Vol. 137, No. 1JouRNAL oF BAoCCRIOLOGY, Jan. 1979, p. 256-2630021-9193/79/o1-0256/08$02.00/o

Specificity and Biological Distribution of Coenzyme M (2-Mercaptoethanesulfonic Acid)

W. E. BALCH AND R. S. WOLFE*Department ofMicrobiology, University of Illinois, Urbana, Illinois 61801

Received for publication 18 October 1978

The specificity of the growth requirement ofMethanobacterium ruminantiumstrain Ml for a new coenzyme, 2-mercaptoethanesulfonic acid (HS-CoM), wasexamined. A variety of derivatives, analogs, and potential biosynthetic precursorsof coenzyme M were tested; only a restricted range of thioether, thioester, andthiocarbonate derivatives of the cofactor were found to replace the HS-CoMrequirement. Bromoethanesulfonic acid (BrCH2CH2SO3 ), a halogenated analogof HS-CoM, potently inhibited the growth response. No coenzyme was detect-able in a wide range of nonmethanogenic eucaryotic tissues and procaryoticorganisms. However, all methanogens available in pure culture exhibited highlevels of coenzyme M which ranged from 0.3 to 16 nmol/mg of dry weight.

Taylor and Wolfe (21) identified 2-mercapto-ethanesulfonic acid (HS-CoM) as a new co-factor of methyl-transfer reactions in methano-gens; three active forms of the coenzyme weredescribed: 2,2'-dithiodiethanesulfonic acid,(S-CoM)2; 2-mercaptoethanesulfonic acid,HS-CoM; and 2-(methylthio)ethanesulfonicacid, CH3-S-CoM. It was observed that Meth-anobacterium ruminantium strain Ml requiredcoenzyme M for growth (20), and recent studieshave detailed a sensitive and quantitative rela-tionship of coenzyme M, final cell yield, andtotal methane production (2). A half-maimalgrowth yield at 25 nM HS-CoM was observed,representing a level of sensitivity similar to thatof other vitamin bioassays. The requirement forHS-CoM could be replaced by CH3-S-CoMon an equimolar basis or by the disulfide forn(S-CoM)2 at a one-half molar equivalent con-centration of HS-CoM.

Since cofactors generally have been found tobe ubiquitous, and since methanogens have beenshown to be only distantly related to typicalprocaryotes (8), it was of interest to establishthe distribution of coenzyme M in nature. M.ruminantium Ml provided the basis for a bioas-say to determine coenzyme M levels in otherbiological material. To establish the specificityof the assay, we tested a variety of derivativesand structural analogs of HS-CoM, as well aspotential biosynthetic precursors or alternativemethyl-donor compounds, for their ability toreplace the coenzyme as a growth factor. Herewe assess the specificity of the coenzyme as agrowth factor and document its uniqueness tothe methanogens.

MATERIALS AND METHODS

Organisms and growth conditions. Methano-bacterium ruminantium strain PS, Methanobacte-rium strain M.o.H., Methanobacterium formicicumstrain MF, and Methanosarcina barkeri strain MSwere obtained from M. P. Bryant, Department ofDairy Science, University of Illinois. Methanobacte-rium strain M.o.H.G. was obtained from S. Schoberth,Institut fir Mikrobiologie der Universitit Gottingen,Gottingen, West Germany (Deutsche Sammiung vonMikroorganismen, DSM-862). Methanobacterium ar-bophilicum strain DHI was obtained from J. G. Zeikus,Department of Bacteriology, University of Wisconsin.Methanobacterium strain AZ was obtained from A.Zehnder, Swiss Federal Institute for Water PollutionControL Diibendorf, Switzerland. Methanospirillumhungatu strain JF1 (R. S. Wolfe) and the methano-gens listed above (except Ml) were grown in thefollowing medium (values in g/liter): K2HPO4, 0.45;KH2PO4, 0.45; (NH4)2SO4, 0.22; NaCl, 0.45;MgSO4.7H20, 0.09; CaCl262H20, 0.06; FeSO4.7H20,0.002; resazurin, 0.001; sodium formate, 3.0; sodiumacetate, 2.5; NaHCO3, 6.0; L-cysteine HCl.H20, 0.5;Na2S.9H20, 0.5; trace mineral solution and vitaminsolution (5), 10 ml each; and yeast extract (Difco) andTrypticase (BBL), 2.0 g each. Methanobacterium mo-bile strain BP and Methanococcus strain PS wereobtained from Paul Smith, Department of Microbiol-ogy, University of Florida, Gainesville. M. mobile wascultured as deseibed previously (2); the medium wassupplemented with a partially purified unidentifiedcofactor found in rumen fluid. Methanococcus strainPS was cultured in the following medium (values ing/liter): NaCl, 18; K2HPO4, 0.14; KCI, 0.35;MgCl262H20, 2.7; MgSO4.7H20, 3.5; NH4C1, 0.25;CaCl262H20, 0.14; Fe(NH4)2SO4, 0.002; sodium acetate,0.75; resazurin, 0.001; NaHCO3, 6.0; L-cysteineHCl.H20, 0.5; Na2S.9H20, 0.5; trace mineral solution(5), 10 ml; trace vitamin solution (5), 20 ml; and yeast

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SPECIFICITY AND DISTRIBUTION OF COENZYME M 257

extract (Difco) and Trypticase (BBL), 2 g each. Ly-ophilized cells of two new marine isolates from theBlack Sea (Black Sea isolate JR-1) and the CariacoTrench (Cariaco isolate JR-1) were provided by J.Romesser, Department of Microbiology, University ofIllinois, and were cultivated in the medium describedfor Methanococcus strain PS. Methanococcus vannie-lii was provided by H. Hippe from the DeutscheSammlung von Mikroorganismen (DSM-1224).Growth conditions were as described previously (12).Procedures for preparation of media and for growth oforganisms have been described previously (2).

Bioassay. Assay culture medium, anaerobic pro-cedures, and growth conditions for M. ruminantiumstrain Ml were as described by Balch and Wolfe (2).Each routine bioassay consisted of 72 tubes of culturemedium which included an HS-CoM standard curve(0, 6, 12, 25, 50, and 638 nM HS-CoM) and 17 levelsof material to be assayed, each known level and un-known additive being assayed in triplicate. Prior totransfer to an anaerobic chamber, each solution to beadded to the basal medium was degassed for 20 minby bubbling with N2 at a flow rate of 20 ml/min. Drypowders were allowed to degas for at least 6 h in theanaerobic chamber prior to the addition of degassedwater. Each supplement to the basal medium wasassayed in the presence and absence of a knownamount of HS-CoM. Growth response (absorbanceat 660 nm, Am6) was followed with a Bausch & LombSpectronic 20 spectrophotometer, an 18mm light pathbeing used.Extraction of coenzyme M from tissues. The

following procedures were used to extract coenzymeM from dry, powdered tissues: procedure I (21) in-volved hot water extraction followed by methanolextraction of the dried water extract under aerobicconditions; procedure II included hot water extraction(10 ml of water to 1 g [dry weight] of tissue) at 1000Cfor 30 min under an N2 atmosphere. When procedureII was modified by addition of 10 mM dithiothreitol,10 mM 2-mercaptoethanol, 50 mM NaBH4, 1 N HCl,or 1 N NaOH, this is indicated in Results. The water-soluble or methanol-soluble fraction was lyophilizedto dryness and resuspended in a minimal volume ofwater prior to addition to the bioassay. Addition ofextract supplements to the basal medium never ex-ceeded 5% of medium volume. Each tissue was ex-tracted in the presence and absence of a known quan-tity of HS-CoM to determine efficiency of recoveryfor a given tissue and extraction procedure.

Synthesis of mixed disulfides. Mixed disulfidesof HS-CoM and 2-mercaptoethanol, cysteine, cys-teamine, and glutathione were synthesized by the fol-lowing procedure: 6.3 pmol ofHS-CoM and 6.3 ymolof R'-SH were combined in 200 ,ul of water and 20i1 of concentrated NH40H under a 100% oxygen at-mosphere. The reaction was allowed to proceed for 1h at room temperature. Subsequently, samples fromthe reaction mixture were chromatographed in thefirst dimension on cellulose-coated sheets (10 by 10cm) with fluorescent indicator (no. 13254, Eastman,Rochester, N.Y.) in a solvent system of acetone-wa-ter-concentrated NH40H (16:2:1). The dried chromat-ogram was subjected to electrophoresis in the seconddimension (Desaga-Brinkman thin-layer electropho-

resis apparatus) at pH 2.0 in formic acid-aceticacid-water (13:58:1,000) at 400 V, 12 mA, for 15 to 60min. Under these conditions the mixed disulfidesshowed Rf and electrophoretic mobilities distinct fromstarting products and other intermediates. The mixeddisulfides were located by use of UV light, scrapedfrom the chromatogram, and eluted with water whichcontained 1% NH40H. Sulfhydryl content was quan-titatively determined with Ellman's reagent (7) afterreduction with NaBH4. Amino groups were qualita-tively determined with ninhydrin aerosol spray (SigmaChemical Co., St. Louis, Mo.). Table 1 summarizes theproperties of the synthesized mixed disulfides.

Tissues. Dry, powdered beef and hog tissues wereobtained from United States Biochemical Corp.,Cleveland, Ohio. Rabbit tissues were obtained fromPel-Freez Bio-Animals, Rogers, Ark. Algal and fungaltissues were obtained from Carolina Biological SupplyCo., Burlington, N.C. Marine plant and animal tissueswere obtained from the Marine Biological Laboratory,Woods Hole, Mass.

Chemicals. The following compounds were pre-pared as described by Gunsalus et al. (10): HS-CoM,CHs-S-CoM, (S-CoM)2, 2-(ethylthio)ethanesul-fonic acid (CH3CH2-S-CoM), 2-(propylthio)ethane-sulfonic acid (CH3CH2CH2-S-CoM), 3-mercapto-propanesulfonic acid, 4-mercaptobutanesulfonic acid,3-(methylthio)propanesulfonic acid, 4-(methylthio)-butanesulfonic acid, and 2,2'-thiodiethanesulfonicacid (03SCH2CH2-S-CoM). The following com-pounds were provided by J. A. Romesser: 2-(dimeth-

+ylsulfonium)ethanesulfonic acid [(CH3)2-S-CoM](21), 2,2'- (methylenedithio) diethanesulfonic acid[CH2-(S-CoM)2], 2-(carboxymethylthio)ethanesul-fonic acid (-OOCCH2-S-CoM), 2-(hydroxymethyl-thio)ethanesulfonic acid (HOCH2-S-CoM), 2-(formylthio)ethanesulfonic acid (CHO-S-CoM), 2-(acetylthio)ethanesulfonic acid (CH3CO-S-CoM),and 2- (carbomethoxythio)ethanesulfonic acid(CH30CO-S-CoM). (3S-CoM)2 was prepared asdescribed by Balch and Wolfe (3). Other chemicalswere obtained as described previously (10).

RESULTSSpecificity of the HS-CoM requirement.

Potential biosynthetic precursors, knownmethyl-donor compounds, and structural ana-logs or derivatives of HS-CoM were examinedfor growth-factor activity. A 1,000-fold excess ofselected compounds which may be potential bio-synthetic precursors to HS-CoM, or altematemethyl-donor compounds, were assayed forgrowth-factor activity in the presence of agrowth-limiting concentration of HS-CoM(12.5 nM). The growth response to HS-CoM(A6ss = 0.4) was neither stimulated nor inhibitedwithin the range of 0.05 A6w-by the addition of:(i) a variety of amino acids (cysteine, methio-nine, homoserine, cystathionine), CasaminoAcids, cysteic acid, or hypotaurine; (ii) complexorganic mixtures such as yeast extract (Difco),

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TABLE 1. Properties ofHS-compounds and their mixed disulfides analyzed by thin-layer chromatography(TLC) and thin-layer electrophoresis (TLE)a

Electrophoretic migra- Reaction Reactiontion Reaction with EU_ with Ell-

Compound Rf (TLC) with nin- ma, man's re-To anode To cathode hydrin man a t agent after(mm) (mm) agent reductionb

HS--CoM .......................... 0.50 21 - + +(S-CoM)2 ......................... 0.38 22 - - +Cysteine ............................ 0.50 5 + + +Cystine ............................ 0.54 6 + - +,8-Mercaptoethanol (reduced) ......C...D'.8-Mercaptoethanol (oxidized) ......... NDCysteamine ......................... 1.00 37 + + +CystAMine .......................... 1.00 37 + - +Glutathione (reduced) ........ ....... 0.05 3 + + +Glutathione (oxidized) ........ ....... 0.05 3 + - +HS-CoM:cysteine .......... ........ 0.22 8 + - +HS-CoM:cysteamine ........ ....... 0.71 0 + - +HS-CoM:,-mercaptoethanol ........ 0.81 12 - - +HS-CoM:glutathione ........ ....... 0.10 10 + - +

'TLC was performed on Eastman cellulose-coated sheet (no. 13254) in the first dimension with acetone-water-concentrated NH4OH (16:2:1); TLE was performed in the second dimension at pH 2.0 with formicacid-acetic acid-water (13:58:1,000) for 15 min at 400 V, 12 mA.

b Dry chromatogram was exposed to concentrated NH4OH vapors for 5 min and was subsequently sprayedwith fresh NaBH4 (20 mg/ml) in water. After 15 min, unreacted NaBH4 was removed by spraying thechromatogram with 1 M HCO. After drying, the sulfhydryl-containing spot was located by use of ElIman'sreagent (7).

'Volatilized; not detectable.

Trypticase (BBL), peptone or tryptone (Difco),malt extract, beef heart infusion, beef extract, orphytone (BBL); (iii) dithiothreitol, glutathione,djenkolic acid, pyridoxine, biotin, lipoate, coen-zyme A, pantothenate, and folate derivatives;and (iv) known methyl-donor compounds suchas choline, betaine, methyl-BI2, and S-adenosyl-methionine.

Structural analogs of HS-CoM were quanti-tatively evaluated for growth-factor activity. Re-placement of the terminal sulfhydryl by a hy-droxyl, amino, or sulfonate group to form is-ethionic acid, taurine, or ethanedisulfonic acid,respectively, neither stimulated nor inhibitedthe response of strain Ml to a growth-limitingamount ofHS-CoM (12.5 nM, Amo = 0.4), whenadded at a 1,000-fold molar excess (Aeeo = 0.4 ±0.05). Similar results were obtained by replace-ment of the terminal sulfonate group by anamino, hydroxyl, carboxyl, or sulfhydryl groupto form, respectively, cysteamine, 2-mercapto-ethanol, 3-mercaptopropionate, or ethanedi-thiol; or by lengthening of the ethylene carbonbridge by one or two additional methylenegroups to form 3-mercaptopropanesulfonic acidor 4-mercaptobutanesulfonic acid. The followingstructural analogs of CH3-S-CoM showed nocoenzyme activity: N-methyltaurine, 3-(methyl-thio)propanesulfonate, 4-(methylthio)butane-sulfonate, 3-(methylthio)propionate, 2-meth-

ylthioethylamine, and 2-(methylthio)ethanol.Results of addition to the bioassay of

thioether, thioester, and thiocarbonate deriva-tives of coenzyme M are presented in Table 2;CH3-S-CoM, CH3CH2-S-CoM, HOCH2-S-CoM, CHO-S-CoM, CH3CO-S-CoM,and CH3OCO-S-CoM were found to stoichi-ometrically replace the requirement forHS-CoM in the medium. In contrast, the pro-pyl thioether derivative CH3CH2CH2-

+S-CoM, (CH3)2-S-CoM, -OOCCH2-S-CoM, -03SCH2CH2-S-CoM, -03SCH2CH2-SCH2-S-CoM, and adenosyl-S-CoMexpressed coenzyme activity only when presentat 10,000-fold molar excess concentration, thecofactor activity being due to trace levels of HS-CoM present in the preparation.Cofactor activity of mixed disulfides

(R-SS-CoM). Since the oxidized form ofHS-CoM, (S-CoM)2, retained full growth-fac-tor activity (Table 2), we tested mixed disulfidesformed between HS-CoM and other mercap-tans to determine their activity. Such interac-tions might readily occur during coenzyme ex-traction from tissues, possibly masking the pres-ence of the coenzyme. As shown in Table 3, theratios of mdles ofHS-CoM detectable with thebioassay per mole of sulflhydryl or disulfideadded to the bioassay tube were 0.94, 0.51, 0.56,

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SPECIFICITY AND DISTRIBUTION OF COENZYME M 259

0.49, and 0.41 for (S-COM)2, HS-CoM:cys-teine, HS-CoM:cysteamine, HS-CoM:2-mer-captoethanoL and HS-CoM:glutathione, re-spectively. These values are within experimentalerror of the theoretically expected values of 1 for(S-CoM)2 and 0.5 for the mixed disulfides, ifthe bound coenzyme were to exhibit full activity.However, results of thin-layer chromatographyof (S-CoM)2, which was added to the complexgrowth medium containing 0.05% cysteine, indi-cated that the disulfide was unstable and inequilibrium with cysteine, yielding predomi-nantly the reduced form, HS-CoM, and themixed disulfide of HS-CoM and cysteine. Re-duction of other mixed disulfides would be ex-

TABLE 2. Cofactor activity of derivatives ofHS-CoM

Compound Analog concn requiredto yieldAm -0.4 (nM)

H-S-CoM 12.5CH3-S-CoM 13.8

CHsCH2-S-CoM 13.8CH3CH2CH2-S-CoM 9,200.0

+(CH3)2-S-COM 40,000.0

-OOCCH2-S-CoM 66,000.0HOCHr--S-CoM 11.2CHO-S-CoM 12.5

CH3CO-S-CoM 10.9CH3OCO-S-CoM 11.5

-O3SCH2CH2S-S-CoM 6.7O3SCH2CH2-S-CoM 70,000.0

CoM-S-CHg--S-CoM 35,000.0Adenosyl-S-CoM >12,500.0

'All derivatives were filter-sterilized and addedaseptically to sterile medium.

pected to occur, yielding the free, reduced fornof the coenzyme.Inhibition of growth-factor activity by

BrCH2CH2SO3-. Bromoethanesulfonic acid, a

potent inhibitor of methanogenesis in cell-freeextracts (10), inhibited the growth response ofstrain Ml to HS-CoM. As shown in Fig. 1,

inhibition of growth was proportional to themolar ratio of HS-CoM to BrCH2CH2SO3 .

Cells grown in the presence of excess HS-CoM(0.21 to 6.2 uM) were incubated for 24 h prior tothe addition of inhibitor (0.44 ,uM). At aHS-CoM to BrCH2CH2SO3C molar ratiogreater than 15:1, no effect on the growth re-sponse was observed. In contrast, cells exhibitedmarked inhibition of growth below this value,inhibition being complete at a molar ratio of 1:1.When the order of addition was reversed, cellgrowth was completely inhibited for up to 48 hafter the addition of HS-CoM. An identicaleffect was observed with ClCH2CH2SO3 .

Br(CH2)3SO3 did not inhibit growth at a molarexcess of 1,000-fold, a most dramatic finding.Assay ofextracts from nonmethanogenic

tissue. Supplementation of the bioassay me-dium with concentrates from a variety of non-methanogenic tissues (extraction procedures Iand II and modifications of II in Materials andMethods) failed to demonstrate the presence ofHS-CoM activity in these tissues, HS-CoMbeing quantitatively recoverable when added totissues prior to extraction. In addition, assaymedium was supplemented with HS-CoM todetermine inhibition or stimuation of growth bythe extract concentrate. Table 4 lists typicalresults obtained for four tissues: beef kidney

TABLE 3. Cofactor activity ofmixed disulfides, CoM-SS-RA B

Chemicalay Bioassay RatioCompound

Total sulfhydryl HS-CoM (aM) B/A

CoM-S-S-CoM 250 235 0.94H

CoM-S-SCH2CH2C-COO 520 266 0.51I

NH2CoM-S-SCH2CH2NH2 600 333 0.56CoM-S-SCH2CH20H 320 157 0.49

H H NH21/

CoM-S-SCH2CNHCOCH2CH2C 320 133 0.41

CONHCH2COO- COO-'A 50-nmol amount of CoM-SS-R was mixed with 100 gmol of fresh NaBH4 in 200 id of water for 12 h

under N2; the reaction was terminated by addition of 50 ,ul of 6 M HCl and neutrlized with 50 id of 6M NaOH.R-SH was assayed by use of Eiman's reagent (7).

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260 BALCH AND WOLFE

'ILl

I

[PM]

0.44

0.440.44

RATIOHS-COM

I143D47.014.3

4.7

0.441 0.47

Time (hr)FIG. 1. Effect ofBrCH2CH2SO3- on the growth response ofM. ruminantium Ml to HS-CoM. Inhibitor (I)

was added to cells growing in increasing concentrations of HS-CoM at the time indicated. The controlrepresents growth response to each level ofHS-CoM in the absence of inhibitor.

powder, Aeromonas liquefaciens, Acetobacte-rium woodii (1), and a methanogen, Methano-bacterium ruminantium PS. Addition of beefkidney, Aeromonas liquefaciens, and Acetobac-terium woodii failed to produce a significant orconsistent growth response (Table 4). In con-trast, M. ruminantium PS showed a very signif-icant and consistent growth response, definingclearly a minimal coenzyme concentration of 150pmol/mg (dry weight) of cells. In general, theextraction procedure allowed clear resolution of10 pmol of coenzyme/mg (dry weight) of tissuewith the techniques outlined.Distribution of coenzyme M in nonmeth-

anogenic tissues. The tissues in which coen-zyme M was not detectable are listed below;they included a broad range of both eucaryoticand procaryotic cell types. (i) Beef tissues: heart,kidney, liver, spleen, bile salts, thymus, supra-renal, ovarian, orchic, prostate, thyroid, pan-creas, cartilage, stomach, pylorus, mammary,gastric mucin, lymphatic, and duodenal. (ii) Rattissues: spleen, testes, lung, cecum, kidney, co-lon, liver, muscle, brain, epithelium, oviduct,small intestine (washed), thyroid, and heart. (iii)Rabbit tisues: ovaries, testes, prostate, spleen,spinal cord, salivary gland, thyroid, lung, andparathyroid. (iv) Hog tissues: bile and gall. (v)

Procaryotic cell tissue: Bacillus alvei, B. cereus,Clostridium sporogenes, C. pasteurianum, C.thermoaceticum, Acetobacterium woodii, De-sulfovibrio gigas, D. vulgaris, Escherichia coli,Pseudomonas putida, P. fluorescens, Peptococ-cus aerogenes, Leuconostoc sp., Bacteroides sp.,Aeromonas liquefaciens, Myxobacter AL-1,Rhodopseudomonas sphaeroides, Spirochaetaezuelzerae, cyanobacteria strains (R. Y.Stanier-6301, 67, 14, Nostoc), methane oxidiz-ers (two unknown species), and methanol oxidiz-ers (two unknown species). (vi) Archaebacteria(24): Halobacterium halobium and Sulfolobusacidocaldarius. (vii) Algal cell tissues: Chlam-ydomonas, Euglena gracilus, Acrochaetium,Derbesia, Rhodochorton purpureum, Ectocar-pus, Porphyridium, Gymnodinium, Coccolithushuxleyi, Melosira varians, Closterium, Gloeo-cystis, Oedogonium foveolatum, Volvox aureus,Botrydiopsis arhiza, and Achnanthes brevipes.(viii) Fungal cell tissues: Allomyces, Phycomycesblakesleeanus, Entomorphthora coronata,Achyla, Physarum polycephalum, Sordariafinicola, Fusarium oxysporum, Candida pseu-dotropicalis, Rhizopus stolonifer, Aspergillusniger, Penicillium notatum, and Cunningha-mella blakeskeana. (viii) Slime mold: Dictyo-stelium discoidium. (ix) Plant tissue: spinach

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SPECIFICITY AND DISTRIBUTION OF COENZYME M 261

and onion. In addition to those listed, a diverserange of marine plant and animal tissues ob-tained from the Marine Biological Laboratory,Woods Hole, Mass., were examined and foundto lack the coenzyme.Distribution of coenzyme among meth-

ane bacteria. Methane bacteria were quanti-tatively assayed to determine the level of coen-zyme present in cells. As shown in Table 5, allstrains examined contained significant levels ofcoenzyme. Celis had an average concentration of1 to 10 nmol of coenzyme/mg of dry cell weight.Values ranged from 0.3 nmol/mg of dry cellweight for M. ruminantium Ml grown in a me-dium which contained just sufficient HS-CoMfor maximal growth yield to 16 nmol/mg of drycell weight for Methanosarcina barkeri MSgrown on methanol. Methanobacterium mobilewas grown on a medium containing exogenouscoenzyme M. The internal pool may reflect up-take of coenzyme M from the medium (3).

DISCUSSION

Results of growth studies of Methanobacte-rium ruminantium Ml show that the obligaterequirement for coenzyme M is highly specific.Potential biosynthetic precursors, alternatemethyl-donor compounds, and complex organicsupplements, as well as structural analogs ofHS-CoM, do not exhibit growth-factor activity.Only a restricted range of thioether, thioester,and thiocarbonate derivatives of HS-CoMreplaced the cofactor requirement stoichio-metrically; these include CH3-S-CoM,CH3CH2-S-CoM, HOCH2-S-CoM, CHO-S-CoM, CH3CO-S-CoM, CH3OCO-S-CoM, and the disulfide form, (S-CoM)2. Nointermediate levels of activity were observed.BrCH2CH2SO3 potently inhibits the growth re-sponse of M. ruminantium Ml to HS-CoM.Since HS-CoM is synthesized fromBrCH2CH2SO3& (10), it is essential that prepa-

TABLE 4. Typical results obtained when the bioassay was used to search for coenzymeM in tissuesExtraction conditions Growth response (maximal Am.0)

Tisue Amt"d Extraction Extract + Extractionwt) ex- proCedUreb Extract only HS-CoM in with addedtracteda assy HS--CoM

None - 1 - 0.42c 0.5d2 - 0.37 0.463 - 0.36 0.454 - 0.36 0.44

Beef kidney 10 1 0.00 0.75 0.402 0.01 0.55 0.423 0.02 0.26 0.464 0.03 0.60 0.40

Aeromonas liquefaciens 20 1 0.00 0.51 0.602 0.01 0.51 0.303 0.02 0.30 0.234 0.00 0.27 0.30

Acetobacterium woodii 10 1 0.04 0.46 0.522 0.05 0.60 0.383 0.06 0.44 0.304 0.05 0.52 0.52

Methanobacterium ruminantium strain PS 1 1 1.50 1.50 1.502 1.50 1.50 1.503 1.60 1.80 1.804 1.50 1.60 1.60

'A 100-pl amount of clarified supernatant solution (3,000 x g; 10 min), representing the indicated level oftissue, was added to the bioassay tube containing 5 ml of medium.

b Hot-water extraction (1000C for 30 min) under an N2 atmosphere in the presence of the following: (1) noadditions, (2) 10 mM dithiothreitol, (3) 1 M HCI, (4) 1 M NH4OH.

Typical response of HS-CoM (12.5 nM) in the bioassay in the presence of 100 Pi of extraction solvents 1to 4.

d Response of bioassay to HS-CoM heat-treated in water by procedures 1 to 4; a 100d portion was addedin the bioassay to a final concentration of 12.5 nM.

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TABLE 5. Levels of coenzyme M found in methanogenic bacteria (each value represents an independentdeternination from a different cell batch)

HS-CoM activity j cell- HS-CoM activity in whole cellscOrganism free extract' (nmol/mg of Amt (nmol)/mg Amt (mol)/

protein)b of dry wt of proteind

Methanobacterium thermoautotrophicum ...... 3.0,6.1,9.1,12.1 2.0 6.7M. fornmcicuwn ............................... 3.2, 31.2 8.4 17.5Methanospirillum hungatii ........ ........... 7.1 1.2 3.0Methanobacterium strain M.o.H. 17.8,19.0 6.0 12.1Methanosarcina barkeri MSH2:CO2 grown .. .................. 15.0, 20.0, 20.0, 22.0 1.5 3.0CH3OH grown ... ... .................. .. 50.0 16.2 44.4

Methanobacterium ruminantium PS ........... _ 5.0 8.3M. ruminantium Ml .......................... 0.3, 0.48 0.5 0.7M. arbophilicum ............................. - 3.3 -

M. mobil.....e.. - 0.3 1.26Methanobacterium strain AZ .................. - - 5.1Cariaco isolate JR-1 ............. ............. - 0.75Black Sea isolate JR-1........................i - 0.32Methanococcus vannieUi ......... ............. - 0.5 -

Methanococcus strain PS ..................... - 2.0

a Cell-free extracts prepared by use of a French pressure cell at 15,000 psi followed by centrifugation for 30min at 40,000 x g.

b Protein determined by the method of Lowry et al. (16).c Whole cells were extracted in water under N2 for 30 min at 100°C.d Biuret protein assay (9).'Not determined.

rations be free from the inhibitor.It is presently difficult to assess whether the

specificity of the coenzyme requirement bystrain Ml for HS-CoM is imposed at the mem-brane level or reflects the specificity of the in-tracellular enzymes. Cell-free extracts of M.thermoautotrophicum cleave a similar spectrumof active coenzyme derivatives to yieldHS-CoM (J. A. Romesser, Ph.D. thesis, Uni-versity of Illinois, Urbana, 1978). Other coen-zyme M derivatives and andogs exhibit onlyweak inhibition of CH4 production by theCH3-S-CoM methyl reductase of M. ther-moautotrophicum (10), the exception beingBrCH2CH2SO3 , a potent inhibitor of methyl-coenzyme M reductase activity. These studiesreflect the structural specificity of the enzymecomplex. The relationship between the specific-ity of intracellular enzymes for the coenzyme,the properties of the bioassay, and the transportsystem for HS-CoM in M. ruminantium Ml isdiscussed in detail elsewhere (3).At the outset ofthis investigation, we expected

to find coenzyme M in other tissues, since thereis an abundance of methyl-transfer reactions innature. We found instead a unique specificity forcoenzyme M in cells of methanogenic bacteria.During the early stages of the survey, negativeresults obtained with other tissues led us tomodify the extraction procedure to insure thatwe were not mising the coenzyme. The use of

anaerobic procedures, reduction with NaBH4,protection of thiol groups with dithiothreitol, orgentle hydrolysis in acid or base did not liberatedetectable levels of coenzyme. The low reducingpotential of the medium precluded the possibil-ity of mixed disulfides masking the coenzymeactivity, as full activity was obserTred for foursynthetic mixed disulfides added to the bioassay.The sensitivity of the bioassay was thoroughlydocumented; the assay would unequivocally de-tect 10 pmol of HS-CoM/mg (dry weight) oftisse. Ifcoenzyme M were to be found in tissuesin a comparable concentration to biotin, presentin liver at a concentration of 0.0001% (50pmol/mg of dry weight) (15), then the bioassaywould have elicited a very positive growth re-sponse. Indeed, tissues of all methanogenic bac-teria available and in pure culture exhibit highlevels of coenzyme activity ranging from 0.3 to16.2 nmol of HS-CoM activity per mg of dryweight. Assuming a cell water content of 3 pl/mgof dry weight, this corresponds to an averageintracellular coenzyme pool of 2 mM. A mini-methane system reported by Postgate (19) sug-gested that Desulfovibrio may be a candidatefor the coenzyme. Since activity wasnot detected, it appears that a phenomenondifferent from that found in methanogenic bac-teria is occurring in these celLs. The results pre-sented here clearly indicate that in the broadrange of tissues exainied a soluble form of the

J. BACTERIOL.262 BALCH AND WOLFE

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SPECIFICITY AND DISTRIBUTION OF COENZYME M 263

coenzyme is not found in levels characteristic ofmethanogenic species; it appears unlikely thatthis coenzyme plays a general role in methyltransfer.

In another light, the results presented hereare in keeping with the recent work of Fox et al.(8) and Woese and Fox (23). Using the 16SrRNA as a tool to systematically relate orga-nisms of diverse origin, these workers demon-strated that the methanogenic bacteria areclearly a unique group. Results of comparativecataloging of 16S rRNA indicate that the meth-ane bacteria are not closely related to typicalbacteria (including the cyanobacteria). In addi-tion, the coenzyme was not detected in Halo-bacterium halobium or Sulfolobus acidocalda-rius. These organisms are classified as membersof the archaebacteria (17, 18, 24), a proposedphylogenetically distinct biological groupingwhich includes the methanogens. It may not beunreasonable to expect to discover an array ofmetabolic features such as coenzymes which areconfined to the methanogens. Indeed, a numberof lines of evidence are accumulating which in-dicate that the methane bacteria have propertiesnot found in other organisms (4, 6, 8, 11-14, 18,21, 22). The conclusion appears clear-cut; theimportance of coenzyme M lies not in its widedistribution in nature but in its high specificityfor and absolute requirement by methyl-coen-zyme M reductase, which is a pivotal enzyme innormal biodegradation in the cecum, rumen,intestine, sludge digestor, and aquatic sediments.

ACKNOWVLEDGMENTSWe gratefully acknowledge the excellent technical assist-

ance of Margaret Cavenaugh, Victor Gabriel, and DebbieSzurgot. We thank M. P. Bryant, J. A. Romesser, P. H. Smith,A. Zehnder, S. Schoberth, J. G. Zeikus, D. Boone, and H.Hippe for kindly providing cultures used in this study. Wethank J. A. Romesser and R. P. Gunsalus for providing thecoenzyme M derivatives used in this study.

This work was supported by Public Health Service grantAI-12277 from the National Institute of Allergy and InfectiousDiseases and by grant PCM 76-02652 from the NationalScience Foundation.

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2. Balch, W. E., and R. S. Wolfe. 1976. New approach tothe cultivation of methanogenic bacteria: 2-mercapto-ethanesulfonic acid (HS-CoM)-dependent growth ofMethanobacterium ruwinantium in a pressurized at-mosphere. Appl. Environ. Microbiol. 32:781-791.

3. Balch, W. E., and R. S. Wolfe. 1979. Transport ofcoenzyme M (2-mercaptoethanesuJfonic acid) in Meth-

anobacterium ruminantium. J. Bacteriol. 137:264-273.4. Best, A. N. 1978. Composition and characterization of

tRNA from Methanococcus.vannielii. J. Bacteriol. 133:240-250.

5. Bryant, M. P., B. C. McBride, and R. S. Wolfe. 1968.Hydrogen-oxidizing methane bacteria. I. Cultivationand methanogenesis. J. Bacteriol. 95:1118-1123.

6. Eirich, L D., G. D. Vogels, and R. S. Wolfe. 1978.Proposed structure for coenzyme F420 from Methano-bacterium. Biochemistry 17:4583-4593.

7. Ellman, G. L 1958. A colorimetric method for determin-ing low concentrations of mercaptans. Arch. Biochem.Biophys. 74:443-450.

8. 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.

9. Gornall, A. G., C. S. Bardawill, and M. M. David.1949. Determination of serum proteins by means of thebiuret reaction. J. Biol. Chem. 177:751-766.

10. Gunsalus, R. P., J. A. Romesser, and R. S. Wolfe.1978. Preparation of coenzyme M analogues and theiractivity in the methyl-coenzyme M reductase system ofMethanobacterium thermoautotrophicum. Biochemis-try 17:2374-2377.

11. Jones, J. B., B. Bowers, and T. C. Stadtman. 1977.Methanococcus vannielii: ultrastructure and sensitivityto detergents and antibiotics. J. Bacteriol. 130:1357-1363.

12. Jones, J. B., and T. C. Stadtman. 1977. Methanococcusvannielii: culture and effects of selenium and tungstenon growth. J. Bacteriol. 130:1404-1406.

13. Kandler, O., and H. Hippe. 1977. Lack of peptidoglycanin the cell walls of Methanosarcina barkeri. Arch.Microbiol. 113:57-60.

14. Kandler, O., and H. Konig. 1978. Chemical compositionof the peptidoglycan-free cell walls of methanogenicbacteria. Arch. Microbiol. 118:141-152.

15. Koser, S. A. 1968. Vitamin requirements of bacteria andyeast, p. 39-57. Charles C Thomas, Publisher, Spring-field, Mll.

16. 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.

17. Magrum, L. J., K R. Luehrsen, and C. R. Woese.1978. Are extreme halophiles actually "bacteria." J.Mol. Evol. 11:1-8.

18. Makula, R. A., andM. E. Singer. 1978. Ether-containinglipids of methanogenic bacteria. Biochem. Biophys.Res. Commun. 82:716-722.

19. Postgate, J. R. 1969. Methane as a minor product ofpyruvate metabolism by sulphate-reducing and otherbacteria. J. Gen. Microbiol. 57:293-302.

20. Taylor, C. D., B. C. McBride, R. S. Wolfe, and M. P.Bryant. 1974. Coenzyme M, essential for growth of arumen strain of Methanobacterium ruminantium. J.Bacteriol. 120:974-975.

21. Taylor, C. D., and RI S. Wolfe. 1974. Structure andmethylation of coenzyme M. J. Biol. Chem. 249:4879-4885.

22. Tornabene, T. G., R. S. Wolfe, W. E. Balch, G. Holzer,G. E. Fox, and J. Or6. 1978. Phytanyl-glycerol ethersand squalenes in the archaebacterium Methanobacte-rium thermoautotrophicum. J. Mol. Evol. 11:259-266.

23. Woese, C. R., and G. E. Fox. 1977. Phylogenetic struc-ture of the prokaryotic domain: the primary kingdoms.Proc. Natl. Acad. Sci. U.S.A. 74:5088-5090.

24. Woese, C. RI, L. J. Magrum, and G. E. Fox. 1978.Archaebacteria. J. Mol. Evol. 11:245-252.

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