JOURNAL OF BACTERIOLOGY, Nov. 1989, p. 5866-58710021-9193/89/115866-06$02.00/0Copyright © 1989, American Society for Microbiology

Vol. 171, No. 11

Transport of Coenzyme M (2-Mercaptoethanesulfonic Acid) andMethylcoenzyme M [(2-Methylthio)Ethanesulfonic Acid] in

Methanococcus voltae: Identification of Specific andGeneral Uptake Systems

MICHAEL DYBAS AND JORDAN KONISKY*

Department of Microbiology, University of Illinois, Urbana, Illinois 61801

Received 27 April 1989/Accepted 6 August 1989

A transport system for coenzyme M (2-mercaptoethanesulfonic acid [HS-CoM]) and methylcoenzyme M[(2-(methylthioethanesulfonic acid (CH3-S-CoM)J in Methanococcus voltae required energy, showedAkinetics, and concentrated both forms of coenzyme M against a concentration gradient. Transport re d

hydrogen and carbon dioxide for maximal uptake. CH3-S-CoM uptake was inhibited by N-ethy m e aDdmonensin. Both HS-CoM and CH3-S-CoM uptake showed sodium dependence. In wild-type M. vokae,HS-CoM uptake was c ntration dependent, with a V__ of 960 pmol/min per mg of protein and an appaentKmof 61 FM. Uptake of CH3-S-CoM showed a V_ of 88 pmot/min per mg of prtein and a K. of 53 FM. Amutant of M. voltae resistant to the coenzyme M analog 2-bromethanesulfonic acid (BES) showed no uptakeof CH3-S-CoM but accumulated HS-CoM at the wild-type rate. Whie the higher-affiniy uptake systm wasspecific for HS-CoM, the lower-affinty system mediated uptake of HS-CoM, CH3-S-CoM, and BES. Analysisof the intraceflular coenzyme M pools in metabolizing cells showed an intraceflular HS-CoM concentration of14.8 mM and CH3-S-CoM concentration of 0.21 mM.

The methanogenic bacteria are strictly anaerobic archae-bacteria which produce methane from H2 and CO2 andsimple organic compounds such as formate, methanol, ace-tate, and methylamines (24). (2-Methylthio)-ethanesulfonate(CH3-S-CoM) is the methyl-donating substrate for the termi-nal reduction in methanogenesis (9, 24). This reaction hasbeen characterized by using a highly purified methylcoen-zyme M reductase from Methanobacterium thermoau-totrophicum (strain Marburg) which has recently been foundto catalyze the reduction of CH3-S-CoM with mercaptohep-tanoyl-O-phospho-L-threonine to form CH4 and CoM-S-S-HTP. The mixed disulfide is subsequently reduced to yield2-mercaptoethanesulfonic acid (HS-CoM) and mercaptohep-tanoyl-O-phosphO-L-threonine (7). While all methanogensexamined have been found to contain coenzyme M (2), onlythe coenzyme M-requiring ruminal methanogen Methano-brevibacter ruminantium (formerly Methanobacterium ru-minantium) has been shown to transport both HS-CoM andCH3-S-CoM (3). [35S]HS-CoM was shown to be transportedwith an apparent Km of 73 nM and a Vm,, of 312 pmol/minper mg (dry weight), while uptake of [35S]CH3-S-CoM had anapparent Km of 50 nM and a Vm. of 320 pmol/min per mg(dry weight) (3). Uptake of coenzyme M at lower rates hasbeen observed in only two of the other methanogenic bac-teria tested, Methanospirillum hungatei and Methanobacte-rium mobile, which show HS-CoM uptake at 30 and 10%o ofthe M. ruminantium rate, respectively (3).The existence of a coenzyme M uptake system in Metha-

nococcus voltae was suggested by analysis of 2-bromoet-hanesulfonic acid (BES)-resistant mutants isolated in thislaboratory (19). [35S]BES was shown to be taken up by thewild type, while uptake did not occur in BES-resistant cells.Energy-dependent uptake of [35S]BES was found to beinhibited by addition of HS-CoM and CH3-S-CoM, suggest-

* Corresponding author.

ing that these three coenzyme M derivatives were accumu-lated in M. voltae by a common carrier-mediated uptakesystem. To further characterize this uptake system and todefine further the phenotypic differences between BEST andwild-type M. voltae, the uptake of CH3-S-CoM and H-S-CoM was studied. This analysis directly demonstrated anenergy-dependent uptake system for coenzyme M deriva-tives in M. voltae and provides further evidence that BESresistance in M. voltae is due to decreased uptake of thisinhibitor into cells exhibiting the mutant phenotype.

MATERIALS AND METHODS

Bacterial strains and growth conditions. Methanococcusvoltae strain PS (DSM 1537) was grown in the definedmedium of Whitman et al. (25) under a pressurized atmo-sphere of 80%o H2-20o CO2 (vo/vol). Cultures were shakenat 80 rpm in 1-liter Wheaton bottles containing 200 to 300 mlof growth medium at 30°C. Methanobrevibacter ruminan-tium Ml (DSM 1093) was grown as described by Balch andWolfe (3) in medium supplemented with 2% rumen fluid and630 nM HS-CoM under a pressurized atmosphere of 80%oH2-200o CO2. The cultures were shaken at 100 rpm in 125-mlserum vials which contained 20 ml of growth medium at420C.

Anaerobic procedures. Anaerobic manipulations were per-formed in a Coy anaerobic glove box (Coy Laboratories,Ann Arbor, Mich.) with an atmosphere of 80%o N2-20% CO2supplemented with 3 to 5% H2 (16). The oxygen level wasmonitored continuously with a Coy gas detector. Solutionswere made anaerobic by flushing with N2 or 80% N2-20%oCO2 or passage through the anaerobic interlock of theanaerobic glove box.

Synthesis of ammonium 2-([3H]methylthio)ethanesu1fonate.[3H]CH3-S-CoM was synthesized by the method ofGunsaluset al. (9) with the following modifications. 2-Mercaptoeth-anesulfonic acid (HS-CoM, sodium salt; 6.3 mmol; Sigma)

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and 10 ml of ammonium hydroxide (reagent grade) weresparged with N2 for 30 min to render them anaerobic. Theammonium hydroxide was anaerobically transferred to aflask containing the HS-CoM. [3H]iodomethane (4.9 Ci/mmol; Amersham Corp., Arlington Heights, Ill.) (5.1 jxmol)was diluted into 6.295 mmol of iodomethane (Sigma) andtransferred to the flask containing HS-CoM in base. Afterincubation overnight under nitrogen in the dark, the flaskwas vented to remove unreacted iodomethane, and theproduct was evaporated to dryness. The ammonium salt wasnext dissolved in a minimal volume of water and recrystal-lized five times with acetone. The crystals were dissolved inwater and passed through a Sephadex SP-C25-120 column(0.7 by 10 cm), and the second 1-ml fraction to elute waslyophilized, dissolved in 200 p1l of water, and purified with aWhatman CF-11 cellulose acetate column (1 by 25 cm;acetone-water, 16:3). The [3H]CH3-S-CoM eluted as a sharppeak which comigrated with nonlabeled CH3-S-CoM stan-dards in two thin-layer chromatography systems, acetone-water (16:3) and acetone-water-concentrated ammonium hy-droxide (64:10:1). The thin-layer sheets were dried andexposed to Kodak XR-5 film at -70°C. Label was deter-mined to be 94% radiochemically pure. The specific activityof the label as synthesized was 4 mCi/mmol.

Synthesis of [2-35S]ammonium 2-mercaptoethanesulfonate.[35S]HS-CoM was synthesized by the procedure of Taylorand Wolfe (22) with the following modifications. Sodium2-bromoethanesulfonate monohydrate (Sigma) (42 mg) wasdissolved in 2 ml of anaerobic ammonium hydroxide. Thismixture was transferred anaerobically to a Douseal vialcontaining 9.25 mg of [35S]sodium sulfide nonahydrate (26mCi/mmol; Amersham) and 38 mg of unlabeled sodiumsulfide nonahydrate (Sigma). The mixture was shaken for 3 hat 25°C in the dark. The vial was flushed with N2 at 5 lb/in2for 30 min to remove ammonium hydroxide and ammoniumsulfide, and the contents were transferred anaerobically to ateardrop flask filled with N2 and then lyophilized. Theproduct was dissolved in 1 ml of water and crystallized with15 ml of acetone at 25°C. Crystals were harvested bycentrifugation for 15 min at 5,000 x g. The crystals weredissolved in 1 ml of water and applied to a Whatman CF-licellulose-acetate column (1.5 by 40 cm) equilibrated withacetone-water (16:3). [35S]HS-CoM eluted as a broad peak,which was pooled and lyophilized. The product was 96%radiochemically pure as determined by thin-layer chroma-tography in acetone-water (16:3) and acetone-water-concen-trated ammonium hydroxide (64: 10: 1), followed by exposureto Kodak XR-5 film at -70°C. The specific activity of thelabel as synthesized was 5 mCi/mmol.Measurement of [35S]HS-CoM uptake. Cells were grown to

mid-logarithmic growth phase (OD660, 0.3 to 0.5), anaerobi-cally harvested by centrifugation (15 min, 3,000 x g, 150C),and washed and suspended in anaerobic buffer containing0.4 M sucrose, 100 mM HEPES (N-2-hydroxyethylpipera-zine-N'-2-ethanesulfonic acid), 10 mM KCl, 10 mM MgCl2,pH 6.5 (11, 19). Cells were suspended to 0.6 to 0.8 mg ofprotein per ml in 1.5 ml of anaerobic transport buffer inserum vials, flushed with appropriate headspace gas, andthen pressurized to 30 lb/in2. The redox potential of thesuspensions was next lowered to that occurring undergrowth conditions by addition of 5 ,umol of Na2S 919H20. Inlow-sodium assays, 1 ,umol of H2S gas was used as thereducing agent. Suspensions were equilibrated for 15 min at25°C with shaking at 80 rpm. Assays were initiated byaddition of labeled substrate. At each time point, 1 ml ofassay mixture was removed and rapidly diluted into 1 ml of

reduced transport buffer containing 10 mM nonradioactiveHS-CoM, followed by vacuum filtration through a Nucle-opore PC filter (0.45-pLm pore size; Nucleopore Corp. Pleas-anton, Calif.) over a Whatman GF/F glass fiber filter. Thefilters were rinsed with 1 ml of reduced transport buffercontaining 10 mM nonradioactive HS-CoM. Filtration andrinse steps took less than 20 s. Radioactivity was determinedin a liquid scintillation spectrometer.Measurement of [3H]CH3-S-CoM uptake by M. voltae. To

determine uptake, cells were grown, harvested, and washedas described for HS-CoM uptake experiments. Samples (5ml) of cells at 0.4 to 0.6 mg of protein per ml were transferredto 12.5-ml serum vials, flushed with desired headspace gas at30 lb/in2, and reduced by addition of 5 ,umol of Na2S. Cellswere equilibrated at 30°C by shaking at 80 rpm for 15 min.Transport assays were initiated by addition of labeled sub-strate. At each time point, a 1.5-ml volume of headspace gaswas removed, equilibrated to atmospheric pressure by ex-pansion in a 5-ml syringe, and assayed for radioactivemethane. Tritiated methane was detected by a liquid scintil-lation system (26) with the following modifications. A 7 mMbutyl rubber hypo vial septum (Pierce) was installed in astandard glass scintillation vial (25 ml) to allow gas injection.A 15-ml sample of toluene-PPO (2,5-diphenyloxazole) scin-tillation cocktail was flushed for 1 min at 5 lb/in2 withmethane to achieve a constant partial pressure of methaneabove the liquid phase and ensure uniform methane solubil-ity in the scintillation fluid. Gas samples were added byinjection, vials were shaken for 5 min at 60 rpm at 25°C, andradioactivity was determined by scintillation counting. Up-take of [3H]CH3-S-CoM was calculated from the rate ofsubstrate conversion to radioactive methane. Counting effi-ciencies for [3H]CH4 (27%) and [14C]CH4 (56%) were deter-mined. [3H]CH3-S-CoM levels in cells were assayed byremoval of 1 ml of buffer at each time point, followed byrapid dilution of the sample into buffer containing 10 mMCH3-S-CoM, followed by filtration over Millipore EH (0.4,um) or Nucleopore polycarbonate (0.4 ,um) membrane fil-ters. Filters were soaked overnight in Biofluor scintillationcocktail (NEN Research Products) before radioactivity wasdetermined.Measurement of [3H]CH3-S-CoM uptake by Methanobrevi-

bacter ruminantium. Cells were grown to mid-logarithmicphase (OD660, 0.4 to 0.8) and anaerobically harvested bycentrifugation (15 min, 5,000 x g, 150C). Cells were sus-pended in coenzyme M-free growth medium (pH 7.1) (3, 22),and 5-ml portions were dispensed into 12.5-ml serum vials.Transport was assayed and uptake was calculated as de-scribed for M. voltae, except that cell suspensions wereshaken at 120 rpm and 42°C (3).

Transport assays in low sodium. Low-sodium transportassays were performed as described for standard assaysexcept with a very low sodium buffer. This buffer is identicalto standard transport buffer except for omission of NaCl andwas prepared with deionized distilled water and stored inplastic vessels. Cell suspensions were reduced by addition of1 ,umol of H2S. Sodium was added as anaerobic stocksolutions of NaCl in assay buffer. The contaminating sodiumlevel in this buffer was determined to be 0.14 ,uM by atomicabsorption spectroscopy. The amount of sodium carriedover with washed cells increased the sodium concentrationin the assay buffer to 0.21 ,uM.

Determination of intracellular concentrations of HS-CoMand CH3-S-CoM in M. volite. M. voltae was grown asdescribed for transport assays, anaerobically harvested, andwashed once with transport buffer as described above. Cells

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5868 DYBAS AND KONISKY

were incubated under transport assay conditions for 15 minin 25-ml Balch tubes under an H2-C02 atmosphere. Cellswere pelleted at 3,000 x g for 15 min in an InternationalEquipment Co. HN-SII table-top centrifuge while metabo-lizing H2-C02 at 25°C. Cells were then suspended in anaer-obic distilled H20 (40 ml/g [wet weight]) and heated at 100°Cfor 15 min under a nitrogen atmosphere. The lysed cellsuspension was then cooled and centrifuged twice anaerobi-cally at 540,000 x g for 20 min at 4°C in a Beckman TL-100ultracentrifuge. The supernatant was pooled and lyophilizedanaerobically overnight. The dry supernatant, designatedthe heat-stable extract, was then analyzed for HS-CoM andCH3-S-CoM. The HS-CoM concentration was determinedby dissolving the lyophilized supernatant in 400 RId of anaer-obic H20, which was applied to a Whatman CF-11 cellulose-acetate column (4 by 1.1 cm) equilibrated and developedwith acetone-H20 (16:3). H-S-CoM eluted as a broad peakand was detected with Ellman reagent (8). The identity ofEllman-positive material was confirmed by thin-layer chro-matography in acetone-H20-NH4OH (64:10:1) and acetone-H20 (16:3). Cells supplemented with HS-CoM (3.2 ,mol)were extracted by this procedure to determine the percentrecovery. A total of 70% of the added HS-CoM was recov-ered by this procedure. The level of CH3-S-CoM in theheat-stable extract was determined by following its conver-sion to CH4 with cell extracts prepared from M. thermoau-totrophicum (10). The heat-stable extract was dissolved in 50mM phosphate buffer, pH 6.8, containing 10 mM MgCl2 and2 mM ATP in calibrated vials. Cell extract (100 ,ul) wasadded to 500 ,u of heat-stable extract in the above buffer,and the sealed vials were incubated for 90 min at 63°C underan H2 atmosphere to allow complete reduction of CH3-S-CoM to methane. Methane was detected by gas chroma-tography as previously described (19), and the amount ofCH3-S-CoM present was calculated from the amount ofmethane formed. An average of 92% of added CH3-S-CoMwas converted to methane by the M. thermoautotrophicumcell extract, as determined by conversion of [3H]CH3-S-CoM to [3H]methane and by conversion of CH3-S-CoMstandards to methane. Cells supplemented with CH3-S-CoM(55 nmol) were extracted and assayed by this procedure, andthe recovery was determined to be 97%.

Other analytical methods. Methanogenesis assays wereperformed on cells cultured, harvested, and washed asdescribed for CH3-S-CoM uptake assays. Cell suspensions(2 to 3 mg of protein) in anaerobic transport buffer weredispensed in calibrated vials and flushed with H2-C02 for 1min. Methanogenesis rates were determined by gas chroma-tography as described previously (19). Protein was deter-mined by a modified Lowry et al. assay (15) with bovineserum albumin as a standard. Concentrations of Na+ and K+were determined by atomic absorption spectroscopy.[3H]CH4 standards were prepared by using a Methanobac-terium thermoautotrophicum cell extract (9, 10) under an H2atmosphere to reduce 500 nmol of [3H]CH3-S-CoM (specificactivity, 4 RCi/4mol) to [3H]CH4. A total of 88% of thesubstrate was converted to methane yielding 443 nmol of[3H]CH4; the specific activity was 4 ,uCi/,Imol. [14C]CH4standards (Amersham) were a generous gift of R. S. Wolfe.

RESULTS AND DISCUSSION

Kinetics of uptake of coenzyme M and methylcoenzyme M.Uptake of [35S]HS-CoM was found to be linear for up to 10min following addition of labeled substrate, while [3H]CH3-S-CoM uptake was linear for up to 45 min (Fig. 1). Uptake of

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FIG. 1. Time course of HS-CoM and CH3-S-CoM uptake. Up-take was assayed under H2-CO2 at 30°C, and the substrate concen-tration was 30 ,uM. Data points without error bars are averages ofduplicate assays; all other points are averages of six assays showingstandard deviation. Uptake was determined as described in Materi-als and Methods.

both HS-CoM and CH3-S-CoM was found to be concentra-tion dependent and showed saturation kinetics in wild-typeM. voltae. A linear least-squares fit of a plot of substrateconcentration/uptake velocity versus substrate concentra-tion (Hanes-Woolf plot) over a concentration range of 10 to300 p.M yielded an apparent Km of 53 p.M (range in separateassays, 49 to 54 p.M) and Vmax of 88 pmol/min per mg ofprotein for CH3-S-CoM. A similar plot of HS-CoM uptakedata over a substrate concentration range of 30 to 100 p.Mshowed a Km of 61 p.M (range in separate assays, 60 to 63p.M) and a Vmax of 960 pmol/min per mg of protein.The initial rate of [35S]HS-CoM uptake was determined to

be 260 pmol/min per mg of protein at 30 p.M substrate. Sincethe average number of cells per mg of protein was 1.23 x1010, as determined by Petroff-Hausser chamber court, theobserved uptake rate corresponds to an average rate of 210molecules of HS-CoM taken up per cell per s at a substrateconcentration of 30 p.M. Similarly, the rate of [3H]CH3-S-CoM uptake at 30 p.M substrate (32 pmol/min per mg) wascalculated to correspond to 25 molecules of CH3-S-CoM percell per s. For comparison, the rate of [35S]BES uptake in M.voltae at 10 p.M substrate was reported to be 5.7 pmol/minper mg in wild-type cells (19), corresponding to 5 moleculesof BES taken up per cell per s.The initial rate of [35S]HS-CoM uptake was determined by

a filtration assay as described in Materials and Methods.However, this method was not successful for examining theinitial rate of [3H]CH3-S-CoM uptake. While preliminaryattempts to measure [3H]CH3-S-CoM uptake by a filtrationassay detected very little accumulation of [3H]CH3-S-CoM,the [3H]CH3-S-CoM in the assay buffer was found to bereduced rapidly to [3H]CH4, which was detected by liquidscintillation counting (26). The rate of radioactive methaneformation was found to be linear and dependent on theconcentration of cells and concentration of [3H]CH3-S-CoM.We reasoned that the formation of [3H]methane was due touptake of [3H]CH3-S-CoM, followed by rapid reduction ofthe tritiated methyl group to tritiated methane. As we hadbeen unable to detect an increase in the intracellular labelconcentration by a filtration assay similar to that used toassay HS-CoM uptake, we concluded that the intracellularpool of tritiated CH3-S-CoM was kept at a low and constantlevel. These observations led us to use the steady-stateassumption to determine the rate of [3H]CH3-S-CoM uptake.

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TABLE 1. Effect of coenzyme M derivatives on [3H]CH3-S-CoMuptake and [35S]HS-CoM uptake'

Assay and addition Concn Uptake(>LM) (% of control)

CH3-S-CoM uptakeHS-CoM 10 56

50 36100 29

BES 10 6350 41

HS-CoM uptakeCH3-S-CoM 10 100

50 92100 83

BES 10 9850 99

For [3H]CH3-S-CoM uptake, cells were grown and harvested as describedin Materials and Methods and incubated with the indicated inhibitor for 5 minunder H2-CO2 prior to addition of 10 ILM [3H]CH3-S-CoM. Control uptakevalue (no addition) was 26 pmol/min per mg of cellular protein. Values areaverages of four assays and were calculated from uptake over 30 min. For[35S]HS-CoM uptake, cells were incubated with the indicated inhibitor for 5min under H2-CO2 prior to the addition of 10 FM [35S]HS-CoM. Controluptake value was 200 pmol/min per mg of cellular protein. Values are averagesof duplicate assays and were calculated from uptake over 10 min.

We concluded that the rate of [3H]methane formation,d[3H]CH4/dt, is equal to the rate of [3H]CH3-S-CoM uptake.To evaluate the [3H]CH3-S-CoM conversion-[3H]methaneformation assay, M. ruminantium Ml, a ruminal methano-gen with an obligate growth requirement for coenzyme M(21), was assayed for uptake of [3H]CH3-S-CoM. The rate ofuptake (108 pmollmin per mg [dry weight]) was found to besimilar to the rate reported for [35S]CH3-S-CoM uptake (80pmol/min per mg [dry weight]) by Balch and Wolfe at thissubstrate concentration (10 ,uM) (3). This fact verified theability of this coupled assay to determine [3H]CH3-S-CoMuptake.Uptake of [3H]CH3-S-CoM by M. voltae required a gas

phase of H2 and CO2 for maximal transport. Cells assayedunder H2 alone showed a 32% lower rate of uptake, and cellsassayed under N2-CO2 or air showed no uptake activity.[35S]HS-CoM uptake also required H2-CO2, and cellsshowed no significant uptake under N2-CO2 or air. N-Ethylmaleimide (1 mM) completely inhibited uptake of[3H]CH3-S-CoM. This concentration of N-ethylmaleimidestill allowed methanogenesis to proceed at 23% of controlrates. This blockage of uptake suggests that transport of[3H]CH3-S-CoM is carrier mediated; however, we cannotrule out the alternative possibility that N-ethylmaleimidereduced methanogenesis and thereby reduced cellular en-ergy below some threshold level required to support trans-port.

Competition of HS-CoM and BES with [3HICH3-S-CoMuptake and competition of CH3-S-CoM and BES with [35 ]HS-CoM uptake. The effect of HS-CoM and BES on [3H]CH3-S-CoM uptake was examined, as was the effect of CH3-S-CoM and BES on [35S]HS-CoM uptake. As can be seen inTable 1, uptake of [3H]CH3-S-CoM was reduced with in-creasing molar ratios of both HS-CoM and BES. These data,combined with the observation that both HS-CoM andCH3-S-CoM protect cells from growth inhibition in thepresence of BES (19), suggests that a common uptakesystem exists for these three compounds. However, additionof CH3-S-CoM at up to 10:1 molar ratios to [35S]HS-CoMresulted in only a 17% reduction in [35S]HS-CoM uptake,and addition of BES had no significant effect on HS-CoM

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FIG. 2. (A) Uptake of [3H]CH3-S-CoM by BEST-1 and wild-typeM. voltae assayed under H2-CO2 at 30°C. The concentration of thesubstrate was 30 ,uM. Data points without error bars representaverages of duplicate assays; all other points represent the averageof four assays. Uptake was calculated as described in Materials andMethods. (B) Uptake of [35S]HS-CoM by BESr_1 and wild-type M.voltae assayed under H2-CO2 at 30°C. The concentration of sub-strate was 30 F.M. All data points for BESr_1 represent averages ofduplicate assays. Uptake was determined by the rapid dilution-filtration assay described in Materials and Methods.

uptake (Table 1). These results suggest the existence of twotransport systems: an HS-CoM-specific high-Vm. system(960 pmol/min per mg of protein), and a general, low-Vmaxsystem (88 pmol/min per mg of protein) which takes upHS-CoM, CH3-S-CoM, and BES.Uptake of [35S]HS-CoM and [3H]CH3-S-CoM by BES-

resistant M. voltae. Since we had previously determined thattwo BES-resistant mutants of M. voltae showed decreasedrates of accumulation of [35S]BES (19), it was of interest todetermine the capacity of a BES-resistant mutant to trans-port [35S]HS-CoM and [3H]CH3-S-CoM. The mutant tested(BESr_1) did not show transport of [3H]CH3-S-CoM, but didtransport [35S]HS-CoM at approximately the wild-type rate(Fig. 2). We interpret these results, in conjunction with theresults of the competition assays described above, as evi-dence for two coenzyme M uptake systems in M. voltae. Wepostulate the existence of a highly specific, high-Vma. HS-CoM uptake system which is present in both the mutant andwild-type cells, and a low-Vmax general uptake system forBES, HS-CoM, and CH3-S-CoM which is present in wild-type but not in the BES-resistant cells.

Role of sodium in uptake and methanogenesis. Sodium hasbeen demonstrated to play important roles in bioenergeticsand transport in M. voltae (6, 11, 12). To examine a possible

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TABLE 2. Effect of sodium concentration on CH3-S-CoM andH-S-CoM uptake

Derivative Reductant' Sodium Uptakebconcn (,uM) (% of control)

CH3-S-CoM Na2S 4,000 100H2S 4,000 100

70 4440 90.1 0

HS-CoM Na2S 4,000 100H2S 32 26

Reductant was Na2S (5 p.mol) or H2S (1 iLmol). Cells were incubated for15 min at 30°C prior to addition of substrate to 30 ,uM.

b Control uptake values were 32 (CH3-S-CoM) and 260 (HS-CoM) pmol/minper mg; sodium concentration, 4,000 ,M.

role of Na+ in uptake of coencyme M derivatives, uptakewas monitored in low-sodium anaerobic buffer. Both theuptake rate (Table 2) and the rate of methanogenesis de-creased as the sodium concentration was decreased (data notshown).Uptake of [3H]CH3-S-CoM was blocked and uptake of

[35S]HS-CoM was reduced 42% by 20 ,uM monensin, acompound which mediates Na+/H+ exchange across mem-branes and collapses sodium gradients (17, 18). Monensin(20 ,uM) also blocked methanogenesis from H2-CO2. Fromthese data, it is clear that any role of sodium in uptake ofCH3-S-CoM cannot be separated from its requirement inmethanogenesis. While the reduction in HS-CoM uptakeobserved in low-sodium buffer also might suggest a directrole of sodium ions in uptake, we cannot rule out that theseresults derive indirectly from the concomitant reduction inmethanogenesis causing a decrease in cellular energy levelscoupled to methanogenesis.

Composition of intracellular coenzyme M pools. To deter-mine whether accumulation of HS-CoM and CH3-S-CoMoccurred against a concentration gradient, we analyzed thecomposition of intracellular coenzyme M pools. From thetotal amount of HS-CoM extracted from metabolizing cells,the intracellular concentration of HS-CoM was determinedto be 14.8 ± 0.93 mM (n = 3). This was based on an averageof 20.4 nmol of HS-CoM per mg (dry weight) and a volumeof 1.37 pul/mg (dry weight) (11).To determine the intracellular concentration of CH3-S-

CoM in M. voltae, we determined the capacity of a heat-stable extract to serve as the substrate for the CH3-S-CoM-dependent formation of CH4 by cell extracts of M.thermoautotrophicum under H2. CH3-S-CoM is heat stableand is therefore not destroyed by heating to 100°C under100% N2. When the M. voltae extract was assayed in thisway, methane production required H2, was a function of theamount of heat-stable extract added, and was not stimulatedby addition of HS-CoM. The amount of methane producedfrom the heat-stable extract added was significantly greaterthan the background amount of methane produced from thesmall amount of CH3-S-CoM present in the M. thermoau-totrophicum extract. Using this assay, we determined theintracellular concentration of CH3-S-CoM to be 0.21 ± 0.03mM (n = 3). This was calculated from an average of 0.27nmol of methane formed per mg (dry weight) of M. voltaeextract.Uptake of CH3-S-CoM and HS-CoM by M. voltae shows

characteristics of a carrier-mediated, energy-dependent pro-cess. HS-CoM, CH3-S-CoM, and BES were accumulated atdifferent rates; the relative rates of uptake were HS-CoM >

CH3-S-CoM > BES. Based on our determination of intra-cellular levels of HS-CoM and CH3-S-CoM in a typicaltransport assay with 30 puM substrate, transport is active,occurring against an HS-CoM concentration gradient of493:1 and a CH3-S-CoM concentration gradient of 7:1 (intra-cellular-extracellular concentration). M. voltae has also beenshown to accumulate BES against a concentration gradient(19).We observed that transported [3H]CH3-S-CoM equili-

brated with the intracellular CH3-S-CoM pool, and this[3HICH3-S-CoM served as the substrate for the methylre-ductase complex, resulting in the formation of [3H]methane.That Gunsalus et al. (9) found that CH3-S-CoM was the onlycompound which the methylreductase could reduce to CH4suggests that chemical modification coupled to an energy-consuming group translocation is not the mechanism oftransport. While our results are consistent with a mechanismwhereby the substrates are taken up in an unmodified form,we are not able to rigorously exclude a mechanism based ongroup translocation. In M. ruminantium, transported HS-CoM and CH3-S-CoM could be detected intracellularly inunmodified form, with CH3-S-CoM as the major stableintracellular form of transported labeled coenzyme M (3). InM. voltae, the composition of coenzyme M pools wasexamined by detecting coenzyme M synthesized by the cellsunder metabolizing conditions instead of examining thecomposition of transported coenzyme M pools. We foundHS-CoM to be the major component of the intracellular pool(14.8 mM), with CH3-S-CoM present at lower levels (0.21mM).Sodium has been shown to have an important role in

bioenergetics and transport of amino acids (6, 11, 12) in M.voltae. [35S]BES uptake has been shown to be slightlystimulated by increasing Na+ concentrations (19). We exam-ined the role of Na+ in uptake of [35S]HS-CoM and [3H]CH3-S-CoM by using a transport assay mix containing a lowconcentration of sodium. Using this procedure, we were ableto show a sodium dependence of conezyme M uptake, inagreement with the inhibition of uptake observed in thepresence of monensin. These results raise the possibility thatthe uptake of coenzyme M derivatives is coupled mechanis-tically to a sodium gradient. However, we believe that adetermination of the exact basis for the observed Na+dependence will require the development of a responsivemembrane vesicle system. Such a system would allow us todistinguish between effects of sodium ions on uptake ofcoenzyme M per se and effects of Na+ on methanogenesisand cellular metabolism.The physiological significance of coenzyme M uptake

under natural conditions is unknown. Autolysis of M. voltaeoccurs under laboratory conditions, and given osmotic andtemperature fluctuations, autolysis probably occurs in itsnatural estuarian environment. The coenzyme M transportsystem would allow this organism to take advantage oflocally concentrated pools of coenzyme M released by celllysis.The uptake of coenzyme M may also provide a sulfur

source for M. voltae in its natural environment. CoenzymeM has the highest percentage of sulfur (45%) of the organiccoenzymes (25) and has been detected in marine sedimentsat low concentrations (.0.5 p.M) (16). M. voltae has beenfound to excrete methionine (20), which has been suggestedto be an important intermediate in the formation of organicthiols in marine sediments (16). It is possible that M. voltaeaccumulates and uses organic thiols as sulfur sources, andperhaps coenzyme M is transported by a system which in

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HS-CoM AND CH3-S-CoM TRANSPORT IN M. VOLTAE 5871

nature accumulates this or some other organic thiol. Furtherwork is needed to determine whether coenzyme M and otherorganic thiols found in marine sediments are involved innatural sulfur cycles.

ACKNOWLEDGMENT

This study was supported by the Office of Naval Research, grantN0014-86-K-0224.

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