JOURNAL OF BACTERIOLOGY, Apr. 1979, p. 99-1040021-9193/79/04-0099/06$02.00/0

Vol. 138, No. 1

Enzymology of Butyrate Fornation by Butyrivibriofibrisolvens

TERRY L. MILLER* AND SUSAN E. JENESELDivision ofLaboratories and Research, New York State Department ofHealth, Albany, New York 12201

Received for publication 17 November 1978

Butyrivibrio fibrisolvens is a major butyrate-forming species in the bovine andovine nrmen. The enzymology of butyrate formation from pyruvate was investi-gated in cell-free extracts of B. fibrisolvens Dl. Pyruvate was oxidized to acetyl-coenzyme A (CoA) in the presence of CoA.SH and benzyl viologen or flavinnucleotides. The bacterium uses thiolase, ,B-hydroxybutyryl-CoA dehydrogenase,crotonase, and crotonyl-CoA reductase to form butyryl-CoA from acetyl-CoA.Reduction of acetoacetyl-CoA to f8-hydroxybutyryl-CoA was faster with NADHthan with NADPH. Crotonyl-CoA was reduced to butyryl-CoA by NADH, butnot by NADPH, only in the presence of flavin nucleotides. Reduction of flavinnucleotides by NADH was much slower than the flavin-dependent reduction ofcrotonyl-CoA. This indicates that flavoproteins rather than free flavin partici-pated in the reduction of crotonyl-CoA. Butyryl-CoA was converted to butyrateby phosphate butyryl transferase and butyrate kinase.

Butyrivibrio fibrisolvens is a major butyrate-forming species in the bovine and ovine rumen.Some rumen protozoa produce butyrate as acarbohydrate fernentation product (10). Theonly other known important bacterial speciesthat form butyrate in the rumen are Megas-phera elsdenii and Eubacterium ruminantium(10).Bryant and Small (4) reported considerable

variation in the characteristics of the strainsthey designated as B. fibrisolvens. B. fibrisol-vens ferments a wide variety of carbohydrates,including starch and xylans, but only a fewstrains ferment cellulose. There is a great dealof variation in the proportions of carbohydratefermentation products formed by differentstrains. In addition to butyrate, B. fibrisolvensproduces acetate, formate, H2 and lactate. Somestrains produce large amounts of lactate andlittle ofthe other products, whereas other strainsproduce no lactate and form mainly acetate,formate, and butyrate.

Nutritional conditions are also reported toaffect the production of lactate versus butyrate(7). Strains also differ in their ability to eitherproduce acetate or show a net utilization ofacetate during fermentation (4, 18).

B. fibrisolvens Dl is a strain that fermentsglucose to mainly butyrate, acetate, and formateand only small amounts of lactate (4). The pres-ent study was initiated to examine the enzymol-ogy of butyrate formation by B. fibrisolvens D1,to establish a basis for future studies of the

factors that control the production of butyratein this and other strains of the species. Thisreport demonstrates that the pathway of bu-tyryl-coenzyme A (CoA) formation from acetyl-CoA is very similar to that previously shown forClostridium kluyveri (2). The pathway of for-mation of butyrate and ATP from butyryl-CoAis the same as that previously shown in thesaccharolytic and glutamate-fermenting clos-tridia (20-22).

MATERIALS AND METHODSCulture conditions. B. fibrisolvens, type strain

Dl, was obtained from the culture collection of M. P.Bryant, University of Illinois, Urbana. The organismwas grown at 370C under 100% C02 using the serum-bottle modification of the Hungate technique (11) asdescribed by Miller and Wolin (17). The medium hadthe following composition: glucose, 5 g; Trypticase, 5g; yeast extract, 2 g; KH2PO4 and K2HP04, 0.238 geach; (NH4)2S04, 1 g; NaCl, 0.475 g; MgS04. 7H20, 0.1g; CaCl2*2H20, 63 mg; butyric acid, 0.58 ml; isobutyric,2-methylbutyric, valeric, and isovaleric acids, 0.1 mleach; resazurin, 1 mg; Na2CO3, 4 g; dithiothreitol(DTT), 15.4 mg; cysteine.HClH20, 0.375 g; Na2S.9H20, 0.375 g; and distilled water to a final volume of1 liter.The organism was grown in 1.5 liters of the above

medium in serum bottle-modified 2-liter growth ves-sels (blue-print no. 07524, Wheaton Scientific, Mill-ville, N.J.) as described by Miller and Wolin (17). Afterreduction and equilibration at 370C, the medium wasinoculated with 15 ml of an overnight culture. Thecells were harvested in late log phase (after 5 to 7 h)by centrifugation atz5°C for 15 min at 16,000 x g in a

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Sorvall RC-5 centrifuge. The cell pastes (3 to 4 g, wetweight, per 1.5 liters) were stored frozen at -76°C.

Preparation of cell extracts. All steps in theprocedures for preparing cell extracts were carried outunder argon at 50C. Either 0.05 M Tris-hydrochloride(pH 7.0) containing 10- M DTT, or 0.05 M piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES, pH 7.0) con-taining 10-3 M DTT, 10-2 M sodium pyruvate, and13% (wt/vol) glycerol, was used as the resuspensionsolution. The PIPES (containing glycerol) and Trissolutions were depleted of 02 by boiling and rapidcooling before the addition of the other components.Three grams (wet weight) of frozen cell paste was

rapidly thawed in 10 ml of buffer. The thawed cellshad a tendency to clump and were resuspended bysonic disruption at 50% of maximum power in a Bron-will Biosonik 11A sonic oscillator. The thawed cellswere cooled in an ice-water bath and sonically dis-rupted for a total of 90 s with a 1-min cooling intervalafter each 30 s of disruption. The cell suspension wasbroken in a French press at 1,111 kg per cm2 andcentrifuged for 1 h at 39,000 x g. The supernatant(containing 17 to 24 mg ofprotein per ml) was collectedas the Tris or PIPES extract and stored at -76°Cunder argon. Protein was measured by the method ofLowry et al. (13) using crystalline bovine serum albu-min as a standard.Enzyme nomenclature. Enzymes used were: thi-

olase (acetyl-CoA:acetyl-CoA acetyltransferase, EC2.3.1.9); ,B-hydroxybutyryl-CoA dehydrogenase (L-3-hydroxyacyl-CoA:NAD+ oxidoreductase, EC 1.1.1.35);crotonase (L-3-hydroxyacyl-CoA hydro-lyase, EC4.2.1.17); phosphate butyryltransferase (butyryl-CoA:orthophosphate butyryltransferase, EC 2.3.1.19); bu-tyrate kinase (ATP:butyrate phosphotransferase, EC2.7.2.7); phosphate acetyltransferase (acetyl-CoA:or-thophosphate acetyltransferase, EC 2.3.1.8); and ace-tate kinase (ATP:acetate phosphotransferase, EC2.7.2.1).Enzyme assays. Under aerobic conditions, ex-

tracts actively oxidized NADH. This activity was neg-ligible when air was removed from stoppered cuvettesby evacuating and refilling with argon 10 times. Spec-trophotometric measurement of the rate of anaerobicoxidation of NADH was used to measure the activityof the enzymes involved in the formation of butyryl-CoA. The reported values were corrected for reducedpyridine nucleotide oxidation in the absence of theCoA substrate. Unless otherwise indicated, all spectro-photometric assays were conducted at room temper-ature under argon in 1-cm cuvettes in a GCA/Mac-Pherson EU-700 series spectrophotometer in the dou-ble beam mode.

,B-Hydroxybutyryl-CoA dehydrogenase was assayedby measuring acetoacetyl-CoA-dependent NAD(P)Hoxidation as described by Maden et al. (14) exceptpotassium citrate was omitted from the potassiumphosphate buffer. The reaction was initiated by theaddition of extract (18.6 ,ug of protein, Tris extract).

Thiolase was assayed indirectly by coupling theformation of acetoacetyl-CoA from acetyl-CoA withthe reduction of acetoacetyl-CoA with endogenous ,8-hydroxybutyryl-CoA dehydrogenase. The 2.0-ml re-action mixture contained 100 mM potassium phos-phate (pH 6.5), 0.12 mM NADH, and 0.6 mM acetyl-

CoA. The reaction was initiated by the addition ofextract (18.6 to 74.4 lAg of protein, Tris extract).

Crotonase was determined by measuring the for-mation of,-hydroxybutyryl-CoA from crotonyl-CoAwith the fB-hydroxybutyryl-CoA dehydrogenase-cou-pled assay ofFong and Schultz (6). The 2.0-ml reactionmixture contained 25 mM Tris (pH 7, 8, or 9), 25 mMKCI, 0.3 mM NAD, and 0.5 mM crotonyl-CoA. Thereaction was initiated by the addition of extract (ca.0.2 to 2.0 mg of protein; Tris extract), which contained,B-hydroxybutyryl-CoA dehydrogenase and crotonase.

Crotonyl-CoA reductase was assayed in a 2.0-mlreaction mixture containing 100 mM PIPES (pH 7.0),0.3mM NAD(P)H, 0.5mM crotonyl-CoA, and 0.1 mMflavin adenine dinucleotide (FAD) or flavin mononu-cleotide (FMN). The reaction was initiated by theaddition of extract (190 ,ug of protein; PIPES extract).The stoichiometry ofNADH:FAD oxidoreductase wasmeasured in a similar reaction mixture in which cro-tonyl-CoA was omitted and the concentration of FADwas 0 to 0.2 mM.The capability of extracts to catalyze the conversion

of acetyl-CoA to butyryl-CoA was measured by deter-mining the stoichiometry of NADH oxidation in thepresence of a limiting concentration of acetyl-CoA.The 2-ml reaction mixture contained 100 mM PIPES(pH 7.0), 0.2 mM acetyl-CoA, and 0.24 mM NADH.The reaction was initiated by the addition of extract(252 ug of protein, PIPES extract). The decrease inabsorbance was continuously monitored until nochange was observed over a 15-min period. At thistime 0.025 mM FAD was injected, and the decrease ofabsorbance was followed until no change occurredover a 10-min period.

Phosphate acetyltransferase and phosphate butyr-yltransferase were assayed in air by measuring thearsenolysis of acetyl phosphate and butyryl phos-phate, respectively. The 1-ml reaction mixture, modi-fied from that of Stadtman et al. (19), contained 200mM Tris (pH 8.0), 3 mM DTT, 0.2 mM CoA.SH, 50mM potassium arsenate, and ca. 10 mM acyl phos-phate (lithium salt). After equilibration at 300C for 10min, protein (0.975 or 1.95 mg, PIPES extract) wasadded. Portions were removed at 5-min intervals andanalyzed for the disappearance of the acyl phosphateby the hydroxamate method of Lipmann and Tuttle(12). Hydroxamate standards were prepared from ace-tyl phosphate after preliminary studies showed thatthe extinction coefficients of acetyl hydroxamate andbutyryl hydroxamate standards were essentially iden-tical. The absorbances at 540 nm of the hydroxamatestandards and samples were measured in 18- by 150-mm tubes in a Spectronic 70 spectrophotometer(Bausch and Lomb).

Butyrate kinase was assayed by a modification ofthe method of Twarog and Wolfe (21). The 1-mlreaction mixtures contained 50 mM Tris (pH 7.4), 4mM MnSO4, 10 mM ATP, 300 mM potassium butyr-ate, and 350mM neutralized hydroxylamine, placed inserum bottles closed with butyl rubber stoppers whichwere sealed with aluminum seals. The bottles wereevacuated and refilled 10 times with argon and incu-bated at 300C for 10 min. Extract (180 or 360 ,ug ofprotein; Tris extract) was injected; the reactions werestopped after 30 min by the addition of 1 ml of 10%o

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BUTYRATE PATHWAY OF B. FIBRISOLVENS 101

trichloroacetic acid, and the amount of hydroxamateformed was determined as described above. Acetatekinase was assayed under the same conditions exceptthat 300 mM potassium acetate replaced potassiumbutyrate as the substrate.

Pyruvate oxidation activity of PIPES extracts wasassayed spectrophotometrically as described by Miller(16). The extracts were prepared from cells grown inthe medium described above supplemented with asolution of 1.0% FeSO4 in 0.12 M HCl to a finalconcentration of 5 mg per liter of medium. The reac-tion conditions for measuring '4C02:pyruvate exchangeactivity of PIPES extracts and the determination ofradioactivity incorporated into pyruvate carboxyl werealso as described by Miller (16). The reported ex-change activity was corrected for nonspecific retentionof radioactivity in a boiled-extract control.

Materials. Butyryl phosphate (lithium salt) was agift from R. K. Thauer, Philipps Universitat, Marburg,West Germany. Potassium butyrate was prepared byneutralization of the free acid with KOH. Benzyl viol-ogen was obtained from BDH Chemicals Ltd. and wasrecrystallized as described by Michaelis and Hill (15).All other electron acceptors, substrates, cofactors, andbuffers were obtained from Sigma Chemical Co. So-dium ['4C]bicarbonate was obtained from New Eng-land Nuclear Corp.

RESULTSButyryl-CoA formation. Extracts of B. f-

brisolvens contained the four enzymes necessaryfor the conversion ofacetyl-CoA to butyryl-CoA,i.e., thiolase, f)-hydroxybutyryl-CoA dehydro-genase, crotonase, and crotonyl CoA-reductase.Except for crotonase, the specific activities areshown in Table 1. Both NADH and NADPHwere electron donors for the reduction of aceto-acetyl-CoA by,B-hydroxybutyryl-CoA dehydro-genase. The specific activity with NADH wasca. ninefold higher than with NADPH (Table1).Extracts contained crotonase activity as indi-

cated by the reduction of NAD in the presenceof crotonyl-CoA. Formation of ,B-hydroxybu-tyryl-CoA from crotonyl-CoA and oxidation by,8-hydroxybutyryl-CoA dehydrogenase wouldcause reduction of NAD by crotonyl-CoA. Noactivity was observed with NADP. The rate ofcrotonyl-CoA-dependent NAD reduction was

TABLE 1. Enzymes involved in butyrate formationSp act

Enzyme (umol/min permg of protein)

Thiolase ......................... 1.18,8-Hydroxybutyryl-CoA dehydrogenaseNAD-linked.3.14NADP-linked.0.36

Crotonyl-CoA reductase, NAD-linked 0.79Phosphate butyryltransferase.0.70Butyrate kinase.0.44

nonlinear. NAD reduction stopped when ca. 0.04,umol of NAD was reduced. The rate and extentof NAD reduction decreased as the pH wasdecreased from 9 to 7. Adding exogenous fl-hy-droxybutyryl-CoA dehydrogenase, increasingsubstrate concentrations twofold, or decreasingextract protein concentration had no effect onthe nonlinearity ofNAD reduction. Because cro-tonase activity was nonlinear under the assayconditions employed, a specific activity for thisenzyme was not calculated.The reduction of crotonyl-CoA to butyryl-

CoA with NADH by extracts required the ad-dition of a flavin nucleotide (Fig. 1). Under thesame conditions no activity was observed withNADPH. In the absence of flavin nucleotide, therate of NADH oxidation was ca. twofold lower,and NADH oxidation stopped after 1 min when0.019 mM NADH had been oxidized (Fig. 1).The specific activity of crotonyl-CoA reductasein the presence of FAD or FMN was 0.79 and0.40 ,mol of NADH oxidized per min per mg ofprotein, respectively. In the absence of crotonyl-CoA, there was a slow rate of oxidation ofNADH which was dependent on the presence offlavin nucleotide (Fig. 1). This rate was sub-tracted from the rate of the complete reactionto calculate the specific activity of crotonyl-CoAreductase shown in Table 1. Although not shownin Fig. 1, the final concentration of oxidizedNAD in the absence of crotonyl-CoA was ap-proximately equivalent to the amount of FADadded as an electron carrier. This result dem-onstrated the presence of NADH:flavin nucleo-tide oxidoreductase activity. The specific activ-ity with FAD and FMN was 0.17 and 0.022 ,Lmolof NADH oxidized per min per mg of protein,

Complete

-0.8

0

-0.4 Minus crotonyl-CoA

Minus FAD

00 2 4

MINUTES

FIG. 1. Flavin nucleotide-dependent crotonyl-CoAreductase. The complete reaction mixture, describedin the text, contained 0.3 mM NADH, 0.5 mM cro-tonyl-CoA, 0.1 mM FAD, and 95 pg ofprotein per ml.

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102 MILLER AND JENESEL

respectively.Additional experiments were conducted to

further establish the stoichiometry of NADH:FAD oxidoreductase activity. The stoichiometryof NADH oxidation with different concentra-tions of FAD as electron acceptor is shown inFig. 2. The ratio of the molar amounts ofNADHoxidized to FAD added was ca. 1:1 at or below0.1 mM FAD and slightly lower at higher con-centrations of FAD. It is possible that an equi-librium was achieved as the ratio of reducedFAD to NAD increased.An experiment was designed to determine if

extracts could catalyze cell-free synthesis of bu-tyryl-CoA from acetyl-CoA. In the presence offlavin nucleotide, the conversion of 2 mol ofacetyl-CoA to 1 mol of butyryl-CoA requires 2mol of NADH. In the absence of flavin nucleo-tide, 2 mol of acetyl-CoA would be metabolizedto crotonyl-CoA and only 1 mol ofNADH wouldbe oxidized. The stoichiometry of NADH oxi-dation in the presence ofa limiting concentrationof acetyl-CoA is given in Fig. 3. In the absenceof FAD, the molar ratio of NADH oxidized toacetyl-CoA added was 0.45. Upon addition ofFAD, an additional 0.08 mM NADH was oxi-dized, and the final molar ratio was 0.85. Thesevalues are slightly lower than the expected val-ues of 0.5 and 1.0. The acetyl-CoA used wasassumed to be 90% pure as indicated by thesupplier, and the concentration was adjustedaccordingly, but a small amount of hydrolysiswould explain the discrepancy between observedand theoretical values.Butyrate formation. Extracts catalyzed the

arsenolysis of butyryl phosphate and the for-mation of butyryl phosphate from potassiumbutyrate and ATP (Table 1). Separate experi-ments showed that arsenolysis of butyryl plios-

0.2

iE

a

0.1 0

00

z

0 0.1 0.2

FAD ADDED (mM)

FIG. 2. Stoichiometry of NADH:flavin nucleotideoxidoreductase. The reaction mixtures, described inthe text, contained 0.3 mM NADH, 95 pg ofproteinper ml, and FAD as the electron acceptor. Theamount ofNADH oxidized was calculated from thedecrease in absorbance at 340 nm after the absorb-ance change reached a final, constant value.

012

oI/

I 006_z

FAD

0o 20 40 60

MINUTES

FIG. 3. Effect of FAD on the stoichiometry ofNADH oxidation with limiting acetyl-CoA. The re-action mixture, described in the text, contained 0.24mMNADH and 0.2mM acetyl-CoA. The addition of0.025mMFAD is indicated by the arrow.

phate was dependent on the addition of CoA.SH, potassium arsenate, and protein. These re-sults show the presence ofenzymes that catalyzethe interconversion of butyryl-CoA and butyrylphosphate and the reversible formation of bu-tyryl phosphate and ADP from butyrate andATP.

Extracts also had acetyl transferase and ace-tate kinase activity. The specific activities were0.23 and 0.34 itmol per min per mg of protein,respectively. The arsenolysis of acetyl phos-phate was also dependent on CoA.SH, potas-sium arsenate, and protein.Pyruvate oxidation. Crude extracts cata-

lyzed a CoA.SH-dependent oxidation of pyru-vate with benzyl viologen, FAD, or FMN as theelectron acceptor (Table 2). No activity wasobserved with pyridine nucleotides as electronacceptors for pyruvate oxidation. Extracts alsocatalyzed the exchange of ['4C]bicarbonate intothe carboxyl group of pyruvate (Table 2). For-mate was not detected as a product of pyruvatecleavage, and extracts did not catalyze [14C]for-mate:pyruvate exchange (data not presented).

DISCUSSIONThe results of this study show that extracts of

B. fibrisolvens have the enzymatic capability toconvert 2 mol of acetyl-CoA to 1 mol of butyrateand 1 mol of ATP by the following reactions:(1) 2 acetyl-CoA -- acetoacetyl-CoA + CoA SH(2) acetoacetyl-CoA + 2H - f,-hydroxybutyryl-CoA(3) fi-hydroxybutyryl-CoA - crotonyl-CoA + H20(4) crotonyl-CoA + 2H -. butyryl-CoA(5) butyryl-CoA + Pi - butyryl-P + CoA. SH(6) butyryl-P + ADP - butyrate + ATPAcetyl-CoA, the precursor of butyrate, is

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BUTYRATE PATHWAY OF B. FIBRISOLVENS 103

TABLE 2. Pyruvate oxidoreductctseSp act

Reaction+CoA *SH -CoA * SH

Pyruvate oxidationaBenzyl viologen ...... ... 1.65 0.040FAD ............... 0.52 0.007FMN....... 0.42 0.005

'4C02:pyruvate exchangeb 115 NECa Pyruvate oxidation was determined under argon

in reaction mixtures containing 75 mM KH2PO4 (pH7.5), 1.5 mM DTT, 0.05 mM thiamine pyrophosphate,0.05 mM MnCl2, 0.04 mM CoA.SH, 10 mM sodiumpyruvate, 10 mM benzyl viologen or 0.05 mM flavinnucleotide, and 84 ,ug of protein per ml. Specific activ-ity is expressed as micromoles of electron acceptorreduced per minute per milligram of protein.

b '4C02:pyruvate exchange was determined underargon in reaction mixtures containing 75mM KH2PO4(pH 6.5), 1.5 mM DTT, 5 mM thiamine pyrophos-phate, 5mM MnCl2, 0.02mM CoA *SH, 20mM sodiumpyruvate, 1 mM sodium ['4C]bicarbonate (0.05 ,LCi perttmol), and 4.5 mg of protein per ml. Specific activityis expressed as disintegrations per minute per micro-mole of pyruvate carboxyl per 10 min per milligram ofprotein.

c NE, Not examined.

formed by the CoA. SH-dependent oxidation ofpyruvate, which is coupled to the reduction offlavin nucleotides or benzyl viologen. Extractsalso catalyzed the exchange of 14CO2 into thecarboxyl group of pyruvate. A pyruvate car-boxyl:4CO2 exchange reaction was previouslydemonstrated with extracts of Bacteroides suc-cinogenes, which also oxidized pyruvate in thepresence of viologen dyes or flavin nucleotides(16). The physiological acceptor for pyruvateoxidation by B. fibrisolvens may be a flavinnucleotide or flavoprotein whose reduced fornwould be oxidized by crotonyl-CoA.The enzymology of crotonyl-CoA formation

from acetyl-CoA (reactions 1 to 3) by B. fibri-solvens is similar to that described for Clostrid-ium kluyveri by Barker (2). The pyridine nu-cleotide specificity ofthe ,B-hydroxybutyryl-CoAdehydrogenase of B. fibrisolvens is similar tothat reported in C. pasteuranium, C. butyricum,C. butylicum, C. tetanomorphum, C. roseum,and C. rubrum, in which NADH is the preferredelectron donor for acetoacetyl-CoA reduction(9). C. kluyveri, however, has two distinct ,B-hydroxybutyryl-CoA dehydrogenases: a soluble,NADPH-specific enzyme and a particulateNADH-specific enzyme (8, 14). The NADPH-specific enzyme has a 30-fold-higher specific ac-tivity than the NADH-specific enzyme. Madenet al. (14) suggested that the NADPH-specificenzyme of C. kluyveri functions in butyrate for-

mation whereas the NADH-specific enzyme isinvolved in caproate formation. B. fibrisolvenshas a NAD-specific glyceraldehyde-3-phosphatedehydrogenase (unpublished data), suggestingthat NADH generated during glucose catabo-lism is the primary electron donor for acetoace-tyl-CoA reduction. This is also the case for car-bohydrate-fermenting clostridia (9). The reduc-tion of NADP by the ,B-hydroxybutyryl-CoAdehydrogenase of B. fibrisolvens and the sac-charolytic and lysine-fermenting clostridia mayserve as a NADPH-generating system for bio-synthetic reactions.Crotonase was present in B. fibrisolvens. The

enzyme was assayed by coupling the hydrationof crotonyl-CoA to ,8-hydroxybutyryl-CoA withthe oxidation of the latter compound to aceto-acetyl-CoA and NADH. The observed nonline-arity of NAD reduction may have been due tothe reversibility of the dehydrogenase reaction,i.e., the reoxidation of NADH by acetoacetyl-CoA. It was not possible to measure crotonasedirectly by following the formation of the doublebond of crotonyl-CoA upon dehydration of ,B-hydroxybutyryl-CoA, due to the high absorb-ance of crude extracts at 263 nm.

B. fibrisolvens reduces crotonyl-CoA withNADH but not NADPH by a flavin nucleotide-dependent crotonyl-CoA reductase. The reduc-tase is more active with FAD than with FMN.Although NADH:flavin nucleotide oxidoreduc-tase activity is present in extracts, the rates ofreduction of FAD and FMN are much slowerthan the flavin-dependent reduction of crotonyl-CoA. This indicates that protein-bound ratherthan free flavin nucleotides participate in thereduction of crotonyl-CoA by NADH. Baldwinand Milligan (1) showed that a partially purifiedbutyryl-CoA dehydrogenase from the bacteriumMegasphera elsdenii required an electron-trans-ferring protein fraction to reduce crotonyl-CoAwith NADH. The butyryl-CoA dehydrogenasefrom this organism was further purified by Engeland Massey (5) and shown to contain FAD asits prosthetic group. Two electron-transferringflavoproteins have also been isolated from M.elsdenii and shown to mediate the reduction ofpurified butyryl-CoA dehydrogenase withNADH as the electron donor (3, 23). One of theelectron-transferring flavoproteins can also usereducing equivalents derived from lactate to re-duce the butyryl-CoA dehydrogenase enzyme(3). Purification of the B. fibrisolvens crotonyl-CoA reductase is necessary to establish the na-ture of the component proteins and to clarifythe role of flavin nucleotides. B. fibrisolvens andM. elsdenii are the only two butyrate-producingbacteria in which the cell-free formation of bu-tyryl-CoA from crotonyl-CoA has been demon-

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strated. In extracts of C. kluyveri, the enzymehas been measured by following the butyryl-CoA-dependent reduction of artificial electronacceptors (2, 8).The reactions leading to the formation of bu-

tyrate from butyryl-CoA by B. fibrisolvens ap-pear to be smilar to those described in C. butyr-icum and C. tetanwmorphum (20-22). Butyryl-CoA is metabolized via butyryl phosphate tobutyrate and ATP by reactions catalyzed byphosphate butyryltransferase and butyrate ki-nase. Extracts of B. fibrisolvens also had phos-phate acetyltransferase and acetate kinase activ-ity. Phosphate butyryltransferase and butyratekinase were purified from C. butyricum (20, 22).Neither enzyme was active with the respectiveacetyl substrates.

In generaL this study shows that the pathwaysof butyrate formation from acetyl-CoA by B.fibrisolvens and the saccharolytic clostridia areessentially the same. Studies of the activities ofthe individual pathway enzymes in strain Dland other strains of B. fibrisolvens should helpto elucidate the enzymatic basis for the largedifferences in concentrations of specific fermen-tation products found between srains and withdifferent growth conditions, and also the differ-ences between strains that do and do not showa net uptake of acetate during fermentation.

ACKNOWLEDGMENTSThis investigation was supported by National Science

Foundation research grant PCM75-15326 and Public HealthService grant AI-12461 from the National Institute of Allergyand Infectious Diseases.

LITERATURE CITED1. Baldwin, R. L, and L P. Milligan. 1964. Electron

transport in Peptosreptococcus elsdenii. Biochem. Bio-phys. Acta 92:424-432.

2. Barker, H. A. 1966. Bacterial fermentations. John Wiley& Sons Inc., New York.

3. Broekman, He L*, and W. A. Wood. 1975. Electron-transferring flavoprotein ofPeptostreptococcus eLsdeniithat funtions in the reduction of acrylyl-coenzyme A.J. Bactenol. 124:1447-1453.

4. Bryant, ML P., and N. Small. 1966. The anaerobic mon-otichous butyric acid-producing curved rod-shapedbacteria of the rumen. J. Bacteriol. 72:16-21.

5. EngeL P. C., and V. Masey. 1971. The purification andproperties of butyryl-coenzyme A dehydrogenas fromPeptostreptococcus eisdenii. Biochem. J. 125:879-887.

6. Fong, J. C., and H. Schultz. 1977. Purification andproperties of pig heart crotonase and the presence ofshort chain and long chain enoyl coenzyme A hydra-tases in pig and guinea pig tissues. J. Biol. Chem. 252:542-647.

7. Gill, J. W., and K. W. King. 1958. Nutritional charac-teristics of a butyrivibrio. J. BacterioL 75:666-673.

8. Hillmer, P., and G. Gott8chalk. 1972. Particulate natureof enzymes involved in the fermentation of ethanol andacetate by Clostridium kluyveri. FEBS Lett. 21:351-354.

9. Hugo, H. V., S. Schoberth, V. K. Madan, and G.Gottschalk. 1972. Coenzyme specificity ofdehydrogen-ase and fermentation of pyruvate by clostridia. Arch.Mikrobiol. 87:189-202.

10. Hungte, R. E. 1966. The rumen and its microbes. Aca-demic Press, New York.

11. Hungate, R. E. 1969. A roll tube method for cultivationof strict anaerobes, p. 117-132. In J. R. Norris and D.W. Ribbons (ed.), Methods in microbiology, vol. 3B.Academic Press Inc., New York.

12. Llpmann, F., and L, C. Tuttle. 1945. A specific micro-method for the deternination of acyl phosphates. J.Biol. Chem. 159:21-28.

13. Lowry, O. H., N. J. Rosebrough, A. L Farr, and R. J.Randall. 1951. Protein meaurement with the Folinphenol reagent. J. Biol. Chem. 193:266-275.

14. Maden, V. K., P. Hillmer, and G. Gottschalk. 1973.Purification and properties of NADP-dependent L(+)-3-hydroxybutyryl-CoA dehydrogenase from Clostrid-jum kluyveri. Eur. J. Biochem. 32:51-56.

15. MlchaelHs, L, and E. S. Hill. 1933. The viologen indica-tors. J. Gen. Physiol. 16:859-873.

16. Miller, T. L 1978. The pathway of formation of acetateand succinate from pyruvate by Bacteroides succino-genes. Arch. Microbiol. 117:145-152.

17. MI}ler, T. L, and M. J. Wolin. 1974. A serum bottlemodification of the Hungate technique for cultivatingobligate anaerobes. Appl. Microbiol. 27:986-987.

18. Shane, B. S., L Gouws, and A. Kistner. 1969. Cellulo-lytic bacteria occurring in the rumen of sheep condi-tioned to low-protein teft hay. J. Gen. Microbiol. 55:445-457.

19. Stadtman, E. R., G. D. Novelli, and F. Llpmann. 1951.Coenzyme A function in and acetyl transfer by thephosphotransacetylase system. J. Biol. Chem. 191:365-376.

20. Twarog, R., and R. S. Wolfe. 1962. Enzymatic phospho-rylation of butyrate. J. Biol. Chem. 237:2474-2477.

21. Twarog, R., and R. S. Wolfe. 1963. Role of butyrylphosphate in the energy metabolism of ClostridiumtetanomoiphwL J. Bacteriol. 86:112-117.

22. Valentine, R. C., and R. S. Wolfe. 1960. Purificationand role of phosphotransbutyrylase. J. Biol. Chem. 235:1948-1952.

23. Wh eld, C. D., and S. G. Mayhew. 1974. Purificationand properties of electron-tranderring flavoproteinfrom Peptostreptococcus eldenji. J. Biol. Chem. 249:2801-2810.

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