Vol. 137, No. 1JOURNAL OF BACTERIOLOGY, Jan. 1979, p. 332-3390021-9193/79/01-0332/08$02.00/0

Acetate Assimilation Pathway of Methanosarcina barkeriP. J. WEIMERt AND J. G. ZEIKUS*

Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706

Received for publication 2 November 1978

The pathway of acetate assimilation in Methanosarcina barkeri was deter-mined from analysis of the position of label in alanine, aspartate, and glutamateformed in cells grown in the presence of ['4C]acetate and by measurement ofenzVrme activities in cell extracts. The specific radioactivity of glutamate fromcells grown on [1-"C]- or [2-14C]acetate was approxiimately twice that of aspartate.The methyl and carboxyl carbons of acetate were incorporated into aspartate andglutamate to similar extents. Degradation studies revealed that acetate was notsignificantly incorporated into the Cl of alanine, Cl or C4 of aspartate, or Cl ofglutamate. The C5 of glutamate, however, was partially derived from the carboxylcarbon of acetate. Cell extracts were found to contain the following enzymeactivities, in nanomoles per minute per milligram of protein at 370C: F420-linkedpyruvate synthase, 170; citrate synthase, 0.7; aconitase, 55; oxidized nicotinamideadenine dinucleotide phosphate-linked isocitrate dehydrogenase, 75; and oxidizednicotinamide adenine dinucleotide-linked malate dehydrogenase, 76. The resultsindicate that M. barkeri assimilates acetate into alanine and aspartate viapyruvate and oxaloacetate and into glutamate via citrate, isocitrate, and a-ketoglutarate. The data reveal differences in the metabolism of M. barkeri andMethanobacterium thermoautotrophicum and similarities in the assimilation ofacetate between M. barkeri and other anaerobic bacteria, such as Clostridiumkluyveri.

The methanogenic bacteria are a morpholog-ically diverse group of anaerobes which catabol-ize a variety of one-carbon compounds and ace-tate to methane. Phylogenetic studies (5, 23)indicate that these microbes are a distinct lifeform separate from other bacteria and from alleucaryotic organisms. Little is known about thebiochemical details of methanogenic metabo-lism. All described species oxidize H2 and reduceC02 (25), employing unique coenzymes (e.g.,F420, a low reduction-oxidation potential electroncarrier, and coenzyme M, a one-carbon carrier).Many species can utilize C02 as the sole carbonsource for growth (25).

Short-term 14002 fixation products in twophylogenetically diverse methanogens, Meth-anobacterium thermoautotrophicum and Meth-anosarcina barkeri, are similar and include co-enzyme M derivatives and alanine, aspartate,and glutamate (4). Detailed studies of C02 fixa-tion products (4) and enzyme activities (26) inM. thermoautotrophicum revealed the absenceof the Calvin, serine, or hexulose phosphatepathways of one-carbon assimilation. Enzymestudies (26) and analysis of the position of label

t Present address: Central Research and Development De-partment, The Experimental Station, E. I. DuPont de Nem-ours and Co., Wilmington, DE 19898.

in alanine, aspartate, and glutamate formed from[U-"C]acetate by whole cells (6) demonstratedthat acetate is assimilated by M. thermoautotro-phicum via a reductive carboxylic acid pathway.M. barkeri is the most metabolically diverse

4"methanogen known (15, 21a, 21b, 25). It differsfrom other described species in its ability togrow not only chemoautotrophically on H2-C02,but also chemoorganotrophically with methanol,methylamine, or acetate as energy sources. Thisspecies, like M. thermoautotrophicum, lacks theCalvin, serine, or hexulose phosphate pathwaysof one-carbon assimilation (4, 21a). M. barkerigrown on methanol-C02 derives half of its cellcarbon from each substrate (21a), employingunique one-carbon carriers (4) for both catabo-lism and initial anabolic reactions (i.e., synthesisof a two-carbon internediate). Acetate is a keyintermediate in the metabolism of M. barkeriand can contribute up to 60% of the cell carbonformed during heterotrophic growth on acetateor methanol, and during autotrophic growth onH2-C02 (21a, 21b). We report here on the path-way of acetate in the synthesis of cell carbon byM. barkeri.

MATERIALS AND METHODSChemicals, enzymes, and gases. All chemicals

were reagent grade. Commercial enzymes and bio-332

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ACETATE ASSIMILATION IN METHANOSARCINA 333

chemicals were obtained from Sigma Chemical Co.,St. Louis, Mo. The radioactive chemicals [1-"C]- and[2-"C]acetate (55 mCi/mmol), ["C]sodium carbonate(60 mCi/mmol), ["Cjmethanol (50 mCi/mmol), and[U-"C]alanine, -aspartate, and -glutamate (each 75mCi/mmol) were purchased from Amersham/Searle,Arlington Heights, Ill. Chemicals for scintillationcounting were obtained from Research Products In-ternational, Rochester, N.Y. Gases were obtained fromMatheson, Joliet, Ill., either as pure gases or as amixture of H2 and CO2 (80:20, vol/vol).Organisms and cultivation. M. barkeri strains

MS and UBS (21a) were cultivated under strictlyanaerobic conditions in PBBW, a phosphate-buffered,vitamin-supplemented mineral medium, as describedpreviously (21a). The medium was supplemented withmethanol (50 to 200 mM) and acetate (1 to 5 mM) asindicated below. A gas phase of N2 or H2-CO2 wasused.

Clostridium tetanomorphum strain X405 was cul-tivated in neoprene-stoppered Erlenmeyer flasks at30°C in a medium that contained, per liter: sodiumglutamate, 5 g; yeast extract, 2 g; MgCl2.6H20, 0.2 g;trace mineral solution (3), 10 ml; and reazurin, 0.002 g.After the medium was autoclaved for 15 min at 15lb/in2, sterile potassium phosphate was added to afinal concentration of 50 mM (pH 7.4). The mediumwas gassed with N2 and reduced with 1.5 mM Na2Sbefore inoculation. Approximately 1.5 g (wet weight)of cells were produced per liter of culture in 10 h.

Escherichia coli strain WB5-H52 was cultured at37°C in cotton-plugged shake flasks which contained,per liter: KH2PO4, 6 g; K2HPO4*3H20, 14 g;MgSO4.7H20, 0.2 g; (NH4)2SO4, 1 g; sodium acetate,5 g; sodium citrate, 0.5 g; and trace mineral solution,lO ml.Incorporation of 14C-labeled substrates into

cell material and isolation of "4C-labeled gluta-mate, aspartate, and alanine. M. barkeri was grownin sealed 2-liter Erlenmeyer flasks that contained 1liter of PBBW supplemented with 50 mM methanol,1 mM sodium acetate, and a H2-C02 gas phase. Indi-vidual flasks also contained 45 to 120 ,uCi of one of thefollowing "4C-labeled substrates: [1-_4C]acetate, [2-"4C]acetate, [14C]carbonate, or [14C]methanol. Flaskswere inoculated with 50 ml of log-phase culture (grownunder similar conditions without 14C label) and wereharvested after 6 days of incubation at 37°C. Cellgrowth and the incorporation of '4C-labeled substratesinto cell material was determined as described previ-ously (21a). Protein-containing cell residue was iso-lated after fractionation of the cells by the procedureof Roberts et al. (13). Cell residue from each labeling(-40 mg) was suspended in 5 ml of 6 N HCI anddistributed into four 2-ml lyophilization ampoules(Wheaton Scientific). The ampoules were sealed undera vacuum and autoclaved at 1210C for 6 to 8 h. Theresulting hydrolysate was centrifuged, and the super-natant was evaporated to dryness and suspended in1.3 ml of distilled water.The protein hydrolysate was applied to a Dowex

AG1-X10 acetate column (24 by 1 cm [ID]). Thesample was eluted with 0.5 N acetic acid and 3-mlfractions were collected, 50 Ad of which was counted ina scintillation counter to localize peaks. Nonacidic

amino acids, glutamate, and aspartate eluted as sepa-rate peaks at volumes of 9 to 15, 30 to 39, and 66 to 72ml, respectively. Fractions corresponding to individualpeaks were pooled, rotary evaporated to dryness at30°C, and suspended in 1.2 ml of water. The identityand purity of glutamate and aspartate were confirmedby thin-layer chromatography and electrophoresis (4).The quantities of these two amino acids were deter-mined by the ninhydrin method of Bishop and Sims(2).

Alanine was separated from other nonacidic aminoacids by enzymatic conversion to lactate, with subse-quent purification by column chromatography by theprocedures of Fuchs et al. (6). The quantity of lactaterecovered was determined enzymatically (10).Degradation of glutamate, aspartate, and lac-

tate (- alanine). The C1 of glutamate was specificallyremoved by using Clostridium welchii glutamate de-carboxylase. The reaction was conducted in open scin-tillation vials that contained 50 pl (- 100 nmol or 6,900to 37,000 dpm) of ['4C]glutamate, 0.30 U of enzyme,and 0.60 ml of 0.80 M potassium acetate buffer (pH4.9). The reaction mixture was incubated at 37°C for5 h before gassing with a gentle stream of CO2 for 1min. After addition of 10 ml of toluene-Triton scintil-lation solution (21a), radioactivity in the vials wasmeasured by scintillation counting. The amount of"CO2 removed was calculated as the difference be-tween the initial and residual disintegrations per min-ute. [U-"4C]glutamate was used as a control in thedegradation procedure.The C5 of glutamate was removed by treatment

with a cell suspension of C. tetanomorphum (9, 21).Log-phase cells from 1 liter of culture were harvestedby centrifugation and washed with an anaerobic solu-tion of 20mM K2504. The suspension was centrifugedagain, and the pellet was suspended in 6 ml of 50 mMpotassium phosphate (pH 7.0) reduced with 1.5 mMNa2S. Experiments were conducted in Warburg flaskssealed with neoprene stoppers at both the main andsidearm openings. The reaction mixture (main com-partment) contained 50 pl of ["4C]glutamate sample,60 ,ul of 50 mM L-glutamate, 0.60 ml of C. tetanomor-phum cell suspension, and an argon gas phase. After6 h of incubation at 30°C, 0.40 ml of80% methanol-20%ethanolamine was added to the sidearm, and 0.50 mlof 4 N H2SO4 was added to the main compartment.After a 6-h equilibration at 4°C, the contents of thesidearm were removed by syringe and placed in ascintillation vial that contained 5 ml of toluene scin-tillation solution (4). It was assured that all trapped"4CO2 was removed and counted by four successive 1-ml washes of the sidearm with methanol-etha-nolamine. Residual 14C was calculated after counting0.20 ml of the reaction mixture (main compartment)in 10 ml of toluene-Triton scintillation solution. [U-"4C]glutamate was used as a control in the degradationprocedure.The C1 and C4 of aspartate were removed by a

modification of the chloramine T procedure of Kembleand McPherson (11). The reaction was performed inWarburg flasks as described above; the reaction mix-ture contained 50 ul (4-100 nmol or 10,000 to 35,100dpm) of [14C]aspartate sample and 1 ml of chloramineT reagent (6). [U-"4C]aspartate served as a control.

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The C1 of lactate was removed as C02 by theprocedure of Fuchs et al. (6). The reaction was per-formed in Warburg flasks as described above; thereaction mixture contained 200 p1 (-30 to 60 nmol or2,200 to 9,200 dpm) of ["C]ilactate in 0.53 ml of waterand 1 ml of 6 mM K2Cr207 (in 9 N H2SO4). [U-"C]-lactate, derived enzymatically from [U-"4C]alanine,served as a control.

Preparation of cell extracts and measurementof enzyme activities. Cell extracts of M. barkeriwere prepared as described previously (21a). Extractsof C. tetanomorphum and E. coli were prepared sim-ilarly, except that dithiothreitol was omitted from thereaction mixture. Protein was measured by the pro-cedure of Lowry et al. (12), using bovine serum albu-min as standard.

All assays were performed at 370C. Spectrophoto-metric assays were performed under anaerobic condi-tions by the general procedures of Zeikus et al. (26).Pyruvate synthase, a-ketoglutarate dehydrogenase,fumarate reductase, and malate dehydrogenase weremeasured by the methods of Zeikus et al. (26).

Isocitrate dehydrogenase was measured spectro-photometrically by following the isocitrate-dependentreduction of pyridine nucleotides at 340 nm (ew - 6.22mM-' cm-'). The reaction mixture contained, in 1ml, 95 mM tris(hydroxymethyl)aminomethane-hy-drochloride (pH 7.8), 5 mM MgCl2, 0.25 mM oxi-dized nicotinamide adenine dinucleotide phosphate(NADP+) or oxidized nicotinamide adenine dinucleo-tide (NAD+), 5 mM dithiothreitol, and 10 pl of extract(-0.1 mg of protein). The reaction was initiated by theaddition of 1 mM threo-D.L.-isocitrate.

Aconitase was measured spectrophotometrically bycoupling the conversion of citrate to isocitrate withthe isocitrate dehydrogenase reaction. The reactionmixture contained, in 1 ml, 95 mM potassium tricine(pH 8.6), 5 mM MgCl2, 0.25 mM NADP+, 0.44 U ofisocitrate dehydrogenase (from pig heart), and 10 pl ofextract. The reaction was initiated by the addition of2 mM sodium citrate.

Citrate synthase was measured by determination of[4C]aspartate conversion to ["4C]citrate by the pro-cedure of Gottschalk and Barker (7). Before the assay,cell extracts were eluted with distilled water througha Sephadex G-25 column (bed dimensions, 15 by 1.1cm) contained within an anaerobic glovebox (CoyManufacturing Co., Ann Arbor, Mich.). The reactionmixture contained, per 1.05 ml under an argon gasphase, 50 mM potassium phosphate buffer (pH 7.4), 1mM MgSO4, 25 mM ,B-mercaptoethanol, 0.13 U ofavidin, 20 mM L-malate, 2.5 mM sodium citrate, 0.5mM coenzyme A, 20 mM sodium acetyl phosphate,17.6 U of phosphotransacetylase, 15mM a-ketoglutar-ate, 17 U of glutamate/oxaloacetate aminotransferase,and protein solution (1.39 mg of M. barkeri extractprotein and/or 186 U of pig heart citrate synthase).The reaction was initiated by the addition of 2.5 mM[U-'4C]aspartate (600 dpm/nmol) and terminated byboiling the reaction mixture for 3 min. Upon comple-tion of the assay, ["4C]citrate formation was measuredafter separation of compounds by thin-layer chroma-tography and electrophoresis, localization of 14C labelby autoradiography, and measurement by liquid scin-

tillation counting, as described by Daniels and Zeikus(4).

Citrate lyase was measured spectrophotometricallyby following the rate of citrate-dependent NADHoxidation in the presence of exogenous malate dehy-drogenase (18). Extracts were prepared as describedabove or were preincubated in the presence of 1.75mM acetic anhydride, as described by Beuscher et al.(1).The limits of assay sensitivity for the enzyme activ-

ities examined were as follows, in nanomoles per min-ute per milligram of protein: NADH- or NADPH-coupled assays, 0.5; methyl or benzyl viologen-coupledassays, 0.2; fumarase, 2.0; phosphoenolpyruvate car-boxylase, 0.2; and citrate synthase, 0.04.

RESULTSIncorporation of ["C]acetate into gluta-

mate, aspartate, and alanine. The quantityand position of 14C label in glutamate, aspartate,and alanine isolated from cell protein of "C-labeledM. barkeri cells were analyzed to provideinformation on the pathway of acetate assimi-lation. Cells were grown in the simultaneouspresence of two energy sources (methanol andH2-C02) to maxnmize cell yield. An initial ace-tate concentration of 1 mM was selected topermit adequate incorporation of acetate withhigh specific activity. Under such conditions ap-proximately 18% of the total cell carbon wasderived from added acetate, with the remainderderived from C02 and methanol. For both glu-tamate and aspartate, similar quantities ofamino acid were derived from the methyl andcarboxyl carbon atoms of acetate (Table 1). Theamount of acetate incorporated per mole of glu-tamate was approximately twice the amountincorporated per mole of aspartate when thedata were corrected for total carbon recovery.The results of partial degradation of gluta-

mate, aspartate, and lactate (=alanine) isolatedfrom cells grown in the presence of "4C-labeledacetate or C02 are shown in Table 2. Significantquantities of acetate were not incorporated intothe C1 of glutamate, aspartate, or alanine or intothe C4 of aspartate. However, the C5 of gluta-mate was partially derived from the carboxylcarbon of acetate. Incorporation of "4C02 intocarbon atoms of glutamate, aspartate, and ala-nine was quantitatively similar to the expectedrandom distribution ofC02 into all carbon atomsof the molecule (i.e., it was similar to that ofuniversally labeled "4C-amino acid standards).The quantities of 14C removed from the U-14C-labeled standards were close to expected theo-retical values, indicating the validity of the deg-radation procedure.Acetate assimilation enzymes. Cell ex-

tracts of M. barkeri contained a pyruvate syn-

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ACETATE ASSIMILATION IN METHANOSARCINA 335

TABLE 1. Specific radioactivities of aspartate and glutamate obtained from protein ofM. barkeri cellsgrown in the presence of["4C]acetatea

Specific radioactivity (dpm/nmol) of: Amt of carbon atomAmino acid [C40]acetate carbon incorporated (nmol

Aminoacid Substrate per nmol of aminoAmino acid Substrate acd bacid)bAspartate C1 (carboxyl) 22.0 100.0 0.28

C2 (methyl) 46.6 260.0 0.23Glutamate C1 (carboxyl) 29.1 100.0 0.48

C2 (methyl) 89.0 260.0 0.57a Cells were grown for 6 days in separate 2-liter flasks, as described in text. The fraction of total cell carbon

derived from each substrate was: Cl acetate, 0.076; C2 acetate, 0.107; C02, 0.598; methanol, 0.218.b The data were corrected for total label recovery based on incorporation of [1-'4C]acetate, [2-'4C] acetate,

'4C02, and '4CH30H and were calculated as follows: number of carbon atoms incorporated per amino acidmolecule = ((number of carbon atoms per amino acid molecule) ([specific radioactivity of amino acid]/[specificradioactivity of substrate]))/1([specific radioactivity of amino acid]/[specific radioactivity of substrate]) for allfour substrates.

TABLE 2. Partial degradation ofglutamate, aspartate, and lactate (=alanine) isolated from cell protein ofM. barkeri grown in the presence of['4C]acetate or '4CO2a

% Of 14C recovered as 14CO2Position of carbon 14Clbld btt * ted Control (U-"C-labeled stan-Compound degraded atoms recovered -aee susrae ncorpora dard)

[1-'4C]acetate [2-'4C]acetate 14C02 Experimental TheoreticalGlutamate C1 0 0 18.5 18.4 20

C5 27.2 3.6 19.8 19.6 20Aspartate Cl + C4 3.4 2.2 41.4 41.7 50Lactate (=alanine) C1 2.8 4.5 37.0 30.8 33.3

a Degradation procedures are described in the text. Results are expressed as mean values of duplicate ortriplicate experiments.

thase activity. Enzyme activity, measured in thedirection of pyruvate oxidation, used F420 ormethyl viologen as the electron acceptor. Activ-ity was dependent upon coenzyme A, pyruvate,and extract. Exposure of extracts to air beforeor during the assay resulted in complete loss ofactivity. The activity was stabilized by additionof pyruvate to the cell suspension before cellbreakage. Pyruvate-stabilized extracts exhibitedhigher activities and retained activity longerthan did unstabilized extracts. The linearity ofthe reaction rate with time was extended byaddition of arsenate, which established a coen-zyme A-regenerating system due to the presenceof endogenous phosphotransacetylase activity.A maximum specific activity of 170 nmol/minper mg of protein was observed for pyruvatesynthase, when methyl viologen was used as theelectron acceptor.M. barkeri extracts contained a citrate syn-

thase activity that was dependent upon the ad-dition of active extract; no activity was observedwhen boiled extract was substituted for activeextract. The [14C]citrate produced comigratedwith ["4C]citrate produced from commercial cit-

rate synthase. The average rate of citrate for-mation, calculated over the 1-h reaction period,was 0.7 nmol/min per mg of protein.

Cell extracts catalyzed the conversion of cit-rate to isocitrate. The Vm. of the aconitaseactivity was 55 nmol/min per mg of protein.Activity was not affected by exposure of extractsto air, but was inhibited 50% by 1.0 mM DL-fluorocitrate (at a citrate concentration of 2mM). Inhibition by fluorocitrate was partiallyovercome by addition of large amounts (50 mM)of citrate.

Cell extracts contained an NADP+-linked iso-citrate dehydrogenase; no activity was observedwhen NAD+ was substituted for NADP+. In thedirection of a-ketoglutarate synthesis, the en-zyme displayed a pH optimum of 7.5 to 7.7 intris(hydroxymethyl)aminomethane -hydrochlo-ride buffer. The activity was not affected by air.Substrate concentrations yielding half-maximalreaction rates for threo-DnL.-isocitrate andNADP+ were 35 and 45 ,uM, respectively. TheVma,, of the enzyme in extracts was 75 nmol/minper mg of protein. Activity in the reverse direc-tion (isocitrate synthesis) was less than 1% that

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336 WEIMER AND ZEIKUS

of the rate found in the direction of a-ketoglu-tarate synthesis.

Cell extracts contained a malate dehydrogen-ase activity. NADH, but not NADPH, served asan electron donor. In the direction of malatesynthesis, the activity displayed a broad pHoptimum at 7.9 in tris(hydroxymethyl)-aminomethane hydrochloride and a V,,. of 76nmol/min per mg of protein. Substrate concen-trations yielding half-maximal reaction rates foroxaloacetate and NADH were 66 and 45 ttM,respectively.

In addition to the above enzymes, smarizedin Table 3, the following activities were alsoobserved in cell extracts; phosphotransacetylase,phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase (NADPH preferredover NADH), glutamate/pyruvate aminotrans-ferase, and glutamate/oxaloacetate aminotrans-ferase. Attempts to demonstrate an acetate ki-nase activity were unsuccessful, due to a veryactive (113 nmol/min per mg of protein) adeno-sine triphosphatase which interfered with theassay.The following enzymes were not detected in

cell extracts (positive controls are listed in pa-rentheses): a-ketoglutarate dehydrogenase/syn-thase, fmarase, f*marate reductase, phospho-enolpyruvate carboxylase (M. thermoautotro-phicum); malate synthase, citrate lyase (E. coli).Addition ofM. barkeri extracts to these positivecontrols did not affect the reaction rate.

DISCUSSIONAcetate is a key compound of intermediary

metabolism in M. barkeri. It is interconvertiblewith, if not identical to, a two-carbon anabolicintermediate, even during autotrophic or meth-

TABLE 3. Enzyme activities in cell extracts ofM.barkeria

Sp act at

Enzyme Substrate 37°C (nmol/min permg ofprotein)

Pyruvate synthase Pyruvate + coen- 170zyme A + MVb

Citrate synthase Oxaloacetate + 0.7acetyl coenzymeA

Aconitase Citrate 55Isocitrate dehy- Isocitrate + 75drogenase NADP+

Malate dehydro- Oxaloacetate + 76genase NADH

a The assay conditions are described in the text.b Methyl viologen (MV) was used as electron accep-

tor.

J. BACTERIOL.

ylotrophic growth (21a). Acetate also contrib-utes up to 60% of the cell carbon formed duringgrowth both in mineral-acetate media supple-mented with H2-CO2 and/or methanol and incomplex media with acetate as the primary en-ergy source (21b).The data presented here indicate that M. bar-

keri incorporates acetate into glutamate, aspar-tate, and alanine by the pathway shown in Fig.1 and 2. The proposed pathway is based uponthe following observations: (i) the C5 of gluta-mate is partially derived from the carboxyl groupof acetate; (ii) acetate is not significantly incor-porated into the Cl of glutamate, aspartate, oralanine or into the C4 of aspartate; and (iii) cellextracts contain significant quantities of pyru-vate synthase, citrate synthase, aconitase, andisocitrate dehydrogenase.The acetate assimilation pathway of M. bar-

keri is similar to that reported for certain non-methanogenic anaerobes, such as Clostridiumkluyveri (7, 16, 17). However, fundamental dif-ferences between M. barkeri and C. kluyveri areapparent with respect to characteristics of indi-vidual enzymes. The pyruvate synthase of M.barkeri, like that of M. thermoautotrophicum(26) is F420 linked, whereas the pyruvate syn-thase of C. kluyveri is ferredoxin linked. C. kluy-veri also contains a citrate synthase of unusualstereospecificity (R type), which results in theincorporation of the carboxyl group of acetateinto the Cl (rather than the C5) of glutamate(7, 17, 20). The incorporation of the carboxylcarbon of acetate into the C5 of glutamate byM. barkeri suggests that this organism containsa citrate synthase of normal (S type) stereospec-ificity (8). However, the possibility that M. bar-keri contains an R-type citrate synthase and astereospecifically unusual aconitase cannot beexcluded by the present data. Although the cit-

*ET1E

AAETh-PcDl

MMA1EA 4.- (brALNTA1E C1IM7EFAA 2 1 3CIM

W4E ISOCIThAT

"-IrA.urwE --- QUTN4TEFIG. 1. Pathway of acetate assimilation into ala-

nine, aspartate, and glutamate by M. barkeri. Solidlines indicate enzymatic reactions demonstrated incell extracts. Broken lines indicate the direction ofconversions suggested by labeling data.

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ACETATE ASSIMILATION IN METHANOSARCINA 337

14*2 AAmiIE

C-30HOOCH&214lf1

> f

COOH

I

HDCOOH

I,

OOH*C=O

*d2*aJoH

cnH

*COOHi*~~~~~~~~~~~~~~~~~

71*42

CLUTNPTE

FIG. 2. Fate of "C-labeled acetate incorporatedinto alanine, aspartate, and glutamate by the path-way proposed in Fig. 1. Citrate synthase and aconi-tase are assumed to have normal stereospecificities.

rate synthase activity in M. barkeri extractsappears to be quantitatively small, it is never-theless almost twice that reported for C. kluyveri(7). Measured activity could undoubtedly beenhanced by optimizing the reaction conditionsand by measuring the initial, rather than theaverage, reaction rate.The acetate assimilation pathway of M. bar-

keri differs from that of M. thermoautotrophi-cum, which synthesizes glutamate via the reac-tions malate -. fumarate -_ succinate -. a-ke-

toglutarate (6, 26). The findings presented hereclearly establish that major differences in inter-mediary metabolism exist among diverse meth-anogenic genera. It is interesting to note thatMethanosarcina and Methanobacterium havebeen recognized as separate subdivisions of themethanogenic group on the basis of wide vari-ance in ribosomal RNA oligonucleotide se-quences (5, 23).

Several other differences in the intermediarymetabolism of M. barkeri and M. thernoauto-

trophicum should be noted. Succinate can beforned from malate in M. thermoautotrophicumvia fumarate reductase (26). Succinate may bean important metabolite in M. barkeri, whichsynthesizes corrinoids in quantities sufficient tocolor cell extracts red (15). However, the path-way ofsuccinate synthesis in M. barkeri remainsobscure in the absence of fumarate reductaseand a-ketoglutarate dehydeogenase. Certainother anaerobes (e.g., C. kluyveri) that lackthese enzymes (16) synthesize succinate by adifferent pathway. The absence of a fumaratereductase eliminates the possibility of a mecha-nism ofATP generation involving fumarate-suc-cinate interconversion coupled to electron trans-port-mediated phosphorylation (19). Such a sys-tem has been suggested to operate in some meth-anogens (G. Gottschalk, S. Schoberth, and K.Braun, In Abstracts of the International Sym-posium on Microbial Growth on Cl compounds.Scientific Center for Biological Research,U.S.S.R. Academy of Science, Pushchino,U.S.S.R., 1977). In addition, the specific enzymesinvolving the interconversion of pyruvate, phos-phoenolpyruvate, and oxaloacetate remain to beelucidated in methanogens. Phosphoenolpyru-vate carboxylase is present inM. thermoautotro-phicum (26), but was not detectable in M. bar-keri.No methanogenic species examined to date

contains a complete tricarboxylic acid cycle(either oxidative or reductive). It is interestingto note, however, that M. barkeri and M. ther-moautotrophicum together contain all the nec-essary enzymes for a complete tricarboxylic acidcycle. This fact may be of evolutionary signifi-cance because these species are distinguished asmembers of separate phylogenetic subdivisions(5) among the methanogen group. Thus, it willbe of interest to examine the tricarboxylic acidcycle enzymes of other methanogens in thesetwo major phylogenetic subdivisions to see howtheir enzymes compare with those ofM. barkeriand M. thermoautotrophicum.

Considerable discussion has been generated inthe past concerning the definition and biochem-ical basis of autotrophy in the microbial world,including the recent suggestion (22) that auto-trophs should be regarded as microorganismsthat can synthesize all of their cellular constit-uents from one or more C1 compounds (i.e., thisgroup should include methylotrophs). In thisregard the biochemical features of M. barkeriandM. thermoautotrophicum are worth discuss-ing. Both of these species can grow lithotrophi-cally on H2-CO2 as an energy source and, asautotrophs (28, 21a), can synthesize all cellularintermediates from CO2. Neither species em-

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ploys the Calvin, serine, or hexulose phosphatepathways (4, 21a), and both species lack a com-plete tricarboxylic acid cycle. The absence of acomplete tricarboxylic acid cycle has been sug-gested as the biochemical basis of obligate au-totrophy (14). Neither M. barkeri nor M. ther-moautotrophicum is an obligate autotroph, asboth species can synthesize a significant portionof cell carbon from acetate when grown chemo-lithotrophically on H2-C02 (4, 6, 21a, 27, 28).However, M. barkeri differs significantly fromM. thermoautotrophicum because, in additionto chemolithotrophic growth on H2-C02, it alsogrows methylotrophically on CH30H alone (21a)or heterotrophically on acetate as the majorenergy source in a complex medium (21b). Thebiochemical features that unite the catabolicmachinery of these diverse species is the pres-ence of unique one-carbon carriers (4) that areused for growth on Cl compounds (includingH2-C02, C0, CH3NH2, and CH30H) and on ac-etate (21a, 21b). M. barkeri, but not M. ther-moautotrophicum, contains the necessary cata-bolic enzymes for growth on methanol or ace-tate. Furthermore, detailed labeling studies in-dicate thatM. barkeri employs these one-carboncarriers anabolically for synthesis of a two-car-bon intermediate when grown on methanol(21a). Thus, both catabolism and anabolism ofmethanogens involves biochemical pathwaysthat appear to be unique to this microbial group.The reason methanogens are not able to growon substrates other than one-carbon compoundsor acetate may ultimately prove to be a reflec-tion of the absence of specific transport mecha-nisms for the uptake of organics rather thanfrom the absence of a complete tricarboxylicacid cycle.

ACKNOWLEDGMENTS

This research was supported by the College of Agriculturaland Life Sciences, University of Wisconsin-Madison, and inpart by grant 73-01511-A01 from the National Science Foun-dation. P.J.W. was supported by a Cellular and MolecularBiology Training Grant (5T32GM07215) from the NationalInstitutes of Health.We thank P. Hegge and J. Lobos for excellent technical

assistance, W. Kenealy and L. Daniels for stimulating discus-sions, J. Lindquist for providing cultures of C. tetanomorphumand E. coli, and R. K. Thauer for communicating unpublishedresults.

LITERATURE CITED

1. Beuscher, N., F. Mayer, and G. Gottschalk. 1974.Citrate lyase from Rhodopseudomonas gelatinosa: pu-rification, electron microscopy, and subunit structure.Arch. Microbiol. 100:307-328.

2. Bishop, R., and A. Sims. 1975. Problems in the deter-mination of specific radioactivity of uniformly '4C-la-beled amino acids after their reaction with ninhydrin.Analyst 100:369-376.

3. Daniels, L., G. Fuchs, R. K. Thauer, and J. G. Zeikus.1977. Carbon monoxide oxidation by methanogenic bac-teria. J. Bacteriol. 132:118-126.

4. Daniels, L., and J. G. Zeikus. 1978. One-carbon metab-olism in methanogenic bacteria: analysis of short-termfixation products of "CO2 and "CH30H incorporatedinto whole cells. J. Bacteriol. 136:75-84.

5. Fox, G. E., L. J. Magrum, W. E. Balch, R. S. Wolfe,and C. R. Woese. 1977. Classification of methanogenicbacteria by 16S ribosomal RNA characterization. Proc.Natl. Acad. Sci. U.S.A. 74:4537-4541.

6. Fuchs, G., E. Stupperich, and R. K. Thauer. 1978.Acetate assimilation and the synthesis of alanine, as-partate, and glutamate by Methanobacterium ther-moautotrophicum. Arch. Microbiol. 117:61-66.

7. Gottschalk, G., and H. A. Barker. 1966. Synthesis ofglutamate and citrate by Clostridium kluyveri. A newtype of citrate synthase. Biochemistry 5:1125-1133.

8. Hanson, K. R., and I. A. Rose. 1963. The absolutestereochemical course of citrate biosynthesis. Proc.Natl. Acad. Sci. U.S.A. 50:981-988.

9. Hoare, D. S. 1963. Photoassimilation of acetate by Rho-dospirillum rubrum. Biochem. J. 87:284-301.

10. Hohorst, H.-J. 1965. L(')-lactate. Determination withlactic dehydrogenase, p. 266-270. In H. Bergmeyer (ed.),Methods of enzymatic analysis, 2nd ed. Academic PressInc., New York.

11. Kemble, A. R., and H. T. McPherson. 1934. Determi-nation of monoamino-monocarboxylic acids by quanti-tative paper chromatography. Biochem. J. 56:548-555.

12. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-275.

13. Roberts, R. B., P. H. Abelson, D. B. Cowie, E. T.Bolton, and R. J. Britton. 1957. Studies of biosyn-thesis in Escherichia coli. Carnegie Institute, Washing-ton, D.C.

14. Smith, A. J., J. London, and R. Y. Stanier. 1967.Biochemical basis of obligate autotrophy in blue-greenalgae and thiobacilli. J. Bacteriol. 94:973-983.

15. Stadtman, T. C. 1967. Methane fermentation. Annu. Rev.Microbiol. 21:212-242.

16. Stern, J. R., and G. Bambers. 1966. Glutamate biosyn-thesis in anaerobic bacteria. I. Citrate synthetase path-way of glutamate synthesis in Clostridium kluyveri.Biochemistry 5:1113-1118.

17. Stern, J. R., C. S. Hegre, and G. Bambers. 1966.Glutamate biosynthesis in anaerobic bacteria. II. Ster-eospecificities of aconitase and citrate synthetase inClostridium kluyveri. Biochemistry 5:1119-1124.

18. Takeda, Y., F. Suzuki, and H. Inoue. 1969. ATP-citratelyase (citrate cleavage enzyme). Methods Enzymol. 13:153-160.

19. Thauer, R. K., K. Jungermann, and K. Decker. 1977.Energy conservation in chemotrophic anaerobic bacte-ria. Bacteriol. Rev. 41:100-180.

20. Tominson, N. 1954. Carbon dioxide and acetate utiliza-tion by Clostridium kluyveri. III. A new path of glu-tamic acid synthesis. J. Biol. Chem. 209:605-609.

21. Wachemain, J. T., and H. A. Barker. 1955. Tracerexperiments of glutamate fermentation by Clostridiumtetanomorphum. J. Biol. Chem. 217:695-702.

21a.Weimer, P. J., and J. G. Zeikus. 1978. One carbonmetabolism in methanogenic bacteria. Cellular charac-terization and growth of Methanosarcina barkeri. Arch.Microbiol. 119:49-57.

21b.Weimer, P. J., and J. G. Zeikus. 1978. Acetate metab-olism in Methanosarcina barkeri. Arch. Microbiol. 119:175-182.

22. Whittenbury, R., and D. P. Keiley. 1977. Autotrophy:a conceptual phoenix, p. 121-149. In Symposia of theSociety for General Microbiology, vol. 27. Cambridge

J. BACTERIOL.

on February 13, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

ACETATE ASSIMILATION IN METHANOSARCINA 339

University Press, London.23. Woese, C. R., and G. E. Fox. 1977. The phylogenetic

structure of the procaryotic domain: the primary king-doms. Proc. Natl. Acad. Sci. U.S.A. 74:5088-5090.

24. Wolfe, R. S. 1971. Microbial formation of methane. Adv.Microbial Physiol. 6:107-146.

25. Zeikus, J. G. 1977. The biology of methanogenic bacteria.Bacteriol. Rev. 41:514-541.

26. Zeikus, J. G., G. Fuchs, W. Kenealy, and R. K.

Thauer. 1977. Oxidoreductases involved in cell carbonsynthesis of Methanobacterium thermoautotrophicum.J. Bacteriol. 132:604-613.

27. Zeikus, J. G., P. J. Weimer, D. Nelson, and L. Daniels.1975. Bacterial methanogenesis: acetate as a methaneprecursor in pure culture. Arch. Microbiol. 104:129-134.

28. Zeikus, J. G., and R. S. Wolfe. 1972. Methanobacteriumthernoautotrophicum sp.n., an anaerobic, autotrophic,extreme thermophile. J. Bacteriol. 109:707-713.

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