Advances in
MICROBIAL PHYSIOLOGY
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Advances in
MICROBIAL PHYSIOLOGY
This Page Intentionally Left Blank
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
ACADEMIC PRESS - LONDON and NEW YORK
ACADEMIC PRESS INC. (LONDON) LTD. B E R K E L E Y SQUARE HOUSE BERKELEY SQUARE LONDON, W l X 6 B A
U.S. Edition published by ACADEMIC PRESS INC.
111 FIFTH
AVENUE
NEW YORK, NEW YORK
Copyright
10003
0 1971 By ACADEMIC PRESS INC. (LONDON) LTD.
.411 Rights Reserve11 N o part of this hook may hc reproduced
111
any form by photostat, microfilm, or any othrr means, without written permission from tho publishers Library of Congress Catalog Card Number: 67- 19850
SBN: 12-027705-0
PRINTED I N GREAT BRITAIN BY WILLIAM CLOWES
&
SONS LIMITED
LONDON, COLCEESTER AND BECCLES
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
.
.
v
Utilization of Aliphatic Hydrocarbons by Micro-organisms M. J. KLUG and A. J. MARKOVETZ
I. Intl.octuction
.
11. Organisms . A. Ycasts . B. Filamentous Fungi . C. Bacteria 111. Ecological Studies
.
.
IV. Growth as Indicator of Substrate Specificity A. Bacteria B. Yeasts . C. Filamentous Fungi . D. Comments .
.
V. Induction and Repression of Hydrocarbon Oxidation VI. oxidation without Assimilation
VIII. Mechanisms of Oxidation A. 72-Alkanes . B. Alk-1-enes .
.
.
.
IX. Occurrence and Biosynthesis of Aliphatic Hydrocarbons . X. Aclinowledgement References .
. vii
1
. .
2 2
.
4
.
5
.
G
. . .
.
7 7 9 13 14
.
17
. .
17
.
.
VII. I’athways of Hydrocarbon Oxidation A. Bacteria . 13. Yeasts . C. IMamentoiis Fungi .
.
.
.
. . .
18 19 24 29
30 30 37
. .
39
. .
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
.
Subject Index
.
. .
.
275
.
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
.
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 .
.
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
-
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 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'
UTILIZATION O F ALIPHATIC HYDROCARBONS BY MICRO-ORGANISMS
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 , 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 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, 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
198
DAVID C . WHITE AND PETER R . SINCLAIR
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,
BRANCHED ELECTRON-TRANSPORT SYSTEMS IN BACTERIA
199
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
200
DAVID C. WHITE AND PETER R. SINCLAIR
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.
BRANCHED ELECTRON-TRANSPORT SYSTEMS I N BACTERIA
201
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
202
DAVID C. WHITE AND PETER R. SINCLAIR
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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203
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
BRANCHED ELECTRON-TRANSPORT SYSTEMS I X BACTERIA
206
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