Arch Microbiol (1996) 166 : 275–281
© Springer-Verlag 1996
O R I G I N A L PA P E R
George W. J. Allen · Stephen H. Zinder
Methanogenesis from acetate by cell-free extracts of the thermophilic acetotrophic methanogen Methanothrix thermophila CALS-1
Received: 18 March 1996 / Accepted: 14 June 1996
Abstract High rates of methanogenesis from acetate and ATP were observed from cell-free extracts of the thermophilic acetotrophic methanogen Methanothrix (Methanosaeta) thermophila strain CALS-1 when cultures were grown in a pH auxostat fed with acetic acid. Specific methanogenic activities ranged from 50–300 nmol min–1 (mg protein)–1, which was comparable to those for whole cells. In contrast to results with Methanosarcina spp., the reaction did not require high levels of H2 in the headspace. CO was inhibitory to methanogenesis from acetate. The inhibition by CO and the lack of effect of H2 on methanogenesis from acetate resemble previous results with whole cells of CALS-1. Protein concentrations in extracts > 5 mg/ml were required for good activity, and the optimum temperature for the methanogenesis was near 65° C. ATP was required in substrate quantities and was converted mainly to AMP. The maximum CH4 /ATP stoichiometry obtained was near 1.0, consistent with acetate activation using an acetyl-CoA synthetase mechanism that converts ATP to AMP and pyrophosphate. Methanogenesis in extracts was inhibited by bromoethane sulfonate and cyanide, indicating the involvement of methylcoenzyme M methylreductase and a carbon monoxide dehydrogenase complex with methanogenesis from acetate. These results are consistent with acetyl-coenzyme A (CoA) as the form of activated acetate involved in methanogenesis from acetate in strain CALS-1, but no activity could be obtained from extracts using acetyl-CoA as a substrate. Key words Methanothrix · Methanosaeta · ethanogenesis · Acetate · Carbon monoxide dehydrogenase · Acetyl-coenzyme A synthetase
G. W. J. Allen · S. H. Zinder (Y) Section of Microbiology, Wing Hall, Cornell University, Ithaca, NY 14850, USA Tel. +1-607-255-2415 e-mail:
[email protected]
Abbreviation BES Bromoethane sulfonate
Introduction Acetate is the precursor of nearly two-thirds of the methane produced in most nongastrointestinal methanogenic habitats (Zinder 1993). Only two genera of methanogens, Methanosarcina and Methanothrix [also called Methanosaeta (Patel and Sprott 1990)], are known to decarboxylate acetate to CH4 and CO2. The faster-growing and more versatile Methanosarcina has received more attention. However, Methanothrix is often the dominant acetate-utilizing methanogen observed in microbial populations from anaerobic bioreactors. Ecological studies have demonstrated that low acetate concentrations favor Methanothrix over Methanosarcina (Zinder et al. 1984; Wiegant and De Man 1986). Studies with whole cells have demonstrated that Methanosarcina cultures have a minimum threshold concentration for acetate utilization near 1 mM, while that for Methanothrix CALS-1 is 5–20 µM (Min and Zinder 1989; Jetten et al. 1990 a; Ohtsubo et al. 1992). Thus, while the two genera carry out the same reaction, Methanothrix is able to carry it out at acetate concentrations ca. 100-fold lower than Methanosarcina, suggesting that there are significant biochemical differences between the two organisms. Conversion of acetate plus ATP or activated forms of acetate [acetyl-phosphate or acetyl-coenzyme A (acetylCoA)] to CH4 has been demonstrated in cell-free extracts of Methanosarcina (Krzycki and Zeikus 1984; Krzycki et al. 1985; Fischer and Thauer 1988). It has been found that for activity in extracts, high partial pressures of H2, or another auxiliary reductant such as Ti3+ need to be added (Krzycki and Zeikus 1984; Fischer and Thauer 1990 b). In general, the cell-free extract results and other results with purified enzymes support a pathway (Ferry 1992) in which acetate is activated to acetyl-CoA by acetate kinase and phosphotransacetylase, followed by disassembly of the acetyl moiety to a methyl group and a carbonyl group by a carbon monoxide dehydrogenase (CODH)-corrinoid
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protein complex. The methyl group is transferred to tetrahydrosarcinapterin or a related compound (Fischer and Thauer 1989; Grahame 1991) and can then be transferred to HS-CoM, most likely by a sodium ion-pumping, membrane-bound methyltranferase (Becher et al. 1992). MethylCoM can be reduced by the methylreductase complex to methane using HS-HTP as a reductant. The resulting CoM-S-S-HTP heterodisulfide can be reduced to free thiols electrons derived from oxidation of the carbonyl group of acetate and passed through ferredoxin (Terlesky and Ferry 1988; Fischer and Thauer 1990 a) to other electron carriers, perhaps including H2 (Fischer and Thauer 1990 b). There is considerably less biochemical data on methanogenesis from acetate in Methanothrix. Acetate kinase and phosphotransacetylase are not found in extracts of Methanothrix cells; instead, high amounts of an enzyme with acetyl-CoA synthetase (ACS) activity is present (Kohler and Zehnder 1984; Jetten et al. 1989 a; Teh and Zinder 1992). A two-subunit enzyme with CODH activity has been purified from Methanothrix soehngenii (Jetten et al. 1989 b), as has a methyl-reductase (Jetten et al. 1990 b), indicating that the general methanogenic pathway in the two organisms is similar (Jetten et al. 1992). We have been studying methanogenesis from acetate in the thermophilic acetotrophic methanogen Methanothrix thermophila strain CALS-1 (Zinder et al. 1987; Kamagata et al. 1992). Strain CALS-1 grows only on acetate with a doubling time near 24 h, considerably faster than mesophilic strains, and is free of contaminants (Patel and Sprott 1990). We have found that during methanogenesis from acetate, cultures did not poise H2 partial pressures near 50 Pa, as do thermophilic Methanosarcina cultures, although CO levels were poised near 0.1 Pa. H2 was not inhibitory to methanogenesis from acetate as it is in many, but not all, Methanosarcina cultures (Fischer and Thauer 1990 b), while low levels of CO were inhibitory to strain CALS-1. Moreover, cell-free extracts had low hydrogenase activity (Zinder and Anguish 1992). Thus, there is little evidence for the involvement of H2 in acetate metabolism in strain CALS-1. In this publication, we demonstrate methanogenesis from acetate in cell-free extracts of strain CALS-1, describe some of the basic characteristics of this cell-free system, and show that, unlike the Methanosarcina cell-free system, there is no requirement for H2.
Materials and methods Cultivation and harvesting of cells and preparation of cell-free extracts Methanothrix sp. strain CALS-1 (DSM 3870) was cultivated at 60° C in bicarbonate-buffered anaerobic growth medium containing 50 mM sodium acetate, as previously described (Zinder et al. 1987; Zinder and Anguish 1992). Cultures for cell-free extract experiments were grown in 10-l amounts in a 14-l fermentor (MF-105, New Brunswick Scientific, Edison, N.J., USA) and stirred at 100 rpm. Once methanogenesis had commenced, acetate was converted to CH4 and bicarbonate, thereby increasing the culture pH. Acetic acid (5 M, sterile, anoxic) was fed on demand to the culture
using a pH controller set at pH 6.4, a culture technique called a pH auxostat (Sowers et al. 1984). Culture purity was checked by examining at least 20 fields from the culture at × 400 magnification with a phase-contrast microscope for morphotypes different from the distinctive Methanothrix morphology and by incubation in medium containing 0.2 g/l yeast extract. It was found that unless fermentor vessels were autoclaved for at least 3 h at 121° C, a sporeforming rod was often present. Critical experiments, such as demonstrating the lack of requirement for H2, were done on cultures in which purity was verified. When cultures had reached an OD600 [measured in a 1-cm cuvette using a Beckman DU-50 spectrophotometer (Fullerton, Calif., USA)] of 0.3–0.5, typically after 7–10 days, the cells were harvested. While OD600 values greater than 1.0 could be obtained, these cells usually had lower specific activity. The cells were transferred by pressure through butyl rubber tubing into an anaerobic chamber (Coy Laboratory Products, Ann Arbor, Mich., USA) with a headspace containing 2–4% H2 and the remainder N2. The medium was dispensed into 500-ml Nalgene bottles with O-ringsealing screw caps (Fisher Scientific, Rochester, N.Y., USA), both of which had been kept inside the chamber for at least 24 h to allow O2 to diffuse out of the plastic. The sealed bottles were removed from the chamber and centrifuged at 10,400 × g at 4° C using a Sorvall RC2-B centrifuge (DuPont, Wilmington, Del., USA) and a GSA rotor. The cell pellets were suspended in approximately 250 ml of anaerobic harvest buffer containing 25 mM Hepes (pH7.5) at 60° C, 10 mM MgCl2, and 2 mM dithiothreitol. Glycerol (10%) was included in early experiments, but was not necessary and was deleted in later experiments. The resuspended cells were centrifuged, and the resulting pellet was suspended in approximately 30 ml harvest buffer. The cell suspensions were loaded into a French pressure cell (Aminco, Silver Spring, Md., USA) inside the anaerobic chamber, and the cells were disrupted at 20,000 psi (ca. 133 MPa). The cell extracts were collected in a serum vial that was continuously gassed with N2 scrubbed of O2 with hot copper coils. These extracts were loaded inside the anaerobic chamber into O-ring-sealed centrifuge tubes and were anoxicically centrifuged for 20 min at 39,000 × g at 4° C. The supernatant was saved, and 1.5-ml portions were dispensed inside the anaerobic chamber into 12-ml vials that were then sealed with butyl rubber stoppers. The extracts were rapidly frozen in a dry-ice-acetone bath and were stored at –20° C for subsequent use. The extracts retained activity for at least 30 days at –20° C. The extracts typically contained 30 mg/ml of protein as measured by the Coomassie brilliant blue binding assay using reagents purchased from BioRad (Richmond, Calif., USA) and using lysozyme as a standard; the extracts were dark brown and slightly turbid.
Methanogenesis from cell-free extracts Methane formation from acetate was assayed in butyl-rubber-stoppered 15-ml serum vials containing 89 µl cell-free extract, 1 µl 100 mM Ti3+ citrate, and 10 µl reaction mixture. Standard reaction mixture contained 50 or 100 mM ATP, 50 or 100 mM MgCl2, 100 mM sodium acetate, and 2 mM CoA in harvest buffer. Vials and stoppers were stored in an anaerobic chamber for at least 24 h prior to use. The extract and Ti3+ citrate were added to each vial inside the chamber, and the vials were stoppered and crimped, then removed from the chamber and stored on ice for up to 6 h until needed. Vials containing extract were pre-incubated at 60° C for 30–60 s in a 60° C gyratory water bath (300 rpm, New Brunswick G-76) prior to initiating the reaction by adding 10 µl of reaction mixture to the vials. Gas samples of 0.2 ml were typically withdrawn from the headspace at 20-s intervals throughout the course of the reaction using 0.25-ml A-2 Pressure-Lok series gas syringes (Dynatech, Baton Rouge, La., USA) equipped with 22-g sideport needles. Gas samples were monitored for methane formation using a Varian 2400 series gas chromatograph with an H2 flame ionization detector and a 1.5-m Poropak R column operated at ambient temperature as de-
277 scribed previously (Lobo and Zinder 1988). H2 and CO were quantified using a 550 thermal conductivity gas chromatograph (GowMac, Bound Brook, N.J., USA) in conjunction with an RGD2 reduction gas detector (Trace Analytical, Menlo Park, Calif., USA) as previously described (Zinder and Anguish 1992). In all cases, corrections were made for the gas volume previously withdrawn during time courses. Liquid chromatographic analysis of adenine nucleotides All liquid samples of extract were prepared for liquid chromatographic analysis by centrifugation at 10,000 × g in a microcentrifuge for 10 min to remove soluble debris. ATP, ADP, and AMP were quantified using an FPLC system equipped with a Mono Q HR 5/5 anion exchange column. Samples of extract were eluted by a KH2PO4 gradient (0–85% 0.5 M KH2PO4, then held at 85% until ATP eluted) in distilled water at a flow rate of 1 ml/min. Absorbance was monitored at 254 nm, and quantities were calculated from the peak area using standard curves. The mimimum detection limit for adenine nucleotides was less than 10 µM.
tor operated as a pH auxostat (Sowers et al. 1984) was activity obtained. Figure 1 shows a typical time course for an extract provided with acetate and ATP. There was usually a 10to 30-s lag followed by rapid and linear methanogenesis (during which rates were calculated), followed by abrupt cessation of activity. Typical rates were 50–100 nmol min–1 (mg protein)–1, and rates as high as 300 nmol min–1 (mg protein)-1 were occasionally obtained. Figure 2 shows the effect of extract protein concentration on rates of methanogenesis. There was little activity at concentrations below 10 mg/ml protein, and activity reached a plateau at higher protein concentrations, which led to a drop of specific activity. Figure 3 shows the effect of temperature on the activity. Optimal activity was obtained at 65° C, which was slightly higher than the growth optimum of the culture. Little activity was present at 37 or 80° C.
Results General properties of the cell-free methanogenesis system Initial attempts at obtaining methanogenesis from cellfree extracts of acetate-grown strain CALS-1 involved growing high densities of cells by periodically feeding acetic acid to batch cultures (Grahame and Stadtman 1987) in 1-l bottles or in 10-l carboys. No activity was ever obtained from these cultures, probably because of large pH changes and intermittent starvation of the culture for acetate. Only when cells were grown in a 10-l fermenFig. 2 Effect of protein concentration on total methane produced, and rate of methanogenesis from acetate and ATP by cell-free extracts of Methanothrix sp. strain CALS-1. Extracts were diluted in harvest buffer. These results are typical of four repetitions of this experiment. P Methane produced, p specific activity
Fig. 1 Methanogenesis from acetate by a cell-free extract of Methanothrix sp. strain CALS-1. The vial was incubated at 60° C, contained 3.1 mg of protein, and received an addition of 5 mM MgATP, 10 mM sodium acetate, and 0.2 mM CoA at time zero. This vial was one of five that gave a specific activity of 104.5 ± 7.1 nmol methane min–1 (mg protein)–1 in the linear region of the time course. Methane in this and subsequent figures represents mmol methane produced per liter of extract + reaction mixture, which is equivalent to mM for soluble compounds. The actual aqueous concentration is much lower and is governed by the Henry’s law constant
Fig. 3 Effect of temperature on the rate of methanogenesis from acetate by cell-free extracts of Methanothrix sp. CALS-1. Assays at each temperature point were performed at least in triplicate, and the experiment was repeated three times with similar results
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H2 is required at high partial pressures for methanogenesis from acetate by Methanosarcina extracts. In early experiments, we found little difference between vials that received additions of H2 and those that did not. However, there was 2–4% H2 in the anaerobic chamber atmosphere, so that such vials cannot be considered to be H2-free. Figure 4 shows the results for a vial that was flushed with N2, which brought the H2 partial pressure down to approximately 1 Pa (≈ 10–5 atm); the CO partial pressure was 0.03–0.05 Pa. Methanogenesis in this vial and in others like it was rapid, and the changes in the H2 and CO detected were random fluctuations typically seen in measurements of these gases at such low partial pressures (Zinder and Anguish 1992). The amount of H2 present in the headspace of these vials was about 1% of the amount needed to reduce the equivalent of 3.5 mmol/l of methyl-coenzyme M to CH4. While the addition of up to 1 atm H2 had little effect on methanogenesis from acetate in extracts of CALS-1 (data not shown), CO was a potent inhibitor (Fig. 5). As little as
1% (0.01 atm) CO nearly completely inhibited methanogenesis from acetate. The reaction was also quite sensitive to oxygen, and any breaches in anaerobic technique led to a complete loss of activity (data not shown). ATP utilization by extracts Substrate quantities of ATP were required for methanogenesis from acetate by CALS-1 extracts, and methanogenesis ceased when ATP was apparently consumed. As shown in Fig. 6, subsequent doses of ATP could lead to further bursts of methanogenic activity. Not all vials re-
Fig. 6 Effect of repeated additions of ATP to a cell-free extract. At time zero, 5 mM of ATP and 10 mM of sodium acetate were added to the extract. Further additions of ATP were made at the time points indicated Fig. 4 Methanogenesis from 10 mM acetate and 5 mM ATP by an extract in a vial flushed with N2 to remove residual H2 from the chamber atmosphere. L CH4, G H2, P CO
Fig. 5 Inhibition of methanogenesis from acetate in cell-free extract by carbon monoxide. Assay vials were prepared as described in the text. CO was measured as a percentage of 1 atm. The time courses on the graph are representative of triplicate assays
Fig. 7 The effect of ATP concentration on the rate of methanogenesis from acetate. Assay vials were prepared in the standard manner described in Materials and methods except ATP was omitted from the reaction mixture and was added separately in the amounts indicated prior to starting the assay. The table indicates the stoichiometry of methane production per ATP at each of the initial ATP concentrations shown on the graph
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sponded to subsequent ATP doses. Once methanogenesis had ceased, nucleotide analysis of extracts that had received 10 mM ATP indicated nearly complete conversion to AMP (9 mM AMP, 2.1 mM ADP, 0.2 mM ATP, recovery = 113%). Figure 7 shows the effect of ATP concentration on the rate of methanogenesis and reaction stoichiometry. The rate increased with increasing ATP concentration up to 10 mM but was inhibited at higher concentrations, perhaps due to high salt concentrations (the pH of the ATP stock solution was neutralized to 7.0 to limit pH effects). A Km value for the lower concentrations of ATP was estimated as 3 mM. The stoichiometry was near 1 mol CH4 /mol ATP added at low ATP concentrations, and less at higher concentrations. This stoichiometry was variable between different batches of extract and was typically less than 1 CH4 /ATP. Effects of methanogenic inhibitors Bromoethane sulfonate (BES) is a potent and specific inhibitor of methylcoenzyme M methylreductase (Gunsalus et al. 1978). When 10 µM BES was added to cell-free extracts of strain CALS-1, the specific activity was lowered from 93 ± 12 µmol min–1 (mg protein)–1 to 34 ± 3 µmol min–1 (mg protein)–1, while 50 µM BES completely inhibited methanogenesis from acetate. Cyanide is a potent and specific inhibitor of electron transfer by carbon monoxide dehydrogenase (CODH), an enzyme complex involved in acetyl-CoA cleavage in Methanosarcina (Grahame 1991; Ferry 1992). Cyanide concentrations as low as 100 µM caused rapid and nearly complete inhibition of methanogenesis from acetate when added during a time course (data not presented).
Discussion Studies of the pathway of methanogenesis from acetate in Methanothrix spp. have been hampered by the lack of a cell-free assay. We describe here high activities for extracts from cells grown using a pH auxostat. Indeed, the activities we obtain of 100–300 nmol min–1 (mg protein)–1 are comparable to the activity of whole cells (Zinder and Anguish 1992); this is unusual for methanogen extracts, which typically have much lower activities than whole cells (Krzycki et al. 1985; Fischer and Thauer 1988; Thauer et al. 1993). A major difference between these Methanothrix extracts and those of Methanosarcina is the lack of requirement for high concentrations of H2, which are believed to be necessary for the regeneration of the free thiols from CoM-S-S-HTP mixed disulfide generated by the methylreductase reaction (Thauer et al. 1993). It is not clear whether H2 is an obligatory intermediate in electron transfer from the oxidation of the acetyl-CoA carbonyl group by CODH to methylreductase in Methanosarcina, or whether it simply passively equilibrates with reduced cofactors in the cell via a hydrogenase (Ferry 1993). Other
potential reducing agents in our reaction mixture include Ti3+, which was able to serve as a reductant to support methanogenesis in Methanosarcina extracts (Fischer and Thauer 1990 b). However, the amount of Ti3+ we added is only sufficient to produce 0.5 mmol/l CH4, and we found that higher concentrations of Ti3+ were inhibitory (data not presented). Dithiothreitol was not found to support methanogenesis in Methanosarcina extracts (Fischer and Thauer 1990 b), and would potentially provide enough electrons to produce 2 mmol/l CH4. Another potential role for H2 and other reductants is the reductive activation of enzymes such as CODH and methylreductase to their ready state (Ferry 1993). This might be accomplished by the 1 mM Ti3+ added to the extracts, although early studies were done without addition of Ti3+, and activities were similar (data not presented). We found that Ti3+ addition greatly decreased the variability in methanogenesis in different vials, presumably because of its O2 scrubbing ability. The lack of effect of H2 is similar to results previously obtained with whole cells of CALS-1 (Zinder and Anguish 1992) in which methanogenesis was unaffected by 100% H2, in which there was no evidence for H2 production or consumption during growth on acetate, most likely because of the low hydrogenase levels in strain CALS-1 as compared to Methanosarcina cultures. Another similarity to results with whole cells of CALS-1 was the strongly inhibitory effect of 1–2% CO on methanogenesis from acetate (Zinder and Anguish 1992). In that study, however, we found a poising of CO near 0.1 Pa during methanogenesis from acetate, while no poising was detected in time courses performed using extracts. Presumably CO inhibits methanogenesis from acetate because it is a strong reducing agent and may not allow regeneration of oxidized forms of cofactors needed for methanogenesis from acetate. In whole cells of Methanosarcina, some of the electrons from CO can be released as H2 (Zinder and Anguish 1992). The approximately 50% inhibition of methanogenesis from acetate by 10 µM BES is comparable to the inhibition detected by Gunsalus et al. (1978) for methanogenesis from methylcoenzyme M in cell-free extracts of Methanobacterium thermoautotrophicum and is consistent with a role for coenzyme M. Methylreductase has been purified from the mesophilic Methanothrix soehngenii (Methanosaeta concilii) strain Opfikon (Jetten et al. 1990 b); it forms the largest peak eluting from a Mono Q column when extracts of strain CALS-1 are fractionated (Teh and Zinder 1992). Similarly, the effect of cyanide on methanogenesis implies a role for a CODH complex in this organism. We found a multisubunit CODH complex (D. A. Grahame, G. W. J. Allen, and S. H. Zinder, unpublished results) similar to those described for Methanosarcina spp. (Terlesky et al. 1986; Grahame 1991), rather than the α2β2 complex described by Jetten et al. (1989 b) in strain Opfikon. ATP is required in substrate (as opposed to catalytic) quantities for methanogenesis from acetate, indicating that acetate activation is required and that energy is not conserved in this cell-free system. The maximum stoi-
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chiometry of 1 CH4 /ATP with essentially complete conversion to AMP is consistent with the operation of an acetyl-CoA synthetase (ACS), which is present in high activities in both mesophilic (Jetten et al. 1989a) and thermophilic (Teh and Zinder 1992) Methanothrix species. This enzyme is coupled to pyrophosphatase (Jetten et al. 1992), which is also present in high quantities leading to the following reaction sequence: Acetate + CoA + ATP → Acetyl-CoA + AMP + PPi Pyrophosphatase PPi → 2 Pi
ACS
The net result of this reaction sequence is the conversion of ATP to AMP plus 2 Pi, thereby consuming the equivalent of two phosphodiester bonds for acetate activation. There is some evidence for association of pyrophosphatase with the cell membrane (Jetten et al. 1992), so that some of the energy from pyrophosphate hydrolysis may be conserved as a membrane potential. The acetate kinase-phosphotransacetylase system used by Methanosarcina spp. consumes only the equivalent of one phosphodiester bond per acetate activated (Ferry 1993). The CALS-1 extracts converted nearly all of the added ATP to AMP. This resembles results obtained with whole cells of M. soehngenii (Jetten et al. 1991) in which the energy charge in cells dropped to 0.2 once acetate was depleted, and in which the primary nucleotide present was AMP. Apparently Methanothrix spp. can still carry out reactions at a very low energy charge. The Km for methanogenesis from acetate estimated for ATP, near 3 mM, was similar to that estimated for purified acetyl-CoA synthetase of 5.5 mM (Teh and Zinder 1992). The CH4 /ATP stoichiometries were often less than 1.0; this may be due to the activity of an ATPase, which has been described in another thermophilic Methanothrix strain (Inatomi et al. 1993) and in our strain (Teh and Zinder 1993). We have found that even in cells broken with a French press at 20,000 psi (approximately 130 mPa) and centrifuged at 200,000 × g for 2 h, nearly 50% of the dicyclohexylcarbodiimide (DCCD)-sensitive ATPase activity was still present in the supernatant. These results are consistent with acetyl-CoA being the activated intermediate of acetate involved in methanogenesis from acetate, as is the case for Methanosarcina spp. However, our attempts at obtaining methanogenesis from acetate from acetyl CoA, in the presence or absence of activating quantities of ATP, have failed. Poor activity using acetyl-CoA in Methanosarcina extracts has been explained by buildup of inhibitory levels of CoA (Fischer and Thauer 1988), but good activity from acetyl-CoA was eventually obtained (Fischer and Thauer 1989). We are presently investigating the roles of CoA and other cofactors, and the role of the cell membrane in methanogenesis from acetate in strain CALS-1. Acknowledgements This research was supported by grant DEFG02-85ER13370 from the U.S. Department of Energy. We thank Tim Anguish for assistance in growing the cells.
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