Ethane production by Methanosarcina barkeri during growth in ethanol supplemented medium

Methanosarcina barkeri strain 227 produced ethane during growth on H₂/CO₂ when ethanol was added to the medium in concentrations of 89–974 mM; ethane production varied from 14 to 38 nmoles per tube (20 ml gas phase, 5.7 ml liquid) with increasing ethanol concentrations. Cells grown to mid-logarithmic phase (A₆₀₀ 0.46, protein = 64 g/ml) on H₂/CO₂, thoroughly flushed with H₂/CO₂, then exposed to ethanol, produced maximal ethane levels (at 585 and 974 mM ethanol) of about 215 nmoles per tube, with an ethane/methane ratio of 1×10^-3. Mid-logarithmic-phase cultures of Methanosarcina barkeri strain Fusaro also produced ethane (up to 20 nmoles per tube) when exposed to ethanol. Cultures of strain 227 growing on methanol in the absence of H₂ produced 6 nmoles per tube of ethane when supplemented with ethanol whereas those lacking ethanol but containing H₂ and/or methanol produced 1.6 nmoles per tube. Cultures of Methanococcus deltae strains LH and RC, Methanospirillum hungatei or Methanobacterium thermoautotrophicum produced 5 nmoles ethane per tube when grown in medium containing ethanol. Ethanol concentrations of 177–886 mM were inhibitory to growth of all methanogens examined. Production of ethane by Methanosarcina was inhibited by >62 mM methanol, and both methanogenic inhibitors tested, CCl₄ and Br–CH₂–CH₂–SO _inf3 ^sup- , inhibited ethane and methane production concurrently. The data suggest that ethanol is converted to ethane by Methanosarcina species using the terminal portion of the methanol-to-methane pathway.     

Kinetic studies on ammonia and methane oxidation by Nitrosococcus oceanus

The kinetics of ammonia oxidation and the ability of a marine ammonia-oxidizing bacterium, Nitrosococcus oceanus, to metabolize methane were investigated in semicontinuous batch culture. The effects of inhibitors (acetylene and nitrapyrin) and coreactants were determined in order to elucidate the behavior of the ammonia oxygenase enzyme in N. oceanus. Acetylene and nitrapyrin were potent inhibitors and their effects were not mitigated by increased ammonia concentrations. Oxygen concentration had the effect of a mixed-type inhibitor; reduced oxygen inhibited the rate or ammonia oxidation at high substrate concentration but may enhance the rate at low substrate concentrations. Substrate affinity in terms of NH ₄ ⁺ increased (K _m decreased) with increasing pH. Optimal pH was about 8. Methane inhibited ammonia oxidation; the interaction was not simple competitive inhibition and the presence of multiple active sites on the enzyme was indicated by the behaviour of the inhibited treatments. Half-saturation constants for methane (K _i=6.6 M) and ammonia (K _m=8.1 M) were similar. N. oceanus oxidized methanol and methane linearly over time, with CO₂ and cell material being produced at approximately equal rates.     

Catalysis of an isotopic exchange between CO2 and the carboxyl group of acetate by Methanosarcina barkeri grown on acetate

Cell suspensions of Methanosarcina barkeri (strain Fusaro) grown on acetate were found to catalyze the formation of methane and CO₂ from acetate (30–40 nmol/min·mg protein) and an isotopic exchange between the carboxyl group of acetate and ¹⁴CO₂ (30–40 nmol/min·mg protein). An isotopic exchange between [¹⁴C]-formate and acetate was not observed. Cells grown on methanol mediated neither methane formation from acetate nor the exchange reactions. The data indicate that the isotopic exchange between CO₂ and the carboxyl group of acetate is a partial reaction of methanogenesis from acetate. Both reactions were completely inhibited by low concentrations of cyanide (20 M) or of hydrogen (0.5% in the gas phase). Methane formation from acetate was also completely inhibited by low concentrations of carbon monoxide (0.2% in the gas phase) whereas only significantly higher concentrations of CO had an effect on the exchange reaction. In the concentration range tested KCN, H₂ and CO had no effect on methane formation from methanol or from H₂ and CO₂; however, cyanide (20 M) also affected methane formation from CO. The results are discussed with respect to proposed mechanisms of methane and CO₂ formation from acetate.     

Acetate metabolism inMethanosarcina barkeri

Methanosarcina barkeri was grown by acetate fermentation in complex medium (N₂ gas phase). The molar growth yield was 1.6–1.9 g cells/mol methane formed. Under these conditions 63–82% of the methane produced byMethanosarcina strains was derived from the methyl carbon of acetate, indicating that some methane was derived from other media components. Growth was not demonstrated in complex media lacking acetate or mineral acetate medium containing acetate but lacking H₂/CO₂, methanol, or trypticase and yeast extract. Acetate metabolism byM. barkeri strain MS was further exmined in mineral acetate medium containing H₂/CO₂ and/or methanol, but lacking cysteine. Under these conditions, more methane was derived from the methyl carbon of acetate than from the carboxyl carbon. Methanogenesis from the methyl group increased with increasing acetate concentration. The methyl carbon contributed up to 42% of the methane formed with H₂/CO₂ and up to 5% with methanol. Methanol stimulated the oxidation of the methyl group of acetate to CO₂. The average rates of methane formation from acetate were 1.3 nomol/min ·ml/culture (0.04mg² cell dry weight) in defined media (gas phase H₂/CO₂) and complex media (gas phase N₂). Acetate contributed up to 60% of cell carbon formed under the growth conditions examined. Similar quantities of cell carbon were derived from the methyl and carboxyl carbons of acetate, suggesting incorporation of this compound as a two-carbon unit. Incorporated acetate was not preferentially localized in lipid material, as 70% of the incorporated acetate was found in the wall and protein cell fractions. Acetate catabolism was stimulated by pregrowing of cultures in media containing acetate, while acetate anabolism was not influenced. The results are discussed in terms of the differences between the mechanisms of acetate catabolism and anabolism.Abbreviations CH₃-S-CoM methyl coenzyme M - TCA trichloroacetic acid - CoM coenzyme M (2-mercaptoethane sulfonic acid) - E^o standard potential change (pH 7) - F₄₂₀ Factor 420, a low redox electron carrier - G^o standard free energy change (pH 7) - kJ kilojoules (=0.24 kilocalories) - PBBW Weimer's phosphate-buffered basal medium - X unknown C₁ carrier     

Methanol conversion in Eubacterium limosum

The conversion of methanol by cell-free extracts of the acetogenic bacterium Eubacterium limosum was studied. Incubation of mixed cell-free extracts of both E. limosum and Methanobacterium formicicum resulted in methane formation from methanol in the presence of ATP and 2-mercaptoethanesulfonic acid. The separate extracts were not able to perform this reaction. Addition of ferredoxin obtained from Methanosarcina barkeri to the mixed extracts resulted in increased methane formation. The enzyme, responsible for methanol binding in cell-free extract of E. limosum, was inactivated by FAD under N₂ and exhibited maximal activity under an atmosphere of H₂. This enzyme contains a firmly bound cobalamin which was methylated by methanol in the presence of ATP. It was demethylated in the presence of methylcobalamin: coenzyme M methyltransferase obtained from M. barkeri under concomitant formation of methylated coenzyme M. These properties are similar to those of methanol: 5-hydroxybenzimidazolylcobamide methyltransferase from M. barkeri. It was proposed that methylotrophic acetogens and methylotrophic methanogens use similar enzymes in the first step of methanol conversion.Abbreviations HS-CoM 2-mercaptoethanesulfonic acid - CH₃S-CoM 2-(methylthio)ethanesulfonic acid - BrES 2-bromoethanesulfonic acid - TES N-tris(hydroxymethyl)-methyl-2-aminoethanesulfonic acid - MT₁ methanol: 5-hydroxybenzimidazolylcobamide methyltransferase - MT₂ methylcobalamin - HS-CoM methyltransferase - DMBI 5,6-dimethylbenzimidazole and HBI, 5-hydroxybenzimidazole, are -ligands of corrinoids - (S-CoM)₂ 2,2-dithiodiethanesulfonic acid     

Methanothrix soehngenii gen. nov. sp. nov., a new acetotrophic non-hydrogen-oxidizing methane bacterium

A new genus of methanogenic bacteria is described, which was isolated from a mesophilic sewage digester. It is most probably the filamentous bacterium, earlier referred to asMethanobacterium soehngenii, fat rod or acetate organism. The single non-motile, non-sporeforming cells are rod-shaped (0.8×2 m) and are normally combined end to end in long filaments, surrounded by a sheath-like structure. The filaments form characteristic bundles.Methanothrix soehngenii decarboxylates acetate, yielding methane and carbon dioxide. Other methanogenic substrates (H₂–CO₂, formate, methanol, methylamines) are not used for growth or methane formation. Formate is split into hydrogen and carbon dioxide. The temperature optimum for growth and methane formation is 37°C and the optimal pH range is 7.4–7.8. Sulfide and ammonia serve as sulfur and nitrogen source respectively. Oxygen completely inhibits growth and methane formation, but the bacteria do not loose their viability when exposed to high oxygen concentrations. 100 mg/l vancomycin showed no inhibition of growth and methanogenesis. No growth and methane formation was observed in the presence of: 2-bromoethanesulfonic acid, viologen dyes, chloroform, and KCN. The bacterium has a growth yield on acetate of 1.1–1.4 g biomass per mol acetate. The apparent K _S of the acetate conversion system to methane and carbon dioxide is 0.7 mmol/l. The DNA base composition is 51.9 mol% guanine plus cytosine. The nameMethanothrix is proposed for this new genus of filamentous methane bacterium. The type species,Methanothrix soehngenii sp. nov., is named in honor of N. L. Söhngen.     

Reductive dechlorination of 1,2-dichloroethane and chloroethane by cell suspensions of methanogenic bacteria

Concentrated cell suspensions of methanogenic bacteria reductively dechlorinated 1,2-dichloroethane via two reaction-mechanisms: a dihalo-elimination yielding ethylene and two hydrogenolysis reactions yielding chloroethane and ethane, consecutively. The transformation of chloroethane to ethane was inhibited by 1,2-dichloroethane. Stimulation of methanogenesis caused an increase in the amount of dechlorination products formed, whereas the opposite was found when methane formation was inhibited. Cells of Methanosarcina barkeri grown on H₂/CO₂ converted 1,2-dichloroethane and chloroethane at higher rates than acetate or methanol grown cells.Abbreviations BrES 2-bromoethanesulfonic acid - CA chloroethane - 1,2-DCA 1,2-dichloroethane - F₄₃₀ Ni(II)tetrahydro-(12, 13)-corphin with an uroporphinoid (III) ligand skeleton     

Evidence for the involvement and role of a corrinoid enzyme in methane formation from acetate in Methanosarcina barkeri

Methane formation from acetate in cell suspensions of Methanosarcina barkeri was inhibited by low concentrations (5 M) of propyl iodide. Inhibition was abolished by short exposure of the suspension to light which strongly indicates that a corrinoid enzyme is involved in methanogenesis from acetate. Propyl iodide (5M) had no effect on the exchange reaction between the carboxyl group of acetate and ¹⁴CO₂, and on methane formation from methanol, from H₂ and methanol, or from H₂ and CO₂. These findings indicate that the proposed corrinoid enzyme has a role in methyl group transfer to coenzyme M after C-C cleavage of acetate.Dedicated to Professor N. Pfennig on the occasion of his 60th birthday     

Changes in the kinetics of phosphate and potassium absorption in nutrient-deficient barley roots measured by a solution-depletion technique

From measurements of the rates of depletion of labelled ions from solution in the low concentration range, we described the phosphate and potassium uptake characteristics of the roots of intact barley plants in terms of the kinetic parameters, K _m and I _max (the maximum rate of uptake). In relatively young (13 d) and older (42 d) plants, cessation of phosphate supply for 4 d or more caused a marked increase in I _max (up to four times), without concomitant change in K _m, which remained between 5 and 7 M. By contrast, 1 d of potassium starvation with 14-d plants caused a decline in the K _m (i.e. an increased apparent affinity for potassium) from 53 M to 11 M, without alteration to I _max. After longer periods of potassium starvation, I _max increased (about two times) while the K _m remained at the same low value. Growth of shoots and roots were unaffected by these treatments, so that concentrations of ions in the tissues declined after 1 d or more of nutrient starvation, but we could not identify a characteristic endogenous concentration for either nutrient at which changes in kinetic parameters were invariably induced. The possible mechanisms regulating carriermediated transport, and the importance of changes induced in kinetic parameters in ion uptake from solution and soil are discussed.Symbol I _max the maximum rate of absorption at saturating concentrations     

Detection of gangliosides by direct binding ofLimax flavus agglutinin to thin layer chromotograms

A simple and sensitive method for detecting gangliosides on TLC plates is described. Gangliosides are extracted by phase partition in chloroform/methanol, developed on TLC plates in chloroform/methanol/0.25% aqueous KCl (5/4/1 by vol) and identified by binding of¹²⁵I-labelled, sialic acid-specificLimax flavus agglutinin (LFA) autoradiography and scanning densitometry. The detection limit of the method is below 1 ng (0.5 pmol) for GM3, GM1 and GT1b, and below 0.3 ng (0.2 pmol) for GM2 and GD1a. Binding of¹²⁵I-LFA is not inhibited by 10⁶-fold molar excess concentrations ofN-acetylneuraminic acid or lactose but is decreased in a dose-dependent manner by eitherN-acetylneuraminyllactose or unlabelled lectin. Gangliosides were not detected after their treatment byClostridium perfringens sialidase in the presence of taurocholic acid. Ten gangliosides were detected in human brain and seven in normal human serum. Extracts from 0.2 mg of brain and 20 l of serum were sufficient for the detection of major gangliosides.Abbreviations LFA Limax flavus agglutinin - ELLA Enzyme Linked Lectin Assay - PIM Poly(isobutyl methacrylate) - PVP Polyvinylpyrrolidone mol.wt. 40,000 - PBS Phosphate buffered saline - BSA Bovine serum albumin     

In vitro inhibition of Candida albicans growth by plant extracts and essential oils

The opportunistic Candida albicans yeast strain ATCC 10261 grows at 37 °C and gives germ tubes after 3 h on corn meal agar and blood plasma. It produces chlamydospores and assimilates sucrose, dextrose, galactose, maltose, trehalose and xylose among the tested carbon sources. Other growth characters were also investigated. The agar diffusion cut plug technique revealed that 200 l of Foeniculum vulgare Miller (fennel), Mentha piperita L. (peppermint) and Citrus limon (lemon) essential oils showed inhibitory actions. Fumigation test of the three essential oils had complete inhibition on the growth. The essential oil of Eucalyptus occidentalis End1 (eucalyptus, eucalypt) had no influence on C. albicans growth by the two tests. Meanwhile, methanol extracts of both leaves and male cones of the conifer Thuja orientalis (eastern thuya) had an inhibitory influence on the growth by two tests (cut plug and filter paper disc assay). In comparative tests the antibiotics mycostatin and dermatin had an inhibitory activity on C. albicans at the concentrations of 50–80 g per hole. Crude methanol extract of leaves of eastern thuya (10–80 g/ml) reduced C. albicans growth and intercellular protein nitrogen on liquid media.     

Hydration of 3-cyanopyridine to nicotinamide by crude extract nitrile hydratase

Summary The kinetic and stability characteristics of crude extract nitrile hydratase fromBrevibacterium R-312 were studied for the hydration of 3-cyanopyridine to nicotinamide. The enzyme was substrate and product inhibited and had the following kinetic constants:K _m =28 mM;K _p =36 mM;K _s =155 mM;V _m =5.8 mol/min/mg protein (25°C). Itsmaximum temperature and pH (phosphate buffer) were 35°C and 8.0, respectively and it had half-lives of 50 days, 10 days and 1 day at 4°C, 10°C and 25°C, respectively. The crude extract also exhibited amidase activity on nicotinamide, but it became significant only at nicotinamide concentrations greater than 300 mM. Mathematical models for batch and fed-batch hydrations were developed to account for substrate and product inhibitions and for enzyme decay. They predicted to within 10% experimental results for initial substrate and final product concentrations up to 300 mM; the accuracies decreased at higher concentrations primarily because of the relatively rapid hydrolysis of nicotinamide.     

Formation of thionates by freshwater and marine strains of sulfate-reducing bacteria

The formation of thionates (thiosulfate, trithionate and tetrahionate) during the reduction of sulfate or sulfite was studied with four marine and four freshwater strains of sulfate-reducing bacteria. Growing cultures of two strains of the freshwater species Desulfovibrio desulfuricans formed up to 400 M thiosulfate and 100 M trithionate under conditions of electron donor limitation. Tetrathionate was observed in lower concentrations of up to 30 M. Uncoupler-treated washed cells of the four freshwater strains formed thiosulfate and trithionate at low electron donor concentrations with sulfite in excess. In contrast, only one of four marine strains formed thionates. The freshwater strain Desulfobulbus propionicus transformed sulfite almost completely to thiosulfate and trithionate. The amounts produced increased with time, concentration of added sulfite and cell density. Tetrathionate was detected only occasionally and in low concentrations, and was probably formed by chemical oxidation of thiosulfate. The results confirm the diversity of the sulfite reduction pathways in sulfate-reducing bacteria, and suggest that thiosulfate and trithionate are normal by-products of sulfate reduction.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone     

Comparative studies of hepatotoxicity and fumonisin B1 and B2 content of water and chloroform/methanol extracts of Fusarium moniliforme strain MRC 826 culture material

Fusarium moniliforme has been associated with several diseases including equine leukoencephalomalacia, human esophageal cancer and hepatotoxicity/hepatocarcinogenicity in laboratory animals. The potential health risks to animals and humans posed by F. moniliforme contaminated grains cannot be assessed until the toxins are identified and toxicologically evaluated. As part of a systematic approach to identifying the hepatotoxins produced by F. moniliforme, diets containing aqueous and chloroform/methanol (11) extracts of F. moniliforme strain MRC 826 culture material (CM) and/or the extracted CM residues were fed to male Sprague-Dawley rats for four weeks. Serum alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase activities were increased after two and four weeks and microscopic liver lesions were found in those animals fed aqueous CM extract and the CM residue after chloroform/ methanol extraction. Fumonisins B₁ and B₂ were extracted from the CM by water, but not chloroform/ methanol, and were present in the toxic diets at concentrations of 93–139 and 82–147 ppm, respectively. Nontoxic diets contained 22 ppm fumonisin B₁ and 65 ppm fumonisin B₂.Abbreviations CM culture material - ELEM equine leukoencephalomalacia Mention of a trademark, proprietory name or vendor does not imply its approval by the US Department of Agriculture to the exclusion of others that may also be suitable.     

Molecular analysis of methane monooxygenase from Methylococcus capsulatus (Bath)

Methane monooxygenase (MMO) is the enzyme responsible for the conversion of methane to methanol in methanotrophic bacteria. In addition, this enzyme complex oxidizes a wide range of aliphatic and aromatic compounds in a number of potentially useful biotransformations. In this study, we have used biochemical data obtained from purification and characterization of the soluble MMO from Methylococcus capsulatus (Bath), to identify structural genes encoding this enzyme by oligonucleotide probing. The genes encoding the and subunits of MMO were found to be chromosomally located and were linked in this organism. We report here on the analysis of a recombinant plasmid containing 12 kilobases of Methylococcus DNA and provide the first evidence for the localization and linkage of genes encoding the methane monooxygenase enzyme complex. DNA sequence analysis suggests that the primary structures of the and subunit of MMO are completely novel and the complete sequence of these genes is presented.     

Improvement in cell yield of Methylobacterium sp. by reducing the inhibition of medium components for poly-β-hydroxybutyrate production

The inhibitory effect of the concentrations of medium components on the growth of Methylobacterium sp. for poly--hydroxybutyrate production was investigated by measuring the specific growth rates for various concentrations of each medium component. When the methanol concentration was increased, the cell growth decreased and was strongly inhibited above 6% (v/v) methanol. Ammonia, calcium and iron ion did not significantly inhibit the cell growth while there were some inhibitory effects at high concentrations of sodium, potassium, and magnesium. In particular, phosphate gave most significant inhibition at concentrations higher than 75 mM. By using an automatic feeding control system of methanol, ammonia, phosphate, and minerals, their concentrations were maintained within the level necessary to reduce the inhibition of medium components. The finial dry cell weight of Methylobacterium sp. in such a system was 172 g/l at 84 h.     

Interaction of α-Chymotrypsin with Dimethyl Sulfoxide: A Change of Substrate Could “Change” the Interaction Mechanism

The kinetic behavior of -chymotrypsin was studied in water–DMSO mixtures at concentrations of the organic solvent that do not cause irreversible denaturation of the enzyme. Various substrates (N-substituted derivatives of L-tyrosine) were found to display substantially different kinetic patterns of interaction with -chymotrypsin, which can be described by totally different kinetic schemes. The differences were ascribed to competition between the N-acyl group of the substrate and the DMSO molecule at the S ₂ site of substrate binding to the active site of the enzyme.     

Regulation of methanol oxidation and carbon dioxide fixation in Xanthobacter strain 25a grown in continuous culture

The regulation of C₁-metabolism in Xanthobacter strain 25a was studied during growth of the organism on acetate, formate and methanol in chemostat cultures. No activity of methanol dehydrogenase (MDH), formate dehydrogenase (FDS) or ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisC/O) could be detected in cells grown on acetate alone over a range of dilution rates tested. Addition of methanol or formate to the feed resulted in the immediate induction of MDH and FDH and complete utilization (D=0.10 h^-1) of acetate and the C ₁-substrates. The activities of these enzymes rapidly dropped at the higher growth rates, which suggests that their synthesis is further controlled via repression by heterotrophic substrates such as acetate. Synthesis of RuBisC/O already occurred at low methanol concentrations in the feed, resulting in additive growth yields on acetate/methanol mixtures. The energy generated in the oxidation of formate initially allowed an increased assimilation of acetate (and a decreased dissimilation), resulting in enhanced growth yields on the mixture. RuBisC/O activity could only be detected at the higher formate/acetate ratios in the feed. The data suggest that synthesis of RuBisC/O and CO₂ fixation via the Calvin cycle in Xanthobacter strain 25 a is controlled via a (de)repression mechanism, as is the case in other facultatively autotrophic bacteria. Autotrophic CO₂ fixation only occurs under conditions with a diminished supply of heterotrophic carbon sources and a sufficiently high availability of suitable energy sources. The latter point is further supported by the clearly more pronounced derepressing effect exerted by methanol compared to formate.Abbreviations FDH formate dehydrogenase - FBPase fructose-1,6-bisphosphatase - ICDH isocitrate dehydrogenase - MDH methanol dehydrogenase - PQQ pyrrolo quinoline quinone - PRK phosphoribulokinase - RuBisC/O ribulose-1,5-bisphosphate carboxylase/oxygenase - RuMP ribulose monophosphate - TCA tricarboxylic acid cycle     

Methanogenic degradation of acetone by an enrichment culture

An anaerobic enrichment culture degraded 1 mol of acetone to 2 mol of methane and 1 mol of carbon dioxide. Two microorganisms were involved in this process, a filament-forming rod similar to Methanotrix sp. and an unknown rod with round to slightly pointed ends. Both organisms formed aggregates up to 300 m in diameter. No fluorescing bacteria were observed indicating that hydrogen or formate-utilizing methanogens are not involved in this process. Acetate was utilized in this culture by the Methanothrix sp. Inhibition of methanogenesis by bromoethanesulfonic acid or acetylene decreased the acetone degradation rate drastically and led to the formation of 2 mol acetate per mol of acetone. Streptomycin completely inhibited acetone degradation, and neither acetate nor methane was formed. ¹⁴CO₂ was incorporated exclusively into the C-1 atom of acetate indicating that acetone is degraded via carboxylation to an acetoacetate residue. It is concluded that acetone is degraded by a coculture of an eubacterium and an acetate-utilizing methanogen and that acetate is the only intermediate transferred between both. The energetical problems of the eubacterium converting acetone to acetate are discussed.     

Function of methylcobalamin: coenzyme M methyltransferase isoenzyme II in Methanosarcina barkeri

Methanosarcina barkeri was recently shown to contain two cytoplasmic isoenzymes of methylcobalamin: coenzyme M methyltransferase (methyltransferase 2). Isoenzyme I predominated in methanol-grown cells and isoenzyme II in acetate-grown cells. It was therefore suggested that isoenzyme I functions in methanogenesis from methanol and isoenzyme II in methanogenesis from acetate. We report here that cells of M. barkeri grown on trimethylamine, H₂/CO₂, or acetate contain mainly isoenzyme II. These cells were found to have in common that they can catalyze the formation of methane from trimethylamine and H₂, whereas only acetate-grown cells can mediate the formation of methane from acetate. Methanol-grown cells, which contained only low concentrations of isoenzyme II, were unable to mediate the formation of methane from both trimethylamine and acetate. These and other results suggest that isoenzyme II (i) is employed for methane formation from trimethylamine rather than from acetate, (ii) is constitutively expressed rather than trimethylamine-induced, and (iii) is repressed by methanol. The constitutive expression of isoenzyme II in acetate-grown M. barkeri can explain its presence in these cells. The N-terminal amino acid sequences of isoenzyme I and isoenzyme II were analyzed and found to be only 55% similar.Abbreviations H-S-CoM coenzyme M or 2-mercaptoethane-sulfonate - CH₃-S-CoM methyl-coenzyme M or 2(methylthio)-ethanesulfonate - [Co] cobalamin - CH₃-[Co] methylcobalamin - H₄MPT tetrahydromethanopterin - CH₃-H₄MPT N ⁵-methyltetrahydromethanopterin - MT₁ methyltransferase 1 or methanol: 5-hydroxybenzimidazolyl cobamide methyltransferase - MT₂ methyltransferase 2 or methylcobalamin: coenzyme M methyltransferase - Mops morpholinopropanesulfonate - 1 U = 1 mol/min