JOURNAL OF BACrERIOLOGY, Feb. 1970, p. 339-345.Copyright © 1970 American Society for Microbiology

Vol. 101, No. 2Printed in U.S.A.

Autotrophic and Heterotrophic Metabolism ofHydrogenomonas: Regulation of Autotrophic

Growth by Organic SubstratesPHILIP E. STUKUS' AND B. T. DECICCO

Department of Biology, The Catholic University of America, Washington, D.C. 20017

Received for publication 15 August 1969

The effects of a number of organic substrates on the autotrophic metabolism ofHydrogenomonas eutropha were examined. Dual substrate (mixotrophic) cultivationin the presence of hydrogen plus either fructose or alanine allowed autotrophicgrowth to begin immediately after the exhaustion of the organic substrate. On theother hand, the presence of acetate, pyruvate, or glutamate caused a lengthy lag tooccur before autotrophic growth commenced. With acetate or pyruvate this lag(plateau) in the dicyclic growth curve was due to the repression of ribulose diphos-phate carboxylase (RDPC) synthesis during mixotrophic growth. During hetero-trophic growth with glutamate, RDPC was partially repressed; however, duringmixotrophic growth, RDPC activity was high. Thus the delay of autotrophic growthwas not due to a repression of RDPC by glutamate. The data suggest that glutamateinterferes with autotrophic metabolism by repressing the incorporation of inorganicnitrogen. The repression of these vital autotrophic functions by acetate, pyruvate,and glutamate occurred both in the presence and absence of hydrogen, i.e., duringboth heterotrophic and mixotrophic cultivation. The derepression of the affectedsystems during the plateau phase of the dicycic growth curves was demonstrated.Carbon dioxide assimilation by whole cells agreed well with the RDPC activity ofextracts from cells grown under similar conditions.

A number of organic compounds, utilizable ascarbon and energy sources for heterotrophicgrowth, can affect the autotrophic metabolismof hydrogen bacteria. In cells grown heterotroph-ically, these effects were observed as decreasedlevels of the two vital autotrophic functions, thehydrogen-oxidizing and carbon dioxide-fixingsystems. Among the members of the genus Hydro-genomonas, H. flava (9), H. facilis (13, 20, 23),H. ruhlandii (16), and Hydrogenomonas H 1 (2)possess inducible or repressible hydrogenases.Only H. eutropha seems to lack this regulatorymechanism (Bovell, Ph.D. Thesis, University ofCalifornia, Davis, 1957; Stukus, M.S. Thesis,The Catholic University of America, Washington,D.C., 1966).The carbon dioxide-fixing system plays per-

haps an even more critical role in the control ofautotrophic metabolism. In Hydrogenomonas,as in other autotrophic bacteria, considerableevidence implicates ribulose diphosphate car-boxylase (RDPC, EC 4.1.1.39) as the primary

I Present address: Department of Biology, Denison University,Granville, Ohio 43023.

catalyst for the assimilation of carbon dioxideduring autotrophic growth (1, 3, 7, 8, 14, 17, 19,21). The activity of this enzyme in a number offacultative autotrophs after heterotrophic cultiva-tion was reported to vary from 0 to 100% of theautotrophic level, depending upon the growthconditions employed. Growth on pyruvate (8),acetate, or malate (5) apparently repressesRDPC in Chromatium. A similar repression byacetate was observed with Micrococcus denitrifi-cans (10) and H. facilis (15), suggesting thatRDPC plays a critical role in the control of CO2assimilation during heterotrophic growth. H.ruhlandii, when grown on lactate, possessed only2% of the autotrophic level of RDPC (22),whereas H. eutropha contained much higherlevels (18). Growth on fructose produced de-creasing levels of RDPC with HydrogenomonasH 16 (6) but not with H. facilis (15). More re-cently, other environmental conditions, includingthe degree of aeration, were implicated in themaintenance of high RDPC activity during hetero-trophic growth on fructose (11).We previously described the dicyclic growth

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patterns which occurred when H. eutropha wasgrown under dual substrate (mixotrophic) condi-tions (4). When the organic substrate allowedsimultaneous autotrophic and heterotrophicmetabolism during the initial growth phase, asevidenced by increased cell yields, a rapid change-over to exclusively autotrophic growth occurredat the point of organic substrate exhaustion.With three of the tested organic substrates,glutamate, acetate, and pyruvate, the initialgrowth phase seemed to represent heterotrophicrather than simultaneous growth, and with thesesubstrates a lengthy changeover period of 5 to 8hr preceded the initiation of autotrophic growth.It was suggested that both the lack of simultane-ous growth and the lag (plateau) before auto-trophic growth commenced were due to a repres-sion of some autotrophic function by these threesubstrates. Since hydrogenase is constitutive inH. eutropha, and since decreased levels of RDPCwere reported in some facultative autotrophsafter heterotrophic growth on glutamate, acetate,and pyruvate, it seemed likely that these sub-strates may be repressing RDPC in H. eutropha,both in the presence and absence of the auto-trophic atmosphere.The present investigation was undertaken to

determine whether the growth patterns observedunder dual substrate conditions could be cor-related with RDPC activity. In addition, 14CO2assimilation by cells grown under various condi-tions was compared to their RDPC activities toconfirm that this enzyme is the major catalyst forautotrophic carbon dioxide-fixation in H. eu-tropha.

MATERIALS AND METHODSSpecial chemicals. 3-Phosphoglyceric acid kinase

3-phosphoglyceraldehyde dehydrogenase, and adeno-sine triphosphate (ATP) were obtained from Calbio-chem, Inc., Los Angeles, Calif.; 2, 5-diphenyloxazole(PPO), dimethyl 1 ,4-bis-2(5-phenyloxazolyl)-ben-zene (POPOP), and Triton X-100 were from PackardInstrument Co., Downers Grove, Ill.; NaH"4CO3was from Schwarz BioResearch Inc., Orangeburg,N.Y.; dibarium ribulose-1, 5-diphosphate and re-duced nicotinamide adenine dinucleotide (NADH)were obtained from Sigma Chemical Co., St. Louis,Mo.; Nessler's reagent was from Arthur H. ThomasCo., Philadelphia, Pa.Media and growth conditions. Autotrophic and

dual substrate cultivation of H. eutropha were aspreviously desctibed (4). For heterotrophic growth,organic substrates which had been autoclaved sepa-rately in distilled water were added to the specifiedconcentrations, and incubation was under air. Be-cause of the influence of shaker speed on RDPCactivity noted by Kuehn & McFadden (11), RDPCwas determined in extracts from cultures which hadbeen shaken at both slow (130 gyrations/min) and

rapid (325 gyrations/min) speeds. Growth wasmonitored by following the increase in optical density(OD) at 540 nm by using a Spectronic-20 colorimeter(Bausch and Lomb, Inc., Rochester, N.Y.). An ODof 1.0 corresponds to a cellular dry weight of 0.32mg/ml, and this relationship is linear throughoutexponential growth.

Cell-free extracts. Cells were grown under thespecified conditions, harvested by centrifugation at2 C, and suspended in 0.05 M tris (hydroxymethyl)aminomethane (Tris)-hydrochloride buffer (pH 7.9)containing 12 mm of 3-mercaptoethanol, 50 mmNaHCO3, and 1.5 mM ethylenediaminetetraaceticacid. The cell suspensions were disrupted by sonictreatment for 2 min in an ice bath by using a BiosonikII sonifier (Bronwill Scientific Co., Rochester, N.Y.)at maximum power. The disrupted suspensions werecentrifuged at 20,000 X g for 20 min at 2 C. The super-natant fractions were dialyzed overnight at 4 Cagainst 4 liters of the same buffer solution in whichthe cells had been suspended. These dialyzed ex-tracts were used in the RDPC assay.RDPC assay. The method employed was a modifica-

tion of that described by Hurlbert and Lascelles (8).The assay consisted of two parts. The first part in-volved the formation of 3-phosphoglyceric acid(3-PGA); the second part was the spectrophotometricdetermination of the amount of 3-PGA formed inpart one measured by the oxidation of NADH. Thereaction mixture for part one contained 12 jsmolesof MgSO4, 12 ,umoles of reduced glutathione, 80,umoles of NaHCO3, 1 ,umole of ribulose diphosphate,100,moles of Tris-hydrochloride buffer (pH 7.9),and the amount of cell extract containing 1.0 mg ofprotein, in a final volume of 2.0 ml. The mixture wasincubated at 37 C for 5 min and then placed in aboiling water bath for 3 min to stop the reaction.After cooling, the mixture was clarified by filtrationthrough a Swinnex 13 membrane filter unit (Milli-pore Corp., Bedford, Mass.) with a pore size of 0.45,am. The filtered material was used in the spectro-photometric assay which was performed by using aHitachi Perkin-Elmer 139 spectrophotometer equippedwith a Coleman-Hitachi 165 recorder. For the assay,the cuvettes contained 17 Amoles of MgSO4, 20 Amolesof L-cysteine, 2 Amoles of ATP, 0.4 jAmoles of NADH,150 ,moles of Tris-hydrochloride buffer (pH 7.9),210 ,ug of 3-phosphoglyceraldehyde dehydrogenase,and 1.0 ml of filtered material, in a final volume of3.0 ml. The reaction was started by the addition of2.2 gg of 3-PGA kinase. The oxidation of NADHwas measured as the decline in OD at 340 nm untilthere was no further decrease, and the amount of3-PGA in the sample was calculated from the amountof NADH oxidized. A unit of enzyme activity isdefined as the amount catalyzing the formation of 1nmole of 3-PGA per min which corresponds to adecrease in OD of 0.0045. Specific activity is definedas units per milligram of protein. Protein was de-termined by the biuret method.

Fixation of labeled bicarbonate. Cells were grownunder the specified conditions, harvested at thedesired growth phase by centrifugation at 2 C, andresuspended to 4.0 OD in 0.04 M potassium phosphate

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buffer (pH 7.2). Incorporation of 14CO2 was per-formed by using double side-arm Warburg flasks.The main compartment of the flask contained 1.0 mlof the cell suspension and 1.5 ml of phosphate buffer.Into one side arm was placed a 0.4-ml amount con-taining 4 Ac of NaHl4CO3 (5.3 mc/mmole); the othersidearm contained 0.3 ml of 20% trichloroacetic acid.The flasks were incubated with shaking in a Warburgapparatus at 30 C. One series of flasks was incubatedunder an atmosphere of 80% H2 and 20% 02, with aduplicate series being incubated under 80% N2 and20% 02. After equilibrating for 15 min, the NaH'4CO3was tipped into the main compartment, and the reac-tion was allowed to proceed for 1 hr. The flasks werethen removed from the water bath, and filter papersaturated with 40% KOH was placed into each centerwell. The flasks were stoppered, and the trichloro-acetic acid was tipped into the main compartment.After allowing the remaining dissolved CO2 to evolve,1.0-ml samples were removed from the main com-partment and assayed for radioactivity by using aPackard Tri-Carb liquid scintillation spectrometer.The scintillation fluid in each vial consisted of 3.0 mlof Triton X-100 and 6.0 ml of a toluene solution con-taining 0.4% PPO and 0.01% POPOP. The resultswere corrected for controls incubated under N2 and02.

Nitrogen determination. Inorganic nitrogen uptakewas followed using Nessler's reagent. Cells wereharvested by centrifugation at 2 C, washed, andsuspended to 2.0 OD in media containing 0.01%MgSO4.7 H20, 0.001% Fe(NH4)2SO4.6 H20, and106 ,umoles (NH4)2SO4 in 0.04 M potassium phos-phate buffer (pH 6.0). The cell suspension (50 ml)was added to flasks and incubated under an atmos-phere of 70% H2, 20% 02, 10% CO2. Samples werewithdrawn at the indicated time intervals and chilledin an ice bath to stop nitrogen uptake. The suspensionswere then centrifuged at 2 C to remove the cells, andthe clear supernatant fluid was assayed for nitrogen.Treatment of the suspensions with trichloroaceticacid rather than chilling yielded no significant differ-ence.

RESULTS

The growth curves obtained when H. eutrophawas cultured heterotrophically and mixotroph-ically in a glutamate-salts medium are shown inFig. 1. Similar curves were obtained when acetate(4) or pyruvate (Fig. 2) was substituted forglutamate. With these substrates, heterotrophicgrowth rates and cell yields are about equal to thegrowth rates and yields during the initial phaseof mixotrophic incubation. The fact that glu-tamate was exhausted at the initiation of theplateau period seen in Fig. 1 was confirmed bybioassay. Thus these three substrates probably donot allow simultaneous growth to occur, as wasobserved with some other organic substrates plushydrogen (4, 18). The plateaus observed in thedicyclic curves shown in Fig. 1 and 2 indicate that

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248

FIG. 1. Growth of Hydrogenomonas eutropha inmineral-salts media containing 0.2% L-glutamate.Incubation was under 70% H2, 20% O2, 10% C02 (0)or under air (*). The arrows indicate the points atwhich culture samples were withdrawn and assayedfor their ability to incorporate ammonia (see Fig. 3).Ordinate is a logarithmic scale.

some enzyme(s) required for autotrophic growthis repressed by these substrates.

Cells grown heterotrophically in the presence ofacetate or pyruvate did not possess significantRDPC activity (Table 1). Under dual substrateconditions, there also was no significant activityof RDPC until the acetate or pyruvate was con-sumed; however, as expected, enzyme activitywas high during the second (autotrophic) phaseof growth. During mixotrophic cultivation withpyruvate, the derepression of RDPC during theplateau period can be observed by comparing thepoints at which culture samples were withdrawn(Fig. 2) with their corresponding enzyme levels(Table 1).The addition of acetate or pyruvate to an auto-

trophic extract had little effect on the RDPC ac-tivity (Table 2), providing evidence that acetateand pyruvate do not directly inhibit the enzyme.Of the organic substrates tested, only acetate

and pyruvate repressed RDPC during dual sub-strate incubation. Thus, although RDPC levelswere low during heterotrophic growth on glu-tamate, the high enzyme levels present during thefirst phase of dual substrate incubation precludedthe repression of this enzyme being responsiblefor the plateau observed with glutamate.

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- 1). The results confirmed that heterotrophic andmixotrophic growth with pyruvate and acetate

E F G H prevented autotrophic CO2 fixation, whereas celscultured mixotrophically in the presence of theother organic substrates allowed fixation to occurat high levels.

Since cells cultivated mixotrophically withC_ // B / glutamate contained a high level of RDPC, the

plateau period after glutamate exhaustion must// ,6 be explained in terms of some other function.

One major difference between heterotrophicgrowth with glutamate and autotrophic growth isthe assimilation of nitrogen. During autotrophicgrowth, H. eutropha must produce its organicnitrogen by the assimilation of ammonia, ap-parently to form glutamate. When grown onglutamate, inorganic nitrogen is not required and,in fact, may be somewhat inhibitory to growth(S:renkoski, unpublished data). It was reasoned,therefore, that the presence of glutamate mayinhibit the assimilation of inorganic nitrogen

, , , , , , during both heterotrophic and dual substrate10 20 30 incubation, and that the plateau after glutamate

exhaustion may be the time required for theT I ME(hnrs formation of a nitrogen-assimilating system. To

2. Growth of Hydrogenomonas eutropha under investigate this possibility, cells harvested fromhdic (o), heterotrophic(*) and mixtotrophic various stages of the (glutamate) mixotrophic anditditions. For heterotrophic and mixotrophic htrtohcgot uvs(i.1 eetse).3% pyruvate was added to the mineral-salts heterotrophic growth curves (Fig. 1) were testedIncubation was under air for heterotrophic for their ability to take up nitrogen (Fig. 3).

and under 70% H2, 20% 02, 10% CO2 for Cells harvested during heterotrophic growth or thehic and mixotrophic growth. The arrows indi- first phase of mixotrophic culture showed littlepoints at which culture samples were with- or no nitrogen uptake during the 80-min testid assayedfor ribulose diphosphate carboxylase period. Cells harvested from sequential points,le 1). along the plateau showed increasing abilities to

incorporate nitrogen, and a rate approaching theshaker speed at which cultures were in- autotrophic level was reached during the secondhad no noticeable effect on RDPC levels phase of the dicyclic growth curve. Cells growncetate or pyruvate was present. With cells heterotrophically or mixotrophically with pyru-ither heterotrophically or mixotrophically vate yielded nitrogen uptake rates similar to thosetose, glutamate or alanine levels of RDPC of autotrophic cultures.,enerally somewhat lower when rapid To demonstrate that the suppression of nitro-was used. The activities in Table 1 were gen uptake in cells grown on glutamate was not

re determined with cultures incubated with due to some accumulating inhibitor or otheraking. The specific activity of RDPC from function of the culture medium, cells were har-phic cultures grown with slow shaking vested from various points along the dicyclic.3 -i- 5.1 units per mg protein. Auto- growth curve, washed, and inoculated into freshcultures grown with rapid shaking showed autotrophic media. This procedure had no effectlower specific activities (43.4 ± 5.6). otheduaino th ll before adtnophicsubstrate cultivation with fructose as the on the duration of the total lag before autotrophicsubstrate gave a dicyclic growth curve growth commenced. Cells harvested previous toa plateau and similar to that seen with or at the beginning of the plateau required about(4). The high levels of RDPC present 7 hr before growth began. Cells harvested at themixotrophic cultivation with fructose cr end of the plateau showed little or no additionalare therefore in agreement with the growth lag, whereas cultures from sequential pointss. along the plateau showed decreasing lag times incultured under conditions similar to those fresh media. The total lag between the exhaustionr RDPC assay were also teste I for their or removal of glutamate, and the initiation ofto assimilate labeled bicaroonate (Table autotrophic growth was about 7 hr in all cases.

342

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organiclackingalanineduringalaninepatternm

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TABLE 1. Relative levels of ribulose diphosphate carboxylase (RDPC) and CO2 fixation duringheterotrophic and mixotrophic growth of Hydrogenomonas eutropha

Organic substrate Atmosphere Growth phase p14CrrrInoratRDPCiontivmsP

Autotrophica Exponentialb (A),c 1.00 1 .00Acetate Air Exponential 0.08 0Acetate Autotrophic Id 0 0Acetate Autotrophic II6 0.89 0.64Pyruvate Air Exponential (B) 0 0.01Pyruvate Autotrophic I (C) 0 0.02Pyruvate Autotrophic I (D) NAf 0.04Pyruvate Autotrophic Plateau (E) 0 0.02Pyruvate Autotrophic Plateau (F) 0.14 NAPyruvate Autotrophic Plateau (G) NA 0.43Pyruvate Autotrophic Plateau (H) NA 0.64Pyruvate Autotrophic 11 (1) NA 0.96Pyruva'e Autotrophic I11(J) 0.72 0.88Fructose Air Exponential 0.55 0.64Fructose Autotrophic I 0.89 0.77Fructose Autotrophic II1 0.85 1 .00Glutamate Air Exponential 0.29 0.36Glutamate Autotrophic I NA 0.74Alanine Air Exponential 0.61 0.27Alanine Autotrophic I 0.92 0.84

a70% H2, 20% 02, 10% CO2.Since only one substrate was present, activities were determined with samples from the exponential

portions of the standard growth curves.cLetters A through J correspond to the points in Fig. 2 at which the samples were harvested.d Harvested during the initial (mixotrophic) phase.- of dicyclic growth.,,Harvested during the second (autotrophic) phase of dicyclic growth.

f Not assayed.

DISCUSSIONA number of previous studies showed that

autotrophic bacteria grown in the presence oforganic compounds often contain decreased levelsof autotrophic enzymes. In most cases these de-creased enzyme levels were observed in the ab-sence of the inorganic substrate; it was usuallynot determined if the enzymes would be similarlyrepressed in the presence of toth organic andinorganic substrates. In one study, however, thefacultative autotroph Chromatium contained lowlevels of RDPC during growth on pyruvate, butmaintained high enzyme levels when both pyru-vate and thiosulfate were present. The thiosulfateapparently prevented the repression of RDPC bypyruvate (8). Our results with H. eutrophademonstrate that the repression of RDPC bypyruvate or acetate occurs in the presence as wellas the absence of the autotrophic atmosphere, andthis repression is not due to a direct effect at theenzyme level.During simultaneous cultivation with hydro-

gen and all tested organic substrates except glu-tamate, there was a correlation between thecapacity of the cells to grow autotrophically andtheir RDPC activity. Substrates such as alanine

TABLE 2. Effect of acetate and pyruvate on theactivity of ribulose diphosphate carboxylase(RDPC) in an autotrophic extract of

Hydrogenomonas eutropha

Additions RDPC (specific activity)

None 40.60.05% Pyruvate 33.00. 1% Pyruvate 40.60.2% Pyruvate 42.40.1% Acetate 32.00.2% Acetate 30.2

and fructose which supported high levels ofRDPCduring the first phase of mixotrophic growthallowed autotrophic growth to commence rapidlywhen the organic substrate was consumed. Withacetate and pyruvate the repression of RDPCduring the first growth phase necessitated thesynthesis of this enzyme before autotrophicgrowth could begin.McFadden and Tu (15) reported that when

H. facilis was grown heterotrophically on glu-tamate it contained approximately 3% of theautotrophic level of RDPC. We found, however,that H. eutropha grown on glutamate possessed

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TIME (minutes)Fio. 3. Nitrogen uptake by Hydrogenomonas eu-

tropha cells grown heterotrophically or mixotrophicallyon glutamate. The assays were performed under auto-trophic conditions with cells which had been washed andresuspended in the mineral medium. Nitrogen uptakerepresents ammonia consumed per milliliter of OD 2.0culture [0.64 mg (dry weight) /ml]. Curves I through 7represent uptake by cells harvested at the correspondingpoints in the growth curves of Fig. 1. Curve 8 repre-sents uptake by an autotrophically grown culture.

36% of the autotrophic level ofRDPC, and, whengrown mixotrophically on glutamate plus hydro-gen, the RDPC level increased to 74%. Thus arepression of RDPC by glutamate could notaccount for the plateau observed in the growthcurves during dual substrate incubation. How-ever, heterotrophic and mixotrophic growth onglutamate does directly or indirectly interferewith nitrogen uptake or assimilation, and thisdeficiency may be responsible for the temporaryinability to grow autotrophically.Of the major nitrogen assimilating catalysts,

only glutamate dehydrogenase has been found in

H. eutropha (Strenkoski, unpublished data). Itsproperties are similar to those reported for Thio-bacillus novellus (12), and it catalyzes both thesynthesis and degradation of glutamate in vitro.Since cells grown on glutamate possess highlevels of glutamate dehydrogenase which couldpresumably function biosynthetically after glu-tamate exhaustion, the control point for nitrogenassimlation in H. eutropha is probably not theassimilating enzyme itself but may be either thenitrogen uptake mechanism or the synthesis of2-oxoglutarate. Heterotrophic or mixotrorhic

growth on glutamate might therefore suppresseither of these processes.The correlation between the relative activities

ofRDPC and resting cell CO2 fixation, using cellsgrown both heterotrophically and mixotroph-ically, strongly implicates RDPC as the majorcatalyst for knaligas-supported C02 fixation inH. eutropha. This correlation can be comparedwith the parallel levels of RDPC and cell-freereductive CO2 fixation observed with H. facilis(15) and suggests a similarity between the path-ways of CO2 assimilation and their regulation inthese two species.

ACKNOWVLEDGMENT

This investigation was supported by grant NGR-09-0S-022from the National Aeronautics and Space Administration.

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8. Hurlbert, R. E., and J. Lascelles. 1963. Ribulose diphosphatecarboxylase in Thorhodaceae. J. Gen. Microbiol. 33:445-458.

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12. LeJohn, H. B., and B. E. McCrea. 1968. Evidence for twospecies ofglutamate dehydrogenases in T7hobachllus novellus.J. Bacteriol. 95:87-94.

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15. McFadden, B. A., and C. L. Tu. 1967. Regulation of auto-trophic aid heterotrophic carbon dioxide fixation in Hy-drogenomonas fachlhs. J. Bacteriol. 93:886-893.

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YDROGENOMONAS 345

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21. Trudinger, P. A. 1956. Fixation of carbon dioxide by extractsof the strict autotroph Thiobacillus denitrificans. Biochem.J. 64:274-286.

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23. Wilson, E., H. A. Stout, D. Powelson, and H. Koffler. 1953.Comparative biochemistry of the hydrogen bacteria. I.Simultaneous oxidation of hydrogen and lactate. J. Bac-teriol. 65:283-287.

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