all of those between the point of inhibition and the primary dehydrogenases should be reduced. Cyanide inhibits the oxidation of all pigments and thus inhibits the oxidase. 2-n-Heptyl-4hydroxyquinoline N-oxide inhibits oxidation of 2-dimethylvitamin K 2 and the primary dehydrogenases, and t o a smaller extent the cytochrome b, and thus inhibits between cytochrome b and 2-demethylvitamin K, and between cytochromes b and c . Secobarbital and malonate inhibit oxidation of the primary dehydrogenases but not 2-demethylvitamin K, or the cytochromes (White and Smith, 1964; White, 1965b). It appears from the data with inhibitors that a t least a portion of each of the pigments is involved in the pathway from substrate t o oxidase. If cells with reduced respiratory pigments are suddenly mixed with oxygen in the pulsed flow device, the pseudo first-order rate constant can be derived directly from the proportion of the pigment that is oxidized during the flow period (Chance and Williams, 1955b). The order of oxidation by oxygen based on the rate constants for a number of preparations containing different proportions of cytochromes is a,, c, b (Smith et al., 1070). Since the primary dehydrogenases are the ratelimiting steps in the reduction of the electron-transport system in these bacteria (White, 1964), this measurement of the rates of oxidation specifically defines the order of electron transport. Hnemophilus parainJluenxae contains a t least seven distinct mem-

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branc-bound primary dehydrogenase activities. These are NADH,, NADPH,, formate, succinate, D-lactate, L-lactate and glycerophosphate dehydrogenases (White, 1964, 1966). These dehydrogenases have the spectral characteristics of flavoproteins and, with the exception of forinate dehydrogeiiase, can be assayed by the rate of reduction of ferricyaiiide in the presence of cyanide (White, 1964). Formate dehydrogenase assayed by oxygen utilization has four times the activity per electron as formate-ferricyanidc reductase. The rates of reduction of oxygen and ferricynnidc per electron transferred are equal for the other substrates. As expected, ferricyanide inhibits oxygen utilization. Ferricyanide reductase activity is inhibited by secobarbital and thenoyl trifluoroacetate. illalonate inhibits succinate ferricyanide reductase activity competitively. As expected from the known sites for inhibition, 2-n-heptyl-4hydroxyq~iiiiolineN-oxide aiid cyanide have no effect on the ferricyanide reductase activities. There is no permeability barrier for ferricyanide, as rupture of the cells has no effect on the rate of reduction. There are several pieccs of evidence for the existence of multiplicity of ferricyanide reductases. Thus, ferricyaiiide reductases for the different substrates have different rates of thermal inactivation, different rates of release from incmbranc fragments, and a dependence on growth conditions for the proportioiis of each that are synthesized. I n addition, the change in flavin absorbance between 443 and 500 nm. for each of the substrates added seqiicntially is additive (White, 1964, 1966). The overlap of several electron pathways in the electron-t,ransport system caii be demonstrated. This is seen best in a mutant of H . parain,uP?iawwhich spontaneously appeared. This mutant forms about lO”/b of thc rytochronie c, as the wild type but, in all other aspects yet examined, ttpl’eitrs to be the same (White aiid Smith, 1964).The different substrates each reduce bet’ween 75 and loo./;, of the membrane-bound cytochromc c (cytochrorne 11 in the mutant) in the anaerobic steady state. In general, the faster the rate of oxygen utilization the greater the proportion of cytochromes 2, or c that are reduced in the anaerobic stcady state. Addition of two or three substrates simultaneously leads to reduction of 90-1000,/, of t h o membrane-bound cytochromes b and c in the anaerobic steady state. la cells or membranes from the parental strain containing high levels of cytoehrome c, the total initial rate of oxygen utilization is equal to the sum of the individual initial rates of oxygen utilization. I n the mutant which has much less cytochrome c, the initial rate of oxygen utilization when several substrates are added together is always less than the sum of the individual rates of oxygen utilization (White and Smith, 1964). The substrates that give the fastest rates of ferriryariide reductase activity reduce the largest proportions of 2-deinctIiylvit~riiinK2 in the anaerobic stcady state (White, 1965b).

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The range is from 16% reduction with NADPH, to 61% with formate. Cells can be grown with higher specific activities of formic and L-lactic ferricyanide reductases by varying the growth conditions. The higher the specific activity of the ferricyanide reductasc the higher the proportion of 2-deniethylvitaniin K, that is reduced. Addition of several substratcs together dicl not increase the proportion of 2-demethylvitamin K, beyond 64%. The initial rate of reduction of 2-dernethylvitamin K2 was too fast to measure with the assay used (White, 196513). During the aerobic steady state (i.e. the period when the rate of oxygen utilization is pseudo-zero order), from 10 to 30% of the cytochrome b , 40-60y0 of the cytochrome c, and O - l O ~ o of the cytochrome oxidase a 2 that is reduced in the anaerobic steady state is reduced in bacteria with various proportions of pigments with formate, succinate, lactate or NADH, as substrates (Smith ct al., 1970; Sinclair et al., 1970). The fact that, under most conditions, the initial rate of oxygen utilization after simultaneous addition of two or three substrates is less than the sum of initial rates of each substrate added singly, indicates that a step other than the primary dehydrogenase may limit the electron flux. This is seen best in cells with low levels of cytochromc c. The electron-transport system of 11. paminjizcenzae exhibits remarkable branching. Electrons are donated to the membrane-bound respiratory carriers from NADH,, NADPH,, formate, succinate, L - r ~ glycerophosphate, D - and L-lactate and oxygen, nitrate, fumarate, pyruvate and very high concentrations of NAD can act as terminal electron acceptors. In preliminary experiments it was found that the respiratory pigments reduced in the presence of formate can be oxidized by the anaerobic addition of NAD, NADP, fumarate, pyruvate or nitrate. Addition of NAD or NADP to membrane fragments reduced in the presence of formate results in the generation of NADH, or NAUPH,. Oxidation of oytochromes b and c and 2-deniethylvitamin K, by thc anaerobic addition of alternative terminal electron acceptors is inhibited N-oxide and cyanide, by the removal by 2-n-heptyl-4-liydroxyquinolinc~ of caytochrome c aftcr freeze-thaw and washing, and by the extraction of ~-denicthylvitaminK, with acetone. This cvxtainly suggests that multiple electron-transport components are involved in the anaerobic oxidation of the respiratory pigments by alternative terminal electron acceptors. Thus far, reversed electron-transport (i.e. NADII, generation in the presence of succiiiate aiid ADP or ATP) has not been demonstrated. The best studied alternative terminal electron-acceptor has been nitrate. Haemophilus parainjiuenxae is unusual in that both nitrate and oxygen are reduced simultaneously with formate as substrate (Sinclair P t nl., 1970). Oxygen inhibits the rate of nitrate reduction. Addition of nitmtc to cells in which thc respiratory pigments are reduced in the

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presence of forniate results in the very rapid oxidation of a portion of the cytochrome c , and tlic slow oxidation of cytochrome oxidase a2. It appears thiLt nitrate interacts with a part of the electron-transport s~ stern that overlaps with the pathway t o oxygen. Growing tlie cells with iiitmte as tlie alternative electron acceptor, or with low concentrations of oxygen tuid nitrate, induces large increases in the activities of nitrate mid nitritt, rediivta . In the coniplex media necessary for growth of If.parainflueizxae, nitrate and nitrite reductase activities can be detected in cells grown in the absence of added nitrate (Sinclair and White, 1970). Nitrite is not a suitable electron acceptor for growth or oxidation of reduced cytochromes. It appears t o damage cytochrome oxidase a,. I n both intact cells and particles, the critical oxygen concentration (the oxygen concentration a t which the rate of oxygen utilization becomes dependent on the oxygen concentration) has proved to be a very useful value t o determine. Cells with a high concentration of functional cytochronie oxidase a , have a low critical oxygen concentration. The critical oxygen coticmitration can be raised by agents which inhibit cytoclirome oxidase u1 (carbon monoxide or incubation with nitrate which niny darnage cytochrorne oxidase u2; Sinclair and White, 1970). Cells which contain c~ytoc*hronieoxidase o as the terminal electron acceptor have a high critical oxygen concentration (50-1 50 pM-oxygen). It is very likely that the cytochrorne oxidase 0,which is the only oxidase formed under conditions of high aeration (White, 1962), has a lower affinity for oxygen than the cytochrome oxidase a,. Further evidence for the differing affinities for oxygen of the two oxidases, and for branching of thc electron-transport system, comes from complex experiments which exploit the very rapid electron flux produced when forniate is the substrate (Sincalair et al., 1970). The very rapid rate of formate oxidation makes tlie oxidase reaction rate-limiting a t higher oxygen concentrations (i.e. raises the critical oxygen concentration) than those found with thc slower electron donors. With forniate as electron donor all of the cytoclironie:, b and a,, but only a part of the membrane-bound cytoclironir c, is reduced a t the end of the pseudo zero-order oxygen uptake (i e. the
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R.

SINCLAIR

fully active. These cells with cytochrome oxidase a2 have a low critical oxygen concentration. The plateau period is markedly lengthened in cells if' the cytochrome oxidase a2 is damaged with nitrite. The biphasic rediictioii of cytochrome c seen after the plateau period indicates that at least a part of the cytochrome c is not on the same pathway as the rest. 'L'lir portion of cytochronie c reduced after the plateau period is rupidly oxidized by the anaerobic addition of nitrate or fumarate (Si:ic*lairPt uZ., 1970) suggesting that part of the cytochrome c could be involved in the reduction of alternative electron acceptors. We have been unable to detect any differences between the cytochrome c isolated after rupture of the cells (i.e. the non-enzymically reducible cytochrome) and the cytochrome c released by washing the membrane in weak buffer (Smith and White, 1962). The cytochrome c550, detected as a trace component in the difference spectra a t -1 96", has never been isolated and its function is still unknown. There is too little of this cytochrome to account for the biphasic kinetics of the cytochrome cgj2. I n these kinetic studies the overlap between cytochromes 11 and c has been corrected as described on p. 180.

-

Cell sap

FP b

C

Oxidases

FIG.1 . Diagrammatic representation of the electron-transport system of Haeniophilus parainjluenzae. Reproduced from White ( 1 962).

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It appears that H . p a r a i n j u e n m e forms a highly complex electrontransport system that can be most easily considered as a network of 2-demethylvitamin K, and cytochromes b, c and a , connecting the seven primary dehydrogeiiases and the two cytochrome oxidases via pathways that overlap to varying degrees (Fig. 1 ) . I n addition, terminal electron acceptors other than oxygen, such as nitrate, fumarate, pyruvate or NBD, can osidize the network by pathways which operate siniultancously with the oxygen pathways. The overlap between pathways to oxygen and to tlic alternative terminal electron acceptors seems to be controlled by the growth conditions. The activity of alternative electron acceptors with thc electron-transport system of H . paraiujhienzae is very much like the cytochrome-containing anaerobe Bncteroides ruininicolrc (White et nl., 1962) and may be much more common than is generally realized. Preliminary studies performed by Maria Elena Nova and Lucile Smith indicate that the cytochrome network in H . p a r a i ~ ~ j u e m is u ecapable of oxidative phosphorylatioii. which, hopefully, will add a further dimension to these studies.

P. BACTERIA CONTAININGCYTOCHROMES a3 AND o Several bacteria contain two carbon monoxide-combining pigments that have not been discussed so far. Broberg and Smith (1967) found that Bacillus megaterium contains cytochrome a3, which rapidly combines with carbon monoxide, and cytochrome o which combines much inore slowly with carbon monoxide. Both oxidases are reduced by N14DH, and oxidized by air. The cytochrome oxidase o can be released from part icnlntc preparations by pancreatic lipase. The loss of cytochromc o does iiot affect tlie NADH, oxidase activity. Revsin and Brodie (1969) found both cytochromes a3 and o in Mycobacterium phlei. The cytochrome o can also be released by pancreatic lipase. Cytochromes o and n3 apparently have been found in leptospira (Baseman and Cox, 1969) and in illicrococcus denitrificans (Porra and Lascelles, 1965; Sclioles and Smith, 1968; Lam and Nicholas, 1969). Cells of Rhizobium jnponicuni cwntaiii cytochromes o and a3, but tlie bacteroid form of this organism isolated from root nodules does not contaiii cytochromes o and n 3 , but instead two other carbon monoxide-binding pigments. These two pigments are a cytochrome c and a Pa,, cytorhroine which may function as oxidases (Appleby, 1969a,b).

C: . Micrococcus denitrijcans Tlie branching of the electron-transport pathways to oxygen, nitrate and ilkrite in Mic~ococcusdenitrificans has been studied by Lam and

208

DAVID C . WHITE AND PETER R SINCLAIR

Nicholas (1969).This organism can utilize the terminal electron acceptors simultaneously, and the pathways from NADH, do not overlap if the cells are grown aerobically. I n cells grown anaerobically, the rate of oxygen utilization is decreased in the presence of nitrate and nitrite. Analysis indicates that nitrite competes with oxygen for an oxidase. Nitrate also inhibits oxygen utilization by diverting electrons from the oxidase into an alternate pathway. Space limitations prcvent detailed examination of other papers. The authors apologizc in advance for deleting some excellent work in their attempts to emphasize what seems t o be understood about the mechanism and utility of branching in bacterial el(.ctron-transl~ortsystems

V. Acknowledgements Thc authors wish to thank the inany people who supplied data prior to publication. Wc cspecially wish to tliank L u c k Smith of Dartmouth Medical School, New Hampshire for many stimulating discussions. This study was supported by Grant-GB-4795 from the National Science Foundation. REFERENCES Ackrell, B. A. C . and .Jones. C. W. (1970). Baochem. J . 116, 2 1 ~ . Aleem, >I. I. H. (1970).A . Rev. P I . Plqsiol. 21. 67. Appleby, C. A. (1969s). B~OCILZWL. biophys. Actn 172. 7 1 . Appleby. C.A. (196913). Bzochzin. biop?t?ys.Acta 172,88. Arima. K.and Oka. ‘L‘. (1965).J . Bact. 90, 734. Azoulay, E., Priig, J . and Couchoud-Ueaiunon1. P. (1969). Hiochiin. biopl~ys.Rctn 171,238. Barrctt, J. and Sinclair, P. (1967) Biochiria. biophys. Actcr 143, 279. Uartsch, R.G. (1968). A . Rev. Microbiol. 22, 181. 13asemaii. J . B. and Cox, C. D. (1969).J . Brict. 97. 1001. Birdsell, D.C. and Cota-Robks, E. H. (1970). Biochim. bzophys. Acta 216,250. Bovcll, C.R., Packer, L. mid Helgerson. R. (1963). Bzochiin. biophys. Acta 75,257. Brrtgg. P. I>. arid Hoii. C. (1967a).Archs Baochena. Baophys. 119, 194. 13mgg, P. 13. and H o i i , C. (1967b).Archs Biochena. B i o p h y s . 119, 202. Rrobcrg, 1’. L. ttnd Sinith. L. (1967). Biochztt~.6 / O l d l y c s .Acts 131,479. IJroberg, 1’. L. aiid Smith, I,. (1968).I n “Structure mid E’inicttionof Cytochroineb”, (K. Okiiniikl, N.D. Kanim and 1. Sokiizii. eds.), $1. 182. tTni\-erqitjyPark Press, Baltimore. Brodie, A. (1962).1 9 6 “Nethods in Enzyniologv ’, (S. P. Colm ick andN. 0. Kaplan, eds.). Vol. V, p. 51. Acstl~micPre9, New \-'ark. Butler, W. 1,. (1962).J . opt. Soc. Am. 52, 292. 13utrtt,W. 1). and Kc~ilin,D. (1962). Proc. R.Soc. 1560,492. Castor. L.N. aiid Chance. I3. (1955).J . b i d . C h e w 217, 453. Chalice, U . (19510). Keo. Scient. Instruna. 22. 619. Chance. 13. (1951b). Rev. Scient. Instruin. 22. 634.

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Chanco, B. (1954). Science, N . Y . 120,767. Chance, B. (19578). In “Mothods in Enzymology”, (S. P. Colowtclr and N. 0. Kaplan, eds.), Vol. IV, p. 273. Academic Press, New York. Chatice, €3. (1957b). Fedn Proc. Fedn. Am.Socs. e z p . Biol. 16, 671. Chmce, U. (1961a).J . baol. Chem. 236, 1544. Chiince, U. (1961b). I n “Haematin Enzymes”, (J.E. Fallr, R. Lemberg and R. K. Morton, c d u . ) , p. 433. Pergamoii Press, New York. Chatice, 13. and Speiiccr, E. L. (1959). Disc. Paraday Soc. 27,200. Chaiiec, 13. atid Williams, G . R. (1935a). J . biol. Chem. 217, 383. Chaticc, 13. i~iidWilliams, (i. It. (1955b).J . 6eoZ. C7?iem. 217,429. Chitticc, B., Uoiiiier, \V. 1). and Storey, 13. T. (1968). A . Rev. 1’1. P/rys~oZ.19,295. Cheah, I<. S. (1968).Gornp. Biochein. Physaol. 23, 277. Chcah, I<. H. (1969). Baocham. bbophys. Acta 180,360. Chcah, K.S. (1970a). Biochim. biophys. Actu 197,84. Cheah, I<. S. (1970b). Biochim. bioplhys. Acta 205, 148. Cheah, K.S. ( 1 9 7 0 ~ )Biochim. . biophys. Acta 216, 43. Clark, I,. C., Jr., Wolf, R., Granger, D. and Taylor, Z. (1953). J . appl. Physiol. 6, 189. Cole, J. A. ( 1 968). Biocham. biopliys. Acta 162, 356. Cole, ,J. A. and Wlmpmiiy, J. 13‘. T. (1968). Btochim. biophys. Acta 162,39. Cox, G. R., Stioswell, A. ill. arid Gibson, F. (1968a).B i o c h ~ mbiophys. . Acta 153, 1 . Cox, G. 13., Gtbaon, 14’. u t ~ dl’tttard, A . J. (1968b).J . Bact. 95, 1591. Cox, 0. U., Young. 1. G., McCann, L. M.and Gibson, F. (1969).J . Bact. 99, 450. Cox, G.U., Newton, N. A., Gibson, F., Hnoswell, A. M. and Hamilton, J. A. (1970). Biochem. J . 117,551. Estabrook, K. W. ( 1 9 5 6 ) . J . biol. Chem. 223, 781. Estabrook, R. W. (1967). In. “Methods in Enzymology”, (R. W. Estabroolr and M. E. Pidlmitti, eds.), Vol. X, p. 41. Academic Press, New York. Falk, .J. E. (1964). I n “Porphyrins and Metitlloporphyrins”, p. 181. Elsevier, New Y ork . Florktn, 31. and Stotz, E. H. (1963).“Comprehensive Biochemistry”, Vol. 13, p. 21. Elsevicr, New Tork. Frerman, F. E. ;tiid White, D. C. (1967).J . Bact. 94, 1868. Fujtta, T. and Sato, R. (1966).J . Biochem., Tokyo 60,691. Gel’maii, N. S., Lukoyanova, M. A. and Ostrovskii, D. M. (1967).I n “Respiration and Phosphorylatioti of Bactcrta”. Plenum Press, New York. Green, D. E. (1964).Scient.Am. 210, 63. Hamiltoil, J. A . , Cox, G. U., Looney, P. D. and Gibson, F. (1970). Biochem. J . 116,319. Hammond, R. I<. and White, D. C . (1969).J . Chrovnat. 45, 446. Itagakt, E.(1964).J . Biochem, Tokyo 55,432. Jones, C. W. and Redfearn, E. R. (1966). B i o c h i ~biophys. ~ Acta 113,467. Jones, C. W. and Rcdfearn, E. R. (1967a). Biochim. biophys. Acta 143,340. Jones, C. W. and Redfearn, E. R. (1967b). Biochirn. biophys. Acta 143,354. Jones, R.G. W. (1967). BioclLem. J . 103, 714. Jones, R.G. W. atid Lascelles, J. (1967). Biochem. J . 103,709. Jurtshuk, P.and Harpor, L. (1968).J . Bact. 96, 678. Jurtshuk, P., Bednarz, A. J., Zey, P. and Denton, C. H. (1969a). J . Bact. 98, 1120. Jurtshuk, P., May, A. K., Pope, L. M. arid Aston, P. R. (1969b).Can. J . Microbiol. 15,797. Kashket, E. R. and Brodie, A. F. (1963a). Biochim. biophys. Acta 78, 52. Kashket, E.R. and Urodie, A. F. (1963b).J . bio2. Chem. 238, 2564.

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Keilin, I).(1966). “The History of Cell Respiration and Cytochrome”. Cambridge University Pross, London. Keilin, D. arid Htwtree, E. F. (1939). Proc. 12.Soc. B127, 167. Kcilin, D. and Hartree, E. P.(1949).Nature, L o n d . 164,254. lilingeiiborg, M. and Eucher, T. (1959). Biochern. 2. 331, 312. Knowles, C.J. and Redfearn, E. R. (1968). Biochinz. biophys. Acta 162,348. Kroger, A. arid Iilirigenberg, M. (1967). Current Topics i n Bioenergetics 2, 151. Lam, Y.mid Nicholas, D. .J. D. (1969). Biochi))~.. biophys. Actcc 172,450. Laiiyt, J. I<. (1968). A r c h Biochem. Biophys. 128, 716. Lariyi, J. l i . (1969).J . bid. Chem. 244, 2864. Lester. It. L. itnd Crane, F. L. (1959). BiochCm. biophys. d c t a 32, 492. Lestcv, R. I,., White, D. C. and Smith, S. 1,. (1964). Biockenvistry, N . 1’. 3, 949. Lindeberg, G., Lode, A. and Somme, R. (1963). Acta chem. scand. 17,232. Mizushirna, S. and Arima, K. (1960).J . Biocheni., 2’okyo 47, 837. Nicholls, P. and Malviya, A. N. (1968). Biochemistry, N . Y . 7, 305. Oka, T.arid Arima, K. (1965).J . Bact. 90,744. Pinchinoty, F.arid D’Ornano, L. (1961). Nature, L o n d . 191,879. Polglase, W.J., Pun, W.T. and Withaar, J. (1966). Biochina. biop?,ys. Acta 118, 425. Porra, R . .J. and Lascelles, J. (1965). Biochem. J . 94, 120. Pullman, &I.E. and Schatz, G. (1967). A . Rev. Biocheni. 36, 539. ltevsin, B.t ~ n dBrodie, A. F. (1969)..I. biol. Che m 244,3101. Rickard, P. A. D., Moss, E’. J. and Roper, G. H. (1967). Biotechnol. Bioengng 9,223. Ruiz-Herrera, J. and DeMoss, J. A. (1969).J . B a t . 99,720. Ruiz-Herrera, J., Showe, M. K. and DeMoss, J. A. (1969).J . Bact. 97, 1291. Schindler, F. J. (1967). I n “Methods in Enzymology”, (R. W. Estabrook and 31. E. Pullman, eds.), Vol. X, p. 629. Academic Press, New York. Scholes, P. 15. and Smith, L. (1968). Biochim. biophys. Acta 153,363. Showe, M. I<. and DeMoss, J. A. (1968).J . Bact. 95, 1305. Sinclair, 1’. R . and White. D. C. (1970).J . B a t . 101,365. Sinclair, P. R., White, D. C. arid Smith, L. (1970). J . biol. CILem. in press. Skerman, V. B. D. and Macrae, I. C. (1957). C a n . J . Microbiol. 3,505. Smith, L. (1961). I n “The Bacteria”, (I. C. Gunsalus and R. Y. Stariier, eds.), Vol. 2, p. 365. Academic Press, New York. Smith, L. (1968). I n “Biological Oxidations”, (T. Singer, ed.), p. 55. Interscience Pub., Now York. Smith, L. arid White, D. C. (1962).J . biol. Chem. 237, 1337. Smith, L., IVhito, D. C., Sinclair, P. R. and Chance, 13. (1970). J . biol. C?iem. in press. Snoswell, A. M. and Cox, 0. €3. (1968). Biochim. biophys. Acta 162,455. Swank, 12. T. and Bnrris, R. H. (1969). Biochim. biophys. Acta 180,473. Temperli, A. and Wilson, P. W. (1960). Biochem. 2. 320, 195. Warburg, 0.(1926). Biochem. 2. 177,471. Whistanco, G. R. and Threlfall, D. R. (1968). Biochem. J . 108,505. Whistance, G.R . , Dillon, J. F. and Threlfall, D. R. (1969). Biocheni. J . 111, 461. White, D. C. (1962).J . Bact. 83, 851. White, D.C. ( 1 9 6 3 ~ )J. . Bact. 85, 84. White, D.C. (1963b). J . biol. Chem. 238, 3757. White, D.C. (1964).J. bio2. Chem. 239, 2055. White, D. C. (1965a).J . Bact. 89,299. White, D. C. (1965b). J . b i d . Chem. 240, 1387.

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White, D. C. (1966). Antonie van Leeuwenhoek 32, 139. White, D. C. (1967). J. Bact. 93, 567. White, D. C. and Cox, R. H. (1967). J . Bact. 93, 1079. White, D. C. and Smith, L. (1962). J . biol. Chem. 237, 1332 White, D. C. and Smith, L. (1964). J . biol. C h e m 239, 3966. \\"nite, D. C. and Tucker, A. N. (1969). J . Bact. 97, 199. \Vhito, D. C., Bryant, M. P. and Caldw-ell, D. R. (1962).J . Bact. 84, 822. Wimpenny, J. W. T. (1969). S y m p . Soc. gen. Microbiol. 19, 161. Wright, E. A. and White, D. C. (1966). J . Bact. 91, 1356. Yokata, I<. and Yaniazaki, I. (1965). Biochim. biophys. Acta 105, 301. Yang, C. C. and Legallais, V. (1954). Rev. Scierit. Instruin. 25, 801. Yamashita, S. and Racker, E. (1969). J . biol. Chem. 244, 1220.

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The Generation and Utilization of Energy During Growth W. W. FORREST* and D. J. WALKER

CSI RO, Division of Nutritional Biochemistry, Kintore Avenue, Adelaide, South Australia, Australia I. Introduction . 11. The Requirement for Energy . A. Lithotrophic Carbon Dioxide Fixation B. Synthesis of Monomers. C. Polymerization . D. Total Synthesis of Bacterial Cells . 111. The Generation of Energy . A. Lithotrophic Metabolism . B. Organotrophic Metabolism . . IV. The Usage of Available Energy A. Molar Growth Yields . B. Thermodynamic Assessments . V. Conclusions . References .

.

. .

. . . .

.

. . . . . .

213 214 214 217 221 223 227 227 236 249 249 264 267 269

I. Introduction Despite the great diversity of their metabolic activity, the details of the energetic behaviour of micro-organisms have in large measure yielded t o the probing of the biochemist. Consequently, a very large number of mechanisms necessary for life support and reproduction in these organisms are known in great detail. As this knowledge has accumulated there has arisen a growing interest in the relationships that exist between the synthesis of bacterial cell material and the energy available to perform synthetic work. It is generally agreed among biologists that the energy requirement of a living cell can be couched in terms of adenosine triphosphate (ATP), since this compound, either directly or by some intermediate formed

* Present address : The Australian Wine Research Institute, Waite Road, Urrbrae, South Australia. 213

214

W.

W. FORREST AND

D . J. WALKER

with its participation, is responsible for coupling the energy-yielding reactions of metabolism to provide the bond-forming power necessary for a great many synthetic reactions. There is little evidence for energy transfer apart from the formation of such intermediates, so that the function of this chapter is to review the major reactions for which the cell needs ATP and from which ATP is derived and t o quantitate these reactions as far as is possible. Following this, the efficiency of usage of available ATP and of coupling between these processes will be assessed in the light of what is known of the extent of bacterial growth, Recent reviews bearing on this subject have appeared from Stouthamer (1969)) Peck (1968) and Forrest (1969a).

11. The Requirement for Energy

Microbes, depending upon the milieu in which they exist, use widely varying synthetic mechanisms for maintenance and growth. Such synthetic reactions are either energy-requiring or involve the use of metabolic intermediates which otherwise could be further degraded in energy-yielding reactions. It is self-evident then that the extent t o which the sum of these syntheses is capable of producing new microbial material is dependent upon the total production of energy from catabolic processes and the efficiency of energy utilization. Ultimate requirements for the formation of new bacterial cell material lie chiefly in the synthesis of protein, nucleic acids, lipid and cell-wall materials. Synthesis of smaller components such as cofactors for enzyme activity demands an insignificant proportion of the total energy involved. For the autotrophic organism, the path t o complete cell synthesis is long and complex since carbon dioxide provides the sole source of carbon, whereas the strict heterotrophs, such as the lactic acid bacteria, exist in environments in which the building blocks for cell synthesis are already present, so that their major task is the polymerization of preformed units into structural and functiocal materials. The range of organisms between these extremes is wide and an attempt will be made to examine the energy costs involved in the synthesis of major cell components by the various groups of bacteria. A. LITHOTROPHIC CARBONDIOXIDE FIXATION The organisms which use carbon dioxide as sole source of carbon for growth can be divided int.0 chemolithotrophs, which use oxidation of inorganic substrates t o generate energy, and photolithotrophs which rely upon light as the primary energy source.

THE GENERATION AND UTILIZATION OF ENERGY DURING GROWTH

215

1. Photolithotrophs

It is now firmly established that the majority of the carbon fixed into the cell material of photosynthetic bacteria arises via the cyclic process elucidated by Calvin and his coworkers (see Elsden, 1962 for review). Glucose is the product of the Calvin cycle operation and the remainder of the cell constituents must be synthesized from this compound. Briefly, carbon dioxide is fixed by reaction with ribulose-l,5-diphosphatc under the influence of the enzyme carboxydismutase with the format ion of two cquivalents of phosphoglyceric acid. Adenosine triphosphatc is then required for the conversion of the latter compound to 1,3-dipliosphoglyceric acid and reduced nicotinamide nucleotide is used to form glyceraldehyde 3-phosphate by reversal of triose phosphate deliydrogenase. Condensation of the triose phosphate results in fructose 1,6diphosphate formation, one-sixth of which represents the newly synthesized carbohydrate and the other five-sixths of which is transformed via transaldolase and transketolase reactions t o ribulose 5 phosphate Expenditure of further ATP regenerates the ribulose 1,5-diphosphate necessary for further carbon dioxide fixation. The overall equation for the fixation is : G COa

+ 18 ATP + 12 N.4DHz

+ Fructose 6 phosphate

+ 18 ADP + 12 NAD + 17 Pi

I n addition to the ATP required for the direct phosphorylation of intermediates in the Calvin cycle, a considerable amount of reduced nicotinamide nucleotide is required. Present indications are that this is formed by ATP-driven reversal of electron transport (Gest, 1966; Vernon, 1 968). In preparations derived from the non-sulphur purple bacterium Rhodospirillum rubrum, photosynthetic electron transport may be coupled to pyrophosphate rather than ATP formation (Baltschevsky et al., 1966) and pyrophosphate can serve in place of ATP in driving NAD reduction (Keister and Yike, 1966). However, this mechanism is not likely to represent energy conservation since pyrophosphate is thought to be formed by transfer of phosphate from a high-energy intermediate to phosphate rather than ADP during electron transport. Unfortunately, there is no clear idea of the stoichiometry of the NAD reduction reaction in photosynthetic organisms. I n chemolithotrophs, estimates of 1.3-5 ATP per equivalent of NAD reduced have been obtained (Peck, 1968). Whilst it is evident that most phototrophic bacteria so far studied employ the Calvin cycle as the chief mechanism for carbon dioxide fixation, there has been discovered in Chlorobium thiosulphatophilum a very different pathway (Evans et al., 1966). Basically, the new pathway involves firstly the formation of pyruvate by pyruvate synthetase : Acetyl-CoA + COz + Ferredoxinred. + Pyruvate + CoA + Ferredoxin,,.

W. W.

216

FORREST AND D. J. WALKER

Pyruvate is then converted t o oxaloacetate : Pyruvate

+ ATP + COz

+ Oxaloacetate

+ ADP + Pi

Oxaloacetate is then launched into a reversed tricarboxylic acid cycle. Reduced ferredoxin is involved again in the carboxylation of succinylCoA to yield a-ketoglutarate : Succinyl-CoA + C 0 2

+ Forredoxinred. + a-Ketoglutarate + CoA + Ferrodoxin,,.

After further carbon dioxide incorporation in transforming a-ketoglutarate to oxalosuccinate, citrate is finally formed which is split t o yield oxaloacetate and acetate. The net result of the new reaction sequence is t,he fixation of four molecules of carbon dioxide and production of one equivalent of oxaloacetate for each turn of the cycle. There is a requirement for 3 ATP in the activation of acetate, the carboxylation of pyruvate and the activation of succinate. The reduced ferredoxin required can arise in C . thiosulphatophilum by light-driven mechanisms (Buchanan and Evans, 1965), but it is not known whether reduced ferredoxin can supply via reduced nicotinamide nucleotides and flavins the remainder of the reducing power required. 2 . Chemolithotrophs

As with the phototrophs, the chief mechanisms responsible for carbon dioxide fixation in those organisms known as chemolithotrophs is by way of the Calvin cycle (Peck, 1968). The difference is that, whereas photoautotrophs obtain their energy from light under anaerobic conditions, the chemautotrophs oxidize inorganic compounds to obtain energy. Thus, in terms of ATP as such for the phosphorylation of intermediates in the Calvin cycle, the requirements of the chemautotrophs are the same as those of the photosynthetic bacteria. As the source of reduced NAD required in hexose synthesis, it would seem a t present that most chemautotrophs must rely upon reversed electron flow driven by ATP (Peck, 1968). The ATP required for this function is open to question. Aleem (1966a) has reported that 1.3 ATP is needed for each equivalent of NADH, produced by Thiobacillus novellus, whilst the corresponding figures for Nitrosomonas europaea (Aleem, 196613) and a Nitrobacter species (Aleem, 1965a) are reported as 5 and 2 respectively. Froin the foregoing, it is obvious that autotrophic bacteria of both photo- and chemosynthetic types have a large, if not precisely known, energy requirement for the fixation of carbon dioxide into simple precursors of cell material. Use of the Calvin cycle : 6 CO:!

+ 18 ATP i- 12 NADHB + Fructose 6-phosphate + 18 ADP + 12 NAD + 17 Pi

involves an expenditure of a t least 30 equivalents of ATP per hexose

THE GENERATION AND UTILIZATION

OF ENERGY DURING GROWTH

217

synthesized and possibly more if NAD reduction does not have a 1:1 relationship t o ATP expenditure. Hempfling and Vishniac (1967),after making a number of assumptions, found that the measured yield of Thiobacillus neapolitanus cells was remarkably close to the calculated value if NAD reduction required only one equivalent of ATP. B. SYNTHESIS OF MONOMERS

For those organisms not fortunate enough to live in nutritionally rich environments, the task, after the synthesis or assimilation of a carbon source, is firstly the synthesis of the monomeric units ultimately t o comprise the cell material. The major requirements are for amino acids, nucleotides, fatty acids and sugars. 1.

Amino Acids

a. Aromatic tcmino acids. Pathways for the formation of phenylalanine, tyrosine and tryptophan are much too complex to be discussed here in detail, and an excellent review is offered by Gibson and Pittard (1968). Briefly, there is a common path for all three amino acids from glucose to chorismic acid. I n these reactions glucose has to give rise t o erythrose 4-phosphate and phosphoenolpyruvate prior t o condensation to form 3-deoxy-D-arabino-heptulosonicacid 7-phosphate, and A'I'P and phosphoenolpyruvate are used in the further conversion to chorismic acid. Formation of tryptophan from chorismic acid involves condensation of phosphoribosyl pyrophosphate with anthranilate with elimination of pyrophosphate (i.e. equivalent t o a loss of two high-energy bonds) and the recovery of an equivalent, of triose phosphate in the final condensation of indoleglycerol phosphate with serine t o yield tryptophan. Serine formation from glucose requires one ATP and triose phosphate can give rise to one ATP and one phosphoenolpyruvate. Therefore, the overall formation of tryptophan from glucose requires the expenditure of four high-energy bonds and one equivalent of NADH,. Conversion of chorismate t o phenylalanine and tyrosine requires no energy expenditure, therefore the synthesis of both of these amino acids from glucose requires two high-energy bonds. b. Amino acids arising from aspartic acid. This group comprises asparagine, lysine, threonine, isoleucine and methionine. Aspartate itself arises by transamination involving oxaloacetate and glutamate, so that no energy exchange is involved a t this level. If glucose is the precursor of oxaloacetate, conversion t o pyruvate plus ATP or to phosphoenolpyruvate is necessary followed by a carboxylation reaction. There may or may not be a gain of energy in this reaction,

W. W. FORREST AND D. J. WALKER

218

depending upon which carboxylating system is operative. The most common mechanism, involving phosphoenolpyruvate carboxykinase, results in energy conservation :

+

0.6 Glucose + Pi + NAD + Phosphoenolpyruvate NADHz Phosphoenolpyruvate + COz + Nucleotide diphosphate + Oxaloacetate Nucleotide triphosphate

+

Pyruvic carboxylase activity would result in no net change of the energy balance : 0.5 Glucose + ADP

+ Pi

+ Pyruvate

COa

+ ATP +Oxaloacetate + ADP + P i

as would phosphoenolpyruvate carboxykinase activity : 0.5 Glucose i PI

COa+Pi --f

Phosphoenolpyruvate

____f

Oxaloacetate

+ PPi

Whilst aspartic acid can be synthesized from glucose a t no energy cost, its conversion t o asparagine involves the loss of two high-energy bonds : Aspartate

+ NH3 + ATP

+ Asparagine

+ AMP + PPi

Aspartate conversion to lysine proceeds via a complex path which can be summarized as follows : Aspartate

4

Pyruvate

+ Glutamate + 2 ATP + 2 NADHz Lysine

+

+ a-Ketoglutsrate + COz + 2 ADP + 2 Pi + 2 NAD

Threonine formation from aspartate proceeds by a five-step sequence leading to the following overall reaction : Aspartate

+ 2 ATP + 2 NADHz

+ Threonine

+ 2 ADP + 2 P i + 2 NAD

Threonine can be further metabolized to yield isoleucine in reactions summarized as follows : Threonine + Pyruvate

+ Glutamate + NADHz

-+ Isoleucine

+ a-Ketoglutarate + COz + NAD

The overall synthesis of isoleucine from aspartate would be depicted as follows : Aspartate

+ Pyruvate + Glutamate + 2 ATP + 3 NADPHz + lsoleucine + a-Ketoglutarate + COz + 2 ADP + 2 Pi + 3 NADP

In methionine biosyiithesis aspartate is also the key starting material in a series of reactions which may be summarized as follows : Aspartate

+ 3 ATP + 2 NADHz + Cysteine + “Methyl donor” + Methionine + 3 ADP + 3 P i + 2 NAD + Pyruvate

c. Glutamate-derived amino acids. Apart from glutamate itself, these compounds are glutamine, proline and arginine. Glutamate arises from glucose by production of a-ketoglutarate in the tricarboxylic acid cycle and amination either by transamination or by reversal of glutamic dehydrogenase.

THE GENERATION AND UTILIZATION O F ENERGY DURING GROWTH

219

Glutamine synthesis proceeds as follows : Glutamate

+

NH3 + ATP + Glutainine i ADP i- PI

In proline biosynthesis the series of reactions is summarized. Glutamate i ATP i 2 NADHz + Proline

“Ll

+ ADP + PI 1-

2 NAD

+ HzO

‘I’he arginine pathway is complex resulting in the following overall at 1011: .

+

2 Glutamate t Aspartate + COz 4 ATP t NADHz -> Argminc + a-Ketoglutarate + Funlarate + 3 ADP

+ 3 Pi + AMP + PPI + NAD

Since one glutamate and the aspartate arc recovered as related compounds with the same number of carbon atoms, theF do not affect the energy calculations. It may therefore be said that formation of arginine from glutamate requires the expenditure of five high-energy bonds and one equivalent of NADH,. d. Serine-derived Amino Acids. These amino acids are glycine and cystinc. Serinc itself arises from 3-phosphoglycerate, the overall reaction being : 3-Phosphoglycerate + Glutamate + NAD + Serine + ~ K e t o g l u t a r a t e+ NADHz

+ PI

If then glucose is precursor of the serine molecule, one high-energy bond would be used per equivalent of serine synthesized. Serine is converted t o glycine in a single-step reaction in which the hydroxymethyl group is transferred to a tetrahydrofolate coenzyme. No energy transaction is required, but it should be noted that the methylene tetrahydrofolate formed can serve as “methyl donor” in the methionine synthesis previously outlined. Serine + Tetrahydrofolate + N5,NlO-methylene tetrahydrofolate + glycine

Cysteine synthesis takes place by reaction of serine with H,S : Serine

+ HzS + ATP

+ Cysteine

+ HzO + ADP + Pi

It should be noted that if the H,S has to be generated from sulphate further energy is required :

+

S042- + ATP + Adenylyl sulphate P P i Adenylyl sulphate ATP -+ Adenylyl phosphosulphate ADP Adenylyl phosphosulphate NADPHz Adenosine-3’,5-diphosphate + NADP 9032- + 3 NADPH2 + S2- 3 NADP + 3 HzO

+

SO42-

t 2 ATP

+ +

+ 4 NADPHa Sz-

+

-+

+

+ ADP + Adenosine-3’-5’ diphosphate + PPi + 4 NADP

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FORREST AND

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Consequently, total synthesis of cysteine from glucose a n d suphate would require the expenditure of five high-energy bonds. e. Pyruvate-derived Amino Acids. Alanine, valine and leucine arise from pyruvate. Alanine is formed without energy utilization by simple transamination. The complex of reactions leading to valine has an overall mechanism as follows : 2 Pyruvate

+ Glutamate + NADPHe

+

Valine +- a-Ketoglutarate + COa

+ HzO + NADP

Leucine synthesis conforms to the following overall reaction :

+ +

+

2 Pyruvate + Acetate + Glutamate NADPHz NAD + ATP + Leucine + ~~-Ketoglutarate2 COz 3 HzO NADP NADHz

+

+

+

+ ADP + Pi

f. Histidine Biosynthesis. This amino acid is synthesized by a complex series of reactions giving an overall reaction of: Glucose + 3 ATP + 2 NAD + Glutamine + Glutamate + Histidine + CO2 + 2 HzO + a-Ketoglutarate + 5-Amino-1-(5’-phosphoribosy1)i1nidazole-4-carboxamide -I Pi + 2 PPi -1 2 NADHn + ADP + AMP

Basically, four high-energy bonds together with the cost of glutamine synthesis are required for the synthesis, but the cell would gain in the reaction sequence not only two reduced NAD equivalents, but also an equivalent of 6-amino-l-(5’phosphoribosyl)-imidazole-4-carboxamide, which is a precursor of the purine nucleus. I n view of the very superficial treatment necessarily given to the subject of amino acid biosynthesis, it is recommended that detailed information be obtained from the paper of Umbarger and Davis (1962) and from the book by Mandelstam and McQuillen (1968). 2. Nucleotides

a. Purine Nucleotides. The key compound in purine nucleotide synthesis is ionosine monophosphate (IMP) whose formatiou from glucose in a multistage reaction is summarized by : Glucose + Aspartate + 2 Glutamine + Glycine + 6 ATP i-N5,N”J-Alethenyltetrahydrofolate + “0-Formyl tetrahydrofolate + lnosine monophosphate 2 Glutamate + Fumarate HzO + 5 ADP AMP 4 Pi + PPi + 2 Tetrahydrofolate

+

+

+

+

Energetically, in addition to seven high-energy bonds expended directly from ATP, two must be added for resynthesis of glutamine and two for regeneration of the formyl- and methenyl-tetrahydrofolates. Thus, eleven high-energy bonds are required for synthesis of one equivalent of IMP.

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The following reaction converts I M P t o AMP : IMP + GTP + Aspartate + AMP

+ GDP + Pi + Fumarate

and I M P conversion to guanosine monophosphate (GMP) is depicted by : IMP

+ NAD + NH3 + ATP

+ GRIP

+ NADHz + AMP + PPi

Therefore, synthesis of AMP from glucose would require 12 highenergy bonds and of GMP 13 high-energy bonds. b. Pyrimidine Nucleotides. Uridine monophosphate (UMP) is the primary product of the pyrimidine nucleotide synthet,ic sequence : 1.5 Glucose + Glutamate + NH3 + 2 ATP + 3 NAD + IJMP COz ~Kietoglutarate ADP AMP

+

+

+

+

+ PPi + HzO + 3 NADHz

Further conversion of UMP t o cytidine derivatives requires further energy :

+

UMP 2 ATP + UTP -t 2 ADP UTP NH3 ATP -+ CTP + ADP + Pi CTP + C D P + P i

+

+

For more detailed information on purine and pyrimidine nucleotide biosynthesis see Magasanik ( 1962) and Mandelstam and McQuillen (1968).

C. POLYMERIZATION 1. Synthesis of Protein

I n preparing amino acids for incorporation into protein, each one is activated by a specific activating enzyme a t a cost of two high-energy bonds. Amino acid

+ ATP

-+ Amino acid adenylate

+ PPi

After reaction with transfer-RNA and the elimination of AMP, the amino acid is then attached t o the growing peptide chain with concomitant hydrolysis of GTP. Thus, in direct protein synthesis reactions, three high-energy bonds are used for each equivalent of amino ccid incorporated. However, RNA, particularly messenger-RNA, has a limited lifespan, and Baldwin ( 1 968) considers that two ATP equivalents are required for regeneration of the more labile RNAs for each 100 g. protein formed. Therefore, since the average molecular weight of amino acids is about 100, up to five equivalents of ATP may be required per amino acid incorporat,ed into protein. These estimates are for the formation of the primary structure of protein ; it is generally considered that the secondary and tertiary structures are uniquely determined from the primary structure and arise spontaneously from it without the expenditure of additional

.).)'>

W . W. FORREST AND

dld

D. J. WALKER

ttncrgy. Stability of these higher order structures is considered to be maintained by hydrogen bonding between groups of the primary structure (Hendler, 1968). 2 . Synthesis of Nucleic Acids For both RNA and DNA, the basic polymerization reactions are the same, involving expenditure of two high-energy bonds in converting nuclcosidc monophosphates to thr triphosphate 1% it11 subsequent elimination o f pyrophosphate upon condensation of the nuclcosidc triphosphates into nucleic acid. 71

Nucleoside-P + 2n ATP + )L Nucleoside P-P-P+ 2 n A D P n Nucleoside-P-P-P + (Nucleic acid) + n PPi

3. Synthesis of Lipids

The bulk of lipid in bacteria is associated with the lipoprotein of the cytoplasmic membrane. In terms of energy requirement for synthesis, we must, consider the need for glycerol and for long-chain fatty acids. Glycerol is actually involved in lipid synthesis as a-glycerophosphate which is produced from dihydroxyacetone phosphate. Thus from glucose wc have : 0.5 Glucose + ATP + Dihydroxyacetone phosphate + ADP Dihydroxy acetone phosphate + NADHz + a-Glycerophosphate + NAD

Long-chain fatty acids are synthesized basically from acetyl-CoA. The latter compound is converted by an energy-requiring process to malonyl-CoA. Acetate + CoA + ATP + Acetyl-CoA + A D P + Pi Acetyl-CoA + COz + A T P --f Malonyl-CoA + A D P Acetate

+ COz + 2 A T P + CoA

+ Malonyl-CoA

+ Pi + HzO

+ 2 ADP + Pi

Malonyl-CoA is then added on to either a n odd- or even-numbered fatty acid as the CoA derivative and carbon dioxide and coenzyme A are eliminated in the process : Acyl-H + CoA + ,4TP --f Acyl-CoA + ADP + PI Acyl-CoA + Malonyl-CoA + 2 [2 HI -> Acyl-CHgCO-CoA + COz

+ CoA

Malonyl-CoA units are then used t o add two-carbon fragments until the desired chain length is attained. For polymerization reactions then, GIGand CI7 fatty acyl-CoA would require 15 ATP, CIS and CI9 require 17 ATP and so on and, in addition, for each two-carbon unit added to a fatty acid chain four reducing equivalents are required in the form o f NADPH,.

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In formation of triglycerides, the fatty acyl-CoA chains condense with cc-glyc.c.I.oi)liosphatewith elimination of coenzyme-A and phosphate : 3 Fatty acyl (b.4 I

CL

Glycerophosphate -> Triglyceride t 3 CoA t Pi

I’hospholipids are synthesized by the condensing of two fatty acylc’oA chains with a-glyceropliospliate followed by inositol, serine or cc-glycerol)liosphateresidues being attached a t the expense of conversion of cytidine tripliospliate to cytidine monophosphate and pyrophosph:ttc. 1. Synthesis of Cell Walls

For the purposes of this discussion, i t will be assumed that the bacterial cell wall consists of mucopeptide only. This is because niucopeptide is a constituent of all bacterial cell walls and is believed to be the factor responsible for wall rigidity. In addition, the pathways of synthesis are more clearly defined than those of other compounds such as the tcichoic. acids which are found in some, but not all, bacteria. The first stage in the synthesis of cell-wall material is the formation of UDP-l\’-ac.ctylniuramic acid : Ketohexose I Glutamate + Hexosamine I a-Hetoglutarate Hexosamine + Acetyl- + N-Acetylhexosamine N-Acetylhoxosaminc + ATP 4 N-Acetylhexosamine-1-phosphate 1ADP N-Acetylhoxosamine- 1-phosphate + U T P + UDP-N-acetyl hexosainiiio PPi UDP-N-acetyl muramic acid P i UDP-N-Acetylhexosamine + PEP + NADH2

+

+

+

Ketohexose i Glutamate + Acetyl- + P E P + LJTP + ATP NADHz --f UDP-N-Acetyl muramic acid I ADP + P P i + Pi I a-Ketoglutarate

Thus, so far, high energy bonds from I-’hosphoeiiolpyruvate, active acetyl, UTP and ATP have been used, a total of four. Five further ATY equivalents are used in adding t o the UDP-N-acetyl muramic acid the amino acids alanine (three residues), glutaniic acid and dianiinopimelic acid, in the formation of a pentapeptide derivative. According to Anderson ct al. (1967), UDP-N-acetyl glucosamiiie is then added to form N-acetyl-glucosamine-N-acetylmuramic acid pentapeptide which is thc nionoinc~rfor cell-wall mucopeptide synthesis. Energy costs involved in N-acetylglucosamine synthesis and attachment to the muraniic acid pentapeptide is 5 ATP, resulting in an overall cost of cell-wall monomer synthesis of 14 ATP. From the molecular weight of the cell-wall monomer formed (about lOOO), i t is seen that about 7 1 grams of cell-wall mucopeptide could be synthesized for each mole of ATP used. More detailed information on cell wall biosynthesis may be obtained from Strominger (1962) and Work (1969). U. TOTALSYNTHESIS OF BACTERIAL CELLS In considering the energy requirement for synthesis of total microbial cell material, it will be necessary t o use very generalized values for

224

W . W . FORREST AND D . J. WALKER

microbial cell composition. The cellular proportion of some cell wall constituents can vary widely with the type of organism, e.g. mucopeptide varies from about 2% in Gram-negative organisms to about 20% in Gram-positive organisms. However, the amounts of other constituents appears to be more constant provided the cells do not contain large amounts of reserve materials or capsular substances. Generalized bacterial composition values have been published and will be used in the following computations.

1. Cell Synthesis from Preformed Monomers

In the case of cells growing in nutritionally-rich environments, the chief requirement for energy lies in the need to polymerize preformed monomeric units into cell structures and functional entities. Calculations for energy requirement based upon the estimates of Mandelstam and McQuillen (1968) for cell composition are given in Table 1. TABLE1. Synthesis of Microbial Cells from Preformed Monomers (From d a t a of Mandelstam and McQuillen, 1968) Polymer formed Mucopeptide Protein Lipid RNA DNA Total a

Amount

(g./lOOg. cells)

A T P required (moles/g. synthesized)

Total A T P required (moles/100 g. cells)

0.014 0.045 0.061 c 0.017d 0.021 e

0.21 2.7 0.37 0.26 0.08 3.62

15 60 6 13 4

*

Derived from equations given in the text.

* Average molecular weight of amino acid taken as 1 1 0 . ' Lipids assumed to contain Czo fatty acids.

Three ATP required to ronvert nurleic acid base t o niononurleotide with an average molecular weight of 300. e Four ATP required to convert nucleic arid baae to deoxymononucleotide wlth an average molecular weight of 280. d

A rather more detailed generalized analysis of bacterial cells is given by Morowitz (1968).Deriving from this data the polymer content of the cells, a further estimate of the energy required for polymerization can be obtained (Table 2). I n the table, the amounts of polymer present in the cells were calculated from the monomer composition given by Morowitz (1968) assuming all ribose and deoxyribose to be present as nucleic acid, and all lipid present as phospholipid since the glycerol content is given

THE UENERATION AND UTILIZATION

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TABLE2. Synthesis of Microbial Cells from Preformed Monomers (From data of Morowitz, 1968)

Polymer formed

Amount present (moles x lO-4/g. cells)

Mucopeptide Protein Phospholipid RNA DNA Polysaccharide Total

1.5 54-6 1.4 4.47 0.96 7.26

Q

ATP required for polymerization (moles x lO-4/g. ce1ls)O 21.0 255 57.4 22.3 4.8 14.5 375.0

From equations given in the text.

as exactly half the fatty-acid content. Mucopeptide was assigned an arbitrary va,lue of 15%, the remaining hexose being allotted to polysaccharide. The different sets of data analysed in Tables 1 and 2 yield virtually the same answer, i.e. that 0.036 to 0.037 moles of ATP are required for polymerization reactions in synthesis of 1 gram of bacterial cells. Gunsalus and Shuster (1962) calculated a value of 0.03 moles ATP per gram of cells, the chief reason for their lower value being that they assumed three ATP per amino acid incorporated into protein whereas a value of five has been used in the present calculations (Section C . l , p. 221).

2 . Cell Synthesis from Carbohydrate

Many organisms are capable of growth upon carbohydrate as sole source of carbon and energy and it seems that most autotrophs fix carbon dioxide first into carbohydrate before synthesizing other cell materials. Thus it is of interest to determine the energy cost of synthesizing all cell material from carbohydrate. I n Table 3, the very complete analysis of bacterial cell material given by Morowitz (1968) has been used together with the equations given in the text of this paper to obtain individual and total energy requirements for synthesis of cell materials derived from glucose, ammonia and sulphate. It is very obvious that such synthesis results in little or no overall energy rcquirement on the part of the cell. Carbon skeletons for bhe various

226

W . W. FORREST A N D D . J. WALKER

TABLE3. Energy Required for Synthesis of Cell Monomers from Hexose Monomer synthesized Alariine Argin ino Bspartate Aspzraglnr Cystcine a Qlutamatcb Glritrtmine b Glycine Histidme Isoleucine Leucine Lys1ne Methionine Phenylalaninc Proline SiTlllc

Threoniiie Tryptophan Tyrosine Val1ne Hcxoso

Ribose Deoxyribose Thymine

AMP GMP CMI'

unw (:lycixrol Fatty acid c ntni

Amount

(moles lO-4/g.) 4.54 2.52 2.01 1.01 1.01 3.53 2.01 4.03 0.50 2.52 4.03 4.03 2.01 1.51 2.52 3.02 2.52 0.50 1.01 3.02 10.26 4.47 0.96 0.24 1.40 1.40 1.40 1.15 1.40 2.80

A T P change in synthesis (moles ( x lO-4)/g. cells produced)

+4.54 t12.60 $2.01 -1.01 -4.04 +17.65 +8.04 -4.03 t2.5 0 1-12.09 0 0 -3.02 +10.08 -3.02 -2.52 -2.0 -2.02 f6.04 0 -4.47 -0.96 -0.72 -16.8 -18.2 -7.0 -2.3 -1.4 +5.6 t7.64 ~~~~

Formed from glucose and sulphate. 0 Assumed to be forined from glucose via the tricarboxylic acid cycle. c As acetate.

monomers have been assumed to arise via the Embden-MeyerhofPnrnas pathway and, where applicable, by way of the tricarboxylic acid cycle. It is not likely, if other reaction mechanisms operate for synthesis of some components, that energy requirements would vary greatly. It appears then that provision of monomers preformed confers no energetic advantage to an organism if it is capable of synthesizing monomers from hexose and simple inorganic precursors.

THE GENERATION

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227

3. Synthesis from Carbon Dioxide

Autotrophic organisms must, prior t o cell synthesis, manufacture hexose according to the equation : 18 ATP

+ 12 NADHz + 6 COz

-+ Glucose + 18 ADP

+ 12 NAD + 17 Pi

Assuming that each NAD reduced by reversed electron transport requires one equivalent of ATP, a total of 30 moles of ATP are required per mole of glucose synthesized or 0.167 moles of ATP per gram of glucose. Since bacteria have approximately the same elemental composition as glucose [(CH,O),], and since conversion of glucose t o cell monomers requires virtually no energy (Section D.2, p. 225), only the energy required for polymerization (Section D.l, p. 225) need t o be added to the energy cost of carbon dioxide fixation. Another consequence of the similarity of the cellular C :H :0 composition to that of carbohydrate is that it is not necessary t o invoke further energy requirements for NADHz production; i.e. in production of cell material from glucose, the redox balance is about zero. As a result of these considerations, it is calculated that total cell synthesis from carbon dioxide and inorganic nitrogen and sulphur sources requires about 0.21 (0.036 for polymerization and 0.17 for carbon dioxide fixation) moles of ATP per gram of cells synthesized. Thus, the cost of monomer synthesis from carbon dioxide in relation to polymerization is very large. The relationship of these calculated energy requirements to those found in practice is discussed in Section IV, p. 249.

111. The Generation of Energy A. LITHOTROPHIC METABOLISM 1. Chemolithotrophs

A common feature of the organisms existing as chemolithotsrophs is that, in all cases but one so far studied, the ATP required for cellular metabolism is derived by oxidative phosphorylation. A second feature of this group is the high degree of uncertainty regarding ATP yields associated with their oxidative metabolism due it seems t o the refractory nature of the phosphorylating systems in cell-free preparations. a. Oxidation of Nitrogenous Compounds

i. Nitrite oxidation. A one-step oxidation of nitrite t o nitrate provides the energy required for metabolic process by Nitrobacter species. The enzyme involved is located on a respiratory particle together with cytochromes c , a and a z , and it has been demonstrated that the transfer

228

W. W. FORREST AND

D. J. WALKER

of electrons in the oxidative step is directly to cytochrome c (Aleem and Nason, 1959; Lees, 1962; van Gool and Laudelout, 1966). By comparison with mammalian electron-transport systems then, it would seem that there is a potential for two sites of coupling of phosphorylation in the Nitrobacter system. However, the actual number of coupling sites is not known. Nitrobacter species can also utilize formate as a source of energy for growth (van Gool and Laudelout, 1966, 1967) and it has been shown that formate dehydrogenase, which resides on the same particle as nitrite oxidase, transfers electrons from formate to the transport system used by the nitrite oxidase. I n addition, the Nitrobacter respiratory particle can also oxidize NADH, (Aleem and Nason, 1959; Aleem et al., 1965; van Gool and Laudelout, 1967). The observation that, in Nitrobacter, the initial electron transfer occurs a t the cytochrome c level confirms the assertion of Lees (1962) that the redox potential of the N02--N03- system is too high t o allow direct reduction of nicotinamide nucleotide coenzymes. This situation, which arises in most chemolithautotrophic organisms, presents the cell with the problem of providing reduced NAD for carbon dioxide fixation. It has been shown that such organisms are capable of reducing NAD by an ATP-dependent, mechanism also involving reduced cytochrome and considered by Aleem et al. (1963) and Aleem (1965a) to be reversed oxidative phosphorylation, although Peck ( 1968) proposes an alternative mechanism requiring the intermediate formation of reduced endogenous substrate which could explain the observations. Whatever the mechanism, it would seem that in terms of their energy economy these organisms must expend a t least 1 mole of ATP for each mole of NAD reduced. ii. Ammonia oxidation. I n the bacterial species Nitrosomonas and Nitrosocystis, energy is obtained by the oxidation of ammonia t o nitrite. Both aut,otrophic (Nicholas, 1963, Lees, 1960) and heterotrophic (Fisher et al., 1956; Hirsch et al., 1961) organisms utilize this mechanism, the details of which are probably the least understood of all the major chemoautotrophic energy-generating systems. As a first step in the oxidation, ammonia is considered to be converted to hydroxylarnine and, since such a reaction is thermodynamically unfavourable, Nicholas (1963) considers that it may be energy-driven. Hydroxylamine oxidation has been known for many years, but it was not until Hofman and Lees’ studies in 1953 that direct evidence was obtained for its role as an intermediate in ammonia oxidation, These investigators reported accumulation of a little hydroxylamine when Nitrosomonas cells oxidized ammonia in the presence of hydrazine. Hydraziiie was shown both to inhibit hydroxylainine oxidation and also

THE GENERATION A N D UTILIZATION

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to be oxidized itself (Nicholas and Jones, 1960), and the same workers noted stimulation of hydroxylamine oxidation in cell-free extracts by cytochrome c and by artificial electron acceptors. Subsequently (Aleem and Lees, 1963; Falcone et al., 1962) Nitrosomonas has been shown to contain particles able t o oxidize both hydroxylamine and hydrazine and containing cytochromes a , b and c. Soluble hydroxylamine- and hydrazine-cytochrome c reductases are present in both Nitrosomonas europaea (Hooper and Nason, 1965; Hofman and Lees, 1953) and Nitrosocystis oceanus (Hooper and Nason, 1965). A further piece of evidence concerning hydroxylamine oxidation is that quinacrine inhibits the reaction, suggesting a flavin-mediated electron transfer. These data indicate that a probable energy-generating system in these bacteria is as follows : NHzOH + flavin

->

cyt. 6 + cyt. c + cyt. n + 02

The immediate product of hydroxylamine oxidation is unknown. Anaerobically, with added electron carrier, nitrous oxide and nitric oxide are produced from hydroxylamine (Anderson, 1965 ; Falcone et al., 1963). Also the soluble hydroxylamine-cytochrome c reductases mentioned above do not produce nitrite, and the stoichiometry of hydroxylamine/cytochrome c reduced suggests an intermediate a t the oxidation level of nitric oxide. I n addition, nitrohydroxylamine (H,N,O,) is oxidized to nitrite by both whole cells (Aleem et al., 1962) and extracts (Aleem and Lees, 1963). Whilst it would seem necessary for the participation of a t least one intermediate between hydroxylamine and nitrite, none of the above-mentioned possibilities is a t all firmly established. Furthermore, there is little evidence concerning the energetics of oxidation reactions beyond that of hydroxylamine. Non-conversion of hydroxylamine t o nitrite even with an added electron acceptor, and incorporation of l8O from molecular oxygen into nitrite formed from ammonia (Rees and Nason, 1966). suggest the possibility of oxygenase involvement in the oxidation of hydroxylamine. Consequently, there is little likelihood of ATP production beyond that generated in the initial oxidation of hydroxylamine. Direct reduction of nicotinamide nucleotides has not been observed during oxidation of any of the intermediates postulated for the ammonirtoxidizing pathway and the demonstration by Aleem (1966a) of ATPrequiring NAD reduction indicates that all NADH, required for cell synthesis must be generated a t the expense of ATP. b. Oxidation of Sulphur Compounds, As is the case with ammonia oxidation, the pathways and intermediates involved when the thiobacilli oxidize inorganic sulphur compounds are not altogether established.

230

W.

W. FORREST AND

D . J. WALKER

Oxidation of sulphide has been reported by several workers studying thiobacilli (Peck and Fisher, 1962; London and Rittenberg, 1964; Adair, 1966; Charles and Suzuki, 1966a). However, the heat stability of the system led Adair (1966) t o conclude that the presence of boiled extracts of thiobacilli merely accelerates the non-enzymic oxidation of sulphide and that the reported inhibition of the reaction by potassium cyanide is probably due t o formation of the non-oxidizable thiocyanate. Peck (196s) also considers that the sulphide oxidation pathway may be an artifact in that sulphide can non-enzymically give rise to free sulphur and that this may then be the compound metabolized enzymically. It would seem then in the present state of knowledge that the thiobacilli are unlikely to be able t o obtain energy by oxidizing sulphide to the level of elemental sulphur. Oxidation of free sulphur by the thiobacilli has been abundantly demonstrated. Adair (1966) has shown the conversion of sulphur to sulphate by washed membrane-wall preparations of T. thioxidans, no added cofactors being necessary and cyanide and azide being inhibitory. Sulphite was also rapidly converted to sulphate, but thiosulphate and tetrathionate were inactive in the system. Curiously, although Adair’s particles would fix carbon dioxide if ATP and ribulose 1,5-diphosphate were added, oxidation of sulphur did not stimulate carbon dioxide fixation. Suzuki and Silver (1966) studied T. thioparus and purified an enzyme which oxidized free sulphur t o sulphite. Heavy oxygen was incorporated directly from molecular oxygen into the oxidation products of sulphur suggesting that, in oxidation to the level of sulphite, an oxygenase is responsible and that production of high-energy bonds is unlikely. Of the oxidations of inorganic sulphur compounds, only those involving conversion of sulphite or thiosulphate to sulphate seem to be associated with cytochrome systems (Milhaud et al., 1958; Charles and Suzuki, 1966b). Oxidative phosphorylation during sulphite oxidation has been demonstrated (Charles and Suzuki, 196613;Davies and Johnson, 1967) albeit with low P/O ratios. Oxidation of thiosulphate to tetrathionate is associated with the reduction of endogenous cytochrome or added cytochrome c (Aleem, 1965b; Trudinger, 1961). The latter reaction is involved in the polythionate pathway of oxidation, an alternative pathway for sulphate production :

However, the significance of this pathway is not clear and complete evidence, especially enzymic, for its operation is lacking. Furthermore thiosulphate has been shown to undergo cleavage to sulphite and a

THE QENERATION AND UTILIZATION OF ENERGY DURING GROWTH

231

disulpliide in thiobacilli and in Chromatium (Charles and Suzuki, 1966a ; Bowen et al., 1965; Peck, 1960a; Bowen et aZ., 1966):

which suggests that the major route for thiosulphate oxidation would he through sulphite and sulphur oxidation rather than via polythionates. Another complicating factor in considering the thiobacilli involves the precise mechanism of the conversion of sulphite t o sulphate. Peck and Fisher (1962) reported the production of adenosine phosphosulphate (APS) from sulphite in T . thioparus. At the same time, esterification of Pi into ADP was noted. This suggested that sulphite was oxidized as follows : 2 S 0 3 2 - + 2 A M P -+ 2 A P S + 4 e 2 APS

+ 2 Pi

-+ 2 ADP

+ 2 5042-

Such a scheme could have energetic advantages over a direct sulphite oxidase mechanism for, if the electrons involved were directed through a cytochrome system, oxidative phosphorylation could occur and a further.yield of ATP could arise from ADP by the action of adenylate kinase : 2 A D P + ATP+AMP

Unfortunately APS reductatase, the key enzyme in this pathway, has not been demonstrated in d l thiobacilli, so that two pathways of sulphite oxidation are likely. For a supply of reduced NAD, the thiobacilli apparently rely upon ATP-requiring NADH, production which has been demonstrated (Aleem, 1 9 6 6 ~ ) .I n addition, reduction of nicotinamide nucleotides during oxidation has not been reported. Consequently, with an apparent coupling of electron transport a t a level of cytochrome c , we have, as with organisms oxidizing nitrogenous compounds, a probable maximum P/O ratio of 2 in the thiobacilli. This estimate conflicts with that of Hempfling and Vishniac (1967) who, on the basis of measured molar growth yields during continuous culture of T . neapolitanus, proposed three sites of oxidative phosphorylation associated with thiosulphate oxidation. Since these sites were supposed only to be associated with the oxidation of the “outer” sulphur of S S 0 3 2 - , and not with SO3,- oxidation, then only conversion of free sulphur to sulphite provided electrons for oxidative phosphorylation in their model. I n their calculations, these authors appear t o have assumed that ATP-sulphurylase was the enzyme producing ATP a t substrate level i.e.

sso32- -+ 10

ANIP

S

+PPi

+ so32- +20 + APS +ATP + so42-

232

W. W. FORREST AND D. J. WALKER

Whereas Peck (1960a) observed that ADP-sulphurylase was the most active enzyme in T . thioparus, i.e.

If we assume a maximum P / O ratio of 2 during oxidation of S032to SO,'- and accept Peck's (1960a) assertion that ADP sulphurylase is the dominant enzyme then, for each thiosulphate oxidized, we would have five ATP generated. This is exactly the yield proposed by Hempfling and Vishniac, but is more in line with current concepts of P/O ratios in electron transport and the lack of phosphorylation associated with sulphur oxidation in the thiobacilli. c. Iron Oxidation. A number of chemolithotrophs are capable of obtaining energy for growth from the oxidation of ferrous to ferric ion : 4 FeS04

+ 0 2 + 2 HzS04

--f

+

2 F e z ( S 0 4 ) ~ 2 HzO

There would seem to be some controversy regczrding the taxonomic position of these bacteria in that they also appear to be capable of oxidizing inorganic sulphur compounds (Peck, 1968). Thus, although originally called Ferrobacillus they might be expected ultimately t o be classified as Thiobacillus. Comparatively little information is available regarding the energyproducing mechanisms in these bacteria. Cytochromes c and a , are present and the current hypothesis is that energy-coupling occurs a t cytochrome c (Vernon et al., 1960): Fez+

--f

cyt. c -+ cyt. a +

0 2

Direct demonstration of oxidative phosphorylation is still lacking, and the picture is further confused by the fact that F . ferroxidans requires sulphate for ferrous ion oxidation. From considerations of the rather high potential of the ferrous-ferric couple (+0.77 V), Peck (1968) considers it unlikely that NAD can be directly reduced during ferrous iron oxidation. Alcem et al. (1963) have demonstrated ATP-dependent NAD reduction which tends t o confirm Peck's assertion. From the scanty information available, it would seem that the maximum energy yield from ferrous oxidation would be two ATP per ferric ion produced. d. Ilydrogen Oxidation i. Oxygen reduction. Hydrogenomonas species obtain energy for growth from oxidative phosphorylation coupled t o the reduction of oxygen by molecular hydrogen. So far as is known, these are the only organisms growing chemolithotrophically which reduce nicotinamide nucleotides directly during substrate oxidation rather than relying upon

THE GENERATION AND UTILIZATION

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233

AT€’-dependent NAD reduction. The enzyme system involved in hydrogen oxidation, hydrogenase, occurs in a t least three different forms. Firstly, there is the most common type which varies considerably in its reduction of natural and artificial electron acceptors (Peck et al., 1 % 6 ) , but does not reduce NAD (Kinsky, 1959). Secondly, ATPdependent molecular hydrogen evolution from ferredoxin or reduced methyl viologen is catalysed by an enzyme appearing to be identical to nitrogenase (Bolen et al., 1965; Hardy et al., 1965; Burns and Bolen, 1965).Finally, an enzyme has been extensively purified which specifically reduces NAD (Bone, 1963). Both soluble NAD-reducing, and particle-bound non-NAD-reducing hydrogenases have been reported by Atkinson and McFadden (1954) in H . entropha and by Wittenberger and Repaske (1961) in an unnamed Hydrogenomonas species. It seems possible that Hydrogenomonas uses one type of hydrogenase to generate energy and another t o supply reduced NAD for biosynthesis. This view is strengthened by a report that the activity of the NAD-reducing system is controlled by the intracellular level of reduced NAD (Ahrens and Schlegel, 1965). Demonstration of cytochromes b and c (Packer, 1958) and a quinone (Lester and Crane, 1959) suggests ATP synthesis by more or less conventional oxidative phosphorylation coupled t o electron transport. However, since two NADH,-cytochrome c reductases have been demonstrated (Repaske and Lizotte, 1965))it may be that Hydrogenomonas has a more complete respiratory chain than do the other chemolithotrophs. The picture is further complicated by a report that an NAD-independent hydrogen-oxidizing system, tightly coupled t o phosphorylation, exists in a hydrogenomonad (Bongers, 1967). Consequently, there is no real evidence upon which an estimate of energyyield may be based in these organisms. ii. Nitrate reduction. The only organism which has been studied in any detail whilst growing autotrophically and rcducing NO,- with molecular hydrogen is Micrococcus denitri3cans. Oxidative phosphorylation has been shown t o occur in extracts when nitrate was reduced with molecular nitrogen (Imai et al., 1967; Asano et al., 1967a) as has reverse electron transport for the reduction of NAD (Asano et al., 1967b). Further details of the energy metabolism of this organism remain to be reported. iii. Sulphate reduction. Dissimilatory sulphate reduction is carried out by sulphate-reducing bacteria capable of the anaerobic oxidation of molecular hydrogen with the production of sulphide. Cytochrome c3 is present in Desulphovibrio species (Postgate, 1956) and the electrontransport proteins ferredoxin (Akagi, 1965) and flavodoxin (Le Gall and Hatchikisn, 1967) have been implicated in the sulphate-sulphide

234

W. W. FORREST AND D. J . WALKER

reduction sequence. Peck (1966)has reported evidence for phosphorylation coupled to sulphite reduction with molecular hydrogen in D. gigas, but overall ATP yields in the reduction pathway are unknown. Energy is required for sulphate reduction, because the conversion of this compound to sulphite is preceded by the utilization of ATP and formation of adonosine 5’-phosphosulphate (APS) (Peck, 1959 ;Ishimoto, 1959). Dinit,rophenol inhibition of sulphate, but not sulphite, reduction and methyl viologen inhibition of sulphate, but not sulphite, reduction (Peck, 1960b), both provide further support for electron-transportmediated phosphorylation. iv. Methanogenesis. Oxidation of hydrogen using carbon dioxide as electron acceptor and resulting in methane synthesis is the energy-yielding reaction utilized by bacteria inhabiting the intestines of animals, the black muds of aquatic environments and in the anaerobic tanks of sewage purification works. Being probably the most strictly anaerobic organisms known, these bacteria are difficult to study in detail and relatively little is known of their metabolism. I n terms of free-energy change, Stadtman (1967) has pointed out that there is little likelihood of a net gain of ATP during the reduction of carbon dioxide to a compound a t the oxidation level of methanol suggesting that only the final reduction step is energy-yielding. From figures quoted by Stadtman (1967) on cell yields of ilfethanosarcina barkeri growing on methanol and producing methane as follows : 4 CH3OH + COz

+ 2 HzO + 3 CH4,

the yield of bacteria is about 5 g. dry weight per mole of methane synthesized. Applying growth-yield figures obtained by Bauchop and Elsden (1960) and many subsequent workers (see review by Forrest, 1969a), this would suggest about 0.5 moles ATP per mole of methane. However, since the cells evidently needed t o synthesize cell material from carbon dioxide, and the fixation of carbon dioxide into 5 g.-cell material by the Calvin cycle requires 0.85 moles of ATP (Section IIA, p. 216) it would seem that a t least 1 mole of ATP is generated per mole of methane produced. It is not known if the methanogenic organisms manufacture NADH, by direct reduction with hydrogen or by reversed oxidative phosphorylation. If the latter be the case, then the yield of ATP may be greater than unity for each equivalent of methane formed. 2 . Photolithotrophs

There are three maiii groups of bacteria t o be considered under this heading, the green sulphur bacteria, the purple sulphur bacteria, and

THE GENERATION AND UTILIZATION

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235

the non-sulphur purple bacteria. Green sulphur bacteria (or Chlorobacteriaceae) represented by the genus Chlorobium are strictly anaerobic and fix carbon dioxide into cell material whilst oxidizing hydrogen sulphide to frcc sulphur which transitorily accumulates in the medium. Sulphur can be oxidized further to sulphate and, i n Chlorobium thiosrrlp~iutophili~ni, thiosulphate can also be oxidized (Larsen, 1 952). For complete oxidation of hydrogen sulphide, the stoichiometry for fixation of carbon dioxide is H2S

+ 2 COz + 2 HzO

+

2 [CHzO]

+ HzS04

In the light, hydrogen can serve directly as source of reducing power for fixation of carbon dioxide in Chlorobium washed cells (Gaffron, 1935) and Larsen has shown that Ch. thiosulphatophilum will grow on a CO.,-H, mixture. Like the green sulphur bacteria, the purple sulphur bacteria, or Thiorhodacene, oxidize sulphur compounds in the light with concomitant fixation of carbon dioxide. During oxidation of hydrogen sulphide, elemental sulphur accumulates in the cells, but is subsequently oxidized quantitatively t,o sulphate when the hydrogen sulphide has been dcpleted. I n like fashion to the Chlorobacteriaceae, fixation of carbon dioxide obeys the above equation, thiosulphate can also be oxidized, and molecular hydrogen can be used directly for fixation of carbon dioxide. A major difference between the green and purple sulphur bacteria is the fact that the latter can grow anaerobically in thc light upon organic compounds whereas the former will grow only in concert with the oxidation of inorganic sulphur compounds. Athiorhodaccae, or non-sulphur purple bacteria, chiefly use organic compounds as hydrogen donors, although a number of strains can use molecular hydrogen for direct reduction of carbon dioxide in the light (van Neil, 1944). Tlic metabolism of these organisms is very complex and although carbon dioxide is fixed during photosynthetic growth upon organic compounds, there is great diffirnlty in deciding to what extent this might be exchange reactions (Elsden, 1962). During autotropliic growth, the photosynthetic organisms, like the rlieniolitliotroy~hicorganisms require energy for both carbon dioxide fixation and the production of reduced nicotinamide nucleotides. The result of a great deal of research into the energy-generating mechanisms operating in photosynthetic bacteria leads to the current view that light is iiccessary for ATP production only. For detailed reviews of the ~ncc~liaiiisrns involved Gest (1966) and Vernon (1968) should be con-

236

W.

W . FORREST AND D. J. WALKER

sulted. Briefly, ATP is generated by a “cyclic” photophosphorylation mechanism schematically shown as follows :

ADP cyt. b

LIGHT

4

becteriochlorophyl

TATP

Y& quinone

ADP

The evidence for two sites of pliosphorylation in this cyclic mechanism is reviewed by Vernon (1968). In green plants, there is evidence for a second mechanism for phosphorylation known as non-cyclic photophosphorylation, but it is currently held that such a reaction sequence does not exist in bacteria. As is the case with the chemolithotrophs, the photosynthetic bacteria used reduced NAD rather than NADP as source of reducing equivalents for biosynthesis. Although ferredoxin has been isolated from photosynthetic bacteria, it has not been shown to be involved in reduction of nicotinamide nucleotides (Vernon, 1968). Ferredoxin is however known to be involved in the reversed TCA cycle mechanism for carbon dioxide fixation proposcd for Chlorobium thiosulphatophilum (Evans, Buchanan and Arnon, 1966) discussed in Section IIA (p. 215). At this time, NAD reduction in the photosynthetic bacteria is considered to arise by reverse electron transport (Gest, 1066; Vernon, 1068).

B.

ORGSNOTROPHIC

METABOLISM

Organisms growing hcterotrophically obtain their energy and carbon for growth from orgaiiic substrates more reduced than carbon dioxide. This does not necessarily meail that carbon dioxide fixation does not occur, indeed, it is an essential feature of the metabolism in many heterotroph, but rather that carbon dioxide is fixed only during the synthesis of certain essential compounds. The range of organic compounds catabolized by bacteria is huge and it is possible only to consider the major classes of substrates within the limits of this review. 1. Anaerobic Phase

A full range of carbohydrates, amino acids and heterocyclic compounds is metabolized by bacteria via anaerobic processes.

T H E GENERATION AND UTILIZATION O F ENERGY DURING GROWTH

237

a. Carbohydrates i. Depolyni Prization of polysaccharides. Despite the fact that n wide range of ~~olysaccl1arides-cellulose, hemicelluloses, starch, xylans, a1 c ~ I ) mm s d others-can be degraded by bacteria, there is no evidence that energy changes of high-energy bond magnitude are involved until the level of relatively simple sugars is reached. For the purposes of this discussion therefore, the mechanics of polysaccharide hydrolysis can be ignored. ii. Degradation of simple sugars. A feature of the several alternative pathways for anaerobic dissimilation of carbohydrates is that the energy yield depends upon the manner in which a phosphorylated ketose sugar intermediate is further metabolized. The ketoses involved are fructose 6-phosphatc, fructose 1 ,G-diphosphste, xylulose 5-phosphate and 2-keto-3-dcoxy-G-~~lzosphoglucoiiate. I n all cases, formation of triose phosphate, w1iic.h is fiirthcr degraded by the latter part of the EnibdenMcyerhof-Parnas pathway, plays a role in energy generation, but in addition, there are pathways which involve conversioii of part of the sugar molecule to acetyl phosphate and to pyruvate without the involvement of the triosc phosphates. These latter compounds are of course further potential sources of energy, but the realization of this potential is dependent upon tlie range of products resulting from the entire sugar ferment at’1011. Of key importance in all sugar-utilizing systems, the EmbdenAleyerhof-Parnas (EMP) pathway results in tlie fermentation of hexose to the level of 1Jyruvate with a net J ield of 2 ATP per glucose equivalent degraded. Tlic entire sugar molecule is fermented via triose phosphate, the important ketose intermediate, fructose 1: 6-diphosphate, having been split by aldolase : Hcxose + 2 ATP -> l+’rurtose 1 : G &phosphate + 2 ADP Fructose 1 : 6-cliphosphntr r 2 Tiiose phosphate 2 ‘ h o s e phosphntc i ADP I 2 Pi --f 2 PyIuvato 2 [2 HI i 4 ATP

+

Hexosc

+ 2 Pi

I 2 .ZDP -r

2 Pyruvate i 2 [2 HI

+ 2 ATP

An intercsting variant of hexose-degrading pathways was discovered by Schramni, Klybas and Racker (1958) in Acetobacter xylinum and further studied by de Vries and Stouthamer (1968) in Bi.dobacterium bi$dus. In this organism phosphorylation of the sugar prior to degradation proceeds only as far as fructose 6-phosphate. At this stage a phosphoketolase specific for this substrate catalyses a split of the ketose phosphate yielding acetyl phosphate and erythrose 4-phosphate. Transaldolase and traiisketolase then transform the latter substrate together with a further equivalent of fructose 6-phosphate into two equivalents of xylulose 5-phosphate. This compound, another key

238

W. W. FORREST AND D . J. WALKER

ketose phosphate, is acted upon by a further phosphoketolase yielding ttriosc phosphat~a and further acetyl phosphate. After this lengthy ~)rowss,t>lw 1;Lf tcr part of the EMP pathway is able to convert’ triose phosphate t o t,he level of pyruvate : 2 Hesoso + 2 ATP --> 2 Fructose 6-phosphate + 2 ADP Pructuso O-phosphato iPi --f Erythrose 4-phosphate Acetyl phosphate Fructose 6-phosphate + Erythrose 4-phosphate + 2 Xylulose 5-phosphate 2 S,yliilosc 5-phosphate + 2 Pi --L 2 Triose phosphate + 2 Acetyl phosphate 2 rrriose phosphatc I- ADP + 2 Pi -+ 2 Pyruvate + 4 ATP + 2 [2 HI

+

2 Hexosr I 5 PI I 2 ADP + 2 Pyruvate

+ 3 Acetylphosphate + 2 [ 2 HI + 2 ATP

The energetic advantage of such a pathway is that there is, compared to the EAIP pathway, an extra mole of high-energy product available from each 2 moles of hexose degraded. Formation of ethanol and carbon dioxide from glucose by Zymomonas fyagilis involves a third mechanism for hexose degradation known as the Entner-Doudoroff pathway. After phosphorylation at C-6, the sugar is oxidized to 6-phosphogluconate which in turn is dehydrated to yield 2-keto-3-deoxy-Ci-pliosphogluconate. The latter compound is the key kctose phosphate in the pathway and is split by an aldolase which results in formation of triose phosphate and pyruvate. The conversion of half of the hexose molecule directly to pyruvate with the involvement of triose phosphate results in loss of ATP generation potential. Overall, in the Z .fmgilis fermentation, the net energy yield for glucose degradation is but one ATP per glucose : Hexose I ATP --f Glucose G-phosphate + ADP Glucose 6-phosphate --f 6-Phosphogluconate 2 [HI G-Phosphogluconate + 2-Keto-3-deoxy-6-phosphogluconate + HzO 2-Keto-3-dooxy-6-phosphogluconate --f Triose phosphate + Pyruvate Trioso phosphate + Pi + 2 ADP + Pyruvate 2 ATP 2 2 Pyruvute i 4 [HI + 2 Ethanol 2 COz

+

+

Hrxosp t AI>P t Pi + 2 Ethanol

+

+

+ 2 COz + ATP

Pentos(b sugars are fermented by one of two altcrnative mechanisms. Thc first of thcsc involves il complex series of reactions resulting in conversion of pentose t o ribose 5-phosphate and xylulose 5-phosphate which subsequently undergo condensation and cleavage reactions under the influence of transaldolase and transketolase finally yielding fructose &phosphate. Thereafter, the conventional EMP pathway is responsible for degradation. From the reactions summarized here, it is seen that for each pentose degraded, 1.67 equivalents of ATP are generated. G Pentose + 6 ATP + G Pentose 5-phosphate + G ADP 6 Pentose 5-phosphate --f 4 Xylulose 5-phosphate + 2 Ribose 5-phosphate 2 Sylulosc 5-phosphate + 2 Ribose &phosphate --f 2 Sedoheptulose 7-phosphate + 2 Triose phosphate

THE GENERATION AND UTILIZATION OF ENERGY DURING GROWTH

2 Sedohoptulose 7-phosphate

+ 2 Ribose 5-phosphate

239

+ 2 Fructose 6-phosphete

+

2 Erythrose 4-phosphate 2 Erythrose 4-phosphete + 2 Xylulose 5-phosphate + 2 Fructose 6-phosphate 2 Triose phosphate 4 Fructose 6-phosphate 4 ATP + 4 Fructose 1,6-diphosphate+ 4 ADP 4 Fructose 1,6-diphosphate -> 8 Triose Phosphate 10 Triose phosphate + 10 Pi + 20 ADP + 10 Pyruvate 10 [2 HI + 20 ATP

+

+

+

G Peritosc

+ 10 Pi + 10 ADP

+ 10 Pyruvate

+ 10 [2 HI + 10 ATP

The st:coiid pat)hwayfor pentose fermentation is much simpler. I n this, tmitosc. is phosphorylated and converted to the key intermediate in xylulosc Ci-phosphat,c.Further breakdown of this compound involves a direct split of the molecule catalysed by phosphoketolase and resulting in t'he production of triose phosphate and acetyl phospliatJe. Pentose + ATP + Pentose 5-phosphate + ADP Pentose 5-phosphate + Xylulose 5-phosphate Xylulose 5-phosphate + Pi + Triose phosphate Acetyl phosphate Triose phosphate + Pi + 2 ADP + Pyruvate 2 H 2 ATP

+

Pentosr I ADP I 2 Pi + Pyruvate

+

+

+ Acetyl phosphate + 2 H + ATP

Takcn to thc level of acetate and pyruvate, the fermentation of each pentosc yic.lds the equivalent of 2 ATP. iii. Further metabolism of pyruvate and acetyl phosphate. I n the metabolic pathways of sugar fermentation discussed, we have considered energy yields to the level of pyruvate and in some cases of acetyl phosphate. Now anaerobes will utilize these compounds to produce a wide variety of end-products, and the ultimate yield of energy for growth available to any one organism depends upon the end-products formed. Acetyl phosphate is of courso a potential source of A T P when acetokinase is present : Acetyl phosphate t ADP

--f

Acetate

+ ATP

Howcver, in certain circumstances, such as in the heterolactic fernientation carried out by Leuconostoc mesenteroides, acetyl phosphate produced by phosphoketolase split of xylulose 5-phosphate is used as a hydrogen acceptor and reduced to ethanol. Thus, the potential for A T P synthesis is in this case lost, and the organism must rely upon the fermentation of triose phosphate. also derived from xylulose &phosphate, to lactate for energy generation Tn certain rlostridial fermentations, acetyl phosphate is used as a iirimary substrate for the formation of butyrate and but anol which, being relatively reduced compounds, are the repository of electrons generated during substrate catabolism (Barker, 1956). As a result, the energy held in acetyl phosphate is lost as acetyl groups condense t o form the four-carbon compounds. Exactly how much energy is required for the synthesis of short-chain fatty acids seems to be in doubt. Certainly

240

W. W. FORREST AND D . J . WALKER

it is known that in bacterial systems so far studied for long-chain fatty synthesis, extra energy is required for the formation of malonyl-CoA prior to attaching the acetyl unit onto the growing fatty acid chain (Kates, 1966): Acctyl-CoA + ATP

+ COz

+ Bfalonyl-CoA + ADP

+ PI

On the other hand, Thauer et al. (1968) argue the metabolism of CZ. Kluyveri on the basis of butyrate synthesis requiring merely acetyl-CoA and acetate, despite the fact that the malonyl-CoA pathway is known to operate in long-chain fatty acid synthesis in this organism. Also, the work of Goldman, Alberts and Vagelos (1963) suggests that direct condensation of two-carbon units operates in short-chain fatty acid synthesis. I n the production of butyrate, the penultimate step is the formation of butyryl-CoA from crotonyl-CoA and in the release of free butyrate, the coenzyme-A moiety can be transferred to acetate thus preserving the high-energy bond. If however butanol is the end-product, butyryl-CoA is reduced to butanol and the energy potential of the thioester is lost (Barker, 1956). It can thus be seen that the provision of electron “sinks’ in certain txpcs of fermentation result in substantial losses of energy which would otherwise be available for growth. Pyruvate also acts as hydrogen acceptor, either directly in reduction to lactate or after conversion to some other intermediate prior to reduction, as in the case of ethanol formation. I n such cases, there is no energy gain from the further metabolism of pyruvate. There are organisms however which have the capacity to generate further ciicrgy from pyruvate by conversion t o active acetyl. Thus, coliforni organisms and certain lactic acid bacteria produce acetyl-CoA or acetyl phosphate and formate from pyruvate : Pyruvatc

+ HzO + PI or CoA

--f

Acetyl phosphate or Acetyl-CoA + Formate

Some clostridia on the other hand possess hydrogenase and can thus dispose of unwanted reducing power as molecular hydrogen. I n this case, also, pyruvate can be metabolized to yield a further high-energy bond : Pyruvatr

e

HnO

+ PI

--f

Acetyl phosphate

+ COz + Hz

An alternative route for pyruvate metabolism in Propionibacterium spp. and certain other species is the production of propionic acid. I n most organisms producing propionic acid, pyruvate is first carboxylated to yield oxaloacetate which is then reduced to succinate. The final stage of the reaction is the decarboxylation of succinate t o yield propionate. The energetics of this pathway are not fully understood (Wood, 1962) although there is some indirect evidence suggesting that 3.5 or more ATP

THE QENERATION AND UTILIZATION

OF ENERGY DURING GROWTH

241

per glucose arise from the propionic fermentation carried out by Propionibacterium pentosaceum (Bauchop and Elsden, 1960). This would suggest that, as well as obtaining ATP from the EMP pathway in production of pyruvate, ATP is also generated in the conversion of pyruvate to both acetate and propionate in the fermentation which has approximately the following stoichiometry : 1.5 Glucose

--f

2 Propionate

+ Acetate + COz

Allen et al. (1964) have purified many of the enzymes associated with the Propionibacterium propionate fermentation and have concluded that the following series of reactions accounts for the energy yields thought to occur : 1.5 Glucose

+ 3 ADP -t 3 Pi

+

3 Pyruvate

COA

+ 3 [2 HI + 3 ATP

+

Pyruvatc + Pi +Acctyl phosphate [2 HI + C02 Acetyl phosphate -t ADP + Acetate + A T P 2 Pyruvate + 2 Methylmalonyl-CoA --f 2 Oxaloacetate + 2 Propionyl-CoA 2 Oxaloacetate + 2 [2 HI + 2 Malate 2 RIalate + 2 Fumarate + 2 HzO 2 Fumarate + 2 ADP !- 2 P i 2 [2 HI + 2 Succinate + 2 A T P 2 Succinate + 2 Propionyl-CoA + 2 Succinyl-CoA + 2 Propionate 2 Succinyl-CoA + 2 Bfethylmalonyl-CoA

+

1.5 Glucose

+ 6 Pi + 6 ADP

+

Acetate

+ 2 Propionate + 2 H2O + COz + 6 A T P

A salient feature of this scheme is a saving of energy in the carboxylation of pyruvatt by coupling with the decarboxylation of methylmalonyl-CoA under the influence of a transcarboxylase. A second feature is the suggestion that energy generation occurs during the reduction of fumarate to succinate, a reaction coupled to the oxidation of pyruvate to acetate. S. R. Elsden (quoted by Gunsalus and Shuster, 1962) has pointed out that the redox potential of the fumarate-succinate couple is such that ATP formation is feasible, but actual phosphorylation has not yet been unequivocably demonstrated in anaerobes. I n a number of organisms, notably several genera isolated from the rumen, succinate arises as a n end-product of fermentation. Studies on the enzymology involved shows that these organisms carboxylate phosphoenolpyruvate (PEP)under the influence of PEP carboxykinase with recovery of the high-energy bond as GTP or ATP (Scardovi, 1963, 1964; Scardovi and Chiappini, 1966; Hopgood and Walker, 1969). Phosphoenolpyruvate

+ GDP or A D P + COZ +

Oxaloacetate

+ G T P or A T P

Hopgood and Walker (1969)did not however detect any phosphorylation associated with the reduction of fumarate t o succinate, and growth-yield

242

W . W. FORREST AND D. J . WALKER

studies combined with enzymic data suggested that the only energyyielding reactions were involved in glycolysis, carboxylation of phosphoenolpyruvate and formation of acetyl phosphate from pyruvate. Propioiiibacteria and Jilicrococcus lactyliticus also carry out a propionic fermentation using lactate as substrate. Indeed, M . lactyliticus will not ferment glucose. The scheme proposed above for glucose fermentation by propionibacteria will account for the apparent ATP yield from lactate (Bauchop and Elsden, 1960), but the situation with &I. Zactilyticus is more complex. According to Galivan and Allen (1968) the latter organism does not contain methylmalonyl-CoA-pyruvate transcarboxylase and one must therefore assume that energy is required for the formation of oxaloacetate from pyruvate. Since the energygenerating sites in the fermentation are unknown, the net ATP yield is also in doubt, although indirect evidence based on growth yields would suggest a net yield of one ATP per lactate (Elsden, 1962). At least three organisms, Clostridium propionicum (Cardon and Barker, 1947)) Peptostreptococcus elsdenii (Lewis and Elsden, 1955 ; Ladd, 1969) and Racteroides ruminicola (Wallniifer and Baldwin, 1967) utilize an alternative route for the conversion of lactate to propionate. This involves dehydration of lactate yielding acrylate which is then reduced to propionate utilizing electrons generated by oxidizing further lactate to acetate via pyruvate :

+ +

Lactate --f Acrylate HzO Lactate -+ Pyruvate 2 H Acrylate + 2 H + Propionate Pyruvate + HzO -+ Acetate + 2 H 2 Lactate + Acetate

+ COz

+ Propionate + COz + 2 H

I n P. elsdenii, the 2 H available is partly disposed of as molecular hydrogen and partly in reduced products such as butyrate and valerate but, in C1. propionicum which has no hydrogenase, further lactate is reduced by propionate in disposing of the excess reducing power. Energetically, P . elsdenii is the organism most intensively studied, and it is known that the various transformations involve the coenzyme A derivatives of lactate, acrylate and the volatile fatty acids (Ladd and Walker, 1965). However, a novel dual activation of lactate is said t o be necessary before conversion to acrylate (Schneider and Wood, 1969). The reaction involves firstly the formation of lactyl-CoA and then phosphorylation a t the hydroxyl group to yield phospholactyl-CoA. Acrylyl-CoA is formed by phosphate elimination. The overall reaction sequence in P. elsdenii would then be : Lactate + Pyruvate Pyruvate + H2O + Pi

+2H --f

Acetyl phosphate

+ CO2 + 2 H

T H E QENERATION AND UTILIZATION O F ENERGY DURING QROWTH

Lactate + Propionyl-CoA -+ Lactyl-CoA + Propionate Lactyl-CoA + Acetyl phosphate -+ Phospholactyl-CoA Phospholactyl-CoA + Acrylyl-CoA P i Acrylyl-CoA + 2 H + Propionyl-CoA

+

2 Lactate

--f

Acetate -t Propionate

243

+ Acetate

+ COz + 2 H

This reaction sequence provides no net energy yield, and Anderson and Wood (1969) suggest that anaerobic electron transport may be coupled to phosphorylation in the organism. I n this connection, Ladd and Walker (1959) noted that the fermentations of both lactate and acrylate were completely inhibited by low concentrations of 2,4-dinitrophenol, azide and hydroxylamine, but so far it has not been possible to demonstrate unequivocally the coupling of phosphorylation to electron transport (Baldwin and Milligan, 1964). b. Amino Acids i. Simple fermentation. Amino acids are fermented singly by a group of some twenty anaerobic or facultatively anaerobic bacteria, chiefly clostridia and micrococci (Barker, 1962). I n the main, volatile fatty acids are the products of fermentation and prior formation of highenergy derivatives of these compounds probably serves as energy-source for the bacteria. However, precise pathways are known only for a few amino acids and organisms, making energy yield assessment extremely difficult. Clostridium propionicum is the only organism so far known which ferments alanine, producing acetate, propionate, carbon dioxide and ammonia. The mechanisms involved are unknown, but since Cl. propionicum forms propionate exclusively via acrylyl derivatives it would seem that an early step must be deainination of alanine to form acrylate. Some acrylate must then be converted via lactate to pyruvate in order that acetate may be produced and reducing equivalents made available for acrylate reduction. Considering the mechanisms previously discussed for the acrylate pathway of lactate fermentation, the following series of reactions could account tor alanine degradation :

+

3 A1 ine + 3Acrylate 3 NH3 2 A c s a t e + 2 Propionyl-CoA + 2 Acrylyl-CoA + 2 Propionate Acrylyl-CoA + P i --f Phospholactyl-CoA Phospholactyl-CoA + Acrylate + Lactate + Acrylyl-CoA + P i Lactate + Acrylyl-CoA ---f Pyruvate + Propionyl-CoA Pyruvate + Pi + HzO -+ Acetyl phosphate COz 2 H Acrylyl-CoA + 2 H + Propionyl-CoA

+

3 Alanine

+ H2O + P i

-+

Acetyl phosphate

+

+ 2 Propionate + 3 NH3 + COz

The energy yield would thus rest on the formation of acetyl phosphate unless anaerobic electron transport-associated phosphorylation occurred. Diplococcus glycinophilus and two species of Micrococcus are known

244

W . W. FORREST AND D . J . WALKER

to ferment glycine. Again, full details of the pathway remain to be elucidated, but due to the work of Sagers and Gunsalus (1958), the following reactions are thought to occur :

+

Glycine Tetrahydrofolate --f RIethylene tetrahydrofolate + COz + NH3 Methylene tctrahydrofolate Glycine + Serine + Tetrahydrofolate Serine + Pyruvatc + NH3 Pyruvate + HzO + P i or CoA + Acetyl phosphate or Acetyl-CoA + COz + 2 H 2 Glycine

+

+ HzO + P i or CoA

+ Acetyl phosphate or Acetyl-CoA

+ 2 COz + 2 NH3 +2H

Thus, to this stage, glycine fermentation would seem to yield energy tlirough the formation of high-energy acetyl derivatives derived from pyruvate. However, there remains a problem of an acceptor system for the reducing power generated. I n D. glycinophilus, molecular hydrogen may be formed, but not in the micrococci. Tracer evidence suggests reduction of carbon dioxide t o acetate in glycine fermentation and, although recent work has indicated the mechanism for this reaction (for review see Ljungdahl and Wood (1959)), the energy-requirement is uncertain. Nonetheless, it is certain that synthesis of acetate from carbon dioxide is energy-requiring, and we have a further example of the need for electron acceptors in anaerobic metabolism being responsible for a lowering of the available energy for growth of the cell. Coniplcte fermentations of arginine, citrulline and ornithine are unknown, although Clostridium perfringens and some streptococci can convert arginine to ornithine with a net production of ATP, carbamyl phosphate acting as high-energy intermediates (Kozenovsky and Werkman, 1953). Arginine + HzO --f Citrullino Citrulline Pi + Ornithine carbamyl phosphate Carbamyl phosphate + ADP --f NHa + COz + A T P

+

Arginine

+ H2O + Pi + ADP

+

+ Ornithinine

+ NH3 + COz + A T P

Bauchop and Elsden (1960) have demonstrated that, whilst Xtreptococcus faecalis cannot grow on arginine alone, when arginine is added in substrate quantities to a glucose-containing medium the ATP derived from arginine catabolism is used to produce an extra increment of growth. Cysteine is fermented by a large number of organisms, many of which have been shown to contain cysteine desulphydrase which removes hydrogen sulphide and ammonia from cysteine yielding pyruvate. It is probable that conversion of pyruvate t o acetyl phosphate or acetyl-CoA is the energy-yielding reaction in this fermentation although complete product analysis has been reported only for Peptostreptococcus elsdenii (Lewis and Elsden, 1955). I n this organism, cysteine gives rise to

THE GENERATION AND UTILIZATION OF ENERGY DURING GROWTH

245

equivalent amounts of C 0 2 and NH,, the chief fatty acids formed being acetate and butyrate. Proteus morganii will degrade homocysteine t o hydrogen sulphide ammonia and a-ketobutyrate (Kallio, 1951): HSCHzCHzCHNHzCOOH

+ HzO

.+ HzS

+ NH3 + CH~CHZCOCOOH

The further metabolism of a-ketobutyrate in P . morganii has not been reported, but Peptostreptococcus elsdenii converts this compound t o propionyl-CoA which could serve as a source of ATP for growth (Walker, 1958): a-Ketobutyrate

+ HzO + CoA

--f

Propionyl-CoA

+ 2 H + COz

I n P . elsdenii, the available reducing equivalents are disposed of as molecular hydrogen. Peptostreptococcus elsdenii, C1. propionicum and some micrococci ferment threonine, again with the participation of a-ketobutyrate as an intermediate. Propionate is the main product in all such fermentations, but GI. propionium also produces large quantities of butyrate. The reason for this is probably that the latter organism, not possessing hydrogenase, cannot dispose of the reducing equivalents from cr-ketobutyrate oxidation as gaseous hydrogen. Consequently, there would seem to be some mechanism for the direct reduction of a-ketobutyrate to butyrate. The fermentation of serine is accomplished by a number of organisms. Pyruvate is the key intermediate, being formed as a result of serine dehydrase activity, and the ultimate products of fermentation and energy yield depends upon the pyruvate-handling system possessed by the organism. Thus, Clostridium botulinum produces approximately equal quantities of acetate and ethanol (Clifton, 1940), which indicates that half of the potential of pyruvate t o produce an energy-rich compound in acetyl phosphate is lost by the necessity to use pyruvate as a hydrogen acceptor. I n contrast, P . elsdenii (Lewis and Elsden, 1955) and M icrococcus aerogenes (Whitely, 1957) possess hydrogenase and can therefore convert most or all of the pyruvate formed from serine to acetyl phosphate or acetyl-CoA, disposing of the reducing power as molecular hydrogen. Fusobacterium nucleatum (Jackins and Barker, (1951) channels the reducing equivalents from pyruvate breakdown into ethanol and butyrate, both of which are synthesized a t the expense of energy-rich compounds. A further alternative is used by C. propionicum, which synthesizes propionate as a means of reducing power disposal (Cardon and Barker, 1947). Histidine fermentation is accomplished by a few organisms, but the

W. W.

246

FORREST AND D. J. WALKER

generation of energy for growth does not occur until a series of reactions has yielded glutamate as follows :

+

Histidine -+ Urocanate NH3 Urocanate + 2 HzO -+ Formiminoglutamate Formiminoglutamate + HzO --f Glutamate + Formamide

Three organisms closely studied from the point of view of histidine fermentation also ferment glutamate. They are Cl. tetanomorphum (Wachsman and Barker, 1955),Cl. tetani (Pickett, 1943) and Micrococcus aerogenes (Whitely, 1957). Of these organisms, the pathway of glutamate degradation in Cl. tetanomorphum has been intensively studied (Barker, 1962). The unusual pathway employed by this organism results in coiiversion of glutamate t o citramalate via P-methylaspartate and mesaconate. Citramalate then splits t o yield acetate and pyruvate which latter compound provides apparently the only precursor of a highenergy compound in the entire fermentation of glutamate or histidine. However, the fermentation of glutamate by any of the above organisms does not result in significant quantities of hydrogen being produced, and the reducing power generated by pyruvate oxidation t o acetyl phosphate or acetyl-CoA is channelled into butyrate synthesis. The result is that only half of the potential energy yield residing in pyruvate is realized : 2 Glutamate --f 2 Citramalate + 2 NH3 2 Citramalato + 2 Acetate 2 Pyruvate 2 Pyruvate + 2 HzO + 2 Acetyl phosphate or Acetyl-CoA 2 Acotyl-CoA -1- 2 [2 HI + Butyryl-CoA CoA

+

+

2 Glutamate

+ CoA

--f

2 Acetate

+ 2 [2 HI + 2 COz

+ Butyryl-CoA + 2 COz

Amino acids apart from those discussed above are, like leucine, valine, proline and hydroxyproline, not known t o be fermented, or they are insufficiently well studied to be able to assess adequately the probable energy yields. Included in the latter group are aspartate, methionine, lysine, tryptophan, tyrosine and phenylalanine. ii. The Stickland reaction. A large number of clostridia can gain their energy for growth by utilizing the Stickland reaction, which is a coupled decomposition of a pair of amino acids neither of which is usually degraded when present alone. I n essence, the reaction results in the oxidation of one amino acid to a keto acid with the same number of carbon atoms. No doubt oxidative decarboxylation of the keto acid to a fatty acid with one less carbon atom is energy-yielding in the same fashion as is pyruvate decarboxylation.

+

+

+

RCHNHzCOOH HzO + RCOCOOH 2 H NH3 RCOCOOH -1HzO + Pi or CoA + RCO-phosphate or RCO-CoA RCHNHzCOOH

+ 2 HzO + Pi or CoA

--f

+ 2 H + COz RCO-phosphate or RCO-CoA + COz + 2 [2 HI

OF ENERUY DURING GROWTH

THE GENERATION AND UTILIZATION

247

Thus, from the oxidation there is reducing power to be disposed of which, in Stickland reactions, is directed to the amino acid being reduced. There is no evidence that reduction of the hydrogen acceptor amino acid is energy-yielding, so that it would be expected that one high-energy bond would be generated for each equivalent of the oxidized amino converted t o products. c. Heterocyclic Compounds. Purines, pyrimidine and related compounds are fermented by a limited number of bacteria. The purine fermentations proceed via xanthine which is converted by way of a number of steps not involving high-energy compounds t o formiminoglycine. The latter compound is then split in the presence of tetrahydrofolic acid yielding free glycine and 5-formiminotetrahydrofolate (Barker, 1962)which, after conversion to the 10-formyl compound, is split in the presence of ADP and orthophosphate yielding formate, tetrahydrofolnte and ATP

+

Purine --f Sarithine --f x NH3 y COz + 10-Formyltetrahydrofolate+ Glyclne 10 Formyltotrahydrofolate ADP PI + Formate + Tetrahydrofolate + ATP

+

Purine

--f

Glycine + Formate

+

+ ATP z NH3 + y COz

Since glycine and formate are the major products of purine fermentation in Cl. cylindrosporum, the expected energy yield for this organism would be one ATP per equivalent of purine degraded. Other organisms, notably Cl. acidiurici, produce acetate as a major end-product (Rabinowitz and Barker, 1956). This is apparently accomplished by the conversion of glycine to serine a t the expense of a formyltetrahydrofolate, thus removing the potential for energy generation from this compound. However, fermentation of serine as discussed above would result in active acetyl production thus restoring an energy-yielding mechanism to the system. For both glycine-formate and acetate-type purine fermentations then the energy yield would be one ATP per purine fermented. Decomposition of pyrimidines by the three organisms known to attack these compounds is incompletely characterized. Zymobacterium oroticum converts orotic acid via dihydroorotic acid and ureidosuccinic acid t o aspartate, carbon dioxide and ammonia (Lieberman and Kornberg, 1955). Since the reaction sequence to this stage does not appear to involve phosphorylation, the further metabolism of aspartate must supply the energy requirements of the organism. The details of this aspartate breakdown are not clear, but Barker (1962) suggests that the following mechanism operates :

+

Aspartate + a Ketoglutarate -+ Oxaloacetate Glutamate Oxaloacetato --f Pyruvate COz Glutamate + NAD + a-Ketoglutarate NADHz NH3 __ __ _ _ Aspartate + NAD + Pyruvete COz NH3 NADHz

+

+

+ +

+

+

248

W. W.

FORREST AND D. J. WALKER

The NADH, generated in this reaction is required t o convert further orotic acid to dihydroorotic acid. Pyruvate oxidation t o acetate could supply energy for growth although, since the organism produces no hydrogen, an electron acceptor must be found for reducing power generated in converting pyruvate t o acetate. Since the organism is reported to produce dicarboxylic acids, it is probable that some oxaloacetate produced from aspartate is reduced t o malate and/or succinate. Clostridium uracilicum converts uracil t o p-alanine, carbon dioxide and ammonia. However, the organism requires carbohydrate for growth and uracil does not stimulate growth, so that it is doubtful if energy is generated during uracil decomposition. Other heterocyclic compounds known to be fermented are allantoin by Streptococcus allantoicus and nicotinic acid by an unidentific clostridium. However, so little is known of the reaction mechanisms that it is fruitless at this stage to speculate upon energy yields. 2. Aerobic Phase

Despite a great deal of research which has taken place over a period of many years, there is still no clear picture emerging of energy yields during the aerobic phase of bacterial metabolism. Certainly there is substantial evidence for the operation in many aerobic bacteria of the tricarboxylic acid cycle as the major oxidative pathway (for review see Krampitz, 1962). In addition, a great many organisms have been shown to contain similar electron-transport components such as flavins, quiiiones and cytochromes to those involved in mammalian electrontransport systems ( D o h , 1962). However, the number and type of electron-transport components in bacteria varies with the species and sometimes with conditions of growth. Insofar as phosphorylation coupled to electron transport in bacteria is concerned, the quantitative aspects are unfortunately confused. Whereas in mammalian systems it is reliably established that 3 ATP are generated when reduced nicotinamide nncleotide is oxidized via the cytochrome system, in preparations from bacteria, values ranging from 0.4 t o 1 ATP have commonly been found (Smith, 1962). Suggestions have been made that such low values for ATP synthesis (or P/O ratio) results from damage to the respiratory system during isolation of the respiratory particles. Indirect support for this contention is provided by growth yield studies which indicate, on the basis of cell yield per mole of oxygen consumcd, that P/O ratios of 2 for E. coli and Ps. jluorescens and ratios of 3 for Aerobacter aerogenes and Saccharomyces cerwisiae oxidizing glucose (Stouthamer, 1969). It would seem that finality on the question of P/O ratios for bacterial oxidations must await a great deal more

THE GENERATION AND UTILIZATION

OF ENERGY DURING GROWTH

249

research. Further discussion on estimates of P / O ratios derived from growth-yield studies is included in Section I V of this review (p. 253).

IV. The Usage of Available Energy

A. MOLARGROWTH YIELDS 1. Anaerobic Growth

Jacques Monod studied anaerobic growth of Bacillus subtilis, Escherichia coli and Salmonella typhimurium in minimal medium with a large number of different carbohydrates as carbon and energy sources. He found that, for every substrate, the yield, expressed as the ratio weight of cells produced to weight of substrate degraded had a fixed reproducible value. De Moss et al. (1951) studied the growth of the lactic acid bacteria Streptococcus faecalis, Leuconostoc mesenteroides and Lactobacillus delbrueckii. They found that the growth yield was a linear function of carbohydrate concentration and proposed that the yield for L. mesenteroides was low because it obtained less energy from the substrate than the other oganisms. Further work then showed that this explanation was correct (Hurwitz 1958). The heterofermentative organism, L. mesenteroides, ferments glucose t o lactate and ethanol by the hexose monophosphate pathway, producing only 1 mole of A T P per mole of glucose compared with 2 moles of A T P per mole of glucose in fermentation by the E M P pathway in the homolactic fermenters. Sokatch and Gunsalus (1957) found that S . faecalis gave similar growth yields on glucose and on gluconate as energy source; their conclusion was that the two substrates produce the same amount of biologically utilizable energy. Bauchop and Elsden (1960) then proposed that the amount, of growth of a micro-organism is proportional t o the amount of A T P available to it from degradation of an energy source, and they proceeded to the experimental verification of this hypothesis by measuring yields of several organism growing anaerobically on complex media with limited energy source. They showed that essentially all the substrate added was used as energy source so that cellular carbon was derived from preformed monomers in the complex media. Thus the free energy required for synthesis under these conditions must be required chiefly for polymerization of the monomers t o cellular macromolecules. As other energy-requiring processes would be a t a minimum, the maximum yield of cells would be expected. I n the fermentations studied, they could calculate the amount of ATP produced by known metabolic pathways, 2 moles per mole of glucose degraded by the EMP pathway by S. faecalis and Saccharomyces sp.,

TABLE4. Growth I'ields from Fermentations

Organism

KI"J? yield Ysubstrate (moles ATP/ (g. dry wt/ mole mole Substrate substrate) substrate)

YATP (g. dry wt/ mole ATP)

Number of determinations

3 4 r @ References

La M

2 Streptococcus f d i s

Sts.elpt0coccu.s lactis Streptococcus pyogenes Lactobacillus plantarum

glucose

2.0-3.0

20.0-37-5

10.9 f 0.2

14

gluconate

1.8

17-6-20

10.4

2

2-ketogluconate ribose arginine pyruvate glucose glucose glucose

2.3 1.67 1.0 1.0 2.0 2.6 2.0

19.5 21.0 10.2 10.4 19-5 25.5 18.8

8.5 12.6 10-2 10.4 9.8 9.8 9.4

1

1 1 1 1 1 1

Bauchop and Elsden (1960) Beck and Shugart (1966) Forrest and Walker (196%) Hempfling et al. (1969) Smalley et al. (1968) Sokatch and Gunsalus (1957) Goddard and Sokatch (1964) Sokatch and Gunsalus (1957) Goddard and Sokatch (1964) Bauchop and Elsden (1960) Bauchop and Elsden (1960) Forrest (1965) Boivinet (1964) Davies et al. (1968) Oxenburgh and Snoswell (1965)

5

tr

P Y

2

B PJ

Saccharomyces cereziisiae

glucose

2.0

18.8-22.3

10.2 5 0.3

5

Saccharomyces rosei

glucose glucose

2.0 1.0

22.0-24.6 8.0-9.3

11.6 8.5 8 0.2

2 5

Zymmnonas mobilis

Bauchop and Elsdtn [ 1 960) Battlcy (1960). Buld(t1 (1963) Kormancikova et al. (1969) c;l Bulder (1963) (1966) Bauchop and Elsden (1960) 0 Belaich and S m e z (1965) M Dawes et al. (1966), Forrest (1967) m Senez and Belaich (1965) kBoiviriet (1964), Hadjipotrou et al. 2 0 (1964) Z Hadjipetrou et al. (1964) kzU Hadjipetrou and Stonthamer (1965) d I,. P. Hadjipetrou and A. H. 2 Stouthamti; cited by Stouthamw (1969) Hernandez and Johiison (1967a) Stouthamer (1969) z

a

2

Aerobucter aerogenma

Aerobucter cloacae Escherichia colia Rurninococcus jlavefueiens a Actinomyces imaelii Bi$dobacterium bifidum

glucose

3.0

26.1-29'5

10.7

2

fructose mannitol

3.0 2.5

26.7 21.8

1O.i 10.8

1 1

gluconate

2.5

21.4

11.0

1

1.5-2.5 3.0 2.75 2.0 2.5-3.0

17.7-27.1 25.8 29.1 24.7 37.4

11.9 0.5 11.2 10.6 12.3 13.1

+

3 1 1 1 1

glucose glucose glucose glucose glucose

8 0

Hopgood and Walker (1967) w Buchanari and Pine (1967) M 15'. de Vries and A. H. Stouthamrr; cited by Stouthamer

2

(1969)

a

Corrected for incomplete fermentation.

!4

4,

CJ

w

252

W. W. FORREST AND D . J . WALKER

and 1 mole per mole of glucose degraded by Zymomonas mobilis by the Entner-Doudoroff pathway. They could then compare the yields of different organisms on the basis of a yield coefficient YATP,defined as the number of grams dry weight of cells produced per (calculated) mole of ATP generated from catabolism. The procedure gave a rational basis for comparison in biochemical terms of the efficiency of different organisms and pathways of fermentation on the basis of the energy available for biosynthesis. They found that the organisms they studied gave a common value of about 10 for Y A T p . Since this work, results have been reported with a number of organisms with different metabolic pathways and a variety of substrates. Table 4 lists molar growth yields for fermentations under conditions where the yields of ATP from the fermentations can be calculated from known metabolic pathways. The mean value of YATPis 10-6 f 1.0 g. per mole for 47 determinations. The differences in Y A T p shown for different organisms are real and usually greater than the experimental error of the determinations. Values for heats of combustion of micro-organisms suggest that their gross compositions are approximately the same (Baas-Becking and Parks, 1927; Senez, 1962). I t follows that microorganisms can use the energy available from catabolism with about the same maximum efficiency of conversion, that is, in fermentations a t least the degree of coupling between anabolism and catabolism is the same for widely differing organisms and metabolic pathways. Under conditions of adequate nutrition with energy generated by phosphorylation a t the substrate level, i t appears that biosynthesis can go on rapidly enough to use the energy from catabolism efficiently as i t becomes available. Streptococcus faecalis grown anaerobically with adequate nutrition and pyruvate as energy source gives a molar growth yield of 10-4. The degradation of pyruvate yields one ATP per mole, so that YATpis also 10.4, in excellent agreement with the mean of Tablc 4.However growth is linear, not exponential, and direct measurement of the intracellular ATP pool (Forrest, 1965) shows that the level of the pool is much lower than that found in this organism during exponential growth; indeed the pool falls to the level characteristic of starved cells (Forrest and Walker, 1965a). The level of the intracellular pool of ATP has been shown in S . faecalis (Forrest, 1965), E . coli (Cole et al., 1967), yeast (Polakis and Bartley, 1966), Klebsiella aerogenes (Harrison and Maitra, 1969) and Rhodospirillum rubrum (Schon, 1969) t o correspond to a balance between the input of ATP from catabolism and withdrawal to drive the processes of biosynthesis. The low level in S. faecalis growing on pyruvate then indicates that growth is so thoroughly limited by the availability of energy supply that exponential growth cannot take place (Hess, 1963). It appears then that this is a case of

THE GENERATION AND UTILIZATION

OF ENERGY DURING GROWTH

253

maximum efficiency of coupling, so that a value of Y A T p of about 10.5 is probably an experimental maximum value for this organism. This value of Y A T p is much less than that to be expected if all the ATP produced by catabolism were coupled entirely to biosynthesis of chemical h i i d s . Several calculations have been made of the requirements of ATP for the synthesis of cellular material from simple precursors (Gunsalus and Slzuster, 1962; Lehninger, 1965; Fukiu and Hirata, 1968). Our detailed analysis (Section IID, p. 223, and Tables I and 2) is in general agreenicnt with the earlier calculations ; it clearly shows that one nould expect a value of YATPof about 28 under the conditions where experimentally YATPis 10.6. It is clear that most of the ATP produced by catabolism is used in ways which are not chemically defined. It is perhaps worth emphasizing that there may be unexpected experimental difficulties involved in determinations of YATP.These have been thoroughly discussed by Stouthamer ( 1969). 2 . Gyowth Yields in Aerobic Metabolism

The Iwsition with aerobic organisms is much less clear than with fermentations. Qualitatively, the yields of aerobically grown cells are usually much greater than those for the same organism grown anaerobically ; this is presumptive evidence for a large increase in biologically available energy and is often cited as evidence for oxidative phosphorylation. Smalley rt al. (1968) found that Streptococcus faecalis, which has no cytochromcs, gave considerable increases in yield in aerobic culture, and similar results have been reported by Moustafa and Collins (1969). On the basis of this increased yield a P/O ratio of 0.5 was calculated. However the data for yields from aerobic growth do not cxhibit the same quantitative regularities as the yields during fermentations. a. PI0 Ratios. Reproducible results have been obtained for individual systems in terms of grams dry weight of organisms produced per gram of substrate cntabolizrd (Monod, 1942) but systematic comparisons between organisms and substrates on the basis of Y A T p values are very unsatisfactory. I n fermentations where ATP is produced by phosphorylation a t the substrate level, the amount of ATP produced can be calculated accurately, but the efficiency of production of ATP from oxidative phosphorylation in bacteria is not well defined. Thus comparisons on the basis of tlic P/O ratio, the calculation of YATp from oxygen consumikion during growth, are not reliable. If the converse correlation is attcniptcd of assuming the value of YQTPto be 10 and calculating P/O ratios from growth yield, values ranging from 0.5 to 3 have been reported; 0-6 for oxidative metabolism in 8.faecalis (Smalley et al., 1968)

234

W. W. FORREST AND D. J. WALKER

and Aerobacter oxidans (Whitaker and Elsden, 1963); 1 for succinate oxidation in A . aeroqenes (Hadjipetrou et al., 1964), 2 for E . coli and Ps. jluorescens, and approaching 3 for A . cloacae, Candida utilis (Stouthamer, 1969) and A . aeroqenes with glucose as energy source (Hadjipetrou et al., 1964). With Zymonzonas mobilis though the normal components in the respiratory chain are present, growth on glucose can take place either anacrobically or aerobically with respiration, but the growth yield is unaffected by respiration (Belairh and Senez, 1965). The inference is that no energy is derived from respiration in this organism, a P/O ratio of zero. Chen (1964) calculated from material balances that the yeast Candida utilis gave 60 g. dry weight of cells from the complete oxidation of 0.144 g. of glucose. Assuming complete energetic coupling and the formation of 38 moles of ATP per mole of glucose from the tricarboxylic acid cycle, this corresponds to a YATpof 11.0, a value in close agreement with the yield coefficient for anaerobic organisms. Unfortuiiately the balance did not include arginine which was also present in the growth medium (Hernandez and Johnson, 1967b) so that the true YATp would be somewhat below the calculated value. Chen also calculated a similar balance for Xaccharomyces cerevisiae. It appears that Site I for oxidative phosphorylation is inoperative in Saccharomyces (Schatz and Racker. 1966; Olinishi et al., 1966) so that 28 moles of ATP are produced per mole of glucose oxidized. The corresponding value of YATPfrom Chen's balance is 8.5. Korinancikova et al. (1969) also found with Sacch. cerwisiae growing on a complex medium, assuming YATp t o be 10.5, that the observed yield corresponded t o the production of 28 moles of ATP per mole of glucose catabolized. It seems that this incompleteness or inefficiency of the respiratory chain may a t least partly account for the low yields often reported from aerobic growth. b. Oxygen Uptake. A commonly used parameter in determinations of growth yields of aerobic organisms is the amount of oxygen consumed during growth; Y o (Hadjipetrou et al., 1964) is expressed as the grams dry weight of organisms produced by the consumption of one gram atom of oxygen; the division of Yo by YATpthen gives the P/O ratio. The assumption is made that oxygen uptake is a measure of that part of the substrate which is completely oxidized. However, the further implicit assumption that the ATP produced by catabolism corresponds to the oxygen uptake is not always valid. The oxygen tension in the culture may have a profound effect on metabolism, leading to wide changes in products of catabolism and ATP production as it is varied (Wimpennp, 1969). Anaerobically, Aerobacter aerogcnes forms ethanol, formic acid, butanediol, acetoin, acetic acid and carbon dioxide. As the oxygen tension is increased, these products disappear in the order listed

THE GENERATION AND UTILIZATION OF ENERGY DURING GROWTH

2%

except for carbon dioxide, production of which increases as the othw products are formed in smaller amounts (Pirt, 1957; Harrison and Pirt, IOG7). The situatioii is further complicated by the effects of oxygen t o ~ i c i t y .If too much oxygen is supplied complete oxidation of the substrate may be effected but the growth of cells can often be inhibited (\Visemun Pt al., 1966; Dalton and Postgate, 1967). Hadjipetrou r / al. (1964) found that, with A . awogenes under different conditions of aeration, the oxygen uptake during catabolism of the energy source was always the same but that the yield of cells depended on the aeration ; apparently less ATP was produced per mole of oxygen consumed when the oxygen tension was lower because of the different balance of products. I n these experiments, growing the organisms with limited energy source (glucose) but with more than sufficient aeration to maximize the yield of cells, about 30% of the glucose was not oxidized but remained as acetate a t the end of growth. The acetate was then oxidized by the organisms with further uptake of oxygen but no furt’her growth occurred during this secondary process. Assuming YATpto be 10, a 1’jO ratio of 3 was calculated for the primary growth process. Very similar results were reported by Grangetto (1963) who carried out microcalorimetric experiments on the aerobic growth of A . aerogenes on glucosc and succinate with adequate aeration. The reported growth yields were in w r y good agreement with those found by Hadjipctron et al. (1964). The microcalorimetric records showed that catabolism of succinate was a simple process accompanying exponential growth, and tlie observed heat production agreed 1vell with theoretical calculations for the enthalpy change during the complete oxidation of succinate. The observed yield corresponded to a P / O ratio of less than 1. However, with glucose, the same complex behaviour as reported by Hadjipetrou P t al. (1964) was found, namely a n initial period of exponential growth with accumulation of acetate, a diauxic lag, followed by degradation of ncetnte with no further growth. Thus a t least 207, of the free energy available from tlie oxidation of glucose was not used for growth, and mergetic uncoupling occurred during the filial degradation of acetate. The calorimetric data also showed clearly that, even after the completion of all tho catabolic processes of the organisms, only about half of the calculated entlialpy change for the complete oxidation of glucose was in fact observed. It seems t h a t oxidation of glucose was not in fact complete, so that the calculated P/O ratio of 3 in this system is difficult to reconcile with these observations. Other l?Ieasureme?rts. I n attempts t o obtain a more meaningful parameter to describe growth yields, Mayberry et al. (1967) proposed that the number of “available electrons” would be a more satisfactory index than Yo.This parameter is calculated from tlie number of moles (3.

256

M’. W. FORREST AWD D. J WALKER

of oxygen stoichionietrically required for complete oxidation of the substrate. I n studies of the growth of soil organisms obtaining their energy from oxidation of a large number of energy sources, much more reproducible results were obtained with this calculated parameter than with the experimentally measured Yo. However extrapolation of the yield of cells per mole of “available electrons” t o other systems gave variable results; it is clear that oxygen uptake, either measured or calculated, is not a sufficient description of aerobic catabolism. Mayberry et al. (1967) also suggested the use of the enthalpy of catabolism as an index of growth yields. Only a few microcalorimetric determinations of this quantity are a t present available, but the microcalorimetric data suggest that even in anaerobes enthalpy of catabolisni is not correlated with growth yields (Forrest, 1970). A large part of the experimental problem appears t o be the incomplete definition of catabolism in aerobic systems. Von Meyenburg (1969) grew Sacch. cerevisiae on a synthetic medium and carefully considered the alternative metabolic pathways which may operate under different experimental conditions. His analysis showed that there are four distinct components of catabolism which may vary with diferent dilution rates in continuous culture, and from studies on synchronous growth, variations occur also between catabolism during cell division and in the periods between divisions. His analysis gave a Y,,, value of 12-0 & 0.5, and a P/O ratio of 1.1 & 0.05. Thus the growth yields of Sacch. cereaisiae have been variously reported to correspond t o P / O ratios lying between 1 and almost 3 (von Meyenburg, 1969; Korniancikova et al., 1969; Stouthamer, 1969; Clien, 1964).The data then all suggest that the yields of aerobically grown organisms are quite generally lower than values calculated on the basis of yield obtained during anaerobic growth, but may reach an upper limit corresponding to this calculated yield. This situation is not inconsistent with a yield coefficient ( YATP)the same as that found in anaerobes; rather it appears to correspond to either energetic unc~oupling(Senez, 1962), the inefficient use of the energy available from catabolism, or to incomplete oxidation of the energy sources. It has been proposed (Rottenburg et al., 1967) that coupling in substrate-level phosphorylation between catabolic reactions and generation of ATP is likely to be quite efficient, whereas in oxidative phosphorylation uncoupling may readily occur a t various points in the respiratory chain, so that exact stoichiometry between moles of substrate oxidized and moles of ATP generated is not to be expected. Instead Rottenburg et al. (1967) show from considerations of irreversible thermodynamics that P/O ratios may vary with such factors as the rate of catabolism or oxygen supply. The fractional P/O ratios often cal-

THE GENERATION A N D UTILIZATION

OF ENERQY DURING QROWTH

257

d a t e d (Stouthamer, 1969) suggest that in fact exact stoichiometry is not obeyed. Comparative measurements of the intracellular ATP pool in Escherichia coli grown aerobically and anaerobically (Cole et al., 1967; \\7mpenny, 1969) show that aerobically the level of the pool is about twice the aiiacrobic level, but this is much less than the differences t o be expected if oxidative phosphorylation produced ATP at the maximum calculated rate. However with the very indirect evidence available it is not possible to say at which point in the complex sequence of coupled reactions uncoupling is likely to occur; what is obviously necessary is some more satisfactory measure of production of ATP during aerobic metabolism. 3. Anomalous Growth Yields

The value of 10-5 for the yield coefficient ( Y A T p ) may be considered as an upper limit for cells of normal average composition growing in a complex medium where full energetic coupling occurs. However several factors may operate to modify this yield. Firstly, the cells may vary in composition. This difference in composition may be partly responsible for the different values for Y A T p for different organisms reported in Table 4 (p. 250). Very large differences in composition may also occur under some circumstances, with cells of the same organisms. If limitation of some constituent of the medium causes growth to cease while excess energy source is still present, catabolism of the energy source continues but the ATP so produced is no longer required to drive the biosynthetic processes of growth. The ATP can then be diverted t o synthesize reserve materials. This synthesis of reserves is generally observed to take place only after exponential growth has ceased (Hungate, 1963; Forrest and Walker, 1965a); in E . coli the laying down of glycogen as a reserve has been shown to involve a repression mechanism under genetic control which allows the biosynthesis t o proceed only in non-growing cells (Damotte et al., 1968). A wide variety of organisms lay down such reserves, mainly carbohydrates or poly-P-hydroxybutyrate (Dawes and Ribbons, 1964; Doudoroff, 1966), although in Staphylococcus aureus (Mikucki et al., 1969) and Streptococcus faecalis (Forrest and Walker, 1963) the reserve materials are possibly nucleic acids. The laying down of these reserves can cause very large increases in the dry weight of the orgaiiisms; up to half the dry weight may be reserves (Doudoroff, 1966; Damotte et al., 1968). Substantial amounts of Carbohydrates added primarily as energy sources may be assimilated instead of degraded, and the polymerization reactions go on with high efficiency of use of ATP (Walker, 1968). When cellobiose or other

2.58

W. W. FORREST AND D . J. WALKER

carbohydrates are polymerized by rumen organisms, about one-third of the carbohydrate is degraded to provide energy for the polymerization of the remainder. This process can give apparent molar growth yields of about 2-10 for the increase in dry weight concurrent with the anaerobic clegradation of cellobiose. For m i twcurate deterniiiintion of thc aniouiit of’ ATP lroduced by catabolism, the metabolic pathway must, of course be known. Actinomyces isradii grown anaerobically on glucose carries out a homolactic fermentation in the absence of carbon dioxide with a normal growth yield (Table 4,p. 250) ; but with substrate quantities of carbon dioxide, fixation occiirs with a large increase in growth yield (Buchanan and Pine, 1967). Thc carbon recovery in this system was unsatisfactory, so that calculations of Y A T p are indefinite. Values for Yglucose up to 40 have been reported for anaerobic growth of Streptococcus faecalis (Forrest and TValker, 1966a; Beck and Shugart, 1966; Moustafa and Collins, 1968; Hempfling et al., 1969; Loy and Beck, 1969). It had earlier been coiisidered that this organism always carried out a homolactic ferment at’ion with 2 moles of ATP being produced for each mole of glucose degraded, and thew high yields have brcn cited as evidence for anomalous values of YATP(Jloustafa and Collins, 1968). Careful analysis of the fermentat ion products have established that these may be lactate, ethanol and acetate with the proportions of each varying with growth conditions. Concurrently with this difference in products, the amount of ATP produced per mole of glucose varies, but Y A T p in this organism is invariant over a wide range of growth conditions (Hempfling et al., 1969). Very high yields have been reported with rumeii organisms. Ruminococcus nlbus grown on cellobiose accwmulated large quantities of storage polpsaccharides and gave an apparent molar growth yield of 90.1 (Hixngatc, 1 963). Corrections were made for storage polysaccharides and assimilation of carbon; the products of degradation were 4 moles of acetate per mole of cellobiose and, assuniing a phosphorolytic cleavage, Hungate proposcd a yield of 9 moles of ATP per mole of cellobiose degraded. This gives a normal valuc for Y A T p of 9.6. However, the amount of ATP produced from the degradation of this disaccharide was much more than twice that usually found from the degradation of simple sugars (Table 4, p. 250) because of the different balance of 1)roducts from the disaccharide, so that even with a normal value of YATp, the molar growth yield was very high. Selenomonas ruminantium grown on glucose in batch culture gave a molar growth yield of 17 with essentially a homolactic fermentation. There was some assimilation of carbon, and the YATPvalue was about 9, but in continuous culture the same organism gavc maximum yields of

THE GENERATION AND UTILIZATION O F ENERGY DURIKG GROWTH

859

65 (Hobson, 1965). Though more volatile acids were produced in continuous culture, these could not explain the great increase in yield. Pirt (1965) analysed Hobson’s data to attempt t o determine the energy of maintenance of the organism, but the analysis gave very abnormal results, suggesting the organism behaved unusually in continuous culture. Similar high yields were obtained by Hobson and Summers (1967) in continuous culture of a lipolytic bacterium which gave a Yfructose value of 60 and Bacteroides amylophilus giving a Y maltose value of 130. There is no reasonable explanation of such divergences. However, they occur in continuous culture, under conditions where catabolite repression of the activities of various enzymes is not very effective (Silver and Mateles, 1969). It is possible then that the organisms are using mixed or unusual substrates as energy sources in pathways which they cannot utilize in batch culture. P. N. Hobson (private communication) has analysed the thermodynamics of the degradation of the substrates on which these high yields are observed. It is thermodynamically possible, that is, enough free energy is available, a t intermediate steps in the degradation pathways to allow the generation of more ATP in addition t o that normally produced during catabolism. However there is no biochemical evidence, aside from the anomalous yields, for such increased energy production. Apparently the only report of an abnormally high yield during oxidative metabolism is in the growth of Agrobacterium tumefaciens on sucrose (Fukui and Hirata, 1968). Here the addition of manganese ion t o the medium caused approximately a fourfold increase in yield, and the YATpvalue, assuming a P/O ratio of 3, was calculated to be over 40. It is much more common, and more readily explicable, to find low growth yields. There are several reasons for these. 4. Energy of Maintenance

I n the situation where the specific growth rate of the organisms is less than the maximum rate of which the organisms are capable on the nicdium, the growth yield is commonly decreased in proportion to the lowering of the growth rate. Such lowered yields can often be observed in continuous culture systems operating a t low dilution rates. Analysis of the data obtained in these systems (Pirt, 1965; van Uden, 1969) reveals that there are two components making up the observed rate of catabolism of the energy source by a unit mass of cells. There is a small component independent of the specific growth rate and a much 1a.rger one proportional to the growth rate. Micro-organisms require a supply of energy which can be coupled through ATP to maintain their normal

260

W.

W. FORREST

AND D . J. WALKER

functions (Forrest and Walker, 1963; Strange et al., 1963; Forrest and Walker, 1965b ;McGrew and Mallette, 1965), so that it has been proposed that the larger component of degradation of substrate during growth supplies the energy for biosynthesis, and the smaller the constant requirement of the cells for energy of maintenance (Pirt, 1965). I n fact the “energy of maintenance” is that component of the energy available from Catabolism of the energy source which is not used to drive the biosyntheses of growth, and it may also include components due to energetic uncoupling and losses from catabolism by non-viable cells, so that it is likely to be an over-estimate of the true maintenance requirements of the cells (van Uden, 1969). Pirt has analysed data for the continuous culture of Aerobacter aerogenes, A . cloacae, a lipolytic bacterium and Selenomonas ruminantium (which gave anomalous results). Other determinations of maintenance requirements have also been reported for Escherichia coli (Schulze and Lipe, 1964), Hydrogenomonas (Schuster and Schlegel, 1967), Thiobacillus neapolitanus (Hempfling and Vishniac, 1967) and Debaryomyces subglobosus (Wase and Hough, 1966).A similar analysis for growth of Azotobacter vinelandii (Aiba et al., 1967) shows comparable behaviour, but indicates a very high maintenance requirement though the amount of energy not used for growt,h apparently varies greatly with different organisms. Pirt’s analysis gives by extrapolation a n assessment of the “true growth yield”, the maximum yield possible a t infinite growth rate when all the available energy is coupled t o the syntheses of growth. This situation is practically unattainable, but the yields obtained in batch culture with adequate nutrition should closely approach this limiting value. The analyses also suggest that, typically, several percent of the energy available is not used for growth. Direct measurements of energy of maintenance in growing cells are not practical, but direct determinations are available for non-growing cells of E . coli (McGrew and Mallette, 1965) and S. faecalis (Iqorrcst and Walker, 1963). These determinations suggest that the maintenance requirement is insignificant on the level of the energy requirements for growth, so that energetic uncoupling may be of importance in contributing t o the indirectly determined “energy of maintenance” during growth. 5. Energetic Uncoupling Under normal conditions of growth in complex media, the eficiency of coupling between anabolic and catabolic processes appears t o be at a maximum, at least in anaerobes; but in the extreme case of a washed suspension of organisms supplied with a n energy source but no nutrients, catabolism of the energy source may go on rapidly with no detectable

THE QENERATION AND UTILIZATION

OF ENERQY DURING GROWTH

261

increase in mass of cells. I n washed suspensions of Streptococcus faecalis catabolizing glucose or arginine, direct measurements of the intracellular pool of ATP show that ATP is generated by catabolism and enters the pool at a rate proportional to the catabolic activity of the veils and the metabolic pathway (Forrest, 1965; Forrest and Walker, l965a) so that the size of the pool may rise up to tenfold during the process of catabolism. Thus energetic uncoupling occurs by a failure to make use of the gTP; the only significant anabolic processes occurring are maintenance reactions which require only a small part of the available energy. Similar processes may be observed during growth. Rosenberger and Elsden (1960) grew S. faecalis on a medium which was tryptophanlimited in continuous culture. Limitation caused a decrease in growth yield, but the rate of Catabolism of glucose per unit mass of cells was unaffected, and presumably the rate of production of ATP was constant also. I n batch cultures of S . faecalis with excess energy source, but with growth limited by growth factors, the cessation of exponential growth is not accompanied by a corresponding immediate decrease in the rate of catabolism of energy source. Thus ATP is produced by catabolism which is not required for biosynthesis, and measurements of the intracellular ATP pool show that this rises sharply at the same time that exponential growth ceases (Forrest and Walker, 1965b). Belaich and Senez (1965) grew Zymomonas mobilis with glucose as energy source on various media in batch culture and obtained molar growth yields of ti on complex medium, 5 on synthetic and 4 on minimal medium. The specific growth rates were in a ratio corresponding to that of the growth yields, but the catabolic activity per unit mass of cells was the same on all of the media; thus the rate of production of ATP was the same in all of the media but the coupling to anabolic processes was most effective in the complex medium. I n an extension of this work, Belaicli P t al. (1969) grew 2. mobilis on synthetic and minimal media in batch culture under conditions of pantothenate limitation. The specific growth rate and growth yields were decreased about fourfold but the balance of products and rate of catabolism of glucose were not affected. The effect appears too large to be explained as variations in the requirements of the cells for energy of maintenance. Nor apparently is it due simply to the greater demands for energy to synthesize simple precursors in the minimal medium. Senez (1962) found with Desulphvibrio desulphuricans growing on a simple medium that the growth yield was the same whether or not the organisms had t o synthesize amino acids de novo. Our calculations (Section IID.2, p. 2 2 5 ) confirm that the requirement of ATP for this biosynthesis is insignificant. I n D . desulphuricans (Le Gall and Senez, 1960) the cellular catabolic activity

262

W. W.

FORREST AND D. J. WALKER

was found to be constant even though the growth yield was very different on diffcrent media. Analysis of the products of catabolism indicated that the ATP yield was the same in the different media. A similar constancy of catabolic activity with varying growth yields on different media was reported with Aerobacter aerogenes (Pichinoty, 1960). Detailed analysis of the changes in kinetics of growth arising from such limitations is given by van Uden (1969). Changes in the degree of coupling between anabolic and catabolic processes may also occur when organisms are grown a t different temperatures (Senez, 1962; Forrest, 1967). Palumbo and Witter (1969) found that the pathways of glucose catabolism in Pseudomonas JEuorescens were not affected by temperature, but the yield of cells decreased a t low temperature while the proportion of glucose consumed in continuous culture for purposes other than growth increased. There was a change in the degree of coupling between anabolic and catabolic processes. It is customary to express the thermal increment in the rate of a kinetic process as the energy of activation E of the Arrhenius equation :

-E

1x1 k = - -~ + C RT where k is the rate constant of the process, R is the gas constant, T is the absolute temperature and C is another constant, Values for E are then determined from the slope of a graph of Ink against 1/T.For most mesophiles around their temperature optima, E is about 10-15 kcal. Figure 1 shows plots of the energies of activation for anabolism and catabolism in S.faecalis and 2. mobilis. Over a wide range of temperature the rate of catabolism of glucose by growing cells is affected by temperature only in the same way as a simple chemical reaction with energies of activation in both organisms of about 11 kcal. Near the temperature optima of the two organisms, the specific growth rate parallels the rate of catabolism, there is the same degree of coupling and the growth yield is constant. At temperatures remote from the optima, there is a large change in the thermal increment for growth; the growth yield falls markedly, but the thermal increment for catabolism is unaffected. Thus coupling becomes less effective, and measurements of the intracellular ATP pool suggest a changed energetic balance above and below the “critical temperature” (Senez, 1962) a t which changes in the thermal increment become apparent. I n all these cases, changes in the degree of coupling occur from failure to make effective use of ATP which had been produced by catabolism. However, uncoupling may also occur if the organism does not have sufficient ATP in its pool to transfer the energy available from catabolism a t a sufficient rate. Senez and Belaich (1865) grew E . coli anaerobically

263

THE GENERATIOS A N D UTILIZATION OF ENERGY DURIXG GROWTH

-

02 -

A ', 'A

A'A _I

19 32

12

33

34

\

A'

33

32

Reciprocal of absolute temperature ( x

34

lo3)

FIG.1. Therinal increments of growth yields, anabolism and catabolism of' (left) Zymomonas mobilis arid (right)Streptococcus faecalis. From Forrest (1967).

on ~)liosl)liatc-liiiiitcdmedium in batch culture ; expo~iciitialgrowth took place with a Yglucobe value of28.9 until pl~osphatelimitation became apparent, thcn linear growth continued with a Yplucose value of 10.9 though the rate of catabolism of energy source was not decreased. The effect of this I)hospIiatc limitation a s immediately and completely reversible. ‘rllk appears t o be a ease where uncoupling occurred through lack of the coupling agent. Direct mcasuremeiit of the ATP pool in I)hosphate-liniit~dE . coli (Damaglou and Dawes, 1967) sliows that t h e l e vel of the 1’001 is much lower t h a n normal. The reversibility of the limitation suggests a change in the equilibrium situation between ATP a n d inorganic pliospliate Contrcl systems analogous t o “respiratory control” in initoclioiidrin have not been demonstrated i n bacteria : in iioii-growing cells the absence of sucli controls under conditions of‘17arying dem:uids of e n e r g for biosynthesis and cation transport liavc been established in S. faecalis (Forrest and \Talker, 1965b) and in E . coli (Hempfling et ul., 1967). The work on energetic unroupling coilfirms t h a t , in growing cells also, there appenrs t o Iw no generally operative iiiecliaiiism by which the growing (.ell controls its ratc3 of production of energy according t o the rate of utilization. Catabolism, a t least with constitutive em! m e systems, goes II

264

11

IV. FORREST A S D D. J . W-ALKEK.

on at thc iiiaxiiiiuni rate of which the organism is citpa,ble in the circu111stances regardless of xvliether or not the energy so produced is uscful to the organisms. Under conditions of adequate nutrition, it appears that I)iosyntlicsis can go on fast enough to use the available energy efficiently, I)ut if the rate of biosynthesis is decreased through inndequate nutrition. t liis t1rcrc:isc does not necessarily affect the rate of catabolism 13. r

l

'

~

~ASSESSJIESTS ~ t ~ ~

~

~

~

I . F W PEnergy EfJicieizuy

I n cwiitrast to the biocliemical approach exemplified by measurements of growth yields, assessments of efficiency have been made on a thermo. The percentage free energy efficiency (Baas-Brvking and Parks. 1927) is dcfinrd a s . - iooAE',, Prce energy of growth x 100 __ I b e energy p r o d u ~ db;~catabolism of energy sourcc AT, ~~~

A 8',] is the frec energy usefully emp1o;ved for biosynthcsis of cell niateri a1 dining growt 11. In most c'Lses values of A F , , vannot bc directly assessed, but in the case of autotrophic bacteria obtaining their cncrgy from inorganic cwiripouiids and their carbon requirements for growth from reduction of' (*arbondioxide. a reasonably accurate definition is possible. BaasI3r~cl
~

~

T I W GEXJWATIO\

A \ I) U T I L I Z A T I O ~OF EXERGY U I i I t I h G G N O T~I L

26.i

byiithesis. Thus, Seiiez ( 1962) calculated t h a t the efficiency of geiieratioii of ,4'l'P by the EM€' pathwaj- does not exceed 5076. Each mole of ATP tlicii givcs risc, t o about 1 0 g. of cells, but from the calculations presented c,arlicr this 3 T P is then used for biosyiithesis with about 309, efficiency. s o 1lint the overall efficiency for substrate-level phospliorylation is onl? a h n t lc?%.For oxidative phosplior~~latioii, t h e cficieiic>y overall is ~)robizblycomparable. A tlieoretical efficiency of generation of A T P of up t o 7 0 9 , is balanced by a generally lower coupling efficiency t h a n ill niiaerobes. Tlieoretical cdculations of A F , b y Rlorowitz (1968) give values of below 100 calories per g. of cells. Again substitution of this value into t h c cquatioii gives a free-energy cfficieiicj- of about 5, ,for homo lac tic^ fernieiiters siirli as S.faecalis. IVhilst these determinations show t h a t the efficiency of growth oii this criterion is low, tlierinodyiiamirs iiidicates only what is energetically ~)ossibleaiid givc no inforniation about tlie details of tlie energetics of growth. The difkrence between t h e thermodynamic aiid biochemical iLssessnieiits is illustrated by considering the hoinoluctic fermentation of glucose which is quoted (Seiiez, 1962) as producing ATP with a n efficiency of SOq , , aiid the ethanolie fermentation which, because of t h e difference in tlie balaiice of products, is oiily 40% efficient,. Both of tlicsc 1)athwnys give rise to a yield of cells of about 2 1 g. (Table 4. 1). 2 5 0 ) ; t l i c x quaiitized iiaturc of production of A T P does not allow an). tiiorc biologically usefiil energy t o be extracted from t h e "inore efficient " limncnt a t i o I 1 2. JIicyocaloyimetyic ilIeaszwernenl.\

'I'here have been iiunierous attempts t o determine directly tlic aiiiouiit of eiiergy incorporated into cellular material. I n a n y process there will a difference in energy between tlic initial and final states of tlie system so that, during the process, c'iiergy will be liberated or absorbed in tlie form of heat. Microcnlorimetry is then a completely general way of studying eiicrgy chaiiges during microbial metabolism (Forrest, 1969b) though the qumitity measured is not the free energy change ( A F ) but the enthalpy change ( A H ) . The experimentally observed heat production represents a summation of all of t h e processes which take plact, so t h a t tlie enthalpy change froin t h e endergonica reactions of biosj-iithesis ( A H , , )will subtract from t h e eiithalpy change of t h e cxergonic reactions of cxatabolism ( A H , ) (Prigogiiie, 1961). Experiiiieiitally determined values of the heat production during growth of hcterotrophic cells growing with limited energy source on a nutritionally adequate medium ( t h a t is, similar conditions t o those obtaining when the maximum growth yield is observed) agree t o within

2fifi

\I

. \\

E'ORKIC\T A X I) I )

. J . \\ ALKEI'.

(~)i~)erinieiital error (to abont + 2 " , ) 11 itli A l l , \:iliiw. tlrcb v a l u c ~ c a ciilatcd from tables of t hermodynamic. data for the entlialpy cliaiige for t h e degradation of the e i i ( ~ g ysourcr t o tlie products of catabolism. I his (*lose correspoiidanc~ hct n-ecii obscrvccl heat production a n d c~ttlculated ciitliall~j-( ~ I i i ~ from l l ~ ~ c+atnbolisni lias been rel'orted for m w r o b i c ~groivtli on glucose of Streptococcu.s l a d i s (Boivinct, 1961). a coli (Seiicz a n d Belaicli, 196.5). Zymonzonus wobilis (Belaicli m t l Seiicz, 1967) and Snccharomyces cerpz'isiap (Belaicli et al., 1968) and for aerol)ic qroa-tli of AProOnctPr opt'oqmcc 0 1 1 snccinatc (Grangetto. r ,

1963).

rl'lius, iii these org:anisms, tlie entlialpy of gron th ( A H , , ) is too small t o be mcasnred. It is t o be e x l ~ ~ t tliat e d \ri\lucs of AH,) would be small in the case of 1ieterotrol)lis where tlir carbon s o u i w is assimilated a t tlicl s m i c lcvel of oxidation, C H 2 0 , as the c~ellulnrmaterial, as t h e heats of c~ombustionof glucose and bacterial cells are almost identical. Howcvcr in the case of' nutotrophs :~ssiniilatingcarbon dioxide as tlic cwbon source, this must he reduced t o the cellular level of oxidation : this iiivolvcs a considerable enthnlpy chaiige. Mcyerhof ( 19'4) found t h a t , with nitrifying bacteria, the experimentally measured heat 1)roductioii was zoo less thnn t h e calculated value of A H , for tlie catnbolism of cncrgy S O L I ~ C T ,and the difference is ~)rol)ably attributable t o this rductioii. 1:atlicr large divergences wcrc reported with A'itvohactet. and A'itrosomouus spp. by Laudelout et al. ( LWS). 'I'heoretic.al calculations by Morowitz (1960) give estimates for the cmtlinll)y of growth for E . coli. The estimate depends 011 the conipositioii of the medium; it a~qxoaclieszero in a rich complex medium wliere i~ maximum yield of cells Tvould be obtaiiied ; but w e n in minimal medium. where the growth yield would be cxpec*ted t o be lower, t h e maximum value of AIZ,. is 12.5 calorics per g. of cells, less t h a n 3°,, of t h e ciitlialpy of catabolisni. Both t h e esperimciital and theoretical approaches then show t h a t onlj. n sninll fraction of tlie energy available from catabolisni is actually incorporated into the organisms as energies of c.liemica1bonds. 3 . Entropy P r o d i d i o n

Jlicroc.aloriiiietric. ineasLirenicnts show also tliat expoiiential growth in bntcli culture is balanced. I n a nutritionally adeqnatc medium, there is an exact corrcq)ondeiicc between the rate of catabolism, iiieasured as heat production, and tlie i.iite of increase of cellular material. This has bec.11 demonstrated for&'. fncculis (Forrest et nl., 1 W l ) , S. lactis (Boivinct . I 964), fl. coli (Scnee and Belaicli, 1965), Aerobactei. a e ~ o p z e s(Graiigetto. 1963) and Zymomonas tnobilis (Helnicli et al., 1968).More detailed stitdies with A'. ,fnPcalis (Forrest and TT'alker. 1964), have slionii t h a t the organ-

umn

AS 11 UTILIZATIOL OF EXERGY DURING GROWTH

267

i h t i i h w t i l l ) a stcady state of thermodyriamic fluxes; in particular the vntropy gcnerated internally by each unit mass of cells is balanced by an cquivnlcnt, outflow of entropy to the surroundings, so that the entropic.allj unfavourable Ilrocesses of biosynthesis are made possible by (‘o:iljli11c to the entropy production of catabolism. This requirement for qrov ing organisms to “feed on negentropy” has been exhaustively cliscusscd (T’oplwr, 1 M T ) ; growth requires a very large increase in c.c.llular organization during biosyiithesis, with concurrent large changes I I I cntroly. Since tlic major part of the biologically useful energy avnilable as ATP is iiot utilized for biosyiithesis at the chemical level (Scction II.D, 1’. 2 2 5 ) , and both the enthalpy and free energy of bio3yiitlicsis arc s111illl (Morowitz 1960, 1968), the major thermodynamic ~)rocessis just this outflow of entropy and tlie major process associated with the outflow of entropy from the cells is that of cellular organiz at‘ion (Forrest. 1 0 7 0 ) . tlowevcr, cnerget ic. uncoupling involving wasteful dissipation of the ciiergy arailrzble from catabolism will also increase the outflow of cwtropy. ‘I’able 3 shows the calculated production of entropy during qrowtll of scvcral organisms. The more efficient organisms having higher yicld coeficients c.xliibit a corresponding lower outflow of entropy.

V. Conclusions The v c ~ y dircrsc ~)rocessesby which bacteria generate metabolicallynscfiil cncrpy a i now ~ i n general well documented so that it seems ruilikcly t h a t mnjor ncw metabolic pathways remain to be discovered, though there arc largc gaps in our detailed knowledge of the metabolism of individual organisms. The understailding of fermentative metabolism is quantitative, m d in most cases a value can be assigned to the number of moles of ATI’ or its equivalent which is generated during catabolism. However, in oxidative phosphorylation, the quantitative aspects are not satisfactory : the complete respiratory chain which occurs in the mitoc.11ondrin of higher organisms is not always operative, so that lower cficiencies of generation of ATP may be found in micro-organisms. ‘I’hc energetics of biosynthesis are also well described at the chemical level, so that we can c h a r up detailed balance sheets for the energetic cost of synthesis of cellular components. However there is little evidence nvailablc >\bouteiicrgctic requirements at tlie molecular level, despite the genetic and biophysical evidence for the very complex procedures or orticring whicli are ncccssary ( Pollard, 1967). IYlien we consider the efficiency of the transfer of energy bctweeii the c.iicrgy-generating processes of catabolism and the requirements of energy for synthesis, the evidence is necessarily indirect. Catabolism

‘r-413~13 3.

Thcrmodyii;iniic Data for Growth

hTP yield

Organism

Pat,hway and substrate

Ysubstrate AH, (g. dry observed (moles/ wt,/mole (kca1.i mole molc) snbstratc) substrate)

AFC CAlculatcd (kcal.1 mole)

2’ASc (Bcal./ mole)

AH, (cal.1

degree/ mole)

A S , = 1/Y x (AS, IA H J T ) (cd./ tlegreo/g. cc.lls) References

.

3 r

24

84

8

-a3

24

100

12

-31

-62

31

102

8

8.S

-32

-56

24

106

25

43..5

-174

-340

166

561

24

Streptococcus lactis

Enibden-Meycrhof; glucosc

2

19.3

-26

-50

Strcptococcus faecalis

Embden-Mcyerhof ; glucose

2

17

-3 1

Saccharoniyces Embden-Meyerhof; cerevisiae glucose

2

23

Z y m o ? ~ ~ o ? ~ a Ji~iitncr-Doi~doroff; s

1

8

rnobilis

Aerobacter aerogenes

0

*

I”

glucose oxidative phosphorylatloii ; snccinato” + H2O -17 0 4- 273’ + 4HC03’ 2HR

+

Boivinet (1964), g Senez (1962) Forrest et al. (1961), Seriez (1962) c Belaich et al. 4 d (1968), 4 Seriez (1962) IMeich et al. (1968), Sentz (1962) Uurtoll and Krchs (l953), Granget,to (1063)

8

THE GESERATION AND UTILIZATION O F ENERGY DURING GROWTH

269

may go 011 whether or not the energy can be used by the organisms, and thc cflickncy of coupling may vary widely; but the data all show that a numbcr of organisms operate with about the same maximum efficiency. ‘l‘hc thcrmodynamic assessment of this efficiency has little value as a l:itdic*tivc prameter, indicating oiily that the overall efficiency of tlie g1~1\\ tli process appears to be low, but the biochemical assessments clearly show that tlie critical parameter is the amount of ATP generated by vatsbolisin The term ITATP then is a well-defined biological constant \\hic.h can be used t o predict yields of organisms aiid t o assess the comparative efficiency of growth. However it is clear from the calculatioiis presented that, even under t h e most favourable conditions, the inajor part of the ATP produced by c~ntubolism is used in ways which cannot a t present bc accurately tlrfined.

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270

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AUTHOR INDEX ,Vuiiiberu

iii

itcrlics rqfer to the ptrges

A

or^

wl~iclireferences nrc listed at the erid of ecich article.

AuquiBre, J. P., 5, 13, 42 Austin, D. J., 108, 124 Azoulay, E., 2, 10, 12, 15, 19, 20, 26, 33, 37, 40, 41, 42, 43, 198, 208

Abbott, B. J., 20, 39 Ackrell, 13. A. C., 192, 208 Adair, F. W., 230, 269 Agnihotri, V. P., 113, 131 Ahrcns, J., 233, 269 Aiba, S., 260, 269 Aida, T., 3, 12, 39 Baalen, C., van, 39, 43 Aitken, 1%'. 1 3 , , 115, 124 Akagi, J. ill., 144, 170, 233, 269 Baas-Becking, L. G. M., 252, 264, 269 Alberts, A. W., 240, 271 Sabcock, H. L., 69, 132 Ihbcock, K. I>., 63, 67, 68, 132 Albro, 1'. W., 39, 39, 40, 41 Aleern, 31. 1. H., 173, 208, 216, 228, 229, lhchofen, R., 153, 164 230, 231, 232, 242, 269 Uaig, I. A,, 269 Alexander, Rf., 162, 170, 225, 271 Baker, E. G., 8, 40 Aloxopoulos, C . J . , 47, 49, 63, 64, 67, 68, I3akhuis, E., 16, 40 111, 112,124, 127 Baldwln, H. H., 47, 65, 124, 126 M i Khan, M. Y., 21, 40 Halclwin, It. L., 221, 242, 243, 269, 274 Allen, J. E., 5, 14, 30, 33, 35, 40, 42 Ualtschevsky, H., 215, 270 Allen, S. H. G., 241, 269, 271 Banbury, G. H., 75, 77, 78, 108, 124, 132 Allport, D. C., 122, 124 Raptist, J. N., 19, 21, 30, 40, 41 Anderson, J. H., 229, 269 Bard, R. C., 249, 270 Anderson, J . S., 223, 269 Barker, H. A., 152, 162, 166, 169, 170, Anderson, n1. L., 48,54, 55, 134 239, 240, 242, 243, 245, 246, 247, 270, Anderson, I<,.L., 243, 269 272, 273, 274 Andreoli, T. E., 34, 43 Barkley, D. S., 51, 52, 124, 12.i, 121) Appleby, C. A,, 178, 207, 20X Barksdale, A. W., 105, 106, 124, 1 3 0 Appleman, 11. D., 228, 271 Harnett, H. L., 77, 124 Barrett, J., 178, 206 Appleton, G. S.,1 0 7 , 124 Arima, K., 3, 12, 24, 40, 42, 186, 187, 208, Hartels, T. J., 17, 43 Bartley, W., 57, 97, 131, 252, 273 210 Arnaud, $I., 109, 110, I27 Bartnicki-Garcia, S., 47, 69, 70, 71, 72, 73, Arnon, D. I., 141, 147, 153, 154, 156, 167, 74,124, 130 Bartsch, R. G., 173, 178, 208 169, 170, 171,172,215,236, 270 Barvah, J. N., 6, 42 Amon, D. J., 136, 150, 152, 1G9 Aronson, .J. M., 69, 70, I 2 4 Baseman, J. B., 207, 208 A4rsenault,G. P., 106, 124 Bassler, L. M., 75, 130 Asano, A., 333, 269, 272 Basu, D., 19, 30, 31, 43 Ashworth, J . XI.. 48, 49, 50, 52, 54, 55, Battley, E. H., 251, 270 58, 59, 124, 127, 130, 131, 132 Bauchop, T., 234, 241, 242, 244, 249, 250, Ashworth, K., 49, 51,129 251, 270 Aston, P. R., 182, 190, 209 Baumann, P., 53, 55, 56, 124 Atkinson, D. E., 233, 269 Bean, W. J., 120, 124 Aubert, S., 230, 272 Beck, J. V., 232, 274 275

276

AUTHOR INDEX

Beck, R. W., 250, 258, 270, 272 Bednarz, A. J., 188, 209 Beerstecher, E., 2, 7, 9, 40 Behal, F. J . , 97, 124, 125 Reinert, H . , 147, 171 Belaich, A., 261, 270 Belaich, J.-P.,251, 254, 261, 262, 266, 268, 270, 273 Bell, 0. E., 39, 40 Beneko, E., 113, 116, 133 Benemann, J. I t . , 147, 156, 161, 167, 169, 172 Bennett, E. O., 39, 41 Benson, A , , 143, 171 Bent, K. J., 101, 118, 125 Rernlohr, R. W., 124, 125 Isertrand, H., 104, 125 Bettleheiin, K. A , , 110, 125 Eianchi, D. E., 71, 74, 90, 91, 1 2 j , 129 Biemann, K . , 106, 124 Bigley, D., 97, 129 Bilai, V. J., 4, 42 Hirdsell, D. C., 195, 208 I3lomstrom, P. C., 143, 171 Boor, W. E., do, 15, 16, 40 Boiwnct, P., 250, 251, 266, 268, 2il/ Bolon, W. -4., 233, 270 Hone, D. H., 233, 270 Hongers, I,., 233, 270 Banner, J. T., 47, 49, 51, 52, 53, 55, 125, 129 Bonner, U'. D., 173, 209 Boon, W.I t . , 138, 169 Borek, E., 61, 131 Borrow, A., 118, 125 Bos, P., 3, 15, 16, 40, 43 Bovell, C. It., 179, 208 Bowen, T. J . , 231, 270 Bowers, W.D., 109, 130 Brachet, J., 74, 125 Rracker, C. E., 72, 123, 12S Hradley, S. G., 55, 132 Bradley, S. S., 48, 54, 123 Bradshaw, W.H . , 152, 169 Bragg, P. D., 193, 208 Brand, J., 5, 42 Brandt, W. M.,122. 125 13rasier, C. X.,78, 125 Rrauker, C., 102, 128 Hreiver, E. N . , 65, 22.j Brill, W.J . , 165, 170 Brisbane, P. G., 6, 40 13roberg, P. L., 177, 185, 207, 208 Hrork, T. D., 109, 125 Hrodie, 9.193, , 208 Rrodie, A. F., 192, 207, 209, 210 Hronsweig, R., 53, 127 Brooks, T., 120, 124

f.

Brown, M. E., 122, 131 Bruhmuller, M., 55, 134 Bruyn, J., 3, 26, 40 Bryant, M. P., 177, 207, 211 Buchalo, A. S., 4, 42 Buchanan, B. B., 136, 141, 44, 150, 152, 153, 154, 169, 170, 171, 215, 216, 236, 251, 258, 270 Bucher, T., 180,210 Buhring, U., 20, 25, 33, 43 Bulder, C. J . E. A., 251, 270 Bulen, W. A , , 147, 157, 158, 159, 169, 170 Ru'Lock, J . D., 108, 119, 121, 122, 124, 125 Hurgeff, H., 107, 125 Burnott, J . H . , 75, 77, 78, 108, 119,125 Burns, R. C., 157, 158, 159, 160, 161, 169, 170, 233, 270 Burris, R. H., 140, 155, 157, 159, 161, 169, 170, 171, 188, 192,210 Burton, K., 268, 270 Butcher, R . W . , 52, 132 Butler, P. J . , 231, 270 Butler, W. L., 178, 208 B u t t , W.D., 178, 208

C Caglioti, L., 108, 125 Cain, C. E., 39, 40 Cainelli, G., 108, 125 Caldwell, D. R., 177, 207, 211 Camerino, B., 108, 125 Camici, L., 87, 12.5 Cantino, E. C., 77, 78, 79, 80, 81, 82, 83, 84, 85, 87, 103, 105, 120, 125, 126,127, 128, 129, 130, 131 Cantor, C. R., 144, 171 Caplan, S. R., 256, 273 Cappellini, R. A., 101, 126 Carbonell, L. M . , 70,129 Carchman, R., 60, 61, 228 Cardini, G., 20, 31, 40 Cardon, €3. P., 242, 245, 270 Carlilr, 31. ,J.,75, 77, 120, 126 Carnahan, J. E., 141, 156, 162, 171 Casicla, I,. E., 17, 20, 35, 39, 41 Castle, J . E , 156, 162, 171 Castor, L. N . , 177, 208 Cattaneo, J . , 257, 270 Cazin, J., 5, 14, 33, 42 Ceccarini, C., 53, 58, 60, 61, 126, 128 Cerda-Olmdo, E . , 108,128 Chain, E. B., 87, 125

277

AUTHOR INDEX

Chance, W., 34, 40, 173, 175, 176, 177, 178, 179, 181, 182, 187, 202, 204, 208, 209, 210 Chang, Y. Y . ,51, 52, 126, 129 Charles, A. iu., 230, 231, 270 (.‘has(:, H. H., 17, 40 Chttsay, 13. RI., 48, 129 Chaudhuri, S. D., 117, 12s Cheah, I<. S., 184, 185, 208 (!her, K. H., 107, 121, 126 Chen, A. \I1., 110, 123, 126 Chen, S.L., 254, 256, 270 Chernoff, R., 60, 133 Chet, I., 66, 126 Chiappini, &I. G., 241, 273 Chibnall, A. C., 4, 39, 40, 41 Chichester, C. O . , 119, 126 Chin, B., 73, 100, 126 Chiquoine, A. D., 55, 12; Chouteau, J., 19, 20, 33, 37, 40 Chung, A. E., 156, 160 Clark, D. Y., 87, 126 Clark, L. C., Jr., 181, 209 Clayton, 1%.B., 38, 43 Clegg, J., 53, 126 Cleland, S. V., 53, 55, 56, 57, 126 Clifton, C. E., 545, 270 Cochrane, V. W., 75, 90, 91, 107, 726, 134 Coe, E. L., 53, 55, 56, 57, 126 Cohen-Uazire, G., 160, 1/39 Cole, H. A., 262, 257, 270 Cole, J. A., 196, 200 Collins, E. H., 253, 658, 273 Collins, J. F., 97, 12.9 Colman, R. I?., 5 7 , 1 2 7 Combepine, G . , 89, 133 Cooke, R. C., 102,126 Coon, &I. J., 19, 21, 30, 31, 32, 33, 40, 41, 42, 43 Cooney, J. ,J., 4, 40 Coopor, D. Y . , 32,4% Corey, E. J., 38, 40 Coston, 31. li., 57, 58, 59, 126 Cota-Robles, I<., 7 2 , 124 (hta-Robles, E. H.,195, 208 Couchoucl-Beauinorit, I’., 2, 10, 12, 40, 198, 208 Cox, C. D., 207, 208 Cox, G. B., 194, 195, 196,209, 210 cox, It. H., 201, 211 Crane, F. L., 188, 210, 233, 272 Crocken, B., 71, 126 Croes,A.F., 109, 110, 111, 123, 126 Curran, J. F., 57, 132

Dainko, J. L., 109, 132 Dalton, H., 159, 170, 255, 270 Daly, J., 38, 41 Damaglou, A. P., 263, 270 Damotte, M., 257, 270 Daniel, J. W., 65, 67, 68, 126 Dark, F. A., 260, 274 Das, M. L., 38, 10 Davidovics, G., 19, 33, 37, 40 Davies, E. &A,230, 270 Davies, H. C., 650, 270 Davis, B. D., 220, 274 Da\is, J . A., 17, 35, 4 1 Davis, J. B., 17, 40 Dawes, E. A., 251, 257, 563, 270 De Busk, A. G., 89, 126 l)e Busk, B. G., 89, 126 Decker, K., 166, 168, 170, 171, 240, 274 Dee, J., 63, 126 DeHertogh, A. A , 101, I30 Del Vecchio, IT. G., 91, 126 I h n u r k , P. J., van, 250, 253, 258, 271, 273 1)e RIoss, J. A., 180, 196, 197, 210 De Moss, R. D., 249, 270 Dennen, D. W., 115, 126, 130 Denton, C. H., 188, 209 Deshpande, 1L B., 100, 126 De Terra, N., 73,127 Detroy, R. W., 158, 170 D’Eustachio, A. J., 144, 156, 157, 158, 159, 167, 170, 233, 271 Devanathan, T., 144, 170 Deysson, G., 109, 127 Dick, S., 113, 129 Dicker, J. W., 88, 89, 91, 127 Dickerson, A. G. F., 123,130 Dillon, J. F., 193, 210 Dilworth, 11. J., 157, 159, 170 Dingley, J. &I., 122, 127 Dittmer, J. C., 39, 39, 40 Dolin, M. I., 248, 270 Done, J., 122, 127 D’Ornano, L., 182, 210 Dostalek, &I., 3, 15, 16, 40, 42, 4,; Doudoroff, &I., 257, 270 Drews, G., 160, 171 Droogenbroeck, R., van, 264, 266, 272 Drost-Hansen, W., 8, 40 Duncan, D., 5 5 , 1 2 7 Duvnjak, Z., 15, 26, 33, 42 Dworkin, M., 5, 13, 40

D

E

Dahlberg, D., 54, 57, 117, 134

Eakin, R. E., 97,125

278

AUTHOR INDEX

Edington, M. A., 6 , 41 Edmonds, Z . , 4 , 40 Eger, G . , 113, 127 I:glinton, G., 39, 40 I~~leina, 13., 137, I 7 0 l’:lliott, C. G., 107, 121, 127 IClsclen, S. R., 215, 234, 235, 241, 242, 244, 245, 249, 250, 251, 254, 261, 270, 272, 273, 274 Emcrson, R., 120, 127 Eiide, van den, 107, 108, 119, 127 lhgland, I).J. F.,116, 123, 130 Ennis, H . L., 48, 52, 54, 126, 127 Erickson, S . I<., 54, 127 Ernster, L., 38, 40 ISspositb, M. S., 109, 110, 127 Espositb, R. E., 109, 110, 127 Essig, A , 256, 273 Estabrook, 1%.W . , 31, 32, 42, 43, 178, 181, 209 hhans, H. J . , 158, 167, 170 Evans, 31. C . W.,130, 153, 154, 169, 170, 215, 216, 236, 270 Eyk, J . , van, 17, 43

F Falcone, A. IJ., 229, 271 Falcone, G., 7 0 , 130 Falk, J . E., 177, 178, 209 Fergus, C. L., 4 , 40 Fiechter, A , , 100, 129 Filosa, M., 53, 126 Finkelstein, l., 60, 133 Finnerty, W. R., 3, 7 , 9 , 12, 19, 22, 24, 27, 32, 33, 40, 42, 43 Fisher, l? , 228, 230, 231, 271, 273 I‘isher, H. . I . , 165, 370 Fisher, T , 2 2 8 , 271 Fling, Al., 122, 128 ~ l l p p l n li. , s., 4, 40 Florkin, > I . , 178, 20:) Folkrs, 13. P , 118, 131 Forncy, h’. W., 9, 22, 23, 33, 34, 40, 42 Forrest, \V. W., 214, 234, 250, 251, 252, 250, 257, 258, 260, 261, 262, 263, 264, 265, 266, 267, 268, 271 Foster, J . W., 3, 5, 7 , 12, 13, 17, 19, 20, 21, 27, 32. 34, 40, 41, 42, 119, 127 Foust, G. P., 146, 150, 170, 171 Fox, D. I , , 120,127 Franks, F., 8, 40 Franzen, J . S., 156,169 Frear, D . C., 38, 41 Fredricks, K . RI., 20, 21, 35, 40 Fredricks, W . W., 166, 170

Freeman, T . E., 7 8 , 127 Frerman, F. E., 176, 181, 209 Fricclen, C., 57, 127 Friend, J., 120, 126 Frornageot, C., 264, 271 Fiihs, G. W., 2, 6 , 41 Fujita, S., 25, 33, 41 Fujita, T., 196, 209 Fukui, S., 5 , 12, 14, 43, 253, 259, 271 Fuller, M. S., 85, 127, 131

G Gaffron, H., 235, 271 Galbraith, J . C., 87, 88, 94, 95, 96, 97, 110,127 Galivan, J . H., 242, 271 Galliard, T . , 35, 41 Galston, A. W . , 7 7 , 127 Galun, E., 7 7 , 103, 127, 128 Ganguli, B . N., 32, 41 Carton, G. A., 119, 127 Gay, J. I,., 110, 126 Geissler, A. W., 7 1 , 133 Gel’man, N . S., 173, 209 Gelpi, E., 39, 41, 43 Gerisch, G., 53, 127 Gerrits, J . P., 251, 254, 255, 271 Gest, H., 140, 152, 153, 154, 170, 215, 233, 235, 236, 271, 273 Gezelius, K., 54, 55, 127 Gholson, R. K., 19, 21, 30, 40, 41 Gibson, D . T., 38, 41 Gibson, F., 194, 195, 196, 209, 217, 271 Gilby, A. R., 39, 41 Girbardt, M., 7 2 , 127 Gleason, F . G., 8 7 ,1 3 3 Goddard, J. L., 250, 271 Goldman, P., 240,271 Goldstein, A., 7 8 , 83, 126, 127 Gollakota, K . U., 97,127 Gooday, G. W . , 100, 107, 108,124,127 Goodman, E . JI., 66, 67, 127, 132 Goodwin, T . W.,108, 119, 120, 127, 132 Gool, A., van, 228, 271 Gorin, P. A. J., 25, 43 Goto, &I., 32, 43 Gottlieb, D., 107, 132 Gottsberger, G., 63, 127 Granger, D., 181, 209 Grangetto, A,, 255, 266, 268, 271 Grasselli, P., 108, 126 Grau, F. H., 155,170 Gray, C . T . , 140, 170 Gray, W.D., 47, 63, 67, 68, 127 Green, D . E., 200, 209 Green, M., 162, 170

279

AUTHOR INDEX

Greenblatt, C. L., 63, 129 Gregg, J. H., 47, 48, 53, 127 Gressel, J., 77, 103, 127, 128 Grice, R. E., 39, 41 Griffin, D. H., 102,128 Gross, P. R., 46, 59, 61, 128 Grove, S. N., 72, 128 Gruen, H. E., 77, 116, 128 Gunness, ill.,97, 132 Gunsalus, 1. C., 32, 41, 225, 241, 244, 349, 250, 253, 270, 271, 273 Guttos, E., 65, 66, 128 Giittes, S., 65, 66, 128, 130

H Hadjipetrov, L. P., 251, 254, 255, 271 Hadley, G., 103, 117, 128 Hagihara, B., 54, 130, 254, 273 Haidle, C. W., 70, 71, 128 Hall, A. N., 21, 40 Hall, D. O., 136, 170 Hdl, E. >I., 51, 52, 125 Halvomon, H. O., 109, 110, 127 Halvorson, H . U., 97, 127 Hamilton, I. It., 155, I70 Hamilton, J. A., 194, 195, 196, 209 Hamilton, R. J., 39, 40 Hammond, R. K., 200,209 Hankin, L., 19, 41 Happold,.!I C., 231, 270 Hardy, K. W. F., 136, 144, 146, 155, 157, 158, 159, 160, 161, 167, 170, 233, 271 Harper, L., 188, 209 Harris, E. J., 263, 271 Harrison, D. E. F., 252, 255, 271 Harrold, C. E., 103, 117, 128 Hart, L. T., 20, 41 Hartman, G. R., 38, 41 Hartrcc, E. F., 177, 178, 2f0 Haskin, hl. A., 223, 269 Haskitis, It. H., 107, 121, 128, 132 Hatch, S. C., 70, 128 Hatchikian, F.C., 233, 272 Hawker, L. E., 63, 75, 116, 117, 128 Hawtrey, E., 7, 9, 12, 32, 33, 40 Haxo, I?. T., 120, 133 Ha,,yaishi, O., 38, 41 Hayea, W. A , , 113, 128 Heftman, E., 51, 1 2 s Heinz, E., 35, 41 Heisenberg, M., 108, 128 Heldt, H . W., 215, 270 Helgerson, R., 179, 208 Hempfling, W. P., 217, 231, 250, 258, 260, 263, 271 Hendler, R. W., 222, 271

Hendrie, M. R., 107, 127 Hendrix, J. W., 107, 121,128 Henney, H. R., Jr., 65,128 Henney, M. R., 65,128 Henning, F. A., 18, 35, 41 Henninger, H., 240, 274 Henry, L., 115,130 Hernandez, E., 251, 254, 271 Hersh, R. T., 144, 170 Hess, B., 252, 271 Heydeman, &I. T., 19, 41 Hill, P., 123, 128 Himes, R. H., 144, 170 Hinckson, J. W., 147, I70 Hirata, A., 253, 259, 271 Hirsch, H. M., 90, 122, 128 Hirsch, P., 228, 271 Hirschberg, E., 60, 61, 128, 133 Hobson, P. N., 259,271 Hoch, G. E., 228,269 Hodgson, G. W., 8 , 4 3 Hofer, iM.,263, 271 Hofman, T., 123,132, 228, 229, 272 Hohl, H. R., 48, 128 Holliday, R., 104, 128 Hollinger, G., 34, 40 HoIzer, H., 100, 128 Homer, R. F., 138, 170 Hoogerheide, J . C., 137, 140, 170 Hooper, A. B., 229, 272 Hopgood, M. F., 241, 251, 266, 268, 271, 272 Hopkins, S. J., 4, 41 Hopton, J. W., 269 Horenstein, E. A., 78, 81, 82, 103, 120,126, 125 Horowitz, H . N., 122, 128 Hoshino, Y., 25, 33, 41 Hostak, M. B., 51,128 Hou, C., 193, 208 Hough, J. S., 260, 274 Howe, R., 25, 28. 33, 35, 36, 41 Hughes, D. E., 252, 257, 270 Hughes, D. H., 113,128 Hungate, &I.1 ' . G., 120, I28 Hungate, R. E., 257, 258, 272 Hurwitz, J., 249, 272 Huston, C. K., 39, 41 Huxley, J. S., 46, 128 Huybregtse, R., 9, 17, 22, 33, 38, 41, 43 H y a t t , M. T., 80, 81, 103, 120, 126

I Ichihara, K., 19, 25, 31, 42 Iguchi, T., 10, 42

280

AUTHOR INDEX

Iida, RI., 24, 25, 33, 41 Iizuka, H., 24, 25, 33, 42 Imai, K., 233, 269, 272 Inselburg, .J , 60, 128 15hl1, It., 3, 10, 43 Tahikura, T., 3, 27, 37, 4 1 Ishimoto, M., 234, 272 Itada, N., 38, 41 Itagaki, E., 193, 1!)4, 197, 209

J Jackins, H. C., 245, 272 Jackson, R. L., 141, 1 7 1 Jacob, F., 45, 128 Jagger, J., 76,128 Jahrline, P., 250, 253, 273 Jefferys, E. G., 118, 125 Jerina, D., 38, 4 1 Jicinska, E., 100, 101, 122, 128 Jinks, J . L., 104, 128, 130 Johnson, E. J., 230, 270 Johnson, 31. J., 2, 3, 8, 10, 15, 33, 34, 41, 42, 141,170, 251, 254,271 Jones, A. C., 19, 32, 43 Jones, C. W., 154, 170, 177, 182, 188, 189, 190, 191, 192, 208, 209 Jones, D. F., 25, 28, 33, 35, 36, 41 Jones, J. G., 6, 41 .rones, 0. T. G., 229, 273 Jones, K . G. W.,103, 2/19 .Takes, T. H., 144, 7Tf .Jump, .4.,66, 129 .Jungermann, K., 166, I W , 170, / T I , 240, 274 .Iunk, .(I \V., 32, 42 Jurtshuk, I’., 3, 5, 12, 13, 20, 31, 40, 42, 182,158, 190,209 ,rust, F., 2, 4 1

K Kallio, R . E., 2, 3, 7, 9, 10, 12, 17, 19, 22, 27, 32, 33, 38, 4 0 , 41, 42, 43, 245, 272 Kamen, $1. D., 140, 152, 170 Kamisaka, S., 110, 129 Kanetsuma, P., 70, 229 Karlsson, J. L., 162, 1711 Karush, F., 250, 270 Kashket, E. It., 192, 209 Katagiri, M., 32, 4 1 Kates, M.,240, 272 Katsuya, N., 2, 3, 10, 41, 43 Kawabata, Y., 3, 10, 43 Kawaguchi, IZ., 54, 130 Kawaguchi, T., 254,273

Keilin, D., 175, 177, 178, 208, 210 Keister, D., 154, 170 .Keister, D. L., 215, 272 Kellermyer, R.W., 241, 269 Kerr, N., 65, 129 Kessell, I<. H. J . , 118, 125 Kester, A . S., 5, 7, 13, 20, 4 1 lihouw, 13. T., 85, 86, 87, 129 Kinsky, S.C., 233, 272 Klebs, G., 75, 116, 129 Klein,D. A., 17, 18, 35, 41 Klemner, H. W., 107, 129 Klieber, R. J., 107, 124 Klingenberg, M., 180, 182, 210, 215, 270 Klucas, R. V., 158, 167, 170 Klug, M. J., 3, 5, 9, 11, 12, 20, 23, 25, 27, 28, 33, 41, 42 Kluyver, A. J . , 137, 140, 170 Klybas, V., 237, 273 Knight, E., 233, 271 Knight, E., Jr., 143, 1 7 1 Knight, E. J., 136, 144, 146, 155, 158, 159, 170 Knight, S. G., 73, 100, 126 Knights, B. A., 107, 127 Knowles, C. J., 189, 210 Kobayashi, S., 38, 41 Kobr, M. J., 71, 89, 90, 91, 129, 133 Koch, B., 158, 170 Koch, J . R., 38, 41 Koepsell, J. H., 141, 170 Kogane, F., 98,134 Kok, R . , 138, 1 7 1 Kolattukudy, P. E., 19, 39, 4 1 Kolderie, M. D., 55, 12s Komagata, K., 2 , 10, 41 Konijn, T. M., 51, 52, 126, 129 Konovaltschikoff-hlazoyer, M., 9, 19, 41, 43 IGmnancikova, I-.,251, 254, 256, 272 Korn, E. D., 63, 129 Kornberg, A., 247, 272 Kornberg, H. L., 97, 129 Kotyk, A., 15, 42 Kovac, L., 251, 254, 256, 272 Koval, E. Z., 4, 42 Kozenovsky, JI., 244, 272 Krampitz, L. O., 248, 272 Krause, F. P., 4, 5, 42 Krebs, H. A., 57, 129, 268, 2711 Krichevsky, M.I., 48, 129 Krivanek, J. O., 55, 129 Krivanek, R. C., 55,129 Kroger, A., 182, 210 Krynitsky, J. A., 4, 42 Krzeminski, Z., 257, 272 Kudo, R. R., 63,129 Kuenzi, M. T., 100, 129

281

AUTHOR INDEX

Kunisawa, R., 160, 169 Kuno, S., 38, 41 Kusunose, E., 19, 21, 25, 30, 31, 33, 42, 43 Kusunose, M., 19, 21, 25, 30, 31, 33, 42, 43

L Lacld, J. N., 6 , 40, 242, 243, 272 Lam, K. W., 34, 43 Lam, Y., 207, 208, 210 Lan, N. T., 109, 127 Lange, W., 4, 5, 42 Lanyi, J. K., 183, 184,210 Larsen, H., 235, 272 Lascelles, J . , 193, 207, 209, 210 Laseter, J . L., 39, 42 Last, F. T., 113, 128 Lata, hf., 90, 91, 131 Laudelout, H., 228, 204, 266, 27J, 272 Leach, C. &I., 76, 129, 132, 13.3 Leadbetter, E. It., 19, 41, 32, 34, 42 Leak, I,. V., 120, 126 Lebeault, J. M., 15, 26, 33, 42 I,e Conte, J . I3 , 157, 158, 1.59, 169, 233, 270 Lees, A. M., 63, 129 Lees, H., 228, 220, 232, 264, 269, 272 Le Gall, J., 233, 261, 272 Legallais, V.,175, 211 Lehninger, A. L., 2.53, 272 Lenney, J. F., 107, 129 Leonard, T. J., 113, 129 Lessie, P. E., 85, 129 Lester, R. L., 188, 200, 210, 233, 272 Levine, L., 53, 132 Lewis, D., 242, 244, 245, 272 Liber, A. F., 39, 42 Lciberman, I., 247, 272 Liddel, G. U., 51, 53, 128, 129 Lie, S., 2, 10, 42 Lieth, H., 68, 129 Lijinsky, W.,119,127 Lilly, V. G., 77, 78, 79, 124, 129 Lindeberg, G., 182, 210 Linden, A . C., van der, 2, 9, 17, 19, 22, 33, 37, 38, 41, 43 Lipe, R. S., 260, 273 Lipmann, F., 141,171 Lippman, E., 72,124 Lizotte, C. L., 233, 273 Ljungdahl, L. G., 244,272 Lloyd, E. C., 118,125 Lloyd, P. B., 118,125 Locksley, H. D., 39, 41

Lode, A., 182,210 London, J., 230, 272 Loomis, W. F., Jr., 49, 51, 57, 58, 59, 126, 129,134 Looney, F. D., 195, 209 Lord, K. E., 38, 43 Lorenz, S., 71, 133 Love, L. L., 48,129 Lovenberg, W., 141, 144, 1 7 1 Lovett, J. S., 81, 82, 83, 85, 103, 120, 128, 129, 130 Lowery, C . E., 3, 5, 12, 13, 42 Lowry, o., 62, 129 Loy, J . I., 258, 272 Lu, A. Y. H., 32,42 Lucas, H., 65,129 Luderitz, I., 53, 127 Ludvik, J . , 15, 16, 42 Luke, H. H., 78,127 Lukens, R. J., 77,129 Lukins, €1. B., 7, 21, 34, 42 Lukoyanova, M. A . , 173, 209 Lynch, T., 65,12X Lyric, R., 229, 26.') Lythgoe, J . N., 77, 120, 126, 129

M IIrAulifTe, C., 8, 42 McCann, L. &I., 194, 209 JIcClary, D. O., 109, 130 McClure, W. K., 72, 130 BIcCurdy, H. D., 85, 86, 87, I29 hIcCurdy, H. D., Jr., 81, 130 McDougall, K. J., 104, 125 McDowell, L. L., 101,130 McFadden, B., 233, 269 McGrew, S. B., 260, 272 hlachlis, L., 105, 112, 129, 130 McKenna, E. J., 2, 17, 31, 32, 33, 42, 43 MacKinney, G., 119, 126 hlclaren, G. W., 4, 42 MacLeod, H., 122,128 AfacMillan, A., 118, 130 MeMorris, T. C., 106, 124, 130 McMurrough, I., 73, 130 McQuillen, K., 220, 221, 224, 272 hIacrae, I. C., 182, 210 Magasanik, B., 221, 272 Mainzer, S. E., 250, 258, 271 Maitra, P. K., 252, 271 Makman, R. S., 51, 130 Makula, R., 9, 24, 42 Malchow, D., 53, 127 hfalkin, R., 136, 141, 144, 1 7 1 Mallette, M. F., 260, 272 Malviya, A. N., 180, 210

282

AUTHOR INDEX

Alaiidelli, K . , 108, 125 Mandelstani, J., 220, 221, 124, 272 Illangum, J. H., 232, 274 Mankowski, Z.,71, 130 Marchant, It., 72, 130 Markovetz, A. J., 2, 3, 5, 9, 10, 11, 12, 14, 20, 22, 23, 25, 27, 28, 30, 33, 34, 35, 40, 41, 42 Marsh, P. 13., 7 5 , 130 Afarshall, B., 123, 1 3 1 Martin, S. M., 123, 128 Rlaruyaiiiu, Y., 98, 130 Mason, J. W., 51, 52, 125 Massey, \'., 14G, 150, 170, 1 7 1 Masuda, Y., 110, 129 Mateles, It. l . ,6, 42, 259, 273 Mather, K., 104, 130 Matsubara, H., 144, 1 7 1 Matsuhashi, M., 223, 269 Matsumoto, J., 19, 25, 31, 42 May, A. I<., 182, 190, 209 Mayberry, W. R., 255, 2.56, 272 Mayhew, S. G., 146, 150, 170, 171 Aloes, G. G., 138, 170 Meyenburg, H. K., von, 256, 272 Meyerhof, O., 266, 272 Illicetich, R. G., 107, 121, 128 Nichals, bl., 91, 131 Illickelson, M.N., 4, 40 Mikucki, J., 257, 272 Miles, P. G . , 71, 73, 132, 233 Rlilhaud, G., 230, 272 Milko, A. A., 4, 42 Miller, G. R., 109, 130 Miller, J. J., 105, 109, 110, 123, 126, 130, 131, 132 Miller, T. L., 2, 3, 10, 42 Bliller, Z. I., 48, 130 Millet, J., 230, 272 Milligan, L. P., 243, 26!) Mihe, H., 55, 130 hlinagawa, T., 91, 130 Miyoshi, M., 4, 42 Mizuno, &I., 101, 42 Mizushima, S., 18G, 210 Mohberg, J., 65, 130 Rlonod, J., 45, 128, 263, 272 Morowitz, H. J., 224, 225, 265, 266, 267, 272, 273 Rforre, D. J., 72, 128 Mortenson, L. E., 141, 155, 157, 158, 161, 170, 171 Rlortlock, R. P., 141, I 7 1 Morton, A. U., 47, 74, 88, 101, 116, 117, 123, 125, 130 Jloseley, B. E. B., 70, 130 Moss, F. J., 178, 210 Moustafa, H. H., 253, 258, 273

Mower, H. F., 143, 1 7 1 Mullins, J. T., 74, 132 Mumford, F. E., 156, 162, 1 7 1 Munk, V., 3, 15, 16, 40, 42, 43 Rlunson, T. O., 157, 170 Murgier, & 266, I., 268, 270 Murphy, &I. N., 83, 85, 103, 13U Miitch, J . T., 76, 133

N Nagasaki, S., 98, 99, 130 Nagel, L., 109, 130 Nakase, T., 2, 10, 41 Nakashima, T., 143, 1 7 1 Nason, A., 228, 229, 269, 272, 273 Neil, C. E., van, 235, 273 Nelson, D. R., 39, 42 Nelson, N., 72, 124 Newell, P. C., 58, 130 Newman, D. A., 78, 130 Newton, J. W., 140, 1 7 1 Newton, N. A,, 194, 196,209 Nicholas, D. J. D., 91, 133, 207, 208, 210, 228, 229, 232, 269, 271, 273 Nicholls, P., 180, 210 Nickerson, W. J., 47, 69, 70, 71, 72, 73, 124, 1 3 0 , 1 3 2 Niederpruem, D. J., 113, 115, 117, 124, 126, 130,131, 133 Niehaus, W. G., Jr., 38, 42 Nishi, A., 98, 130 Nishizawa, Y., 260, 269 Nixon, I. S., 118, 125 Noel, E., 52, 132 Nomachi, Y., 123, 1 3 4 Novelli, G. D., 124, 125 Novotny, H. M., 91, 1.41 Nozaka, J., 19, 31, 42 Nozaki, M., 154, 1 7 1 Nutting, W.H., 105, 130 Nygaard, 0. F., 65, 130 Nyns, E. J.,5, 13, 42

0 Ogina, S., 3, 12, 24, 40, 42, Ohama, H., 123, 130 Ohja, M. N., 89, 90, 91, 130 Ohnishi, T., 54, 130, 254, 273 Ohrloff, C., 166, 168, 170 Oka, T., 186, 187, 208, 210 O'Kane, D. J., 141, I 7 1 O'Keefe, 111 G., 51, 52, 125 Omura, T., 32, 42

283

AUTHOR INDEX

Onodera, M., 260, 269 Orinerod, G., 153, 170 Ormerod, K. S., 153, 170 Orb, J., 39, 41, 42, 43 Orrenius, S., 38, 40 Ortiz d e Montellano, P. It., 38, 40 Osborn, &I. J., 58, 59, 132 Osnos, M., 60, 61, 128 Ostrovskii, 1).M., 173, 209 Otsuka, R., 3, 10, 43 Oulcvey, N., 71, 91, 129 Oulevey-Matikian, N., 88, 89, 90, 91, 93, 127,131 Overrein, L., 228, 271 Owens, 0. V. H., 138,171 Owens, R. G., 91,131 Oxonburgh, &I. S.,250, 273 Oye, I., 52, 132

Pittenger, T. H., 104, 125 Plempel, M., 107, 108, 120, 125, 131 Polakis, E. S., 57, 97, 131, 252, 273 Polglase, W. J., 193, 210 Pollard, E. C., 267, 273 Pontefract, R . D., 109, 131 Pop?, L. hl., 182, 190, 209 Popper, K. R . , 267, 273 Porra, R . J., 207, 210 Postgate, J. R., 159, I70, 233, 255, 270, 273 Pressman, B. C., 263, 271 Prieto, A , , 108, 125 Prigogine, I., 265, 273 Prince, A. E., 4, 43 Prochazka, G. J.,255, 256, 272 Proctor, hI. H., 19, 43 Puig, J., 198, 208, 257, 270 Pullmt~n,31. E., 180, 210 Pun, W. T., 193, 210

P Packer, L., 179, 208,233, 273 Page, R . &77, I., 103, 131 Palumbo, S. A., 262, 273 Pandhi, P. N., 82,131 Pannbacker, R . G., 56, 60, 131 Parejko, R . A , , 158, 170 Park, D., 72, 103, 130, 131 Park, .J. Y., 113, 131 Parker, P . L., 39, 43 Parker, W., 107, 127 Parks, G. S., 252, 264, 269 Passonnuau, J . V.,62, 121) Payne, W. J., 107, 124, 255, 256, 272 Pcake, E., 8, 43 Pcat, A , , 102, 117, 131 Peck, H. D., 214, 215, 216, 228, 230, 231, 232, 233,234, 273 Pecka, K., 3, 40 Pengra, It. M.,138, 161, 171, 172 Penn, l., 255, 274 Perry, J. J., 6 , 17, 21, 43 Peterson, J. A , , 19, 30, 31, 32, 33, 43 Peterson, J. L., 4, 43, 101, 128 Phillips, W. ,J., 143, 171 Pichinoty, F., 182,210, 262, 273 Pickett, &I. J., 246, 273 Pidoplichko, N. M., 4, 42 Pillinger, D., 61, 131 Pine, L., 251, 258, 270 Piper, S. H., 39, 40 Pirt, S. J., 102, 117, 131, 255, 259, 260,271, 273 Pitt, D., 102, 131 Pittard, A. J., 194, 209 Pittard, J., 217, 271

Q Quanco, J., 48, 130 Quilico, A., 108, 125

Rabinowitz, J. C., 136, 141, 144, 152, 169, 171, 247, 273 Racker, E., 192, 211,237,254,273 Haeburn, H., 152, 171 Rafelson, hl. E., Jr., 108, 132 Rakoczy, L., 68, 131 Rainirez, C., 110, 123, 131 Hanby, € 3 . , 54, 127 IZantlle, P . E., 113, 128 Rao, P. S., 115, 131 Raper, J . R., 106, 112, 113, 129, 131 Haper, K. 13., 48, 51, 126 Rapoport, H., 105,130 Ratledge, C., 5, 43 Raymond, 1Z. L., 17, 40 Razin, hf., 65, 129 Redfearn, E. It., 177, 182, 188, 189, 190, 191,209,210 Rees, D. A,, 251, 270 Rees, M.,229, 273 Reichle, R . E., 85, 131 Repaske, R . , 233, 273, 274 Revsin, B., 207, 210 Ribbons, D. W., 251, 257, 270 Rickard, P. A. D., 178, 210 Righelato, R . C., 102, 117, 131 Rittenberg, 8 . C., 230, 272

284

AUTHOR INDEX

Roberts, E., 255, 274 Robertson, N . F., 7 0 , 7 4 , 131 Robinson, D. S., 21, 40 Robinson, P. &7I 2. , 103, , 130, 131 Roche, B., 15, 26, 33, 42 Itomano, A., 69, 71 , 1 3 1 Roper, G. H . , 178, 210 Rose, A. H., 73, 130 Rose, H . G., 39, 42 Rosen, 0 . &I., 56, 131 ltosenberger, F., 261, 273 Rosenthal, O., 32, 4 2 ltosness, P . A., 07, 58, 131 R o s , I. I<., 66, 131 Roth, K . AT., 58, 59, 131 Rottenburg, H . , 256, 273 Rowe, A , , 55, 127 Rudd, J. H . , 250, 270 Ruiz, Herrera, J . , 180, 197, 210 Runyon, E . H . , 51,1 3 1 Rupprecht, E., 166, 168,170, 171 Iturainski, €1. J . , 138, 171 Rusch, H . P., 47, 63, 65, 66, 67, 68, 69, 121, 126, 127, 128, 130, 131, 732 ltussol, D . W., 122, 127, 131 Hussel, S. A , , 168, 170 Hussey, W. Is., 35, 40 Rynearson, 1'.K . , 4 , 43

S Sackin, M. J., 50, 52, 124 Segers, R. D., 244, 273 Sakamoto, Y . , 32, 43 Salton, 31. It. J., 123, 131 Salvatori, T . , 108, 125 Samuel, E . W., 51, 131 Sanadi, D . R., 34, 4 3 San Pietro, A., 233, 273 Sanwal, 13. D., 90, 91. 94, 131, 132 Sarje, B . D., 100, 126 Sato, R., 32, 42, 196, 209, 233, 269, 272 Sauer, H . W . , 63, 67, 68, 6 9 ,1 2 7 , 132 Sauer, L., 67,127 Scardovi, V . , 241, 273 Schaeffer, P., 116, 119, 132 Schatz, G., 180, 210, 254, 273 Scheda, R., 3, 43 Scheinmann, F., 39, 41 Scheld, H . W . , 6 , 17, 43 Schindler, F . J . , 181, 210 Schisler, L . C., 113, 132 Schissler, D. O., 19, 32, 43

Schlegel, H. G., 233, 260,269, 273 Schlenk, F., 109,132 Schlosser, E., 107, 132 Schnabel, W . , 2, 41 Schneider, D . L., 242, 273 Schneider, H . J., 39, 41 Schon, G., 160,171, 252, 273 Scholes, P. B., 207, 210 Scholhorn, R., 159, 171 Schramm, M . , 237, 273 Schroepfer, G. J . , Jr., 38, 42 Schulze, K . I., 260, 273 Schuster, E., 260, 273 Schwalb, M. N., 7 1 , 132 Selva, A., 108, 125 Senez, J . C., 2 , 9 , 10, 12, 19, 20, 33, 40, 41, 43, 251, 252, 254, 256, 261, 262, 264, 265, 266, 268, 270, 271, 272, 273 Senoh, S., 10, 42 SentheShanmuganathan, S., 7 3 , 1 3 2 Sermonti, G., 87, 125 Seydoux, J . , 9 7 ,1 3 3 Shaffer, B. M . , 5 1 ,1 3 2 Shafia, F. M . , 232,274 Shaw, D. A,, 9 4 ,1 3 4 Shaw, D . S., 85, 126 Sheng, C., 120, 132 Sheng, T . C., 120,132 Shethna, Y . I., 147, 1 7 1 Sheu, C. W., 160, 171 Shibata, R., 32, 43 Shimizu, A., 5, 14, 43 Shiniojima, Y . , 10, 42 Showe, &I. K . , 196, 197, 210 Shug, A. L., 229,271 Shugart, L. R., 250, 258, 270 Shuster, C . W., 225, 241, 253, 271 Sietsma, J . H., 107, 121, 132 Sigal, N., 257, 270 Sih, C . J., 37, 43 Silver, M., 230, 274 Silver, R. S., 259, 273 Simon, W . , 6 2 ,1 3 4 Simonart, P.-C., 264, 266, 272 Simon-Pietri, P., 261, 270 Sinclair, P., 178, 208 Sinclair, P . R., 179, 180, 181, 182, 189, 199, 200, 202, 204, 205, 206, 210 Sinden, J . W., 113,132 Sinha, U., 49, 50, 132 Sistrom, W. R., 160, 169 Skerman, V . B . D., 182,210 Smalley, A. J., 250, 253, 273 Smillie, R. M., 148, 171 Smith, C., 4 , 40 Smith, D., 7 2 , 130 Smith, J . E., 55, 87, 88, 94, 95, 96, 97, 110, 127, 133

285

AUTHOR INDEX

Smith, L., 173, 176, 177, 179, 180, 181, Takeda, I., 10, 42 182, 185, 189, 198, 199, 200, 201, 202, Takeuchi, I., 55,130,132 Talal, N., 57, 132 203, 204, 205, 206, 207, 208, 210, 211, Tamelen, E. E., van, 38, 43 248, 273 Tamura, G., 3, 12, 24, 40, 42 Smith, H.. R., 39, 40 Smith, S. L., 200, 210 Tanaka, A., 5, 12, 14, 43 Snider, I. S.,110, 132 Tanaka, M., 143,171 Snos\r-e11, A. M., 194, 196, 209, 210, 250, Tannenbaum, S. R., 6, 42 E. L., 71, 73,126,127 ‘rat-, 2 73 Sokatch, t J . H., 250, 271 Tausson, W. O., 2, 4, 43 Taylor, A., 122, 127 Sokatch, J. T., 249, 250, 273 Taylor, B. P.,231, 270 Sommo, R., 182, 210 Taylor, E. E., 75, 130 Sonneborn, D. R., 51, 53, 132 Taylor, J. H., 105, 132 Spencer, E. L., 178, 209 Taylor, Z., 181, 209 Spencer, J. F. T., 25, 35, 41, 43 Temperli, A., 188, 210 Srapinka-Kwasze~~ska, S.,257, 272 Srere, P. A , , 57, 62, 132 Terenzi, H. F., 71, 132 Teulings, F. ,4. G., 251, 254, 255, 271 St.achow, C. S., 94, 132 Stadtman, E. Et., 166, 170 Thangamani, A., 123,132 Thauer, R., 166, 168,170 Stadtman, T. C., 234, 274 Thauer, R.K., 168, 171, 240, 274 Staniei-, R. Y., 160, I 6 9 Thijsse, G. J. E., 2, 9, 17, 19, 22, 43 Stevenson, D. P., 3, 9, 19, 22, 27, 32, 43 Thomas, D. If.,108, 132 Stewart, J. E., 3, 9, 19, 22, 27, 32, 43 Thomas, D. S. D., 74, 132 Stine, G. J., 94, I32 Threlfall, D. R., 193, 210 Stjernholm, R. L., 241, 269 Tinker, K., 153, 1 7 1 Stokes, J. L., 97, 132 Storck, R.,70, 71,128, 132 Tomkins, G., 57, 132 Tomlinson, T. E., 138, 170 Storey, B. T., 173, 209 Tomonaga, G., 123, 130 Stotz, F. H., 178, 209 Stouthamor, A. H., 214, 237, 248, 251, Torigoe, Y., 5, 13, 29, 43 253, 254, 255, 256, 257, 271, 274 Tornabene, T. G., 39, 43 Towler, D. A., 116, 130 Strandbcrg, G. W., 161, 171 Strange, R. E., 260, 274 Toyoda, S., 24, 4 1 Strauss, 13. S.,91, 130 Trecanni, V., 19, 43 Strominger, J. TJ., 223, 269, 274 Treschow, C., 113, 132 Trinci, A. P. J., 75, 77, 78, 102, 117, 131, Stumpf, P. K., 35, 41 132 Subramanian, D., 157,170 Trione, E. J., 76, 132, 133 Sulya, L. L., 39, 40 Trudinger, P. A , , 230, 274 Summers, R., 259, 271 Sunshine, L. D., 65,131 Truesdell, L. C., 85, 126 Sussman, If., 47, 48, 51, 52, 53, 54, 55, 57, Tucker, A. N., 200, 211 58, 59, 60,124,125, 127, 128,129, 130, Tulloch, A. P., 25, 35, 38, 41, 43, 107, 121, 131,132,133,134 128 Sussman, R., 51,132 Turian, G., 47, 71, 74, 75, 77, 78, 88, 89, 90, 91, 92, 93, 97, 112, 120, 121, 125, Sussman, R. R., 47, 48, 64, 57, 58, 60, 132 Sutherland, E. W., 51, 52, 130,132 126,127,129,130,131, 133 Turner, J. F., 70, 128 Sutter, R. P., 108, 132 Turner, N. A., 107, 121,126 Suzuki, I., 230, 231, 270, 274 Tuttle, L. C., 141, 1 7 1 Svihla, G., 109, 132 Swank, R. T., 188, 192, 210

T Taber, W. A,, 113,132 Tagawa, K., 141, 154, I 7 1 Takahashi, J., 3, 10, 43

U Uden, N., van, 259, 260, 262, 274 Udenfriend, S., 38, 41 Ullmann, S., 2, 41 Umbarger, E., 220, 274

286

AUTHOR INDEX

Unaini, Y., 24, 25, 33, 4 1 Unestam, T.. 87,133 Updike, J., 115, 130 Urayama, T., 113, 133

Wessels, J. G. H., 74, 113, 114, 115, 117, 130,133 Whiffen, A. J., 120, 133 Whistance, G. R., 193, 210 Whitaker, A. &I., 254, 274 White, D. C., 174, 176, 177, 179, 180, 181, 182, 189, 198, 199, 200, 201, 202, 203, V 204, 205, 206, 207, 209, 210, 211 White, G. J., 48, 51, 53, 54, 132, 133 White, H. B., 39, 40 Vagelos, P. R., 240, 271 Valentinc, It. C., 136, 138, 141, 147, 151, Whitely, H. R., 245, 246, 274 Wiaux, A. L., 5, 13, 42 153, 156, 160, 167, 169, 171, 172 Wilhelms, H., 53, 127 Van Dalfsen, J . W., 137, 170 Willett, J. D., 38, 43 Van de AIeene, J. G. C., 51, 52, 129 Williams, G. R., 181, 182, 202, 209 Varncr, J. E., 228, 26.9 Williams, M. W., 105, 130 Velmelage, lt., 90, 130 Willmer, J. S., 120, 127 Vernon, L. P., 154, 170, 171, 215, 232, Wilson, P. W., 138, 140, 147, 165, 158, 161, 235, 236, 274 162,169,170,171,188,210 Vestal, J. R., 21, 43 Wimpenny, J. W. T., 196, 209, 211, 252, Vidova, &I., 251, 254, 256, 272 254, 257,270, 274 Villanueva, J. R., 69, 133 Winstanley, D. J., 108, 124 Violago, F. C., 255, 274 Winter, H. C., 159, 1 7 1 Vishniac, W., 217, 231, 260, 271 Winters, K., 39, 43 VolfovQ, O., 3, 15, 40, 42, 43 Wiseman, G. M., 255, 274 Volz, P. A., 113, 116, 133 Withaar, J., 193, 210 Von Stedingk, L. V., 215, 270 Witkop, B., 38, 41 i’ries, W., de, 237, 274 Wittenberger, C. L., 233, 274 Witter, L. D., 262, 273 Witz, D. F., 158, 170 WoIf, R., 181, 209 Wolfe, P. B., 51, 52, 125 Wolfc, R. S., 141, 171 Wachsman, tJ. T., 246, 274 Wong, P. S., 119,126 Wada, F., 32, 43 Wood, H. G., 241, 244, 269, 272 Wade, H. R., 260, 274 Wood, W. A., 240, 242, 243, 269, 273, 274 Wagner, 13., 91, 130 Woodruff, H. E., 118, 119, 121, 133 Wagner, F., 20, 25, 33, 43 Work, E., 223, 274 Walker, C. G., 91, 133 Wright, B., 62, 134 Walkcr, D. J., 241, 243, 245, 250, 251, Wright, B. E., 46, 47, 48, 51, 63, 54, 55, 252, 257, 258, 260, 261, 263, 266, 268, 56, 57, 60, 62, 63, 88, 117, 124, 127, 271, 272, 274 128, 129, 131, 133, 134 Wallnofer, P., 242, 274 Wright, E. A., 201, 211 Walsh, B. T., 62, 134 Wyss, D., 162, 171 Wang, C. S., 73, 133 Wyss, M. B., 162, 1 7 1 Warburg, O., 71, 133, 177, 210 Ward, C., 55, 13.1 Ward, J. M., 68, 133, 134 Wase, D. A. J., 260, 274 Y Watanabe, Y., 122, 128 Watson, K., 55, 133 Yamada, K., 3, 6, 10, 13, 29, 43 Weaver, P. F., 153, 160, 171 Yamaguchi, K., 3, 12, 39 Weber, D., 39, 42 Yamashita, S., 192, 211 Weinstein, I. B., 60, 133 Yamazaki, I., 178, 211 Weiss, 13., 74, 90, 133 Yanagisawa, K., 51,134 Weiss, D., 229, 269 Yanagishima, N., 110, 129, 134 Wenning, J., 240, 274 Yanagita, T., 98, 123, 130, 134 Werkman, C. H., 244, 272 Ytmg, C. C., 175, $10

w

287

AUTHOR INDEX

Yang, C. S., 143, lYl Yano, K., 3, 12, 24, 40, 42 Yasunobu, I<. T., 143, 171 Yernni, E. W., 118, 134 Yielding, K. L., 57, 132 Yiko, N. J., 154, 170, 215, 272 I’och, D. C., 147, 156, 161, 167, 169, 171, 172 Yokata, K., 178, 211 Young, I. G., 194, 209

Z Zahn, W., 20, 25, 33, 43 Zalokar, RZ., 89, 90, 91, 93, 9 4 , 119, 134 Zaltzman-Nirenberg, P., 38, 41 Zeldrin, M. H., 6 8 , 134 Zey, P., 1 8 8 , 2 0 9 Zimmerman, E. J., 90, 129 Zink, M. W., 9 4 , 134 Zwilling.de Vries, J. T., 9, 43

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SUBJECT INDEX A

Adenosine triphosphate-cont inued production by micro-organisnis, 249 Acellular slime moulds, differentiation in, requirement for synthesis of bacterial 63 cells, 225 Acetabularia mediterranea, morphogenesis Aeration, effect of, on yeast-niycelium in, 74 dimorphism in fungi, 71 Acetate, effect of, on conidiation in Xeuro- Aerobncter aerogenes, aerobic production of spora crassa, 89 energy by, 248 Acetobacter xyliiaum, hexose degradation in, cytochrorne a 2 of, 188 237 energetic uncoupling in, 262 Acetylglucosarninidase in development of maintenance energy of, 260 Dictyostclium discoideum, 59 molar growth yields of, 251 Acetyl phosphate, energy from, 239 thermodynamic data for growth of, 268 Acetylene reduction by nitrogenase, 159 A . clocrcnp, molar growth yields of, 261 I r h l y n , morphogenesis in, 74 A . ozidrcits, P / O ratios in, 254 sexual reproduction in, 10G Aerobic ferredoxins, 150 sporangial development in, 102 Aerobic metabolism, growth yields in, 253 Achromobacter spp., electron-transport Aerobic nitrogen fixation in Azotobacter, systems in, 186 electron flow in, 154 Aconitate hydratase activity and differen- Aerobic phase of growth, energy produc. tiation in Blristocladiella emersoiaii, 86 tion during, 248 .4crasiales, biochemistry of differentiation Agaricus bisportis, differentiation in, 113 in 50-47 Agglutination in Hansenula wiiLgei, 109 -$erasin, action of, on cellular slime moulds, Aggregation of amoebae of Dictyosteliu~n 51 discoideum, effect of drugs on, 60 chemical nature of, 51 Agrobacteriurn tumefaciem, cell yields of, Acremoniuni sp., utilization of alkanes by, 259 4 Alanine, requirement for energy in Actirzomyces israelii, molar growth yields synthesis of, 220 of, 251 Alanine fermentation by bacteria, 243 Actinoinycin D, effect of, on morphogenesis Aldolase and Neurospora conidiation, 93 in Uictyosteliuin discoideum, 58 Aliphatic hydrocarbons, utilization of, by Activation of amino acids, energy requiremicro-organisms, 1 ments of, 221 Alkaline phosphatase in development of rlcytostelium leptosonwna, action of yohimDictyostelium discoideum, 59 bine on, 51 Alkanes, mechanism of oxidation of, by Adenine nucleotides as inhibitors of yeasts, 24 nitrogenase activity, 162 oxidation of, 1 Adenosine monophosphate, energy requirepathways of oxidation of, in bacteria, 19 ment in synthesis of, 221 n-Alkanes, ability of yeasts to use,2 Adenosine phosphosulphate in conversion utilization of, by filamentous fungi, 4 of sulphate to sulphite, 231 Alkenes, mechanism of oxidation of, 22 Adenosine triphosphate, as an energy curmechanism of oxidation of, by yeasts, 26 rency in micro-organisms, 213 production of, from alkanes, 20 involvement of, in nitrogenase action. Alk-1-enes, microbial utilization of, 3 159 oxidation of, 1 pool of, in micro-organisms, 262 utilization of, by fungi, 5 289

290

SUBJECT INDEX

Allantoin, fermentation of, 248 AZZomyces, differentiation in, 105 .4.jnuaiiicus, carotenoid pigments in, 120 .-I lteriinria s o h r i , flavins in conidial formation in, 77 Amino acid, and peptide derivatives in fungi, 121 composition of flavodoxins, 146 sequence of clostridial ferredoxin, 144 .$mino acids, energy production from, 243 energy requirement in synthesis of, 217 Ainnionia, inhibitory effect of, on nitrogenase, 161 oxidation by micro-organisms, 228 Anaerobes, cnergy production by, 239 Anaerobic bacteria, electron carriers in, 136

electron chains in, 150 Anacrobic growth, molar growth yields during, 249 ilnaerobic phase in organotrophic metabolism, 236 Anomalous growth yields, 257 Anthcridiol, formula of, 106 .-I plutironiyces nstctci, biochemistry of differentiation in, 87 Apical localization in fungal growth, 72 Apoprotein of flavodoxin, 146 Argininr, action of ( 'lostridiu?rbperfri)igeirs on, 244 cnergy requircment in synthesis of, 219 Aromatic amino acids, energy requirement in synthesis of, 217 Artificial electron donors, use of, in studies on bacterial electron transport, 183 ilscobolzcs ntercontrius, sexual reproduction in, 112 Ascogoniuin, differentiation of, 1 1 1 Ascomycetes, sexual reproduction in, 108 Ancortmte, iisc of, in studies on hacteriul electron transport, 185 Aaexual reproduction, and honnones in fungi, 104 in Pilobolus, 103 of fungi, biochemistry of, 79 Aspartic acid, energy required in synthesis of amino acids derived from, 217 A spergillus gigaiiteus, carotcnogenesis in, 75 conidiophore extension in, 75 -4. glnucus, production of conidia by, 104 A . niger, utilization of paraffin wax by, 4 Aspergillus spp., differentiation in, 94 Assay for high-enorgy electron carriers, 149 Assimilation, oxidation of hydrocarbons without, 17 Autotrophic organisms, energy requirements of, 227

Available electrons in niic ro-organisms 255 Azide, effect of, on Escherichia coli, 195 reduction by nitrogenase, 159 use of, in studies on bacterial electron transport, 190 Azotobacter, electron carriers from, 147 electron flow in aerobic nitrogen fixation in, 154 ferredoxin, 148 pathways for ATP synthesis in, 192 Azotobacter spp., electron-transport system in, 188 A . wimlandii, effect of redox dyes on, 139 high-energy electron carriers in, 136 maintenance energy of, 260 production of temperature-sensitive mutants of, 164 separation of nitrogenase enzymes from, 149 Azotoflavin, 147

B Bacillus nregaterium, cytochromes of, 207 reaction of cytochromes in, with carbon monoxide, 185 B. subtilis, cell yields of, 249 Bacteria, growth of, on hydrocarbons as an indicator of substrate specificity, 7 high-energy electrons in, 135 pathways of hydrocarbon oxidation in, 19 regulation and genetics of electron chains in, 160 utilization of alkanes by, 5 Bacterial branched electron-transport systems, 173 Bacterial cells, analyses of, 224 total synthesis of, 223 Bacterial ferredoxin, 140 Bacterial urate reductase, 152 Bncteroides amylophilus, cell yields of, 259 B. ruminicola, carbon monoxide-combining pigments in, 177 conversion of lactate to propionate by, 242 Basidiomycetes, sexual reproduction in, 112 Benzoquinone in Escherichia coli, 192 Benzylviologen, use of, with bacteria, 138 Bicarbonate and morphogenesis in Blnntocladiella emersonii, 77 Bicarbonate trigger in Blastocladiella emersonii, 79 bifidum, molar growth Bi'dobtrcterium yields of, 251

SUBJlECT INDEX

8. byidus, hexose degradation in, 237 Uinding in high-energy electron carriers from bacteria, 151 Biochemistry of asexual reproduction in fungi, 78 r i f fungal differentiation, 48 Udogical reclox eriergy scale, 136 Blukeslen trisporcr, carotenoids in, 191 ~l(tstocladielltr,effect of light on, 77 britcrnnicrr. photorertsptor in, 78 c u w r s o u i i , biocnhrwistry of re~mxluction in, 79 main events in diflwcntiation in, 84 Hotrytis cinerecr, utilization of alkanes by, 4 I3ranched electron-transport systems in bacteria, 173 12reuihncterium sp., oxidation of propane by, 21 I h t a n e , growth of filainentoiis fungi on, 1J

C Calvin cycle, operation of, 216 ('nnditla nlbicccns, yeast-mycelium dimorphism in, 70 C'. i i i t e n i ~ e d i autilization , of alkanes by, 2 6'. lipolytica, ability to attack alkanes, 2 mechanism of alkilnc oxidation by, 25 G'. rigidrr, utilization of kerosone by, 3 C. rugosa, mechanism of oxidation of alkanes by, 24 C. tropicalis, ability of, t o attack alkanes, 2 C . wtilis, P / O ratios in, 254 Carbohydrate, cell synthesis from, 225 metabolism, during morphogenesis in Acrasiales, 53 in meiosis in yeast, 110 of SehiZOp/LylllLTlL co?lk7lLzLlle, 1 14 synthesis during rnorphogenesis in Dictyosteliwm discoidewm, 54 Carbohydrates, energy production from, 237 Carbon dioxide, and yeast-mycelium dimorphism in Mwcor rousii, 70 fixation, lithotrophic, 214 synthesis of bacterial cells from, 227 Carbon monoxide, complexes of cytochrorncs, 177 use of, in study of bacterial electron transport, 177 Carboxydismutase, activity of, 215 Carotenogenesis in Aspergillus gigccnteus, 76, 78 Carotenoids, and sexual development in fungi, 108

29 1

Carotenoids-cont iisued as possible photoreceptors in fungi, 75 in fungi, 119 Cary spectrophotonieter iu the study of bacterial electron transport, 175 Cell adhesion in cellular slimc moulds, 53 Cell aggregation in cellular slime moulds, 51 Cell division, interference with, in Cnndidu rrl6iccr1~s,70 Cell synthesis, from carbohydrate, 225 from ireformed monoincrs, 224 Cells, bacterial, total synthesis of, 223 Cellular slime mould differentiation, 47 Cellulosespore case of ccllular slime moulds 49 Cellulose synthesis during niorphogetlcsis in Acrasiales, 54 Cell-wall composition and reproduction i n fungi, 69 Cell-wall construction in Eumycotina, 69 Cell walls, energy requirements for synthesis of, 223 Cell yields of micro-organisms, 249 Changes in Carbohydrates during tnorphogenesis of Acrasialr.s, 5 3 Chcmolithotrophs. 214 euergy metabolism in, 2 I 6 generation of energy in, 227 Chemotaxis in cellular slime moulds, 5 1 Chlorate resistance in Eschcrichin coli, 198 Chlorin haem in bacteria, 178 Chlorobiu?n thionulph~rtophilici,a, carbon dioxide fixation in, 215 oxidation of thiosulphate by, 235 reductive assimilation of carbon dioxide in, 153 Chloroplast photo-NADP reaction, 147 Choonephorn spp., morphogenesis in, 77 Choico of wavelength in double-beam spectrophotometry. 179 G'hron~ntiumslip., oxidation of disulphide in, 231 Chytridiomycetes, sexual reproduction in, 106 Citrate-condensing enzyme and differentiation in Blrrstoclndiella e?nersonii, 86 Cladosporiuna sesinnc, alkane utilization by, 14 Ckrdo.sporium sp., utilization of alkanes by, 4 Clostridia and the Sticltlmd reaction, 246 Clostridial fermentations, 239 Clostridial ferredoxin, 141 in Azotobacter systems, 157 Clostridium iicidi-wrici, urate fermentation by, 151

292

SUBJECT INDEX

C. kluyveri, energy production by. 240 P. pnsteuriai~uni,ferredoxin and, 141

flavodoxin from, 144 C . propionicum, conversion of lactate to

propionate in, 242 C'. tetanomorphum, fermentation of histidine by, 246 6'. uracilicuna, uracil formentation by, 248 Coenzyme Q8 in Azotobucter sp., 188 Coenzyinc-Q in Esclterichiu coli, 192 c:~)liforniorganisins, iiictabolisrn of pyriivate in, 240 (;ollectotric/wiii ~ r t ~ ~ c ~ t t i e i r t c c ~ iuti1izatii)n ritii, of alkanes by, 5 Conidia, fungal, differentiation of, 74 Conidistion, ill Aspergillus Iiiyer, 94 in li'usurium, 103 in S e u r o s p r u crassn, biochemistry of, 8R Conidiophorc exterlsiorl ill rlspeyqillus giganteus, 75 C'oprim~sZogopus, development in, 115 Coremia, morphogenesis of, 73 ~~o1.!/tiebficterizr11~ sp., grow t I i o 1; o I I dot1cc . I -em, 7 cT,,iipletldecoinposit,iuti C J ~ *' t i i i i i i o acids tjy bacteria, 246 Coupling, rnergy, i n iiiicri)-ol.ganisms,214 (!riteria in methodology studies 0 1 ' bacterial electron transport, 183 ('ritical oxygen concentration, collcept of, in bacterial electron transport, 181 Cr?yptococcus , n e o f o r ~ i ~ n i i s ,utilization of kerosene by, 3 ~ ' u ~ i i ai r r ~ h a ~ ~sp., a e ~utilization llm of alkanes

by, 4 Cyanide, effect of, on Hnenaophilus parcrinjuenzae, 202 reduction by nitrogenase, 159 us0 of, in studies on bacterial elcctron t,ransport, 185 Cyclic-AMP, action of acrasin as, 61 Cyclic photophos~horylation in niicroorganisms, 236 Cycloheximide, effect of, on morphogenesis of Dictyostelium discoideum, 58 Cysteiw and morphogenesis in Neurosporn crassa, 74 desulphydrasc in micro-organisms, 244 energy requirenient in synthesis of, 219 fermentation of, 244 Cytidine monophosphate, energy requireincrit in synthesis of, 221 Cytochromc u j in bacteria, 207 Cytochrorne b in bacteria, 179 Cytochrome o in bacteria, 179, 207 Cytochrome absorption, 170

Cytochrome oxidase, and conidiogenesis in Neurospora crassa, 93 in Achroinobacter spp., 186 Cytochromes, and conidiogenesis in Neurospora crassa, 91 in Achrmobacter spp., 186 in Azotobncter spp., 188 in Escherichia coli, 192 in Huemophilus purainjuenz(te, 199 in Halobacterium cutirubrum, 183 in h'itrobtrcter spp., 227

D Ilccldiizarici coticentricci, phenol oxidrtses and, 122 Dcboryomyees spp., ability to attack alkanes, 2 D . subglobosus, maintenance energy of, 260 Of* by fungi, Dehydrogenases in Hnernophilus pnrninjluenzae, 201, 203 Deosyribonuclease activity and developnient in =ispergillus i f iger, 98 Desiccation, offevt of, 011 amoebae, 65 d f w t of, on plasmodium of Physnitcm polycepiialun~,65 l~esulphouibrio desulphuricrcirs, c.nergetiv uncoupling in, 26 I /),.aulphot&rio spp.. ~ ~ l e r gproductioll y in. "33 2.6.1)ichloroindophcnoi, use in studies on bacterial electron transport, 190 ~ i ~ ~effect ) of, ~ on~ enzymes ~ ~ from ~ l Escherichin coli, 194 Dictyoste~iu,,,2.discoidewllb, life cycle of, 48, 49 parasexual cycle in, 50 Difference spectra of Hnemophilus porninjuenzae, 180 iff^^^^^^ spectroscopyin the study of bacterial electron transport, 175 Differential enzyme inactivation in Dictyosteliuna discoideum, 57 Differentiation, and secondary metabolism in fungi, 11 6 definition of, 46 fungal, biochelnistry and physiology of, 45 Dimorphism, and cell.wall construction in fungi, 72 in Eumycotina, 69 Diplococcus glycinophilus, energy metabolism of, 243 Dipyridylium dyes, effect of, on bacteria, 138 Dipyridyls, effect of, on micro-organisms, 138

,

293

SUBJECT INDEX

Dissimilatory sulphate reduction in microorganisms, 233 Diphenylarnine, effect,of, on light-induced sporulation in futigi, 77 1A)ithle-beani spectrophotoiiieters in the study of bacterial electron transport, 175 1)yc permeability of micro-organisms, 140 Dyes, rodox, use of, with micro-organisms, 138

E Eoological studies on hydrocarbon utilization, 6 Electrodes, oxygon, in study of bacterial electron transport, 181 Electron-accepting groiip of ferredoxin, 141 Electron rtwriws in tmctcria, 135 Electron chains i n anaerobic bacteria, 150 Electron flow in ttcrobic nitrogen fixation by Azotobacter, 154 Electron transfer in h'itrobricter spp., 228 Electron-transport systems, branched, in bacteria, 173 in micro-organisms, 246 Electrons, high-energy, in bartoria, 135 T':inhden-Meyerhof-Parnas pathway, 237 Endogenous respiration in Hnemophilus prim i ~ f l u e u t n r ,108 b~:rirlom.yrc~s spp., a hi li t,,, if. to a t tark alkanrs, 8 K ~ l o t h i t rp/ru.sitictr, differentiatioir iu, 10 I Etidotrophic cwnitliation in I'et!ici&un!, 101 Energetic hehaviour of niicro-organisms, 213 Energetic uncoupling, 260 Energy, generation of, in micro-organisms, 227 involved in synthesis of mononicrs, 21 7 of maintenance, 259 requireiiicnt for, 714 us0 of, in micro-organisms, 749 Enthalpy of cat)aholisinin micro-organisms, 256 Entnrr-Doudoroff pathway of energy production, 238 Entropy prodnct,ion in micro-organisms, 266 Environmental factors, effect of, on sporulation in fungi, 75 Enzyme activity, and ronidiation in A spergillus wiger, 96 and clevclopment in Ul~ixtoclndielln emersotiii, 86 (

Enzyme Activity-continued and differcntiation in Rlastoclrrrliellrr emcrsonii, 80 variations in during conidiation in Neurospora c r u s s ~ i6,'3 15nzyme disappearance, regulation of; in Dictyostelium discoideum, 58 Kpoxide formation, possible, in oxidation of alkencs, 27 ZC&erichin coli, cell yields of, 249 cyt'ochrome u2 of, 188 electron transport in, 192 maintenance energy of, 260 P / O ratios in, 254 Ethane, oxidation of, by mycobacteria, 17 Ethanol dehydrogenase and couidiogenesis in Xeurosporn crrissa, 9 3 6-Ethylpurine and conidiation in =Is,ueryill u s ~ i g e r97 , Euascomycetidae, differentiation in, I 1 0 Euniycotina, biochemistry of differentiation in, 69

F Fatty acids, energy requircnicnts ill synthesis of, 222 Feedback inhibition of nitrogenas eactivity, 161 E'eriiientation, of amiuo acids, 243 of cysteine, 244 (if glutamate, 246 of heterocyclic c o t i i ~ ~ o u n ~247 ls. [~f'histidine,245 of pentoscs, 239 E'erredoxin and microbial inetalbolism, 140 Ferredoxin-dependent pyruvate synthase, 153 Perredoxins in bacteria, 135 Ferric ion, oxidation of, by inicro-organisms, 232 Ferricyanide reductase of Hnemophilus pnrainfluenzne, 203 E'crrobacillus, energy production in, 232 Filamentous fungi, growth of, on hydrocarbons as an indica.tor of substrate specificity, 13 mechanism of hydrocarbon oxidation by, 29 utilization of hydrocarbons by, 4 Flavins, as possible photoreceptors in fungi, 75 in ferredoxins, 144 Flavodoxin, 144 Flavodoxins in bacteria, 136 5-Fluorouracil, inhibition of light-induced sporulation by, 77

294

SUBJECT INDEX

Formate oxidase in Escherichia coli, 195 Formate, utilization of, by Nitrobncter spp.,

228 Free energy efficiency in micro-organisms,

264 Fructification in Schizophyllum commune,

114 Fruit body forination during development of Ph?JRfIrl(7lL ~ ~ o ~ y c e ~ i l , h d67 u?n, Funiaratc hydratasc and different,iation in Blnstocladiellu emersonii, 86 Fungal differentiation, biochemistry and physiology of, 45 Pusariuin moniliforine, utilization of alkanes by, 4 Busobacteriuin ~ ~ u c l e u t u m fermentation , of pyruvate in, 245

Glycolytic pathway in Blastocladiella emersonii, 80 Glyoxylate cycle and conidiation in Aspergillus niger, 96 Green sulphur bacteria, production of energy by, 234 Growth, as indicator of substrate specificity with hydrocarbons, 7 generation and utilization of energy during, 2 13 yields, anomalous, 257 in aerobic metabolism, 253 molar, in micro-organisms, 249 Guanosine monophosphate, energy rcquirement in synthesis of, 221

G

H

P-Galactosidase and LYczcrospwa conidiation, 93 Gametes in myxomycetes, 63 Gamones of fungi, 104 Gene expression in relation t,o differentiation, 45 Generation of energy in micro-organisms,

Haemin, effect of on Huemopleilus poru-

227 Gcncration and utilization of energy during growth, 213 Genetics of electron chains in bacteria, 160 G‘eotrichun~,differentiation in, 103 Gibberelln zeue, differentiation in, 101 Glioclndiurn coteniilatirm, growth of, on butane, 13 Glnconeogcnesisin morphogenesis in Dictyostelium discoideum, 56 Glucose 6-phosphatc dehydrogenase and conidiogeriesis in Neurospora crassu. 93 P-Glucosidase in development of DietgoStcliiLnl, discoideum, 59 Glutamate, dehydrogenasc nctivit,y and Neurospora conidiation, 94 energy requirement in synthesis of amino acids derived from, 218 fermentation, 246 Glutamine, energy requirement in synthesis of, 219 Gly-cerol-water, use of, in the study of bacterial electron transport, 176 Glycine, formentation of, 244 Glycogen metabolism in morphogenesis in Acrasiales, 53 Glycolgsis, during morphogenesis in Dictyostc&um discoideune, 55 energy production by, 237

in$uenzae, 199 Hrcemophilus puraii@uenzne, branched electron-transport systems in, 174, 179, 188, 190 electron transport in, 198 Haems in bacteria, 178 Hrilobacteriurn cutirubrum, branched olcctron-transport systems in, 193 H . Iialobium, cytochromes in, 185 H . salinarium, cytochromes in, 185 Halophilic bacteria, branched electrontransport systems in, 183 Hand spectroscopes in the study of bacterial electron transport, 174 Hrcnsenula spp., ability of, t o attack alkanes, 2 H . subpelliculosa, utilization of kerosene by, 3 H . wingei, agglutination in, 109 Heats of combustion in micro-organisms, 264 Hclicostylum sp., utilization of alkanes by, 5 Helminth’osporiuin stcnospiluna, effect of light on, 78 Hemiascomycetidae, sexual reproduction in, 108 2-n-Heptyl-4-hydroxy-quinoline N-oxide, use of, in studies on bacterial electron transport, 184 Heterocyclic compounds, fermentation of, 247 Hexoses, role of, in yeast-mycelium dimorphism in Mucor rouxii, 71 High-energy electrons, in bacteria, 135 in photosynthetic bacteria, 152

295

SUBJECT INDEX

Histidine, energy requirement in synthesis of, 220 fimncntations, 245 tli.stopl~~stntLc i r p s r i l r t l i c t i L , ~cast.-iiiyc:cliuirr dinlorphisin in, 7 0 I 1oiiiobasidioiiiycc~ticlnc~, scxual reiwoduction in, 112 Hoinocysteine, degradation of, 245 Hormoderrd~ro/r sp., utilization of alkanes by, 4 Hormones and asesual reproduction in fungi, 104 Hydrazine, role of, in oxidation of ammonia, 229 Hydrocarbon oxidation, pathways of, 18 repression of, 17 ~lydrocarbon-oxidiziiigorgaiiisnis, 2 tlydrocarbons, aliphatic, utilization of, by micro-orgaiiisins, I Hydrogen osidation in riiic:ro-vrganisiiis, 232 , activity of, in hydrogen bacteria, 233 kiydrogcnlynsc activity of J h 9 ~ c r i c h i ucoli, 198 lIydri~(ienoi~eoiiii,seiitropiiu, cnergy production in, 232, 233 4-Hydroxybenzoio acid, cffcct of, on Eschrrichitr coli, 193 Hydroxylaniiric, formatioil of, during oxidation of ainmonia, 228 products of oxidation of, in niicroorganisins. 229

I lnduction of hydi~carbonoxidation, 17 Inhibitors, effert of, on light-induced sporulation in fungi, 77 use of, in studics o n IJnc%crial electron transport, 182 Initiator cells, nature of, iii cellular slime moulds, 52 Inorganic sulphur conipouiids, oxidation of, by micro-organisms, 230 Inosine monophosphate, energy requirement in synthesis of, 220 Interpretation of data on bacterial electron transport, 182 Iron, effect of, o n ferredoxin synthesis, 160 eRect of, on production of azotoflavin, 147 oxidation by micro-organisms, 232 Iron-sulphur linkage in ferredoxins, 143 Iron-wire model of electron transport in bacteria, 161

Isocitrate dehydrogenase activity anti conidiation in Aspergillus )z iger, 95 lsocitrate tlehydrogenase, and differentiation in l ~ l r i s t o c l n d i ~ 2el n s r r s o i ~ i i86 , in Azotobacter, 156 Isoprcnoids, in fungi, I I 9

K Karyogamy in Physnrum polycephaluin, 64 Kerosene, utilization of, by yeasts, 3 a-Ketobutyrate, fermentation of, 245 Kinetic models of differentiation in Dictyosteliuni discoideum, 62 lilebsiella nerogenes, pool of adenosine triphosphatc in, 252 I i . pnenmonirie, effect of redox dyes o n , 138

L Lnctobtccillris delhriiecl;ii, molar growth yields of, 249 L. plnntiwum, molar growth yields of, 250 Leptospira, cytochromes of, 207 Leucine, energy requirement in synthesis of, 220 Lrziconostoc mesentrroides, energy production by, 239 molar growth yields of, 249 Life cycle, of cellular slime moulds, 4 i of myxornycctes, 63 Light, effect of, on clevelopuient of Physnrum polyceplinlz~in,07 Light-induced sporulation in fungi, 75 Lipids, energy requirements in synthesis of, 222 Lipoprotein antigen in cellular slime-mould aggregation, 53 Liquid nitrogen, use of, in analysis of bacterial cytochronies, 178 Lithotrophic carbon dioxide fixation, 214 Lithotrophs, generation of cnergy in, 227 Localized anaerobiosis in cells, 159 Low-redox chains in photosynthetic bacteria, 152 Lysine, cnergy rcquirenient in synthesis of, 218 Lyxoflavin, effect of, on light-induced sporulation in fungi, 7 7

M &I forms in Eumycotina, 09 Macroconidia formation in Trichophyton mentagrophytes, 100

296

SUBJECT INDEX

llaintrnance energy, 259 Mindeizinlla spp., biochemistry of differhlalate dohydrogenase activity and conidientiation in, 87 ation in Aspergi1lu.s nige.r, 95 RIiracil D, effect of on development of Binliite dehydroganase and differentiation Dictyostelium discoideum, 60 in Hkrstocliitlaclla enaersonii, 86 Mitochondria1 activity during morpho.\ltdate oxidation in I ~ s c h e r i c h i ocoli, 192 genesis in Dictyosteliuin discoideicm, Rlulate syiithaso activity and conidiation 85 i n :tc~pwqiZZu.vniger, 96 Molar growth yields in ~nicro-or~ariisn~s, Jlnlic enzyincx and differentiation i n 249 AYeiirospor(icrnssrr, 94 of Tliiobucil1u.s neapolitcititrs, 23 I >ll&lolltlte,effect Of o i l Hne??aop,hi22i3 /Jtlrtr.\lolybdenuln, in hacterial urate reductase, i/lJfie,tzrlr:, 202 151 use of in studies on hcterial electron role of in nitrogenase, I60 transport, 190 .TloitiZin spp., ability of to attack alkanes, Mating types in HansenuZ~rusi,zyc:i, I ( J ~ 2 Meiosis, biochemistry of in yeast, 109 Monomers, energy involved in synthesis of, in Physczrunz. pol,ficephnluw~, 64 217 Melni?otus, primordia formation in, 78 Morphogenesis in Eumycotina, 69 BIembrane-bound electron-transport, sys- Mucopeptide, energy requirements for tems in bacteria, 173 synthesis of, 223 Membrane components iii Htrc~tmph;Zu.s Mucopolysaccharide formation during p r u i n & e ~ m z e , 201 morphogenesis in Acrasiales, 54 Meiiatlione, rffect of 011 EsrImrI'cILit~ coli, ilfzteor hirmrZis, differentiation in, 100 194 N . ?micedo, sexual reproduction in, 107 hlepacrinc, effect of on light-induced .lf. rouzii, yeast-m)-celiurn dimorphism in, sporulation in fungi, 77 70 Metabolic pathtvtbys involvccl in coiiitlio- M u c o r sp., utilization of allcanes by, 4 genesis i n LVrurosporn cr(iss(r, 92 Mucodes, sexual reproduction in, 107 Metabolism during morphogcnesis of Mushroom, differentiation in, 113 Acrasialos, 53 Mutants of nitrogen-fixing bacteria, 162 Metabolism of photo-induction in fungi, 77 Mycob~cteriutrapunrirffinicuna, oxidation of Metabolism, shunt, in fungi, 119 ethane by, 17 Metabolizing bacteria, high-energy elecM . phlei, cytochroiiies of, 207 trons in, 137 iM. smegmatis, toxicity of hexane to, 7 hfethnwobacteriunl omeliamki, amino acid Mycota, differentiation in, 63 sequence of fcrredoxin from, 144 Mycotorula j n p ~ 1 7 i c nutilization , of kerosene Methanogenesis in micro-organisms, 234 by, 3 Methnnostrrcititr bnrkeri, energy production Myxamoebae, action of acrasin on in in, 234 cellular slime moulds, 52 .\lethionine, energy requirement, in of cellular slime moulds, 47 synthesis of, 218 Rlyxomycetcs, differentiation in, 63 Met,hodology, in stutlying electron trttnshlyxonrycotinw, differentiation in, 63 port in barterin, 174 of differoncr spectroscopy, 176 31ethy1 groul), trimiiriiil, oxidation of, 20 N Methylonc: b l i i ~rotluction , of by );:sc/iw~chi~c coZi, 195 Naphthaquinone in #LsrIieric?Lin coli, 192 BIcthylviologcn, use of \vith bacteria, I38 Ncotetrazolium as an electron acceptor for JIicrocalorimetrir r~ieasicreruonts,265 Hneinophzlus partrinjluenzae, 202 on micro-orgaiiisins, 256 n'rurosporci c m s s n , morphogenesis in, 74 Jlicrococci, allcaiie oxidation by, 7 Seuroaporci spp., biochemistry of asexual !llicrococcus ccrjJicum, growth of on hexareproduction in, 88 dec-1-ene,9 Neutral electron carrier, 141 oxidation of alkenes by, 24 Nicotinamide nucleotides, role of, in auto39. driiitrificu,~~, cytchroines of, 207 trophic metabolism, 227 rlertron transport in, 207 Nicotinic acid, fermentation of, 248 ~ i i o r g yproduction in, 233 Nitrate, and electron transport in AchroM . Znctyliticus, energy production by, 242 mobacter spp., 187

SUBJECT INDEX

297

Oomyeetes, sexual reproduction in, 105 Nitrate-continued reductase, location of, in Escherichia Organisms which attack aliphatic hydrocarbons, 2 coli, 196 Organotrophic metabolism in microreduction, by Eschevichiu coli, 196 organisms, 236 in micro-organisms, 233 Nitrite formation from nitrate by L4c/two- Orotic acid, fcrmentation of, 247 Overlap of electron pathways in Hoewiornobuctev spp., 188 philus paruin$uenzae, 203 Nitrite oxidation by micro-organisms, 227 Oxidase activity and sporulation in Nitrite reduction by Escherichia coli, 196 Physnrum polycephalurn, 68 Sitrobncter spp., energy metabolism of, 216 Oxidation, hydrocarbon, induction of, 1 7 energy production by, 227 of ammonia by micro-organisms, 228 Sitrocystis occanus, energy production by, of hydrocarbons without assimilation, 229 17 Nitrocystis spp., oxidation of ammonia by, of hydrogen in micro-organisms, 232 228 of iron by micro-organisms, 232 Nitrogena.se, a high-energy olcctron accepof nitrogenous compounds, 227 tor, 157 of sulphur compounds, energy prodncAzotobacter, oxygen sensitivity of, 158 tion during, 229 electron carriers coupling to, 147 Oxidation-reduction reactions in bacteria, enzymes, separation of, from Azotobucter vinelandii, 149 135 Oxidative pathways in micro-organisms, levels in Azotobacter, 156 248 relation t o hydrogen uvolution in bacteria, 152 Oxidative phosphorylation in chemolithotrophs, 227 Nitrogenase-deficient mutants, 162 Oxygen, consumption and differentiation Nitrogenases, in reactions, other than nitrogen reduction, 159 in Blastocladiello ernersonii, 81 electrodes in the study of bacterial nature of different, 158 electron transport, 181 Nitrogenous compounds, oxidation of, 227 sensitivity of Azotobacter nitrogenase Nitrosomonas europaen, energy metabolism of, 216 to, 158 uptake by micro-organisms, 254 energy production by, 229 AYitrosomorins spp., oxidation of ammonia utilization, inhibition of, by nitrite, 188 by, 228 Nitrous oxide reduction by nitrogenase, 159 P Nonane, growth of yeasts on, 13 I’nmcoccoicles brnsilioksis, yeast-mycelium Non-haom iron sulphur prosthetic group in ferretloxin, 151 dimorphism in, 70 Paraffin wax, utilization of, by fungi, 4 Non-sulphur purple bacteria,, encrgy production by, 235 Parasexual cycle, in cellular slime moulds, 49 Nucleic acid synthesis during development of Physnvurn polycephalum, 66 in fungi, 104 Nucleic acids, encrgy requirements in Pasteur effect, relationship to condiogenesis synthesis of, 222 in Neurosporn crassa,91 Nucleotides, energy requirement in syn- Pathways of hydrocarbon oxidation, 18 thesis of, 220 Penicillin formation, kinetics of, 117 Nutrient supply and secondary ~netabolisni, Penicillium chrysogenutta, biochemistry of 118 conidiation in, 88 Nutrimts, c+l’wt of, o n conidiation in I’. notctbum, alkane Utilization by, 14 4 spergillus ttfcpr, 96 /’c,nici&wm spp., moryhogenusis in, 74 Pentane, growt,h of yeasts on, 12 I’entose sugars, degradatiun of, 2338 Pcptostreplococcus elsdrnii, conversion I J ~ lactate t o propionate by, 242 0 fla,vodoxin from, 144 Orcurwncr of a.liphatic hyrlroewhons, 39 Perrmosl)orales, sexual reproductioll ill, Ontogeny in Rlrcstocltrdiella wrmrsoiaii, 82 107

298

SUBJECT INDEX

Podosporci, development of senescence in, Phenethyl alcohol, as a microbial inhibitor, 104 71 Polarography in study of bacterial electron Phanol oxidases, processes involving, 122 traneport, 181 Phosphatase activitics and development Polymerization reactions, energy requirei n . - I s p ~ r q i l l i t s,tiger, 98 ments of, 221 T’i,~~~l)hi)diesternst~ producotl by cellular l’olyphosphate metaholism during develop slinic nioulds, 52 rnent of I’hysurwn p o l y c ~ ~ p h d i r 67 ~n, ~ ’ l ~ ~ ~ s ~ ~ l i energy o l i ~ i i ~rqiiircnicwts ~s, iir Polysaccharide in aggregation of cellular synthrsis of, 223 slime moulds, 53 PhospIior(~c1~stic reaction i n bacteria, 141 Polysaccharides, depolynierization of, 237 Phosphorylat,ion in aorobic microbial Pol,y.~pho,idylilrn1.jliii~~ pnllidiunk, cultivation of, nietalmlism, 248 48 sitcs in hact,orial ~~hotophosphorylatioii, Pool of adenosine triphosphate in micro236 organisms, 252 Photochrmical action spectra, use of, in stutly of tiactcrial cloctron transport,, Preformed monomers, synthesis of bacterial cells from, 225 177 Primary alcohols, production of, from Photohydrogen production, 154 alkanes, 19 Photo-induction in fungi, nictabolism of, Primordia formation in Mclcrnotus, 78 57 Processes involving phenol oxidascs, Phnt,olithotrol,hs, 2 14, 2 15 122 cncrgy proclurtion in, 234 Proline, cnergy requirement in synthcsis of, Phi)tonitrog~wproduction, 154 219 l’hotorcccptors in fungi, 75 Propane, oxidation of, by f3rcuibricterium Photosynthetic l)act,cria, 215 sp., 21 high-enrrgy t:lrctrons in, 152 ~ro~io)ribncterirc1,!. slip., encrgy production Photosyntlict,ic elcctron transport in microin, 240 organisins, 2 15 Prosthetic groups of bacterial cytochrornes, 55 P t i ~ ~ t o t r o ~ ~ iins ifungi, ns 178 Pli~jcom?yce.shlrrkesleerrii us, earotrnc formaProteaso activity, and fungal differetitiation in, 120 tion, 118 I’hyconiyrctrs. soxiit~lreproduction in, 105 and sporulatioii induction in penicillia, P l i y s n r u m / ~ ~ i l ? y ~ r . ~ ~ I life i r r l cyrle i i ~ i ~ ,of, 64 101 Physiological nct,ivities and conidiogenesis Protein disulphide reductase in Cnrididn in Scitrosporrr crrrssn, 93 rrlhicrins, 70 I’h,ysiohgy, of fungal diffcrentiatinn, 45 Protein metabolism in yeast sporogenesis, of mc.iosis in fungi, 105 111 I’hytoflnvin, 148 Protein synthcsis, during development of Pltytophtho~rrsspp., effcct of light on reprw P h y s n r u m polycephalum, 66 duction of, 78 energy requirements of, 221 sterols and reprodurtion of, 107 in differentiation in Blnstoclndiella Pichirr sp.. utilization of alkanes by, 2 enkPrSOni i , 83 Pinricitlin, rffcct of, on F?schcrichin coli, 194 Proteus morqnit i i , action of, on homocysPigment,s, rcspiratory. in Hneinophilus teinc, 245 p,/rrrtilt~7tellzne,201 Protohaem in bacteria, 178 Pilciis morphogrnesis in Schizopihyllicnk Pseudun~onns jiflzioi-escens, energetic unc o ? l t ~ ? l l > ~ l t54 l“, coupling in, 262 Pilobolus, a s e x i d reproduction in, 103 P / O ratios in, 254 morphogencsis in, 77 P. pseudomnllei, cytochrome (12 of, 188 f’ithormyces chnrtrrrum, differentiation in, Pseudoplasmodium of cellular slirnc 122 moulds, 48 Plasmodia1 slinic moulds, differentiation Pteridine as a photoreceptor in fungi, 75 in, 63 Pulsed flow apparatus, use of, in bacterial Plasmodium of Ph ysarwn polycephnlum, electron-transport studies, 182 65 Purine fermentations, 247 Plasmogniny, in fungi, 104 Purine nncleotides, energy requirements in in Pli?ysnrum polycephalum, 64 synthesis of, 220 P/O ratios, 253

299

SUBJEC>TINDEX

Puromycin, effect of, on development of Dictyostelium discoideum, 61 Purple sulpur bacteria, production of energy by, 234 Pvrimidine dimers, formation of, in fungi, 70 Pyrimidine nucleotides, energy requirement in synthesis of, 221 Pyrimidines, fermentation of, 247 Pyruvate, as a hydrogen acceptor in encrgy production, 240 carboxlyase and conidiogenesis in NRZWOspom crassn, 93 dehydrogenane in Clostridium acidiurici, 152 energy from, 239 energy requirement in synthesis of amino acids from, 220 Pyruvate-urate electron chain in Clostridiurn acidi-urici, 151 Pyruvic carboxylase activity of microorganisms. 218 Pythium, sterols and, 121 sterols and reproduction of, 107

Q Quinacrinc, effect of, on development of Dictyosteliu?ndiscoideum, 6 1 Quinones in Haemophilus parainfuenzae, 200

R Radiations and morphogenesis in fungi, 75 Redox dyes, use of, with micro-organisms, 137 Reduced-minus-oxidized difference spectra, 176 Reductase, protein dusulphide, in Candidn albicans, 70 Reduction, of cytochromes in Azotobacter spp., 189 of nitrate in micro-organisms, 233 of respiratory pigments in study of electron transport in bacteria, 177 of sulphata in micro-organisms, 233 Reductive assimilation of carbon dioxide by Chlorobium thiosulpl~citophilum,153 Reductive carboxylic acid cycle, 153 Reflectance spectrophotometers, use of, in study of bacterial electron transport, 178 Regulation, of electron chains in bacteria, 160

Regulation-continued of enzyme accumulation in Dictyostelium discoideurn, 58 Repression, of hydrocarbon oxidation, 17 of nitrogenase synthesis, 161 Reproduction and cell-wall composition in fungi, 73 Requirement for energy, 214 Reserves, production of, by microorganisms, 257 Respiratory control in mitochondria, 263 Respiratory metabolism during morphogenesis in Uict?yo~stp.liu~n di.scoidetc?n,54 Reversed electron transport in microorganisms, 215 Rhizobium japonicum, cytochromes of, 207 Rhizomorphs, morphogenesis of, 73 Rhodospirillum rubrum, effect of redox dyes on, 139 electron chains in, 152 energy metabolism in, 215 pool of adenosine triphosphate in, 252 Rhodotorula glutinis, utilization of kerosene by, 3 Rhodotorula, spp.. ability of, to attack alkanes, 2 Riboflavin as a photoreceptor in fungi, 75 Ribonuclease activity and development in Aspergillus niger, 98 Ribonucleic acid metabolism during dcvelopment of Dictyostelium discoideum, 60 Rubredoxin involvement of alkane oxidation, 31 Rumen micro-organisms, energy production by, 241 Rwninococcus albus, cell yields of, 258 R. fiauefaciens, molar growth yields of, 25 1

s Saccharomyces cerewisiae, aerobic production of energy in, 248 molar growth yiclds of, 251 products of fermentation by, 256 sporogenesis in, 111 thermodynamic data for growth of, 268 S. chevnlieri, utilization of kerosene by, 3 S. rosei, molar growth yields of, 251 Snccharomycodes, sexual reproduction in, 109 Salmonella typhimurium, cell yields of, 249 Salt dependence of cytochromes in halophilic bacteria, 183 Saprolegniales, sexual reproduction in, 105 Sapromyces spp., biochemistry of differentiation in, 87

300

SUBJECT INDEX

Scheme for production of clostridial ferred- Spore formation in cellular slime moulds, 48 oxin, 143 Schizophyllum c m m u n e , differentiation in, Spores, fungal, differentiation of, 74 113 in myxomycetes, 63 pileus morphogenesis in, 74 Sporogenesis, biochemistry of in yeast, 109 Sporogenic substances in fungi, 75 Sclerotia, morphogenesis of, 73 Sclerotinia sclerotiorum, differentiation in, Sporogens of fungi, 104 Sporophore formation in fungi, 11 2 102 Sclerotium development in Sclerotinia Sporophore development in Schizophyllum commune, 115 sxlerotiorum, 102 Sclerotium formation in Ph,ysaruin pol?/- Sporulation in Physarunc polyccphalum, 67 cepha,lum, 65 Stability of messenger-RNA in developSecobarbital, effect of, on Haernophilus ment of Dictyostelium discoideum, 61 parainJuenzae, 202 Staphylococcus aureus, reduced-minusSecondary metabolites and differentiation oxidized difference spectrum of, 176 in fungi, 116 Starvation and sporulation of Physarum Seleiaomonas ruminnntium, cell yields of, polycephalum, 68 Stemphylium botryosuna, physiology of 258 maintenance energy of, 260 sporulation in, 76 Sequential enzyme development in mor- Sterols, and differentiation in fungi, 121 in reproduction of P y t h i u m spp., 107 phogenesis in Dictyostelium discoidStickland reaction, 246 eum, 56 Serine, energy requirement in synthesis of Streptococci, action of, on arginiue, 244 amino acids from, 219 Streptococcus ~faecalis, effect of growth temperaturo on growth yields of, 263 fermentation of, 245 Sexual hormones in fungi, 74 energetic uncoupling in, 261 Sexual rnorphogenesis in fungi, 104 molar growth yields of, 249 Sexual reproduction, in ascomycetes, pool of adenosine triphosphate in, 252 P / O ratios in, 253 108 in basidiomycotes, 1 1 2 thermodynamic data for growth of, 268 in phycomycetes, 105 S.Zactis, molar growth yields of, 250 Short circuiting of metabolic pathways in thermodynamic data for growth of, 268 micro-organisms, 138 S. pyogenes, molar growth yields of, 250 Shunt metabolism in fungi, 119 Structure of clostridial ferredoxin, 143 Succinate dehydrogenase activity, and Silicomolybdate, effect of, on Hneinophilus conidiation in Neurospora crassa, 90 parainJuenzae, 210 Simple sugars, degradation of, 237 and hTeurosporaconidiation, 93 Sirenin, formula of, 106 Succinate dehydrogenase and development Sodium dithionite, use of, in study of in Blastocladiella emersoiiii, 86 Succinate oxiduse activity in EscherichicL bacterial electron transport, 177 Solute permcation in Haernophilus paracoli, 193 injZuenztce, 198 Succinate oxidation in Escherichia coli, Sordaria fcmicola, sterols and, 12 1 192 Sorocarp formation in cellular slimemoulds, Sugars, degradation of, 237 48 Sulphate reduction in micro-organisnis, Spectrophotometcrs in the study of 233 bacterial electron transport, 175 Sulphide, energy production during oxidaSpectrophotometry in the study of electron tion of, 230 transport in bacteria, 174 Sulphite, energy production by oxidation Sphaeroplasts of Haemoph.ilus p w a of, 230 inJuenzac, 201 Sulphur compounds, energy production Spicccria sp., utilization of alkenos by, 6 from, 229 Sulphur, elemental, oxidation of, by Split-beam spectrophotometern in the micro-organisms, 230 study of bacterial electron transport, Sulphydryl compounds, effect of, on yeast175 Sporangial development in Achlya, 102 mycelium dimorphism in fungi, 71 Sporangial formation in Blastocktdiclkr Synchronous development of Dictyosteliwn emersonii, 71 discoideum, 56

301

SUBJECT INDEX

Synchronous growth of Physarum polycephalum, 69 Synchrony in life cycle of cellular slime moulds, 48 Syntheses, microbial, need for energy in, 214 Synthesis, of bacterial cells, 227 of lipids, energy requirements in, 222 of monomers in micro-organisms, 2 17 of nitrogenase, regulation of, 160 of nucleic acids, energy requirements of, 222 of proteins, energy requirements of, 221

T Temperature, effect of, on energetic uncoupling during growth, 262 on yeast-mycelium dimorphism in fungi, 71 Temperature-sensitive mutants of Dictyostelium discoideum, 61 Temperature-sensitive nitrogenase mutants of Azotobacter vinelandii, production of, 164 Terminal electron acceptors for Achromobacter spp., 187 Terminal methyl group oxidation, 20 Tetradec-1-ene, utilization of, by yeasts, 3 Tetramethyl-p-phenylenediamine, use of, in studies on bacterial electron transport, 186 Tetrathionate, energy production by oxidation of, 230 reductase activity of Escherichia coli, 198 Thumnidium spp., morphogenesis in, 77 Thermal increments of growth yields in bacteria, 263 Thermodynamic assessments of microbial energy production, 264 Thermodynamic data for growth of microorganisms, 268 Thiobacilli, energy production by oxidation of sulphur by, 230 Thiobacillus neapolitanus, maintenance energy of, 260 T . nevellus, energy metabolism of, 216 Thiosulphate, energy production by oxidation of, 230 reductase activity of Escherichia coli, 198 Threonine dehydrase in development of Dictyostelium discoideum, 59 Threonine, energy requirement in synthesis of, 218 fermentation of, 245 Thymine synthesis and growth of Blastocladiella emersonii, 78

Torulopsis colliculosa, ability of, to attack alkanes, 2 T . magnoliae, mechanism of oxidation of alkanes by, 25 Torulopsis spp., ability of, to attack alkanes, 2 Toxic effect of hydrocarbons on bacteria, 8 Transcription in development of Dictyostelium discoideum, 5 9 Transhydrogenation in Azotobacter, 156 Translation in development of Dictyostelium discoideum, 59 Trehalose metabolism in morphogenesis in Acrasiales, 53 Trehalose 6-phosphate synthetase activity in Dictyostelium discoideum, 57 Tricarboxylic acid cycle in Blastocladiella emersonii, 81 Tricarboxylic acid cycle, operation of, in microorganisms, 248 Trichoderma, physiology of differentiation in, 103 Trichophyton mentagrophytes, macroconidia formation in, 100 Trichosporon spp., alkane utilization by, 2 Tridecane, growth of yeasts on, 12 Trigger mechanisms in differentiation, 46 Triglycerides, energy requirements in synthesis of, 223 Trisporic acid, formula of, 106 Trophocyst formation in Pilobolus spp., 77 True slime moulds, differentiation in, 63 Tryptophan synthetase activity and Neurospora conidiation, 93

U Uncoupling, energetic, 260 Uracil, fermentation of, 248 Urea, inhibitory effect of, on nitrogenase, 161 Uridine diphosphogalactose :polysaccharide transferase activity in Dictyostelium discoideum, 58 Uridine diphosphogalactose pyrophosphorylase activity in Dictyostelium discoideum, 58 Uridine monophosphate, energy requirement in synthesis of, 221 Use of energy in micro-organisms, 249 Utilization of aliphatic hydrocarbons by micro-organisms, 1 Utilization of energy during growth, 213

V Valine, energy requirement in synthesis of, 220

302

SUBJECT INDEX

Vegetative differentiation in Eumycotina, ti9 Verticilliutti, sclwotia in, 122

\'er;icles a t growiug tip of fungi, 72 Vitamin Ii2 in Escherichia coli, 192 \'r)latile fatty acids, production of, by fermentation of unino acids, 243

Yeast-mycelialdimorphismin Eumycotina, 69 Yeasts, ability to oxidize aliphatic hydrocarbons, 2 growth of, on hydrocarbons as an indicator of specificity, 9 mechanism of oxidation of allranes by, 24 Yohirnbine tartrate, hormone action of, 51

W Wall, cell, contruction of, in Eumycotina, 69

Y Y Ovalues, 254 YATPvalues, see Molar growth yiclds Y forms in Eumycotina, 69 Yeast, biochemistry of meiosis in, 110 pool of adcnsoine triphosphate in, 252

Z Zygomycetes, sexual reproduction in, 107 Zymobacterium oroticzcm, decomposition of pyrimidines by, 247 Zynomonas fragilis, energy production in, 238 Z.mobiliu, effect of growth temperature on growth yields of, 263 molar growth yields of, 251 P/O ratios in, 254 thermodynamic data for growth of, 268