specialist phototrophs, lithotrophs, and methylotrophs: a ... · quently assigned to the genus...

BAcT:RIoLOxIcA Rzvizws, June 1977, p. 419-448Copyright © 1977 American Society for Microbiology

Vol. 41, No. 1Printed in U.S.A.

Specialist Phototrophs, Lithotrophs, and Methylotrophs: aUnity Among a Diversity of Procaryotes?

ARNOLD J. SMITH* AND DEREK S. HOAREtDepartment ofBiochemistry, University College of Wales, Aberystwyth, Dyfed, Wales, United Kingdom,* and

Department of Microbiology, University of Texas, Austin, Texas 78712

INTRODUCTION.............................................................. 419Semantic Problems ............................................. 419Occurrence of Versatile and Specialist Phototrophs, Lithotrophs, and Methylo-trophs ...................................................... 421

CHARACTERISTIC ENERGY AND CARBON METABOLISM ....... ............ 423Energy Metabolism ...................................................... 423Carbon Metabolism ...................................................... 423

METABOLISM OF ORGANIC COMPOUNDS BY SPECIALIST PHOTOTROPHS,LITHOTROPHS, AND METHYLOTROPHS ................................. 424

Influence of Organic Compounds on Growth ........... ....................... 424Assimilation of Organic Compounds ................. ......................... 425Requirements for the Assimilation of Organic Compounds ....... .............. 427Fate of Organic Carbon Assimilated by Specialist Strains ....... ............... 427Substrate Transport and the Specialist Condition............................... 432

OTHER POSSIBLE CAUSES OF SPECIALIST CHARACTERISTICS ...... ...... 433Regulation of Enzyme Synthesis and Activity .......... ....................... 433Energy Conservation in Specialist Strains ............. ........................ 434Specialized Nutritional Requirements ................ ......................... 437

CONCLUSIONS ........................................................... 437LITERATURE CITED ......................................................... 440

INTRODUCTIONProcaryotic organisms differ widely in the

range of compounds that support their growth.At one extreme, there are pseudomonads,which can grow on any one of 80 or more or-ganic compounds (208), while at the other,there are organisms that can utilize only one ortwo compounds as a source of carbon and en-ergy for growth (57). Growth on a very limitedrange of substrates is most pronounced amongprocaryotes noted for their ability to exploitspecialized and often unconventional sources ofenergy, namely, phototrophs, lithotrophs, andmethylotrophs (Table 1). The purpose of thisreview is to discuss selected aspects ofthe phys-iology and biochemistry of organisms that ex-hibit just such conservative nutritions, to drawtogether the results of recent work with theseorganisms, and to explore the possibility of alatent unity in their physiology. Though thereare reviews that deal individually with photo-trophs (73, 173, 258), lithotrophs (3, 118, 169,183, 236), and methylotrophs (8, 177, 179), someof which discuss aspects of their restricted nu-trition (118, 183), none has sought to draw to-gether the organisms from these three dispar-ate groups for detailed comparison. The strat-egy adopted in this review involves a detailedconsideration ofthe metabolism oforganic com-t Deceased.

pounds by strains with a conservative nutri-tion, together with aspects of macromolecularsynthesis, electron transport, and energy con-servation. Although the assimilation of C, com-pounds by these organisms and the generationof energy from their characteristic energysources have been thoroughly reviewed else-where (3, 132, 169, 177, 179, 213), these aspectsof their metabolism will be covered briefly, asthey are pertinent to a consideration of theirrestricted physiology. Although nutritionalspecialization is a characteristic of certain pho-tosynthetic bacteria (173), they will be excludedfrom this review because few strains have beenstudied in sufficient detail. For the same rea-son, the more exotic organisms capable of oxi-dizing inorganic compounds such as species ofBeggiatoa, Thiothrix, and Gallionella will notbe considered.

Semantic ProblemsThe meaning of terms commonly applied to

these organisms has been subject to changewith the passing of old theories, with the recog-nition that some organisms can embrace morethan one mode of metabolism, and with therealization that more than one of the growthrequirements of an organism needs to be speci-fied to fully characterize it (183). Furthermore,the division of organisms according to biochem-ical criteria related to carbon assimilation does

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TABLE 1. Organisms exploiting unconventional sources of energy and their heterotrophic potentialHeterotrophic growth

Category Energy source_b~~~~~~

Phototrophsc Light Aphanocapsa 6714Anabaenopsis circularisChlorogloea fritschiiNostoc sp. MACPlectonema boryanumLyngbya lagerheimii

Lithotrophs H2

S2-, etc.

NH4+

NO2-

Methylotrophs

Pseudomonas facilisAlcaligenes eutrophusParacoccus denitrificans

Thiobacillus acidophilaThiobacillus intermediusThiobacillus novellusThiobacillus A2Sulfolobus acidocaldaricus

None

Nitrobacter agilis (Delwichestrain and others)

CH4 and other [CH3-]compounds

[CH3-] compoundsother than CH4

Methylotroph strain XX

Arthrobacter 2B2Bacillus PM6Pseudomonas 3A2Pseudomonas AM1Methylotrophs 5B1, W3A1,S2A1 and W6A

Anacystis nidulansSynechococcus 6301Gloeocapsa alpicolaAphanocapsa 6308Anabaena variabilisAnabaena flos-aquaeAgmenellum quadruplicatum

Methanobacterium thermoauto-trophicus

Thiobacillus denitrificansThiobacitlus ferrooxidansThiobacillus neapolitanusThiobacillus thiooxidansThiobacillus thioparusThiobacillus thermophilicaThiospira pelophila

Nitrosocystis oceanusNitrosolobus multiformisNitrosomonas europaeaNitrosospira briensis

Nitrobacter sp.Nitrococcus mobilisNitrospina gracilis

Methylobacter sp.Methylococcus sp.Methylomonas sp.Pseudomonas methanicaMethylocystis sp.Methanomonas methanooxidans

Methylotroph 4B6Methylotroph C2A1Methylotroph W1

a Growth on organic compounds in the absence of the specific energy source (versatile strains).b No growth on organic compounds in the absence of the specific energy source (specialist strains).c Photosynthetic bacteria are not included in this table as these organisms are not covered by this review.

not correspond to the groupings that originatein the designation of compounds as either or-

ganic or inorganic. It is, therefore, necessary tomake clear the intended meaning ofterms usedin this review. Phototrophs can use light as a

major source ofenergy for growth, (chemo-)lith-otrophs as a group can derive energy from theoxidation of a variety of inorganic compounds,and methylotrophs can obtain energy from theoxidation of methyl groups attached to atomsother than carbon (Table 1). These terms referwholly to energy source and are not intended to

carry any implication about the carbon source.The distinction that is drawn among com-pounds providing the bulk of cell carbon is be-tween C, compounds, on the one hand, andorganic compounds containing more than onecarbon atom on the other. Most phototrophs,lithotrophs, and methylotrophs can utilize C,compounds as a source of the major part of cellcarbon; for the first two groups, CO2 is thespecific C, carbon source (autotrophic growth),whereas for methylotrophs, organic compoundscontaining methyl groups attached to atoms

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PHOTOTROPHS, LITHOTROPHS, AND METHYLOTROPHS 421

other than carbon can satisfy their carbon re-quirements. Many procaryotic organisms canuse organic compounds containing one or morecarbon-carbon bonds and this can be describedprecisely as chemoorganotrophic-heterotrophicor in short as heterotrophic growth (183). Somephototrophs, lithotrophs, and methylotrophsexhibit a degree of flexibility in their metabo-lism that also embraces this mode of nutritionand, as such, will be referred to as versatilestrains. Other strains do not show the sameadaptability and appear unable to grow hetero-trophically in any conventional organic media;this is all the more surprising as they havebeen counted among the most "complete" ofall microorganisms (113, 169), having the abil-ity to synthesize all of their cell constituentsfrom the simplest of carbon compounds.Any discussion of the inability of organisms

to grow on organic media is subject to an histor-ical handicap, especially in the case of litho-trophs. This stems from the characterization ofthe lithotrophic mode of life by Winogradsky onthe basis of his investigations of the nitrifyingbacteria and the effect of organic compounds onthem (253, 256). The features that he singledout as being characteristic were as follows: (i)development in a wholly inorganic mediumcontaining an oxidizable inorganic compound;(ii) close linkage of the life processes to thepresence of the inorganic compound; (iii) oxida-tion of this substance as the only source ofenergy; (iv) no requirement for an organic com-pound either as a source of cell material or as asource of energy; (v) inability to utilize organiccompounds because they inhibit development,and (vi) assimilation of CO2, the sole source ofcarbon, by chemosynthesis.These characteristics provided the founda-

tion for the concept of the "obligate autotroph,"which displays a complete dependence on en-ergy obtained from inorganic oxidations and onCO2 as carbon source together with an inabilityto metabolize or tolerate organic compounds(255). At that time, biochemical investigationwas in its infancy and the possibility ofa molec-ular unity beneath the diversity of life was notyet widely accepted. The compatibility of thechemolithotrophic and heterotrophic modes ofmetabolism was, however, apparent from thegrowth of hydrogen bacteria and of Thiobacil-lus novellus on organic as well as inorganicmedia (110, 188, 209). Furthermore, the demon-stration of the chemolithotrophic heterotrophyofDesulfovibrio desulfuricans (155, 175) and ofThiobacillus perometabolis (144) has shownthat energy generated from inorganic oxidationreactions need not of necessity be coupled withan autotrophic carbon assimilation. Although

no phototroph, lithotroph, or methylotroph hasbeen shown completely incapable of utilizingany organic compound in the synthesis of atleast some of its cell constituents, the concept ofthe "obligate autotroph" has remained largelyunchanged. It has suffered increasingly fromits original connotation, and cogent argumentshave been put forward in favor of consigning itto the conceptual rubbish heap (143, 183, 184).Although the organism defined by Winograd-sky is an hypothetical abstraction with no real-ity in fact, some blue-green bacteria approachthis condition: although they can assimilatemany organic compounds into cell material insmall amounts, they are still dependent on C02as the major source of cell carbon (see Metabo-lism of Organic Compounds by Specialist Pho-totrophs, Lithotrophs, and Methylotrophs). Inview of the conceptual ambiguity of the term"obligate autotroph," our inclination is to sup-port the suggestion that it be discarded. Inrecognition of the existence of organisms of di-verse physiology with the common feature offailure to grow on organic media, we shall referto such organisms as specialist phototrophs,lithotrophs, or methylotrophs. These termsshould not, however, be taken necessarily tosignify a direct causal relationship between theability to exploit energy sources other than con-ventional organic compounds and the failure togrow on organic media.

Occurrence of Versatile and SpecialistPhototrophs, Lithotrophs, and

MethylotrophsThe inability of certain microorganisms to

utilize organic compounds as growth substrateswas first reported by Winogradsky (253) in thecourse of his classical studies of the nitrifyingbacteria; these organisms were shown to becapable of growth in a wholly inorganic me-dium using energy derived from the oxidationof inorganic compounds of nitrogen and werethe first bona fide examples of the now diversecollection of organisms known as chemolitho-trophs. Winogradsky and Omeliansky (256) in-vestigated the effect on nitrifiers of a variety oforganic compounds that supported the growthof other microorganisms and found that nonesupported growth and several inhibited growthand nitrification. In spite ofthe claim by Beijer-inck to have grown a strain of Nitrobacter onorganic media (13), Winogradsky (254) main-tained that, as a group, nitrifying bacteriawould not grow on organic media. Over the past10 years, Watson and his colleagues (see refer-ence 241) have isolated several new species ofnitrifying bacteria; with the exception of cer-

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tain Nitrobacter strains, all ofthese isolates areincapable of growth on organic media in theabsence of the specific inorganic energy source.

Shortly after Winogradsky's work with thenitrifying bacteria, Nathansohn reported theisolation of bacteria able to flourish in an inor-ganic medium like the nitrifiers but with re-duced inorganic sulfur compounds in place ofammonium or nitrite (159); these were subse-quently assigned to the genus Thiobacillus(12). Later, Starkey isolated several putativethiobacilli (209); one of these grew on inorganicmedia but not on organic substrates (Thiobacil-lus thioparus), and another grew both on or-

ganic and on inorganic media (T. novellus).Since then, other strains have been isolated,some of which grow only on their characteristicinorganic carbon and energy sources (119, 193,221) and others that will also grow on organicmedia (142, 193, 215, 219, 252). Among the lat-ter, Thiobacillus intermedius is atypical inthat it will grow on simple organic media aslong as an organic compound containing re-

duced sulfur is also present (206). Several newthiotrophs that are morphologically distinctfrom the thiobacilli have been described: Sulfo-lobus acidocaldaricus grows on organic andinorganic media (26), whereas neither Thiomi-crospira pelophila nor the sporeforming"Thiobacillus thermophilica" will grow on or-ganic substrates in the absence of reduced inor-ganic sulfur compounds (66, 133). Chemolith-otrophs oxidizing H2 with oxygen or NO3- asterminal electron acceptor have, without excep-tion, been shown capable of growth on severaldifferent organic media (53). By contrast, theanaerobic methanogenic lithotroph Methano-bacterium thermoautotrophicus, which growson H2 and C02, will not grow on organic media(259).Early work with cyanobacteria, whose domi-

nant mode of growth is oxygenic photosyn-thesis, suggested that these organisms pos-sessed a capacity for growth in the dark onorganic compounds (80, 257). However, the firstdetailed study of the physiology of this group oforganisms clearly showed that the strains thenavailable were incapable of significant growthunder these conditions (130). Since then, manymore strains of varying types have been iso-lated in pure culture, and many of these havebeen shown incapable of growth in the dark(see reference 202). Over the last 10 years, re-

ports of the growth of specific strains in thedark on carbohydrates and related compoundshave appeared with increasing frequency sug-gesting that, though not universal among blue-green bacteria, growth under these conditions

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is not as unusual as was previously supposed(69, 87, 126, 127, 180, 238).Bacteria that grow on methane have been

known since the early 1900s (161). Detailedstudies over a period ofyears with three strainshave established their absolute requirement formethane or another C, compound such as meth-anol for growth (28, 61, 74). Many new isolatescapable of growth on methane have also provedincapable of growth on conventional organicmedia (249). However, Patt et al. (165) haverecently reported the isolation from lake mud ofoxygen-sensitive strains that will grow not onlyon methane and other C1 compounds but also onsugars and acids. In contrast, most of the orga-nisms isolated that use reduced Cl compoundsother than methane are also able to grow on avariety of other organic substrates as well (seereference 177); a few strains exhibit a restrictednutrition comparable to the majority of themethane users (41, 42, 50).

This division of phototrophs, lithotrophs, andmethylotrophs, which depends on the failure ofspecialist strains to grow on organic media inthe absence of their characteristic energysources (Table 1), introduces several problems.It should be recognized that very few specialistorganisms have been rigorously screened forgrowth on every conceivable organic com-pound. In these tests, it has been common prac-tice to use a few carboxylic and amino acids,and one or two carbohydrates (207, 221, 239,244). In a few cases, a more comprehensive,though still not complete, range of substrateshas been tested (41, 130, 249). It is, therefore,possible that some strains currently classifiedas specialists may, on closer investigation, beshown to grow on organic media. In addition,some organisms may grow on organic media atvery slow rates that have been regarded asinsignificant. Several strains of Nitrobacter,versatile blue-green bacteria, and "restrictedfacultative" methylotrophs grow more slowlyon organic media compared with mineral media(43, 69, 87, 180, 203). This makes the identifica-tion of specialist phototrophs, lithotrophs, andmethylotrophs on the basis of inability to growon organic media all the more difficult andraises the question of what constitutes a validmeasure of such growth. A further problem iswhether all of the existing reports of growth onorganic compounds are adequately substanti-ated; there are cases where, for one reason oranother, doubt remains. Certain strains of theorganism variously known as Thiobacillus fer-rooxidans, Ferrobacillus ferrooxidans, or Fer-robacillus sulfooxidans have been reported togrow on glucose in the absence of Fe2+ (149, 193,


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215). However, adaptation is a slow process andis accompanied by changes in the characteris-tics of the organism; more significantly, pro-longed subculture on organic media results inthe loss of the ability to grow with Fe2l as

energy source and C02 as carbon source (194,215). Several explanations accounting for thelatter observation have been proposed by Tuov-inen and Kelly (228) including the possibilitythat the culture might contain an heterotrophiccontaminant; this was not specifically ruled outin an early report of the growth of this orga-nism on organic media (149), and Zavarzin(259) has isolated an acidophilic heterotrophicorganism with a high requirement for Fe2+ andcapable of growth on glucose from enrichmentsfor T. ferrooxidans. More recently, Guay andSilver (78) have isolated an acidophilic Thioba-cillus from cultures of T. ferrooxidans strainTM after exposure to glucose. This new isolategrows not only on sulfur but also on a widerange of organic media. In addition, the basecomposition of its deoxyribonucleic acid (DNA)is significantly higher than that of DNA iso-lated from T. ferrooxidans. Although some

doubt remains about the ability of this strain ofT. ferrooxidans to exploit organic growth sub-strates, it is significant that other isolates willnot grow on glucose in the absence ofFe2+ (193).Growth of a blue-green bacterium and certainlithotrophs has been reported in organic mediasubjected to continuous dialysis (22, 162).Quantitative aspects of this work have beencriticized (118, 183), and attempts to repeatthese findings have failed (154). It is, neverthe-less, essential to avoid adopting a closed mindto the possibility that organisms, traditionallyconsidered specialist, may grow on organiccompounds under suitable conditions. In thepast, firm convictions that neither blue-greenbacteria (88) nor Nitrobacter strains (254) arecapable of growth on organic media have beenvoiced only to be proved incorrect by subse-quent work (69, 87, 203). It is, however, neces-sary that reports of the successful growth ofputative specialist organisms on organic mediaare substantiated in the most rigorous way pos-sible (see reference 202).

CHARACTERISTIC ENERGY ANDCARBON METABOLISM

Energy MetabolismPhototrophs transduce light energy into bio-

logically useful forms by mechanisms involvingan array of light-absorbing molecules (chloro-phylls, phycobiliproteins, and carotenoids) anda photochemical reaction center with associated

electron transport carriers (see reference 132);energy is conserved in the form of reduced pyri-dine nucleotides and adenosine 5'-triphosphate(ATP). Lithotrophs and methylotrophs arecharacterized by oxidative mechanisms com-prising specific oxidases or dehydrogenaseswith associated carriers for electron transportto an appropriate terminal acceptor such asoxygen or nitrate (3, 169, 177, 179, 213). Phos-phorylation coupled to electron transport hasbeen demonstrated in cell-free preparations ofseveral lithotrophs (see reference 213), but com-parable activities have not yet been found inmethylotrophs. With organisms in which pyri-dine nucleotides are not reduced directly bysubstrate oxidation, this is accomplished by anenergy-dependent transport of electronsagainst a thermodynamic gradient (2, 4, 189,192).A common but not wholly universal feature

of organisms exploiting these unconventionalenergy sources is the possession of complex in-ternal arrays of membranes (5, 52, 158, 176,182, 241), which are considered to be the sites ofenergy conservation within the cell. In blue-green bacteria, the hydrophobic photosyntheticpigments are membrane bound (195), and in asimilar way, specific oxidation reactions havebeen associated with particulate preparationsin lithotrophs (5, 243) and methylotrophs (74,234). Organisms lacking complex internalmembranes include species of thiobacilli (196,224), certain nitrifying bacteria (240, 242), anew cyanobacterium (181), and Cl users that donot grow on methane (50); presumably, in thesestrains the cytoplasmic membrane is the site inthe cell of processes for energy conservation.Such membranous arrays are neither unique tospecialist strains nor a feature of all of them;some phototrophs, lithotrophs, and methylo-trophs that are able to grow on organic mediaalso possess comparable intracellular mem-branes (87, 165, 174) and specialist thiobacilliand methylotroph W1 lack such internal struc-tures (50, 196, 224).

Carbon MetabolismThe synthesis of the bulk of cell constituents

in phototrophs, lithotrophs, and methylotrophscan be accomplished through the assimilationof C, compounds. Phototrophs and lithotrophsassimilate CO2 like green plants via the reduc-tive pentose phosphate cycle (3, 169, 202),whereas the methylotrophs use either the ribu-lose monophosphate route (122, 123, 177) or theserine pathway (14, 177) for the assimilation ofC, compounds into cell material. These special-

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ized assimilatory routes all involve specific en-

zyme-catalyzed reactions that are unique to C1assimilation in conjunction with transforma-tions that are also part of other pathways ofintermediary metabolism. In lithotrophs andphototrophs, the unique enzymes are ribulose5-phosphate kinase and ribulose 1,5-diphos-phate carboxylase. Although alternative routesfor CO2 assimilation have been implicated incarbon fixation by cyanobacteria (59), there is,as yet, no reason to suppose that the reductivepentose phosphate cycle is not the dominantroute for the net assimilation of CO2 in theseorganisms. Supplementary CO2-fixing reac-tions have been demonstrated in both photo-trophs (39, 47) and lithotrophs (214); estimatesattribute 10 to 30% of total CO2 fixation to theoperation of such mechanisms (10, 31). The en-zymes unique to the assimilation of reduced C,compounds include hexulosephosphate syn-thase and phosphohexulose isomerase for theribulose monophosphate route and malyl coen-

zyme A (CoA) lyase and hydroxypyruvate re-ductase for the serine pathway (8). There is noevidence for a correlation between specialistcharacteristics and the type of assimilatoryroute in methylotrophs; although the ribulosemonophosphate and serine routes were first re-

ported in specialist and versatile organisms,respectively, there are now well-establishedcases of specialist methylotrophs exploiting theserine pathway (134) and versatile strains us-ing the ribulose monophosphate route (49).

METABOLISM OF ORGANICCOMPOUNDS BY SPECIALIST

PHOTOTROPHS, LITHOTROPHS, ANDMETHYLOTROPHS

Since the common feature of specialist orga-nisms is an apparent inability to grow on or-

ganic media, interest has centered on their ca-

pacity to metabolize organic compounds in or-der to identify the basis of their specialist char-acter.

Influence of Organic Compounds on GrowthStrains representative of all three groups of

specialist organisms have been screened for theeffect of a variety of organic compounds ongrowth. In general, organic compounds do notstimulate the growth of specialist phototrophs,lithotrophs, or methylotrophs; one of the fewexceptions is the stimulation of the growth ofAgmenellum quadruplicatum by glucose at a

low light intensity that supports marginalgrowth on CO2 (231). Among versatile strains,specific organic compounds stimulate thegrowth of blue-green bacteria (87, 231), hydro-gen bacteria (54), and Thiobacillus interme-dius (142) on their characteristic carbon andenergy sources. It is not uncommon for certainorganic compounds to inhibit the growth of spe-cialist strains. Such inhibitory effects are usu-

ally observed with amino acids. Patterns ofamino acid toxicity vary from organism to orga-nism (Table 2), which suggests that there is no

TABLE 2. Inhibition ofgrowth ofphototrophs, lithotrophs, and methylotrophs by organic compounds

Organism Compounds inhibiting growth' Reference

Phototrophs

A. quadruplicatum L-Phe (0.04) 101Aphanocapsa 6308 L-Thr (1) Hutber andAphanocapsa 6714b L-His (1) ;mithaA. variabilis L-Cys (1), L-val (1), L-prO (1)

LithotrophsT. denitrificans Acetate (25), formate (25), pyruvate (50) 221T. neapolitanus L-Asn (1), L-cys (10), L-his (10), L-phe (1), L-thr (1),

acetate (20) 106,114,145,190T. thiooxidans L-CyS (1), L-his (1), L-met (1), L-ser (10), L-val (10) 145T. thioparus L-Cys (10), L-phe (1) 145N. agilisb DL-Thr (1), L-(HO)pro (1), DL-val (1) 56N. oceanus L-Asn (4), L-his (3), L-thr (4) 239N. europaea L-His (0.02), L-lys (0.02), L-met (0.02), L-thr (0.02),

L-val (0.02) 38MethylotrophsM. capsulatus L-His (1), L-phe (1), L-thr (1) 62a Figures in parentheses are concentrations (millimolar) that reduce the exponential growth rate by more

than 50%.b These organisms are versatile strains; the others are specialist types.c G. Hutber and A. J. Smith, unpublished data.

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susceptible site common to all specialist orga-nisms. Similar effects are encountered withversatile lithotrophs and phototrophs (Table 2),as well as with typical heterotrophic organisms(11, 76, 216). Inhibition of this type, which isusually annulled by other organic compounds(Table 3) including balanced mixtures of aminoacids (145), is attributed to a disruption of thefine control of intracellular metabolic processes(62, 65, 114-116, 145, 229). Although these ob-servations argue against the theory that allorganic compounds prevent the growth of spe-cialist organisms, they do not completely ex-clude the possibility that strain-specific inhibi-tory effects may determine the restricted physi-ology characteristic of specialist strains. Thispossibility is made less likely by the toleranceto toxic compounds that organisms can acquire(Table 4) without the loss of specialist charac-ter; such resistance has been attributed to lossof permeability to the inhibitor, loss of an en-zyme involved in its metabolism, or to an alter-ation in the enzyme whose activity is blockedby the inhibitor. Failure to grow on organiccompounds has been attributed to the accumu-

lation in cultures of inhibitory compounds inamounts sufficient to prevent growth (29); thecompound most commonly implicated is pyru-vate (22, 162). It has been reported to accumu-late in cultures of acidophilic thiobacilli (22,191) and to inhibit the growth of T. thiooxidans(22). There is, however, no conclusive evidencethat the growth of other specialist organisms isprevented by the accumulation of toxic metabo-lites.

Assimilation of Organic Compounds

Inhibition of the growth of specialist strainsby specific organic compounds implies that thecell wall-cytoplasmic membrane complex doesnot completely prevent the entry of organiccompounds into the cell. Similar conclusionscan be drawn from the ability of specialiststrains to utilize organic nitrogen compoundsas nitrogen sources for growth (28, 109, 163,211, 249). Direct evidence for the entry of or-ganic compounds into the cell has come frominvestigations of the assimilation of organiccompounds using isotopic tracer techniques.Because of the sensitivity of these procedures,it is essential to take rigorous precautions toavoid the contamination of cultures with heter-otrophic organisms.

Specialist strains from all three physiologicalgroups have been shown to assimilate organiccarbon from a wide range of substrates into cellmaterial in growing cultures, usually at con-stant differential rates (Table 5). The rates ofassimilation range from about 50-nmol/mgincrease in dry weight for some amino acidsand, with one exception, for carbohydrates toapproximately 2,000-nmol/mg increase in dryweight for acetate. These represent contribu-tions to cell synthesis ranging from less than1% to about 10%o. Glucose assimilation byAna-baena variabilis, which represents a 26% con-tribution to new cell material, is a notable ex-ception. Specialist phototrophs, lithotrophs,and methylotrophs are, therefore, not com-pletely impermeable to organic compounds.

TABLE 3. Abolition of the inhibitory effect of specific organic substrates by other organic compoundsOrganism Inhibitor Compounds annulling growth inhibition Reference

A. quadruplicatum L-Phenylalanine L-Tyrosine, L-tryptophan, L-leucine, L-alanine, 101L-isoleucine, and L-methionine

T. neapolitanus L-Phenylalanine L-Tyrosine, L-tryptophan, L-serine, L-leucine, 114and glycine

M. capsulatus L-Threonine L-Methionine, L-alanine, and L-valine 62

TABLE 4. Effect ofacquired tolerance on the assimilation oforganic compounds that normally inhibit growthDifferential rate of

assimilationOrganism Organic compound (nmollmg [dry wt]) Altered function Reference

Wild type Mutant

M. capsulatus Threonine 66 (M27) 0.5 Transport 63T. neapolitanus Phenylalanine 5.5a (HPT) 0 Transport 106T. neapolitanus Phenylalanine 291 (P4) 316 DAHP synthase 116A. nidulans Propionate 86 (PR) 0 Acetyl CoA syn- 204

thetasea Rate quoted in nanomoles of substrate assimilated/change in optical density at 660 nm of 0.1.

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However, under the conditions used in theseexperiments, none of the substrates makes amajor contribution to the synthesis of new cellmaterial; the greater part of cell carbon is stillderived from the C1 growth substrate. Thesmall contribution of a variety of organic com-pounds to cell synthesis may be due to a slowrate ofentry ofthese organic substrates into thecell. This is implicated where an increase in theconcentration of the organic compound in themedium results in an increase in the differen-tial rate of assimilation. Using high concentra-tions of organic substrates (Table 6) and, wherepossible, reducing the availability of the C1growth substrate (166, 167), the rate of assimi-

lation should approach a maximum. Underthese conditions, low rates of assimilation maybe due to an inability to utilize the organiccompound for the synthesis of more than a fewcell constituents because the organism lacksthe necessary enzymes.With the exception ofA. variabilis, specialist

strains from all three physiological groups as-similate hexoses at very slow rates. Since therate of uptake by blue-green bacteria is concen-tration dependent (Table 6), the utilization ofhexoses may be limited by the relative im-permeability of specialist organisms to thesecompounds (see Substrate Transport and theSpecialist Condition). The rate of acetate as-

TABLE 5. Assimilation of organic compounds by specialist phototrophs, lithotrophs, and methylotrophs

Differential rates of assimilationa(nmol assimilated during exponential growth/mg increase in dry wt)

Organism Reference

Glucose Fruc- Acetate Pyruvate Succinate Malate Aspar- Gluta- Leucinetose tate mate

A. nidulans 41 (5) 83 (5) 2160 (5) 42 (5) 42 (5) 20 (5) 52 (5) 84 (5) 555 (5) 205C. peniocystis 110 (5) 1700 (5) 770 (5) 130 (5) 145 (5) 20 (5) 22 (5) 460 (5) 205G. alpicola 140 (5) 2250 (5) 83 (5) 105 (5) 105 (5) 965 (5) 150 (5) 460 (5) 205A. variabilis 1833 (20) 1200 (20) 166,167

T. thioparus 1910 (2) 360 (2) 1200 (2) 750 (2) 600 (2) 200 (2) 205T. thiooxi- 200 (2) 1820 (0.2) 550 (1) 85 (0.5) 380 (2) 445 (2) 250 (2) 180 (2) 205dans

T. neapoli- 5100 (1) 347 (0.1) 420 (0.5) 107 (0.03) 111, 112,tanus 117

Methylotroph 69 (5) 1040 (5) 972 (5) 208 (5) 104 (5) 464B6

Methylotroph 69 (5) 1458 (5) 1250 (5) 104 (5) 208 (5) 46C2A1a Figures in parentheses are the concentration (millimolar) of the organic compound in the growth medium.

TABLE 6. Influence of substrate concentration on the rate of assimilation by specialist organisms

Substrate

Organism Acetate D-Glucose L-Aspartate L-Leucine Reference

Ca Assimi- a Assimi- C a Assimi- a Assimi-Concn lationbCon" n lationb Concn lationb Concn lationbA. nidulans 1 2160 5 42 0.1 103 205

5 2150 25 195 1 20625 2620 50 588 5 555

G. alpicola 5 2250 5 965 20525 2280 25 4650

T. neapolitanus 0.1 5020 0.05 81 1121 5200 0.2 317

0.5 425

N. europaea 0.05 72 Smithc0.5 2785 278

Concentration (millimolar) of the organic substrate in the growth medium.b Differential rate of assimilation (nanomoles-per-milligram increase in dry weight of the culture).c A. J. Smith, unpublished data.

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similation by Nitrosomonas europaea is notonly low but also shows marked concentrationdependence (Table 6). This could be attributedto limited permeability. In contrast, acetateassimilation by thiobacilli and cyanobacteria isindependent of concentration; it is, however,limited as a result of a restriction imposed byintermediary metabolism (see Fate of OrganicCarbon Assimilated by Specialist Strains). Theincorporation of relatively small amounts ofseveral amino acids by these organisms is dueto the limited capacity of cells to utilize thesecompounds, which are, for the most part, incor-porated unchanged into protein (62, 111, 205).Whether or not organic compounds can pro-

vide the cell with a source ofcarbon sufficient tosupport growth can only be established by elim-inating net Cl assimilation. Such an approachhas been used with blue-green bacteria (180)and involves the indirect inhibition of CO2 as-similation by the herbicide 3-(3,4-dichloro-phenyl-)-1,1-dimethyl urea (DCMU), whichinhibits photosytem II (17). A wide range ofcyanobacteria, including specialist and versa-tile strains, has been screened for the ability togrow in the light in the presence of glucose andDCMU (124, 180); most of the specialist strainstested (42 out of48 isolates) failed to grow underthese conditions. The interpretation of theseresults is unambiguous; most specialist blue-green bacteria cannot use glucose as a principalcarbon source. An extension of these studies toother organic compounds is necessary to estab-lish the general relevance of this conclusion.Another technique that has been used to

screen organic compounds as carbon sources forthe growth ofblue-green bacteria in the light inthe absence of net CO2 fixation involves thecomplete replacement ofC02 in the growth me-dium by organic compounds. Unfortunately,this method discounts any requirement for CO2for reactions other than the primary carboxyla-tion process, which prejudices the significanceof a negative result. Nevertheless, A. quadru-plicatum has been grown on glycerol in theabsence of CO2 (102).The development of comparable techniques

applicable to lithotrophs and methylotrophs isnecessary. The procedure involving the replace-ment ofthe Cl substrate in the growth mediumwith an organic compound is unsuitable formethylotrophs because they derive their energyfrom the catabolism ofthe Cl growth substrate.

Rneuirments for the Assimilation ofOrganicCompounds

Although growth experiments yield informa-tion on the magnitude and, thereby, the likely

significance of organic assimilation, investiga-tions with cell suspensions have identified therequirements of this process (46, 50, 84, 111,112). In general, the incorporation of any or-ganic compound will require both energy andacceptor molecules. Where both of these can beobtained from the organic compound itself, as-similation will be independent of any addi-tional factor. Where neither can be obtainedfrom the metabolism of the organic compound,its incorporation will be dependent on the avail-ability of other substrates that will provide en-ergy and acceptor molecules. With specialiststrains from all three physiological groups, therequirements for the assimilation of acetate arethe most thoroughly characterized. Acetate in-corporation by suspensions of these organismsis absolutely dependent on the specific energysource (Fig. 1; 46, 112, 164, 244): with blue-green bacteria, it is light dependent; for thioba-cilli, thiosulfate dependent; with nitrifiers, itrequires an oxidizable nitrogen compound; andwith methylotrophs, a suitable C0 substratesuch as methanol or trimethylamine. In thesecases, the extent of assimilation is proportionalto the amount of energy substrate available(Fig. 1; 112, 220), which eliminates a depend-ence on the specific energy source merely forthe initiation of substrate assimilation (183).The amounts of acetate assimilated by special-ist strains when related to the quantity of en-ergy source oxidized are of the same order ofmagnitude (Table 7). In contrast, the assimila-tion of acetate by suspensions of the versatilelithotrophs Nitrobacter agilis and ThiobacillusA2 is not wholly dependent on the availabilityofthe oxidizable inorganic compound although,in both cases, it doubles the rate ofuptake (Fig.1; 220). Acetate assimilation by specialist pho-totrophs and lithotrophs is also dependent onC02 (85, 112, 220); in the absence of CO2, thereis a marked reduction in the rate of assimila-tion of acetate by suspensions ofAnacystis nid-ulans and specialist thiobacilli. The depend-ence of acetate assimilation on CO2 in mostspecialist strains suggests that they cannot ob-tain the necessary acceptor molecules for ace-tate assimilation from its further metabolism.With T. denitrificans, oxaloacetate will in partreplace the requirement for CO2 (220).

Fate of Organic Carbon Assimilated bySpecialist Strains

The qualitative significance of the assimila-tion of organic compounds can be assessed bydetermining the distribution of assimilated or-ganic carbon among cell constituents. An inves-tigation of the fate of acetate carbon assimi-lated by specialist organisms from all three

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a E

0E

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w

nI4

I.-

49

Incut-on tdm (M

0Ec

Cti

0-JJ.2rnCI)

CuI-4

4c

MINUTES

FIG. 1. Acetate assimilation by cell suspensions of three specialist organisms and a versatile lithotroph.Acetate assimilation was determined in cell suspensions of (a) A. nidulans (10.7 mg [dry wt] per ml), (b) N.agilis (0.36 mg ofprotein per ml), (c) N. europaea (0.3 mg ofprotein per ml), and (d) methylotroph Wi (1.2mg ofprotein) incubated (a) in the light (0) and dark (a), (b) with NO2- ([0] 5 jumol/ml, [Al none), (c) withNH4+ ([A] 10 tumol/ml, [0120 pimol/ml), and (d) with MeOH ([@] 20 pimol). Utilization ofoxidizable energysources was followed in terms of (b) NO2- disappearance (a), (c) NO2- formation (@,A), and (d) 02consumption (0). A. nidulans is a specialist phototroph, N. agilis a versatile lithotroph, N. europaea aspecialist lithotroph, and methylotroph Wi a specialist methylotroph. References include (a) Hoare and Moore(84), (b) Smith and Hoare (203), (c) A. J. Smith (unpublished data), and (d) Dahl et al. (50).

physiological categories has provisionally iden-tified the metabolic routes involved. It has ledto the division of specialist phototrophs, litho-trophs, and methylotrophs into two distinctgroups (Table 8). Acetate carbon assimilated byorganisms in group A is essentially restricted tolipids and protein with significant amounts inonly four amino acids: leucine, glutamate, pro-line, and arginine. In contrast, the fate of ace-tate carbon assimilated by organisms in groupB is not restricted.The restricted pattern of acetate utilization is

typical of blue-green bacteria, some litho-trophs, and some methylotrophs, includingtype I methane users (46, 50, 64, 84, 112, 164,205, 210, 221). The differential labeling of gluta-mate and aspartate by [1-14C]acetate in theseorganisms is apparent from the magnitude ofthe ratio of their specific radioactivities (Table9). Isotope incorporated from [1-14C]acetate intoleucine is present solely in the Cl atom (85),whereas that in glutamate is in the C5 position(Table 10; 86); such patterns of labeling areconsistent with routes for the biosynthesis of

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TABLE 7. Acetate assimilation supported by theoxidation of energy sources in suspensions of

specialist lithotrophs and methylotrophs

Organism Energy Acetate as- Referencesource similationa

T. neapolitanus S2032- 12 112T. denitrificans S232-b 34 220

S4062- b 41 220

N. europaea NH4+ 3 SmithcM. capsulatus MeOH 5 HoaredM. sporium MeOH 7 HoaredMethylotroph Wi MeOH 8 50

MeNH2 21 50

Methylotroph C2A1 Me3N 9 46Methylotroph 4B6 Me3N 14 46

a For lithotrophs the units are nanomoles assimilatedper micromole of energy source oxidized and, for methylo-trophs, nanomoles assimilated per micromole of 02 takenup.

° NO3- used as terminal electron acceptor in place of 02.c A. J. Smith, unpublished data.d D. S. Hoare, unpublished data.

TABLE 8. Division of specialist organisms on thebasis of acetate metabolism

Group A Group B(restricted distribu- (unrestricted distri-tion of acetate car- bution of acetate

Type of bon: lipid, protein, carbon: most cellorganis and four amino constituents, in-organism acids, leucine, glu- cluding the amino

tamate, proline, acids of the gluta-and arginine) mate and aspartate

families)Phototrophs A. nidulans

G. alpicolaA. variabilisA. flos-aquaeN. muscorum

Lithotrophs T. denitrificans N. europeaT. neapolitanus N. oceanusT. thiooxidansT. thioparusN. multiformis

Methylo- M. capsulatus M. methanooxidanstrophs Methylotroph WI,

4B6, CZA1

these amino acids established in detail in otherorganisms and confirmed in part in blue-greenbacteria (85, 91, 200) and Thiobacdillus neapoli-tanus (112).With organisms that possess a functional tri-

carboxylic acid cycle, acetate carbon is found inseveral amino acids including aspartate; thelabelling of glutamate by [1-14C]acetate is char-acteristic: 66% in C5 and 33% in C, (18). Therestriction of carbon from the carboxyl group ofacetate to the C5 position of glutamate in spe-cialist strains in group A has been attributed toa block in the tricarboxylic acid cycle prevent-

ing the recycling of intermediates (Fig. 2; 86).This also accounts for the apparent upper limitto the amount of acetate assimilated by theseorganisms during growth, the lack of apprecia-ble amounts of label in cell constituents otherthan lipid and protein, the limited distributionof acetate carbon among amino acids, and thefailure of cell suspensions to convert acetate toCO2 (112, 164). The distribution among cell con-stituents of carbon from compounds metabol-ically related to acetate, such as pyruvate andglutamate, is also restricted in these organismsand in a manner that is consistent with themetabolic routes in Fig. 2 (62, 111, 117, 205).The specialist strains which we assign to

group B include type II methane utilizers andthe lithotrophs N. europaea and N. oceanus. Inthese organisms, acetate carbon, though assim-ilated at relatively low rates (Table 5), is dis-tributed among more cell constituents than inthe organisms in group A, including aspartateand metabolically related amino acids (234); in

TABLE 9. Specific radioactivity ofglutamate andaspartate isolated from specialist organisms grown

in the presence of [1-14C]acetateSpecific radioac-

tivity Glutamate/Organism (CPfIAM__ aspartate Reference

Gluta- Aspar- ratiomate tate

A. nidulans 7.5 0.08 93 146A. flos-aquae 6.1 0.06 101 146G. alpicola 99 0.5 198 146T. denitrifi- 11.8 0.08 147 221cans

N. europaea 2.9 0.12 23 Smith andWatsona

N. oceanus 4.7 0.13 35 Smith andWatsona

a Unpublished work by A. J. Smith and S. W. Watson.

TABLE 10. Distribution ofradioactivity in glutamatefrom specialist organisms grown in the presence of

[1-"4C]acetate% of total isotope inthe carboxyl carbon

Organism atoms of glutamate Reference

C, C5Group AA. nidulans <1 97 85G. alpicola <1 92 146T. neapolitanus 0-2 67-74 112T. denitrificans 0 99 221N. multiformis <1 100 Hoare

and Wat-sona

M. capsulatus 0 100 164Group BM. methanooxidans 30-39 26 234

a D. S. Hoare and S. W. Watson, unpublished data.

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Co2 phosphoenolpyruvate

\ / l w~~alaninepyruvate b- valine

\/It " ^ leucineacetylCoA- acetate

oxaloacetate citrate

11aspa rtate

lysine methionine

threonine

isoleucine

isoc it rate

ot-ketogluta rate

guaa

prolineglutamnate--ar ni

FIG. 2. Metabolic routes to glutamate and aspartate in specialist organisms in group A.

addition, the ratio of specific radioactivity ofglutamate to that of aspartate from these orga-nisms is much lower (Table 9). These resultsare to be expected in the case oftype II methaneutilizers, since they assimilate Cl compoundsvia the serine route into, which acetate shouldfeed directly and so contribute to the synthesisof many cell constituents (8). With Methano-monas methanooxidans, label from [1-'4C]acetate incorporated into glutamate is pres-ent in appreciable amounts in the Cl position(Table 10), and CO2 is produced from both car-bon atoms of acetate (234). The distribution ofacetate carbon among the cell constituents ofspecialist strains in group B is consistent withthe operation of the tricarboxylic acid cycle orother routes for the conversion of C2 compoundsto C4 dicarboxylic acids.

Isotopic studies alone are not sufficient toidentify the metabolic routes that operate inthese organisms. It is essential to show that theenzymes involved are present. At the cell-freelevel, some of the organisms in group B havebeen shown to contain all of the enzymes of thetricarboxylic acid cycle, including a-ketoglu-tarate dehydrogenase (Table 11). In contrast,the strains in group A lack a-ketoglutaratedehydrogenase and, in some cases, succinylCoA synthetase as well (Table 11; 244); evenradiochemical methods have failed to demon-strate any of this ketoacid dehydrogenase inblue-green bacteria (168). Although negativeenzymological data are unsatisfactory and, inisolation, questionable, they are, in this case,consistent with the restricted distribution ofacetate carbon among the cell components ofthese organisms and the specific labeling of

glutamate. The significance of the report of a-ketoglutarate dehydrogenase in Thiobacillusthiooxidans (30) will be in doubt until the fateof acetate carbon in this particular strain hasbeen established by isotopic tracer techniques.Similarly, the claim that N. europacea lacksthis enzyme (94, 237) needs to be reexamined inview of the results of labeling studies with thisorganism. It has yet to be established whetherthe lack of demonstrable a-ketoglutarate dehy-drogenase activity in organisms of group A isdue to the absence of all or only part of theenzyme which, in other organisms, is a complexcontaining several distinct enzymically activecomponents (128). In contrast, the homologousmultienzyme complex, pyruvate dehydrogen-ase, has been detected in two specialist methy-lotrophs (50, 164) and one cyanobacterium (23)but not, as yet, in any other organisms in groupA. Leach and Carr have, however, detectedpyruvate:ferredoxin oxidoreductase, which ac-complishes the same overall reaction as pyru-vate dehydrogenase, in the blue-green bacte-rium A. variabilis (137).The negligible amounts of isotope from

['4C]acetate in aspartate isolated from thioba-cilli and cyanobacteria implies not only that thetricarboxylic acid cycle is blocked but also thatcertain other metabolic routes do not operate toa significant extent; these include the ferre-doxin-dependent decarboxylation of a-ketoglu-tarate and the glyoxylate cycle. Blue-green bac-teria have been reported to contain both keyenzymes of the latter (166), as well as a-keto-glutarate:ferredoxin oxidoreductase (24). In ad-dition, isocitrate lyase has been detected in twospecialist methylotrophs (41, 43). However, the

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TABLz 11. Activities ofenzymes of the tricarboxylic acid cycle in specialist organisms

Enzyme activity (nmollmg of protein per min)

Organism AcOn- Isocitric de- a-Ketoglu- Succinic de- Refer-tAconi hydrogen- taric dehy- hydrogen- Fumarase Malic dehy- ences

ase drogenase ase drogenaseGroup APhototrophsA. nidulans -a 36 0 6.9 - 7.8 205C. peniocystis - 36 0 2.1 - 7.1 205G. alpicola - 36 0 3.4 - 17 205A. variabilis 2.1 4.7 0 0.2 3.7 2.0 168

Lithotrophs

T. thiooxidans 80 0 7.9 - 11.2 205T. thioparus 26 204 0 - 80 223 107T. denitrificans 56 61 0 0.5 148 16 220

Methylotrophs

M. methanica 10 15 0 12 34 430 51M. albus 3 19 0 12 37 340 51M. minimus 6 22 0 10 52 440 51M. capsulatus 8 22 0 2 32 74 51Methylotroph Wl - 5 0 0.8 - 69 50Methylotroph C2A1 28 52 0 0 0 6 43Methylotroph 4B6 10 75 0 0.6 9 0 41

Group BLithotrophs

N. oceanus 250 34 11 0.6 480 540 251N. europaea - 56 0 1.6 - 99 94

Methylotrophs

M. trichosporium 32 47 7 51 38 450 51M. sporium 17 53 10 59 43 360 51M. parvus 4 38 8 64 50 480 51

-, Not determined.

activities ofthese enzymes are very low and areinsufficient to contribute significantly to theconversion of acetate to oxaloacetate. They maybe concerned with the formation ofsuccinate fortetrapyrrole biosynthesis in these organisms(148, 166). The significance of the much higheractivity of both key enzymes of the glyoxylatecycle in T. denitrificans (170) is uncertain inview of the failure of acetate to contribute sig-nificantly to aspartate synthesis in this orga-nism (Table 9).The lack of a-ketoglutarate dehydrogenase

in organisms in group A confers on the remain-ing enzymes of the cycle a purely biosyntheticfunction rather than the dual role in biosyn-thesis and catabolism that is a feature of theintact cycle in other organisms (112, 168). It isnot surprising, therefore, that the mechanismsfor the regulation of some of these enzymes inspecialist organisms differ from those in gram-negative bacteria possessing an intact cycle. Inthe latter, citrate synthase is of the large type(molecular weight, ca. 250,000) and is sensitive

to reduced nicotinamide adenine dinucleotide(NADH) (247); in specialist thiobacilli and cy-anobacteria, the enzyme is also of the largetype but is insensitive to NADH and inhibitedby a-ketoglutarate (Table 14). In view of thesimilarity between lithotrophs and phototrophsin the regulation ofthis enzyme, it is surprisingthat it has different allosteric properties inmethylotrophs in group A. In Methylosinustrichosporium it is insensitive to NADH and a-ketoglutarate and only partially inhibited byhigh concentrations of adenine nucleotides (45);in methylotrophs 4B6 and C2A1, citrate syn-thase has similar properties except that it isalso sensitive to high concentrations of a-keto-glutarate. The extension of this work to othermethylotrophs and to lithotrophs in group B inconjunction with molecular weight determina-tions is essential.The block in the tricarboxylic acid cycle, al-

though explaining the features of acetate as-similation by specialist organisms in group Aand deserving an explanation in evolutionary

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terms (see Conclusions), does not, of itself, offera rational explanation for the conservativephysiology of these organisms. Other orga-nisms that exhibit the same defect are capableof heterotrophic growth; these include Aceto-bacter suboxydans (77), Escherichia coli grownanaerobically (7), versatile blue-green bacteria(86, 146), and the "restricted facultative" methy-lotrophs W3A1 and W6A (43). The lesion in thecycle will, however, have the effect of greatlyreducing the potential choice of organic com-pounds theoretically capable of supportinggrowth just as the absence ofpyruvate dehydro-genase greatly restricts the range of organiccompounds capable of supporting the growth ofhyphomicrobia (81). This accounts for the fail-ure of versatile cyanobacteria and the "re-stricted facultative" methylotrophs W3A1 andW6A to grow on common organic substratesother than carbohydrates and related com-pounds (43, 69, 87, 126, 127, 180, 238). Althoughspecialist organisms from group A assimilatecarbohydrates, the rates of incorporation arevery slow (Tables 5 and 6). The failure of thesesubstrates to support the chemoheterotrophicgrowth of these organisms may be due to le-sions that limit their utilization.

Substrate Transport and the SpecialistCondition

Studies of the quantitative significance of or-ganic assimilation have refined the definitionof the specialist blue-green bacterium; in a ma-jority of cases, it is an organism that is able toassimilate many organic compounds but unableto obtain sufficient carbon from this sourcealone to meet all of the carbon requirements ofthe cell.The cause of this effect and its relation to the

specialist character has been investigated bycomparing the ability of specialist and versatilecyanobacteria to metabolize glucose (172). Aph-anocapsa 6714, a versatile strain, converts glu-cose carbon to CO2 in the dark at a high rate,whereas two specialist isolates, Aphanocapsa6308 and Synechococcus 6301, oxidize this sub-strate at very slow rates (Fig. 3). Two otherisolates capable of growth in the dark on carbo-hydrates also convert glucose to CO2 at appre-ciable rates (Table 12).The failure of organisms to grow on organic

media in the absence of their characteristic car-bon and energy sources implies that specialiststrains have a limited ability to metabolize or-ganic compounds. In the particular case ofblue-green bacteria, there is a correlation betweenability to grow on organic media and the capac-ity to convert carbohydrate carbon to CO2. Be-cause cell-free preparations of versatile and

specialist blue-green bacteria contain similaractivities of enzymes for glucose dissimilation,Pelroy et al. (172) have attributed the limitedcapacity of specialist strains to convert glucoseto CO2 in the dark to relative impermeability tothis compound; they suggested that this mightbe due to the lack of a mechanism to mediatethe entry of glucose into the cell. The very lowdifferential rate of carbohydrate assimilation inthe light (Table 5) as well as the low level ofglucose metabolism in the dark by specialistblue-green bacteria (Fig. 3, Table 12) are bothconsistent with the absence of a mechanism forthe effective transport of this compound intothe cell.Although there is, as yet, no direct evidence

for this theory, a corollary to it is that photo-trophs capable of growth on glucose in the dark

TABLE 12. Oxidation of glucose in the dark by cellsuspensions of specialist and versatile blue-green

bacteria

Glucose oxidation(nmol glucose equivalents released as

CO,/mg dry wt per h)Organism

Photoauto- Photohet- Chemohet-trophiCa erotro- erotro-trophic phiCa phiCa

Specialist strainsA. nidulans 0.6 0.6A. flos-aquae 0.4 0.9

Versatile strainsNostoc sp. MAC 22 36 87C. fritschii 7.7 6.0 4.8

a Organisms were grown in the light with CO2 (photoau-totrophic) or with CO2 and glucose (photoheterotrophic) ascarbon sources and in the dark on glucose (chemohetero-trophic). From Lucas (146).

25

a 20E20

1l5i0

° 1050

lo /

I~~ ~ ~~~ ~ ~~~~~~~~~~~---,7/ $10-2m1-0m 5xj0- m 10-3m

[Glucose]

FIG. 3. Production of 14C02 from D-[U-"4C]glucoseby suspensions of specialist and versatile blue-greenbacteria. Rates are expressed as nanomoles of "4CO2released per milligram of organisms per hour. Theorganisms used were Aphanocapsa 6714 (-), a ver-satile strain, and Aphanocapsa 6308 (@) and Syne-chococcus 6301 (0), both of which are specialiststrains. Reference, Pelroy et al. (172).

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should possess a mechanism to facilitate theentry of this compound into the cell. Suspen-sions of the versatile cyanobacterium Aphano-capsa 6714 assimilate 1-glucose in the dark(172), and its uptake is inhibited by the glucoseanalogues 3-0-methyl-n-glucose and 6-deoxy-D-glucose (A. J. Smith and A. A. D. Beauclerk,unpublished data); cell suspensions also takeup 3-0-methyl-n-glucose by a mechanism thatis inhibited competitively by 1-glucose. The ac-cumulation of the analogue takes place againsta concentration gradient and does not involvethe concomitant modification ofthe transportedsubstrate. In addition, analogue uptake is in-hibited by the proton conductors tetrachlorosal-icanilide and carbonylcyanide 3-chlorophenyl-hydrazone. These data suggest that the versa-tile blue-green organism Aphanocapsa 6714takes up glucose and accumulates it within thecell by an active transport mechanism drivenby the proton gradient of the membrane (157,248). In contrast, suspensions of the relatedspecialist cyanobacterium Aphanocapsa 6308take up glucose at less than 1% of the rate withAphanocapsa 6714, and substrate uptake is notreduced significantly by 3-0-methyl-n-glucose,6-deoxy-D-glucose, or any other glucose ana-logue. These results imply that glucose takenup by the specialist strain probably enters thecell by passive, nonspecific diffusion down aconcentration gradient.The specialist characteristics of many blue-

green bacteria may be due to a virtual, thoughnot absolute, impermeability to those organiccompounds that could provide an adequate sup-ply of carbon and energy once inside the cell.These organisms may, therefore, be analogousto cryptic mutants of heterotrophic organismsthat will not metabolize or grow on specificorganic compounds because they have lost theability to transport these substrates into thecell (125). As specialist lithotrophs and methy-lotrophs in group A possess the enzymes for thedissimilation of glucose (44, 51, 107, 154), theslow differential rates of carbohydrate assimi-lation by these organisms (Table 5; 64) may alsobe due to the lack of an effective transportmechanism for these substrates. The failure ofthese organisms to grow on organic media couldbe a further consequence of their crypticity.

This theory is a refined form of that origi-nally proposed by Umbreit (230), who portrayedspecialist lithotrophs as microbial submarines,insulated from their environment by a barrierimpermeable to all organic compounds. How-ever, specialist organisms in group A need onlybe cryptic with respect to the restricted group oforganic compounds that are theoretically capa-ble of supporting growth. These organisms may

assimilate appreciable amounts of a variety ofother organic compounds that do not supportchemoheterotrophic growth because they can-not be used either for the generation of energyor in the synthesis of all cell constituents.Because organisms in group B are not subject

to the same intermediary metabolic restrictionsas those in group A, they should be capable ofchemoheterotrophic growth on a wider range oforganic compounds. Although the specialistcharacteristics oforganisms in group B could beexplained in terms of effective crypticity, itwould require these bacteria to be virtuallyimpermeable to a wide range of organic com-pounds. At the present time, not enough isknown about these organisms to assess the rel-evance of this theory to their specialist charac-teristics.

OTHER POSSIBLE CAUSES OFSPECIALIST CHARACTERISTICS

Besides toxicity and impermeability, it hasbeen suggested that a specialist physiologymight be the consequence of one of the follow-ing factors: an inability to adjust the activity ofkey enzymes to accommodate the needs ofgrowth on organic media (33, 34); the possessionof mechanisms for electron transport and en-ergy transduction that are distinct from thosein heterotrophic organisms (179) or which arenot coupled to phosphorylation reactions (82);an absolute dependence on a metabolite onlyavailable or a transformation only operatingduring growth on the characteristic energysource (113, 160).

Regulation of Enzyme Synthesis and ActivityIn E. coli and other heterotrophic bacteria,

enzyme activity is subject to control at the stageof gene expression, as well as at the level ofpreformed enzyme. Although the activity of avariety of enzymes in specialist strains is notaltered by changes in growth conditions (33, 92,93, 145, 154, 166-168, 220, 245), these organismsare not wholly incapable of regulating the syn-thesis of specific enzymes and other proteins;the exceptions in cyanobacteria include nitro-genase (72), alkaline phosphatase (21, 71), py-ruvate:ferredoxin oxidoreductase (24), and phy-cobiliproteins (6). In addition, Singer and Doo-little (201) have presented evidence that thereis a preferential synthesis of glycogen phospho-rylase and glucose 6-phosphate dehydrogenaserepresenting a 20-fold change in the differentialrate of protein synthesis when A. nidulans istransferred from light to dark. It has been ar-gued that failure to demonstrate regulation ofenzyme synthesis might be due to repression by

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an endogenous metabolite (33). Attempts to cir-cumvent this using auxotrophic strains havegiven conflicting results; the synthesis ofhomo-serine O-trans-succinylase in a methionine-re-quiring strain of A. nidulans is unaffected bymethionine starvation (55), whereas starvationof a tryptophan auxotroph of A. quadruplica-tum for tryptophan resulted in elevated levelsof tryptophan synthetase (103). Such studiesare complicated by the release of amino acidsby the breakdown of phycobiliprotein initiatedby amino acid starvation (200). With hetero-trophic organisms, changes in the relative ratesof synthesis of different ribonucleic acid (RNA)species that accompany alteration in growthrate have been attributed to control of geneexpression at the level of transcription (187); nosuch changes have been detected in A. nidu-lans over a wide range of growth rates (150).A corollary to the theory that the specialist

character is a consequence of an inability toadjust enzyme levels according to the needs ofgrowth on organic media is that versatilestrains should exert control over the expressionof their genetic information and transfer ofthese organisms from their characteristic car-bon and energy sources to organic media shouldbe accompanied by changes in enzyme activity.Versatile strains closely related to specialistspecies should then without exception be dis-tinctive in their ability to control macromolecu-lar synthesis. The activity of ribulose 1,5-di-phosphate carboxylase in versatile blue-greenbacteria is unaltered by growth in the dark onorganic compounds (68, 146) and, with the prob-able exception of glucose 6-phosphate dehydro-genase, enzymes of intermediary metabolismare largely unaffected by growth conditions(146, 172). Although the enzymes for C, oxida-tion in "restricted facultative" methylotrophsare apparently subject to strict control, the ac-tivities of other enzymes of intermediary me-tabolism do not vary markedly with changes ingrowth conditions (43, 44). With other versatilelithotrophs and methylotrophs a different pic-ture emerges. These organisms show amplesigns of regulatory mechanisms operating atthe level of gene expression (36, 41-44, 129, 143,154, 155, 165, 170, 185, 203, 212, 219, 227). It issignificant that there appears to be a correla-tion between ability to regulate enzyme synthe-sis and rate of growth on organic substratesrather than with ability to grow on organicmedia (Table 13); organisms with a limited reg-ulatory ability grow relatively slowly on or-ganic media, whereas much faster growth isachieved by versatile lithotrophs and methylo-trophs that have a comprehensive capacity for

controlling gene expression. In these cases itcould be argued that though the ability to con-trol enzyme synthesis is not essential for heter-otrophic growth it is necessary for rapidgrowth. Whether the versatile nitrifying bacte-riumN. agilis (Delwiche strain) will conform tothis generalization is not clear; growth of thisorganism on acetate-casein hydrolysate ismuch slower than on inorganic media eventhough the synthesis of isocitrate-lyase and ri-bulose 1,5-diphosphate carboxylase is subject tocontrol (203).Peck (169) has proposed that the specialist

condition is a consequence of regulatory mecha-nisms whose primary purpose is the conserva-tion of ATP. Modulation of the activity of pre-formed enzyme by effects of an allosteric typehave been demonstrated in a variety of special-ist strains (Table 14). Apart from differences inthe point at which control is exerted and thenature of the effector molecule(s), there is noreason to conclude that such mechanisms inthese organisms differ in any major way fromthose in versatile strains and heterotrophic or-ganisms. Whether differences in detail haveany causal relationship to the specialist condi-tion has not been resolved.

Energy Conservation in Specialist StrainsThe adaptation of the electron transport ma-

chinery of phototrophs, lithotrophs, and methy-lotrophs for the purpose ofexploiting unconven-tional energy sources has been cited as a possi-ble cause of failure to grow on organic media(33, 113, 164, 179, 183). This aspect of metabo-lism was first implicated in this connection sev-eral years ago with the claim that specialiststrains were unable to oxidize NADH (205).Although the data on which this theory was putforward have been questioned (96, 107, 135, 136,154, 226), an investigation of the pool sizes ofpyridine nucleotide cofactors in a specialistblue-green bacterium has confirmed that thisorganism has a very limited capacity to oxidizeNADH (16). NADPH is probably the principalrespiratory cofactor, since the pool of NADPHincreases and that of NADP+ decreases whenorganisms are transferred from aerobic to an-aerobic conditions in the dark; in contrast, thepools ofNADH and NAD+ do not change signif-icantly.More recently, it has been proposed that spe-

cialist strains will not grow on organic com-pounds because their electron transport ma-chinery, designed solely for the purpose oftransducing light energy or conserving energyderived from the oxidation of C, compounds orofreduced inorganic compounds ofnitrogen and

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TABLz 13. Exponential growth rates of versatile phototrophs, lithotrophs, and methylotrophs on theircharacteristic carbon and energy sources and on organic media

Mean generation time(h)

Organim- ReferenceCharacteristic carbona Oaand energy source O ic media

(A) Organisms growing at similar rates on or-ganic and specialized media

T. intermedius 20 (S2032-/CO2) 20 (glucose/yeast ex- 206tract)

Hydrogenomonas eutropha 3.3 (H1C02) 2.0 (acetate) 54Pseudomonas oxalaticusb 3.2 (HCOOH/CO2) 4.5 (oxalate) 19Bacillus PM6 3 (trimethylamine) 4 (citrate) 43Methylotrophs S2A1 3 (trimethylamine) 3 (glucose) 43Methylotroph 5B1 4 (trimethylamine) 4.5 (glutamate) 43

(B) Organisms growing at a much lower rateon organic media

Aphanocapsa 6714 15 (light/C02) 50 (glucose) 180C. fritschii 26 (light/C02) 144 (sucrose) 146Nostoc sp. MAC 6.5 (light/C02) 48 (glucose) 87N. agilis 20 (N02-/C02) 95 (acetate/casein hy- 203

drosylate)Methylotroph W3A1 2.5 (trimethylamine) 38 (glucose) 43Methylotroph W6A 2.5 (trimethylamine) 32 (glucose) 43a Growth substrates are given in parentheses.b When grown on formate, P. oxalaticus assimilates CO2 via the reductive pentose phosphate cycle.

sulfur, cannot accommodate the needs of heter-otrophic growth (179). But the existence of ver-satile phototrophs, lithotrophs, and methylo-trophs implies that an ability to exploit uncon-ventional energy sources is not a fundamentalobstacle to growth on organic compounds. Lith-otrophs as a group are characterized by highlevels ofcytochromes (67, 156, 223), and markedsimilarities have been noted between the cyto-chrome complement of specialist methylotrophsand their versatile counterparts grown undersimilar conditions (225). However, markedchanges in cytochrome content occur when ver-satile lithotrophs and methylotrophs are trans-ferred to conventional organic media (36, 219,225). It has yet to be established whethergrowth on such media is wholly dependent onalterations in the relative proportions of theconstituent electron carriers.

Failure to couple electron transport to phos-phorylation has also been suggested as a poten-tial cause of the specialist condition (82, 107).However, available information implicates cou-pled phosphorylation in energy conservation byorganisms capable ofexploiting unconventionalenergy sources (15, 120, 121, 186). In addition,cell-free preparations have been shown to cou-ple oxidation reactions to the synthesis of ATP(136, 213).In their natural environment, phototrophs,

Iithotrophs, and methylotrophs are unlikely to

have access to a continuous supply of their spe-cific energy source; their survival will then de-pend on an ability to obtain metabolically use-ful energy from energy sources other than lightor specialized oxidation reactions. Specialiststrains of blue-green bacteria maintain a de-tectable pool of ATP in the dark (25, 100, 171),and starved thiobacilli contain ATP amountingto 0.05 to 0.2% of dry cell weight (120). Limitedamounts of energy could be derived from thecatabolism of certain endogenous reserves. Thenature of these varies from group to group:cyanobacteria contain polysaccharide (35, 75,97), poly /8-hydroxybutyrate (32, 105), or cyano-phycin (199) or combinations of these, whereaslithotrophs and methylotrophs probably accu-mulate either polysaccharide or poly-,B-hydrox-ybutyrate (140, 242, 249). Dense suspensions ofspecialist lithotrophs, phototrophs, and methy-lotrophs take up oxygen at very slow rates (16,20, 89, 131, 166, 198, 232, 246). In the case ofblue-green bacteria, the rate of oxygen con-sumption declines over 24 h in the dark, pre-sumably as a result of the depletion of endoge-nous oxidizable substrate (131, 167, 246). Poly-saccharide reserves, which accumulate in blue-green bacteria during nitrogen starvation (6),decrease when organisms are incubated in thedark under aerobic conditions (139).Various studies have implicated the oxida.

tive pentose phosphate cycle as the route for the

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TABLE 14. Regulatory properties of enzymes in specialist organisms

Effector molecule(s)aEnzyme Organism Reference

Inhibitors Activators

Glucose 6-phosphate dehy-drogenase

PhosphofructokinaseCitrate synthase

DAHP synthetase

Isocitric dehydrogenase

Fructose 1,6-diphosphatasePyruvate:ferredoxin oxido-

reductaseN-acetylglutamate kinaseThreonine dehydratase

Homoserine O-trans-succin-ylase

a-Hydroxyacid synthetase

PEP carboxylasePyruvate kinase

Aphanocapsa 6714A. variabilis

(vegetative)A. variabilisT. denitrificansT. neapo4itanus

Aphanocapsa FNostoc sp.Phormidium sp.

A. flos-aquaeA. nidulansG. alpicola

M. albusM. trichosporiumMethylotroph 4B6Methylotroph C2A1

A. variabilisA. nidulansT. neapolitanusT. neapolitanus

T. neapolitanusA. variabilis

A. nidulansA. variabilisA. quadruplicatumA. nidulans

T. thiooxidansA. variabilisT. thiooxidansT. neapolitanusA. variabilis

RuDPRuDP

ATP, PEP, citratea-Ketoglutaratea-Ketoglutarate, ATP

a-Ketoglutarate, ATPa-Ketoglutarate, ATPa-Ketoglutarate, ATP

a-Ketoglutaratea-Ketoglutaratea-Ketoglutarate

ATPATPa-Ketoglutarate, ATPa-Ketoglutarate, ATP

TyrosineTyrosineTyrosine, phenylalanineATP, ADP, glyoxylate plus

oxaloacetateAMP, PEP

ArginineIsoleucineIsoleucineHomoserine, methionine

Valine, isoleucineValineAspartate, malateCarboxylic acidsCitrate

ATP

ATP

FADAcCoASugar phosphatesSugar phosphates

aAbbreviations: RuDP, Ribulose 1,5-diphosphate; PEP, phosphoenol pyruvate; FAD, flavin-adenine dinucleotide.

catabolism of polysaccharide reserves in blue-green bacteria: cell-free preparations containNADP+-specific glucose 6-phosphate and 6-phosphogluconate dehydrogenases (70, 167,172); loss of these two dehydrogenases by muta-tion virtually eliminates the low rates of endog-enous respiration ofA. nidulans (60); specialiststrains mediate a very slow preferential releaseof the C, atom of glucose as CO2 (167, 250). Thispathway is also the major, if not the sole, routefor the dissimilation of organic substrates inthe dark by versatile cyanobacteria (37, 146,172). Uncertainty remains about the occurrence

of phosphofructokinase, a key enzyme of glycol-ysis, in specialist blue-green bacteria; low mar-ginal levels have been reported in one versatileand two specialist strains (172), whereas vege-tative cells ofA. variabilis are reported to con-

tain significant activity (141, 167).At the present time, the only evidence for the

operation of the oxidative pentose phosphatecycle in other specialist species from group A isenzymological; several lithotrophs and methy-lotrophs have been shown to contain glucose 6-phosphate and 6-phosphogluconate dehydro-genases and to lack phosphofructokinase (44,51, 107, 154). In contrast, glycolysis in conjunc-tion with the tricarboxylic acid cycle is theprobable route for the catabolism of organiccompounds in specialist strains in group B (51);they contain phosphofructokinase and a-keto-glutarate dehydrogenase while lacking glucose6-phosphate and 6-phosphogluconate dehydro-genases. The only organism that does not fitinto either ofthese two categories is Nitrosocys-tis oceanus, which contains the enzymes of theoxidative pentose phosphate route and of thetricarboxylic acid cycle (31, 251). Although spe-cialist organisms in group B contain the en-

zymes of the tricarboxylic acid cycle, it is not

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known how effectively this route can accom-plish the catabolism of exogenous organic com-pounds.The probability that organisms incapable of

growth on organic media can obtain energy formaintenance by the oxidative catabolism of en-dogenous organic reserves raises doubt regard-ing the claim that their electron transport ap-paratus is incompatible with the conservationof energy during the breakdown of at leastsome organic compounds. It is, however, possi-ble that energy obtained by the catabolism ofexogenous organic compounds is sufficient tomaintain the cell but not to support significantgrowth in the absence of the specialized energysource. This might account for the failure ofstrains of blue-green bacteria, which grow onglucose in the light in the absence of net CO2fixation, to grow on this substrate in the dark(124, 180).Organic compounds have little if any effect

on the slow rate of endogenous oxygen uptakeexhibited by suspensions of specialist orga-nisms (16, 20, 138, 139, 170, 171, 197, 239, 246).In the case of blue-green bacteria, the mostmarked effects are obtained with organismsthat have been starved for 24 h by removal ofthe CO2 source or by transfer to darkness (133,170, 171, 253). However, the final rates underthese conditions are similar to the endogenousrate of nonstarved organisms. The weak respi-ratory response of specialist organisms to exog-enous organic compounds is consistent withcrypticity as well as with limited respiratoryactivity.

Specialized Nutritional RequirementsThe inability of specialist strains to grow on

simple organic media could be due to the inabil-ity of such media to meet the requirements ofthese organisms for chemoheterotrophic growth(113). The specialist condition might be due toan inability to assimilate sulfate in the case ofthe thiobacilli or nitrate in the case ofthe nitri-fying bacteria, and blue-green bacteria woulddepend on a light-mediated transformation. Inthis event, it should be possible to formulateorganic media that can satisfy the specific nu-tritional requirements of these organisms andsupport heterotrophic growth. However, at-tempts to grow selected specialist phototrophs,lithotrophs, and methylotrophs on a variety ofcomplex and defined organic media have failed.In addition, nitrifying bacteria contain reduc-tase enzymes that mediate the assimilation ofnitrate and nitrite (235).A lesion in sulfate assimilation prevents the

versatile lithotroph T. intermedius from grow-

ing on glucose in the absence of reduced inor-ganic sulfur compounds (206). It will grow onthis substrate, provided small amounts of me-thionine or related organic sulfur compoundsare added to the medium. However, T. interme-dius is not a specialist lithotroph. It differsfrom specialist thiobacilli such as T. neapoli-tanus and T. thiooxidans in several importantrespects: it assimilates organic compounds atmuch higher rates than specialist strains (205),organic compounds stimulate its rate of growthon reduced inorganic sulfur compounds (142),and it catabolizes glucose via the Entner-Dou-doroff route in conjunction with the tricarbox-ylic acid cycle (153).

CONCLUSIONSThe purpose of this review has been to survey

the features of specialist phototrophs, litho-trophs, and methylotrophs and to identifylikely causes of their conservative physiology.Progress has been made with the characteriza-tion of these organisms, but considerable gapsremain in our knowledge of their physiologyand biochemistry. Various explanations havebeen proposed to account for the specialist con-dition in each of the three physiological groups.With one possible exception, none ofthese theo-ries has yet been adequately substantiated.Certain of these can, however, be discarded onthe basis of available data.The growth of specialist organisms is in-

hibited by specific organic compounds but theseeffects, which are also observed with chemohet-erotrophic microorganisms, do not account forthe failure of specialist strains to grow on or-ganic media.

Unlike many chemoheterotrophs, specialistorganisms in group A (Table 8) lack a-ketoglu-tarate dehydrogenase. This metabolic lesioneliminates the normal catabolic role of the tri-carboxylic acid cycle and leaves the remainingreactions of the cycle with a purely biosyntheticfimction. Although it is clear that the loss of a-ketoglutarate dehydrogenase need not be anobstacle to heterotrophic growth (see Fate ofOrganic Carbon Assimilated by SpecialistStrains), a deficiency of this enzyme has beenresurrected as a cause of the conservative phys-iology of specialist methylotrophs on the basisof studies with the versatile methylotrophPseudomonas AM1 (222); a mutant that haslost this enzyme will not grow on a variety oforganic compounds that support the growth ofthe parent organism. Although it is logical inthis case that specialist growth characteristicswill result from the loss of a-ketoglutarate de-hydrogenase, the mutant is not a strict counter-

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part of organisms in either of the two majorgroups of specialist methylotrophs. Those thatresemble Pseudomonas AM1 in the route for Classimilation (type II) possess a-ketoglutaratedehydrogenase (51) which, in conjunction withthe other reactions of the tricarboxylic acid cy-cle and the glycolytic route, could accomplishthe complete catabolism of a wide range of or-ganic compounds. In contrast, specialistmethylotrophs lacking a-ketoglutarate dehy-drogenase (type I) not only exploit the otherroute for C, assimilation but also possess theenzymes of the oxidative pentose phosphate cy-cle (51), through which a relatively restrictedrange of organic substrates could be catabo-lized. The restricted nutrition of specialistmethylotrophs using the serine route is morelikely to be a consequence of other metaboliclesions. Failure to grow on organic compoundsother than C, substrates could result from alack of pyruvate dehydrogenase and malatesynthase, neither of which are involved in C1assimilation.An inability to regulate the synthesis of

many enzymes, which appears to be a commonfeature of most specialist strains, has been pro-posed as a potential cause of their conservativephysiology. Although this theory has neitherbeen confirmed nor excluded, such effects areconsidered more likely to hinder growth on or-ganic media rather than to prevent it alto-gether.

Certain aspects of the physiology of specialistorganisms suggest that their inability to growon organic compounds may be related to theirenergy metabolism (183). However, failure toobtain energy from organic compounds mayhave a variety of causes, some concerned withthe uptake and metabolism of organic com-pounds as well as others related directly tounique features of the apparatus for energyconservation. There is, as yet, no more thancircumstantial evidence for a direct link be-tween the specialist character and the mecha-nisms for energy conservation in these orga-nisms.The failure of some microorganisms includ-

ing T. intermedius, a versatile lithotroph, togrow on single organic compounds is due tospecific nutritional requirements. There is, asyet, no evidence to suggest that the growth ofspecialist phototrophs, lithotrophs, or methylo-trophs on organic media is prevented by nutri-tional deficiencies.At the present time, the weight of evidence,

albeit indirect, favors crypticity to potentialgrowth substrates as the basis of the specialistcharacter of blue-green bacteria. The develop-ment of techniques for the transformation of

genetic characters among blue-green bacteriashould provide the means for testing this theorydirectly. Specialist strains should gain the abil-ity to grow chemoheterotrophically on specificorganic compounds after the acquisition of thegenetic determinants of the transport mecha-nisms for these substrates.The specialist character of lithotrophs and

methylotrophs in group A can also be explainedin terms of crypticity. At the present time, thisinterpretation is based on circumstantial evi-dence alone. In certain areas oftheir intermedi-ary metabolism, these organisms are very simi-lar to blue-green bacteria. In addition, theyassimilate organic compounds that are poten-tial growth substrates at very low rates al-though they possess reasonable levels of theenzymes for their catabolism.The comparative approach, which has been

applied successfully to blue-green bacteria,may be equally rewarding if applied to litho-trophs and methylotrophs. It would require ver-satile species that are identical to specialiststrains in all respects other than their capacityfor growth on organic media. There are well-established examples of specialist and versatilestrains of thiobacilli (Table 1), and surveys oftheir properties (99) and fatty acid composition(1) suggest close similarities between certainspecies. However, large differences in the DNAbase composition of several isolates (104, 133,152) imply a degree of species variation greaterthan anticipated, which may prove incompati-ble with the comparative approach. With nitri-fying bacteria, detailed studies at the biochemi-cal level of the strains isolated by Watson andhis colleagues are still at a preliminary stage.A comparative approach to the specialist char-acter of certain strains of Nitrobacter may befeasible, as available isolates include both ver-satile and putative specialist types (203, 242).Among methylotrophs, the versatile strainscharacterized by Patt et al. (165) are analogousto type II methane utilizers in terms of mem-brane array, route for C, assimilation, and car-bohydrate metabolism. Zatman's group has iso-lated a variety of methylotrophs (9, 41-43),some of which would be suitable for a com-parative study; such an approach would be ap-propriate with versatile strains W3A1 and W6Aand specialist isolates C2A1 and 4B6, which areanalogous to versatile and specialist blue-greenbacteria, respectively. In spite of recent ad-vances in our knowledge of the physiology oforganisms that exploit unconventional sourcesof carbon and energy, it is not yet certainwhether there is a unitary basis for the special-ist character among all of these organisms.However, the specialist characteristics of pho-

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totrophs, lithotrophs, and methylotrophs ingroup A, despite their diverse carbon and en-ergy metabolism, may be due to their crypticityto organic growth substrates.An ecological interpretation of the existence

of specialist strains of phototrophs, lithotrophs,and methylotrophs is necessary but, at thepresent time, the relevant information is notavailable. The presence of these organisms inthe microbial world, where speed of growth andmetabolic versatility are two of the ingredientsfor survival, is an apparent contradiction. Nev-ertheless, specialist blue-green bacteria, litho-trophs, and methylotrophs can be isolated fromvarious sources, in some cases, together withversatile strains (42, 209, 221). Although versa-tile and specialist types may appear to occupythe same environment, it is possible that theyinhabit separate microniches. In this event, thecomposition of the microenvironment mayprove to be of fundamental importance to thesurvival of specialist strains. It is possible,however, that specialist strains may survive inopen competition with versatile organisms. Theability of specialist strains to compete withtheir versatile counterparts could be estab-lished by investigating the influence of growthconditions, including the availability of variousorganic compounds, on the development ofmixed populations of specialist and versatileorganisms from the same physiological group.Although doubt has been expressed about theability of T. neapolitanus, a specialist litho-troph, to compete under these conditions withthe versatile T. intermedius (184), this has yetto be proved.

It is also relevant to consider the significanceof the specialist character and of the distinctiveintermediary metabolism of specialist orga-nisms in group A (Table 8) and their versatilecounterparts in the context of microbial evolu-tion. Broda (27) and Margulis (151) have re-cently outlined a probable sequence of meta-bolic types during microbial evolution. Fermen-tative organisms with negligible ability forsmall-molecule synthesis are likely to have pre-ceded other physiological types in an anaero-bic environment rich in organic compounds; en-ergy would be obtained by substrate phospho-rylation reactions such as those in glycolysis,and macromolecular precursors would be ob-tained from the environment. The proliferationof such organisms would, in time, limit theavailability of the immediate precursors of cellconstituents and confer a considerable advan-tage on organisms that acquired an ability toproduce the substrates for the synthesis of mac-romolecules. This would involve the develop-ment of many new metabolic pathways; these

would include at least part of the pentose phos-phate route to provide pentoses for the synthe-sis of nucleic acids (95) and the incomplete tri-carboxylic acid cycle (Fig. 2) for the synthesis ofaspartate, glutamate, and related amino acids.Competition for organic substrates for energygeneration would be increasingly intense, espe-cially with the increased demand for metabol-ically useful energy for the synthesis of macro-molecular precursors. At this point in evolu-tion, the continued survival of living organismswould be at its most precarious (27). Such con-ditions would, however, establish a powerfulselective advantage for organisms that devel-oped the ability to exploit alternative sources ofenergy and carbon.Under anaerobic conditions, there would be

few alternatives to fermentation for the pur-pose of energy generation. One possibility con-stituting a major advance would be the develop-ment of a mechanism for the transduction oflight energy into a biologically useful form.Initially, these ancestral photosynthetic bacte-ria are likely to have grown photoheterotrophi-cally using, as carbon source, acids and alcoholsthat were the end products of various fermenta-tions. The corresponding breakthrough in theexploitation of alternative carbon sourceswould come with the evolution of a pathway forthe net fixation of CO2; this is likely to haveresulted from the grafting of new reactionsunique to C02 fixation onto the existing routefor the synthesis ofpentoses. Contemporary an-aerobic organisms with these abilities includemany of the photosynthetic bacteria. The re-ducing power for the net assimilation of CO2would be obtained from inorganic compoundssuch as H2, sulfide, and sulfur. Reserves ofthese reductants would, however, be limited,and ultimately phototrophic organisms capableof exploiting H2O as a source of reducing powerwould have a clear selective advantage; amongthem would be the ancestors of contemporaryblue-green bacteria. Oscillatoria limnetica,which can use either H2S or H2O as electrondonor, in photosynthesis, may represent an in-termediate stage in the transformation fromanoxygenic to oxygenic photosynthesis (40). Or-ganisms exploiting light as primary energysource would also need other mechanisms forthe generation of energy to sustain the cellduring periods of darkness. This could be ac-complished by the dark metabolism of endoge-nous reserve polymers. It is reasonable to sup-pose that the efficient utilization ofendogenouspolysaccharide under aerobic conditions wasmade possible by the acquisition ofthe enzymescatalyzing the two oxidation reactions uniqueto the oxidative pentose phosphate cycle, to-

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gether with a modification ofthe electron trans-port machinery of the cell to couple the oxida-tion of NADPH to molecular oxygen.The development of photosynthetic oxygen

production established the aerobic environment(27), opening the way for the exploitation ofotherwise refractory compounds such as ammo-nium and nitrite, as well as methane and otherreduced C, compounds as sources of energy inconjunction with C1 compounds as carbonsource; organisms with such metabolic abilitiesare likely to have been the ancestors of methy-lotrophs and lithotrophs in group A (Table 8).They could have developed from primitive pho-tosynthetic organisms by loss of pigment sys-tems and diversification of electron transportmachinery to encompass the aerobic oxidationofreduced C, compounds and reduced inorganiccompounds of nitrogen. Sulfur-oxidizing chem-olithotrophs are likely to have evolved fromprimitive photosynthetic bacteria and cyano-bacteria, since these groups would have in-cluded organisms capable of oxidizing reducedinorganic compounds of sulfur. In the absenceof their specialized energy sources, these orga-nisms probably generated maintenance energyby the catabolism of endogenous carbohydratereserves via the oxidative pentose phosphatecycle. In addition, the tricarboxylic cycle wouldstill be incomplete, fulfilling a purely biosyn-thetic role in intermediary metabolism. Theactivities of these phototrophs, lithotrophs, andmethylotrophs will have contributed to therestoration of the stocks of organic compoundsin the environment.The efficient aerobic catabolism of a re-

stricted range of these exogenous organic com-pounds would be possible via the oxidative pen-tose phosphate cycle; the utilization of a muchwider range of organic compounds would de-pend on the development of a complete tricar-boxylic acid cycle. It is likely, therefore, thatorganisms capable ofexploiting unconventionalcarbon and energy sources first appeared beforethe development of a functional cycle. Conse-quently, the incomplete form of the cycle and toa lesser extent the oxidative pentose phosphatecycle are metabolic indicators of the evolution-ary origins of phototrophs, lithotrophs, andmethylotrophs in group A (Table 8). The originof the physiologically related specialist orga-nisms in group B is uncertain; these orga-nisms, together with their versatile counter-parts which also possess a complete tricarbox-ylic acid cycle, may have evolved by quite sepa-rate adaptations to chemolithotrophy andmethylotrophy, postdating the first appearanceof the complete cycle.The first organisms exploiting unconven-

tional carbon and energy sources may, there-fore, have evolved in response to a specific com-bination of circumstances. Organic compoundscontaining more than one carbon atom werevery scarce, whereas Cl substrates, reduced in-organic compounds of nitrogen and sulfur andlight were, by comparison, readily available.The specialist character may have been ac-quired coincidentally during the evolution ofthese organisms by the loss of permeability toorganic compounds capable of supportinggrowth; an ability to metabolize such com-pounds would have been of little benefit at thattime because of their relative scarcity. In addi-tion, the ability to exploit specialized sources ofcarbon and energy may have conferred suffi-cient advantage on these organisms so thateven where organic compounds were available,the ability to exploit them would be ofcompara-tively little benefit. Something akin to this sit-uation must of necessity persist at the presenttime in the ecological niches occupied by thecontemporary forms of these organisms. In afew cases, however, the loss oftransport mecha-nisms may not have been complete, resulting instrains that have retained the potential for het-erotrophic growth on a restricted range of or-ganic substrates.

ACKNOWLEDGMENTSWe wish to thank our collaborators for access to

unpublished data and various publishers and au-thors for permission to reproduce published figures.A.J.S. expresses particular gratitude to MartinKnight and Peter Trudgill for constructive com-ments on the manuscript during its preparation.Sincere thanks are also due to our secretary EveGoodliffe for the skilled typing of several drafts andnot least to the editors for their patience during theprolonged gestation period of this paper.

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acterization of Thiobacillus species by gas-liquid chromatography of cellular fattyacids. Arch. Mikrobiol. 89:257-267.

2. Aleem, M. I. H. 1966. Generation of reducingpower in chemosynthesis. II. Energy linkedreduction of pyridine nucleotides in the che-moautotroph, Nitrosomonas europaea.Biochim. Biophys. Acta 113:216-224.

3. Aleem, M. I. H. 1970. Oxidation of inorganicnitrogen compounds. Annu. Rev. PlantPhysiol. 21:67-90.

4. Aleem, M. I. H., H. Lees, and D. J. D. Nicho-las. 1963. ATP dependent reduction of NADby ferrocytochrome c in chemoautotrophicbacteria. Nature (London) 200:759-761.

5. Allen, M. M. 1968. Photosynthetic membranesystem in Anacystis nidulans. J. Bacteriol.96:836-841.

6. Allen, M. M., and A. J. Smith. 1969. Nitrogen

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