Showing papers on "Methanosarcina barkeri published in 1984"
TL;DR: In this article, it was shown that the C15 and C20 saturated alcohols in Methanobacterium thermoautotrophicum have mainly the all-R configuration.
189 citations
TL;DR: It is demonstrated that methane formation according to the equation CH3OH + H2----H2 ----H2----CH4 +H2O was coupled to ATP synthesis by a chemiosmotic mechanism and was under the control of delta psi: Methane formation only proceeded if the delta psi generated was used for ATP synthesis or if an uncoupler was present.
Abstract: The addition of methanol to a cell suspension of Methanosarcina barken resulted in an increase of the intracellular ATP concentration from 1 nmol/mg to 10 nmol/mg protein and in the formation of a proton-motive force Δp of-130 mV. Δp consisted of more than 90% of the membrane potential ΔΨ. These values were similar under N2 and under H2. The addition of the uncoupler tetrachlorosalicylanilide to the above system under N2 led to a drastic decrease of both, the ATP concentration and the Δp and to a stop of methanogenesis. With methanol and H2, however, methane formation continued, although the effect of the uncoupler on the ATP pool and on Δp was a under N2. The proton-translocating ATPase inhibitor N,N-dicyclohexylcarbodiimide caused a rapid exhaustion of the ATP pool and a discontinuation of methane synthesis, whereas Δp was unaffected. Inhibition of methane formation under these conditions could be relieved by the addition of the uncoupler tetrachlorosalicylanilide. These results demonstrate that methane formation according to the equation CH3OH + H2 CH4+ H2O was coupled to ATP synthesis by a chemiosmotic mechanism and was under the control of ΔΨ: Methane formation only proceeded if the ΔΨ generated was used for ATP synthesis or if an uncoupler was present. Under N2, methane formation according to the equation 4CH3OH CO2+ 3 CH4+ 2H2O was abolished by an uncoupler, because one step in the oxidation of methanol to 1CO2 apparently depended on an energized state of the membrane.
149 citations
TL;DR: These experiments document the ability of the organism to grow slowly and to produce methane and CO2 either on CO as the sole carbon and energy source or by the simultaneous consumption of methanol and CO.
Abstract: Methanosarcina barkeri was adapted to grow on carbon monoxide by sequential transfer of the culture in medium that contained CO (100% of culture headspace). These experiments document the ability of the organism to grow slowly (65-h doubling time) and to produce methane and CO/sub 2/ either on CO as the sole carbon and energy source or by the simultaneous consumption of methanol and CO. During growth on CO as carbon and energy source, net hydrogen formation occurred when the CO partial pressure in the culture headspace was greater than 20% CO, but hydrogen was consumed when the CO concentration was below this value.
131 citations
TL;DR: Diazotrophy by an archaebacterium, the methanogen Methanosarcina barkeri strain 227, is reported, raising interesting evolutionary questions about the divergence of the Archaebacteria, eubacteria and eukaryotes.
Abstract: The ability to fix nitrogen (N2) is found among a wide variety of the prokaryotic eubacteria, but not in eukaryotes1. In addition to the prokaryotic eubacteria and eukaryotes, a third ‘kingdom’—the archchaebacteria—has been defined based on the comparison of 16S ribosomal oligonucleotide sequence catalogues2,3. Included in the archaebacterial kingdom are certain obligate halophiles and thermoacidophiles, and the methanogens, strictly anaerobic, methane-producing bacteria4. Here we report diazotrophy by an archaebacterium, the methanogen Methanosarcina barkeri strain 227. Because it has been proposed that the archaebacteria, eubacteria and eukaryotes diverged at an early stage in evolution2,3, the discovery of diazotrophy (N2 fixation) in a member of the archaebacterial group raises interesting evolutionary questions.1
125 citations
TL;DR: The enzyme was nearly homogeneous after 20-fold purification, indicating that a significant proportion of soluble cell protein was CO dehydrogenase (ca. 5%), and the spectral properties of the enzyme were similar to those published for CO dehydration from acetogenic anaerobes.
Abstract: Carbon monoxide-dependent production of H2, CO2, and CH4 was detected in crude cell extracts of acetate-grown Methanosarcina barkeri. This metabolic transformation was associated with an active methyl viologen-linked CO dehydrogenase activity (5 to 10 U/mg of protein). Carbon monoxide dehydrogenase activity was inhibited 85% by 10 microM KCN and was rapidly inactivated by O2. The enzyme was nearly homogeneous after 20-fold purification, indicating that a significant proportion of soluble cell protein was CO dehydrogenase (ca. 5%). The native purified enzyme displayed a molecular weight of 232,000 and a two-subunit composition of 92,000 and 18,000 daltons. The enzyme was shown to contain nickel by isolation of radioactive CO dehydrogenase from cells grown in 63Ni. Analysis of enzyme kinetic properties revealed an apparent Km of 5 mM for CO and a Vmax of 1,300 U/mg of protein. The spectral properties of the enzyme were similar to those published for CO dehydrogenase from acetogenic anaerobes. The physiological functions of the enzyme are discussed.
114 citations
TL;DR: It is concluded that methanogenesis is sensitive to both the pH of the cytoplasm and the medium, and additions of NH4OH or various NH+4 salts were most effective in causing K+ depletion in media of alkaline pH, suggesting that NH3 was the chemical species crossing the membrane.
105 citations
TL;DR: The data indicate that the isotopic exchange between CO2 and the carboxyl group of acetate is a partial reaction of methanogenesis from acetate, and proposed mechanisms of methane and CO2 formation from acetates are discussed.
Abstract: 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 μM) 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 μM) also affected methane formation from CO. The results are discussed with respect to proposed mechanisms of methane and CO2 formation from acetate.
88 citations
TL;DR: Methanol:5-hydroxybenzimidazolylcobamide methyltransferase from Methanosarcina barkeri has been purified to approximately 90% homogeneity by ion-exchange chromatography on DEAE-cellulose and QAE-A50 Sephadex columns.
Abstract: Methanol:5-hydroxybenzimidazolylcobamide methyltransferase from Methanosarcina barkeri has been purified to approximately 90% homogeneity by ion-exchange chromatography on DEAE-cellulose and QAE-A50 Sephadex columns. The molecular weight, estimated by gel electrophoresis, was found to be 122,000, and the enzyme contained two different subunits with molecular weights of 34,000 and 53,000, which indicates an alpha 2 beta structure. The enzyme contains three or four molecules of 5-hydroxybenzimidazolylcobamide, which could be removed by treatment of the enzyme with 2-mercaptoethanol or sodium dodecyl sulfate. In both cases the enzyme dissociated into its subunits. For stability, the enzyme required the presence of divalent cations such as Mg2+, Mn2+, Sr2+, Ca2+, or Ba2+. ATP, GTP, or CTP was needed in a reductive activation process of the enzyme. This activation was brought about by a mixture of H2, ferredoxin, and hydrogenase, but also by CO, which is thought to reduce the corrinoid chemically. The CO dehydrogenase-like activity of the methyltransferase is discussed.
85 citations
TL;DR: There appeared to be a correlation among the organisms maintaining the highest internal potassium concentrations also had the largest number of acidic ribosomal proteins.
Abstract: The intracellular potassium concentration and percentage of acidic ribosomal proteins were determined for nine methanogenic bacteria representing diverse phylogenetic groupings. Representatives of the Methanobacteriaceae family possessed very high internal potassium concentrations: Methanobrevibacter arboriphilus, 1225 mM; Methanobrevibacter smithii, 1065 mM; Methanobacterium thermoautotrophicum, 1103 mM; Methanobacterium bryantii, 861 mM; and Methanobacterium strain G2R, 886 mM. Members of other families tested maintained much lower internal concentrations of potassium. Methanosarcina barkeri (family Methanosarcinaceae) and Methanospirillum hungatei (family Methanomicrobiaceae) maintained approximately 200 mM internal potassium, while Methanococcus voltae (family Methanococcaceae) possessed an intermediate concentration of 725 mM. Those organisms maintaining the highest internal potassium concentrations also had the largest number of acidic ribosomal proteins. There appeared to be a correlation among the...
65 citations
TL;DR: Evidence is presented which indicates that a protein component may be associated with the heteropolysaccharide cell wall of Methanosarcina barkeri strain 227, and it is proposed that these species remain in separate genera based on differences in ribosomal ribonucleic acid homology and fractional differences in midpoint temperatures.
Abstract: The phylogenetic relationships among seven strains of methylotrophic methane-producing bacteria were determined by ribosomal ribonucleic acid hybridization and deoxyribonucleic acid homology techniques. The strains tested had deoxyribonucleic acid guanine-plus-cytosine contents of 39 to 43 mol% and represented a diversity of phenotypic characteristics. Our results indicate that these strains should be divided into six species within the genera Methanosarcina, Methanolobus, and Methanococcoides. The genus Methanosarcina includes Methanosarcina barkeri strains MS and 227, Methanosarcina sp. strain TM-1, Methanosarcina acetivorans strain C2A, and Methanosarcina mazei strain S-6. The genus Methanolobus is represented by Methanolobus tindarius strain Tindari 3, and the genus Methanococcoides is represented by Methanococcoides methylutens strain TMA-10. Despite phenotypic similarities between Methanolobus tindarius and Methanococcoides methylutens, we propose that these species remain in separate genera based on differences in ribosomal ribonucleic acid homology and fractional differences in midpoint temperatures. The divisions indicated by deoxyribonucleic acid homology experiments complemented the ribosomal ribonucleic acid hybridization results. Phenotypic characteristics were consistent with these phylogenetic divisions; an apparent exception was cell wall composition, which is a conserved trait. Methanosarcina acetivorans had only a thin protein cell wall, but all other strans of Methanosarcina previously studied have been reported to have heteropolysaccharide cell walls. We present evidence which indicates that a protein component may be associated with the heteropolysaccharide cell wall of Methanosarcina barkeri strain 227.
59 citations
TL;DR: A soluble hydrogenase from the methanogenic bacterium, Methanosarcina barkeri has been purified to apparent electrophoretic homogeneity, with an overall 550-fold purification, a 45% yield and a final specific activity of 270 mumol H2 evolved min-1 (mg protein)-1.
Abstract: A soluble hydrogenase from the methanogenic bacterium, Methanosarcina barkeri (DSM 800) has been purified to apparent electrophoretic homogeneity, with an overall 550-fold purification, a 45% yield and a final specific activity of 270 mumol H2 evolved min-1 (mg protein)-1. The hydrogenase has a high molecular mass of approximately equal to 800 kDa and subunits with molecular masses of approximately equal to 60 kDa. The enzyme is stable to heating at 65 degrees C and to exposure to air at 4 degrees C in the oxidized state for periods up to a week. The overall stability of this enzyme is compared with other hydrogenase isolated from strict anaerobic sulfate-reducing bacteria. Ms. barkeri hydrogenase shows an absorption spectrum typical of a non-heme iron protein with maxima at 275 nm, 380 nm and 405 nm. A flavin component, identified as FMN or riboflavin was extracted under acidic conditions and quantified to approximately one flavin molecule per subunit. In addition to this component, 8-10 iron atoms and 0.6-0.8 nickel atom were also detected per subunit. The electron paramagnetic resonance (EPR) spectrum of the native enzyme shows a rhombic signal with g values at 2.24, 2.20 and approximately equal to 2.0. probably due to nickel which is optimally measured at 40 K but still detectable at 77 K. In the reduced state, using dithionite or molecular hydrogen as reductants, at least two types of g = 1.94 EPR signals, due to iron-sulfur centers, could be detected and differentiated on the basis of power and temperature dependence. Center I has g values at 2.04, 1.90 and 1.86, while center II has g values at 2.08, 1.93 and 1.85. When the hydrogenase is reduced by hydrogen or dithionite the rhombic EPR species disappears and is replaced by other EPR-active species with g values at 2.33, 2.23, 2.12, 2.09, 2.04 and 2.00. These complex signals may represent different nickel species and are only observable at temperatures higher than 20 K. In the native preparation, at high temperatures (T greater than 35 K) or in partially reduced samples, a free radical due to the flavin moiety is observed. The EPR spectrum of reduced hydrogenase in 80% Me2SO presents an axial type of spectrum only detectable below 30 K.
TL;DR: A bacterial consortium capable of sucrose degradation primarily to CH(4) and CO(2) was constructed, with acetate as the key methanogenic precursor, and the effect of agar immobilization on the activity of the consortium was determined.
Abstract: A bacterial consortium capable of sucrose degradation primarily to CH4 and CO2 was constructed, with acetate as the key methanogenic precursor. In addition, the effect of agar immobilization on the activity of the consortium was determined. The primary fermentative organism, Escherichia coli, produced acetate, formate, H2, and CO2 (known substrates for methanogens), as well as ethanol and lactate, compounds that are not substrates for methanogens. Oxidation of the nonmethanogenic substrates, lactate and ethanol, to acetate was mediated by the addition of Acetobacterium woodii and Desulfovibrio vulgaris. The methanogenic stage was accomplished by the addition of the acetophilic methanogen Methanosarcina barkeri and the hydrogenophilic methanogen Methanobacterium formicicum. Results of studies with low substrate concentrations (0.05 to 0.2% [wt/vol]), a growth-limiting medium, and the five-component consortium indicated efficient conversion (40%) of sucrose carbon to CH4. Significant decreases in yields of CH4 and rates of CH4 production were observed if any component of the consortium was omitted. Approximately 70% of the CH4 generated occurred via acetate. Agar-immobilized cells of the consortium exhibited yields of CH4 and rates of CH4 production from sucrose similar to those of nonimmobilized cells. The rate of CH4 production decreased by 25% when cysteine was omitted from reaction conditions and by 40% when the immobilized consortium was stored for 1 week at 4°C.
TL;DR: It was proposed that methylotrophic acetogens andmethylotrophic methanogens use similar enzymes in the first step of methanol conversion.
Abstract: 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.
TL;DR: Cell extracts prepared from Methanosarcina barkeri converted acetate into methane and carbon dioxide under a hydrogen atmosphere in a manner consistent with that observed in whole cells and a proposed model for ATP generation from acetate that involves both membrane-bound and soluble enzymatic components such as CO dehydrogenase.
Abstract: Cell extracts prepared from Methanosarcina barkeri converted acetate into methane and carbon dioxide under a hydrogen atmosphere. Methanogenesis by cell extracts required acetate and ATP and, the in vitro rate was 5 to 10% of the rate of methanogenesis observed during exponential growth of cells on acetate. Methane and carbon dioxide produced by cell extracts originated predominantly from the methyl and carboxyl groups of acetate, respectively, in a manner consistent with that observed in whole cells. Acetate degradation activity was detected in the soluble (150000 × g supernatant) fraction and not in the membrane fraction. These results are discussed in relation to a proposed model for ATP generation from acetate that involves both membrane-bound and soluble enzymatic components such as CO dehydrogenase.
TL;DR: A trimethylamine:2-mercaptoethanesulfonate (HS-coenzyme M) methyltransferase has been shown to be present in trimmedethylamines grown cells but not in methanol-grown cells of Methanosarcina barkeri.
Abstract: A trimethylamine:2-mercaptoethanesulfonate (HS-coenzyme M) methyltransferase has been shown to be present in trimethylamine-grown cells but not in methanol-grown cells of Methanosarcina barkeri. The transfer of one methyl group was catalyzed by this enzyme so that dimethylamine and methyl-S-coenzyme M were the products. Enzyme activity required the presence of ATP and preincubation of the protein solution under H2. Fifty percent of the maximum activity was obtained under N2 in the presence of NAD(P)H plus dithioerythritol.
TL;DR: In this article, an uncoupler tetrachlorosalicylanilide (TCS) was added to the uncoupled TCS to improve the inhibition of methane formation from CH2O + H2, but not from CO2+H2.
Abstract: Methane formation from formaldehyde and H2 or from carbon dioxide and H2, as performed by cell suspensions of Methanosarcina barkeri, was coupled to ATP synthesis. In correspondence with this, methane formation was inhibited by N,N′-dicyclohexylcarbodiimide (DCCD), which at the same time, caused a decrease of the intracellular ATP concentration but only a slow decrease of the membrane potential. Addition of the uncoupler tetrachlorosalicylanilide (TCS) led to a relief of the inhibition of methane formation from CH2O + H2, but not from CO2 + H2.
TL;DR: The reducing system of hydrogenase and ferredoxin was able to reduce dithiols, like dithiodiethanesulfonate and cystine to their monomers, in the presence of a corrinoid, which acts as an electron carrier.
TL;DR: Experiments indicate that the biosynthesis of factor III in the “archaebacterium” M. barkeri is similar to the corrinoid biosynthesis in the anaerobic “eubacteria” Eubacterium limosum, Clostridium barkeri, and Clostridgeium thermoaceticum.
Abstract: Exogenous 5-hydroxy-[2-14C]benzimidazole was transformed by Methanosarcina barkeri into 5-hydroxy-[2-14C]benzimidazolylcobamide. Thereby the endogenous biosynthesis of 5-hydroxybenzimidazole was completely blocked.
TL;DR: A gas-vacuolate strain of Methanosarcina barkeri formed protoplast in substrate-depleted cultures and gas vesicles were isolated from the protoplasts and were shown by electron microscopy to have a rib spacing of 4.8 nm.
Abstract: SUMMARY: A gas-vacuolate strain of Methanosarcina barkeri formed protoplasts in substrate-depleted cultures and gas vesicles were isolated from the protoplasts. Vacuolate protoplasts were separated from unvacuolate ones by flotation and the protoplast membrane was removed by Tween 20, liberating the gas vesicles. The gas vesicles were purified by flotation after initial passage through a 0.45 μm filter to remove contaminating material. Gas vesicle membranes were purified by isopycnic gradient centrifugation and were shown by electron microscopy to have a rib spacing of 4.8 nm.
TL;DR: It is requested that an Opinion regarding rejection of the type species Methanosarcina methanica (Approved Lists 1980) and conservation of the genus Methosarcina with MethanosARCina barkeri (App Approved Lists1980) be given.
Abstract: We request an Opinion regarding rejection of the type species Methanosarcina methanica (Approved Lists 1980) and conservation of the genus Methanosarcina with Methanosarcina barkeri (Approved Lists 1980) as the type species, and we provide an emended description of the genus Methanosarcina.
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TL;DR: The 5-hydroxybenzimidazolylcobamide derivatives from Methanosarcina barkeri were isolated and two B12-HBI compounds with unidentified β-ligands were found, of which one constituted a major part of the corrinoid content.
TL;DR: Cell lysates of acetate-grown Methanosarcina barkeri 227 were found to cleave acetate to CH4 and CO2, indicating the aceticlastic activity was not derived from unbroken cells, which are unaffected by proteolytic treatment.
Abstract: Cell lysates of acetate-grown Methanosarcina barkeri 227 were found to cleave acetate to CH/sub 4/ and CO/sub 2/. The aceticlastic reaction was identified by using radioactive methyl-labeled acetate. Cell lysates decarboxylated acetate in a nitrogen atmosphere, conserving the methyl group in methane. The rate of methanogenesis from acetate in the cell lysates was comparable to that observed with whole cells. Aceticlastic activity was found in the particulate fraction separate from methylcoenzyme M methylreductase activity, which occurs in the soluble fraction. Pronase treatment eliminated methylcoenzyme M methylreductase activity in lysates and stimulated aceticlastic activity, indicating the aceticlastic activity was not derived from unbroken cells, which are unaffected by proteolytic treatment.
TL;DR: Heterologous aminoacylation of tRNA with high selectivity for Archaebacterial tRNA and substrate properties of ATP analogues reveals a unique pattern, reflecting the supposed genealogical difference between the urkingdoms of archaebacteria, eubacteria, and eukaryotes.
TL;DR: 2-Bromoethanesulphonate, a specific inhibitor of methanogenesis, completely inhibited the enrichment at a concentration of 10 mumol/l and addition of H2 formate or methanol to the enrichment did not affect the rate of meetinghanogenesis.
Abstract: In a study of the anaerobic utilization of acetate, an enrichment culture of sewage sludge organisms was initiated with calcium acetate as the sole carbon and energy source. A mixed bacterial population became established from which 14 anaerobic species were isolated. Two of the isolates were methanogenic bacteria but only one of these, Methanosarcina barkeri, utilised acetate as an energy source in axenic culture. The other methanogenic isolate, a Methanobacterium sp., utilised H2/CO2 but not acetate. A third methanogen, which was morphologically identical to Methanothrix soehngenii, was detected in the enrichment but was not obtained in monoculture. 2-Bromoethanesulphonate, a specific inhibitor of methanogenesis, completely inhibited the enrichment at a concentration of 10 mumol/l. Addition of H2 formate or methanol to the enrichment did not affect the rate of methanogenesis. An H2-utilizing Desulfovibrio sp. was also isolated from the enrichment.
TL;DR: Measurements of time-resolved photobleaching and nanosecond fluorescence decay from microscopic samples of methanogenic bacteria are reported and active and inactive bacterial cells could be differentiated by following the course of photobLEaching.
01 Jan 1984
01 Jan 1984
TL;DR: A bacterial consortium capable of sucrose degradation primarily to CH/sub 4/ and CO/sub 2/ was constructed, with acetate as the key methanogenic precursor, and the effect of agar immobilization on the activity of the consortium was determined.
Abstract: A bacterial consortium capable of sucrose degradation primarily to CH/sub 4/ and CO/sub 2/ was constructed, with acetate as the key methanogenic precursor. In addition, the effect of agar immobilization on the activity of the consortium was determined. The primary fermentative organism, Escherichia coli, produced acetate, formate, H/sub 2/, and CO/sub 2/ (known substrates for methanogens), as well as ethanol and lactate, compounds that are not substrates for methanogens. Oxidation of the nonmethanogenic substrates, lactate and ethanol, to acetate was mediated by the addition of Acetobacterium woodii and Desulfovibrio vulgaris. The methanogenic stage was accomplished by the addition of the acetophilic methanogen Methanosarcina barkeri and the hydrogenophilic methanogen Methanobacterium formicicum. Results of studies with low substrate concentrations (0.05 to 0.2% (wt/vol)), a growth-limiting medium, and the five-component consortium indicated efficient conversion (40%) of sucrose carbon to CH/sub 4/. Significant decreases in yields of CH/sub 4/ and rates of CH/sub 4/ production were observed if any component of the consortium was omitted. Approximately 70% of the CH/sub 4/ generated occurred via acetate. Agar-immobilized cells of the consortium exhibited yields of CH/sub 4/ and rates of CH/sub 4/ production from sucrose similar to those of nonimmobilized cells. The rate of CH/sub 4/more » production decreased by 25% when cysteine was omitted from reaction conditions and by 40% when the immobilized consortium was stored for 1 week at 4/sup 0/C.« less
01 Jan 1984
TL;DR: Results support CH3-S-CoM asacandidate for the proposed universal intermediate X-CH3 and support Barker'shypothesis ofaunifying mechanism for theterminal step in methanogenesis from all knownsubstrates.
Abstract: Thetransfer ofthemethyl group ofacetate tocoenzyme M (2-mercaptoethanesulfonic acid; HS-CoM) during themetabolism ofacetate tomethane wasinvestigated incultures ofMethanosarcina strain TM-1.The organism metabolized CD3COO to83%CD3Hand17%CD2H2andproduced noCDH3orCH4.Theisotopic composition ofcoenzyme M incells grownonCD3COO-wasanalyzed withanovel gaschromatography-mass spectrometry technique. Thecells contained CD3-S-CoM andCD2H-S-CoM) inaproportion similar tothat of CD3HtoCD2H2.Theseresults, inconjunction withareport (J.K.Nelson andJ.G.Ferry, J.Bacteriol. 160:526-532, 1984) that extracts ofacetate-grown strain TM-1contain highlevels ofCH3-S-CoM methylreductase, indicate thatCH3-S-CoM isanintermediate inthemetabolism ofacetate tomethane inthis organism. Nearly 30yearsago,Barker (5)proposed a common terminal methyl carrier (X-CH3) formethaneproduction fromacetate, methanol, orthereduction ofCO2.Since that time, methylamines havealso beenidentified asmethanogenicsubstrates (9). Coenzyme M (2-mercaptoethanesulfonic acid; HS-CoM), acofactor unique tomethanogenic bacteria (3), isconsidered tobetheterminal methyl carrier for methane production fromH2-CO2, methanol, andmethylamine.'4CH3-S-CoM isformedfrom['4C]methanol, ['4C]methylamine, andthereduction of"4CO2, and2(methylthio)ethanesulfonic acid(CH3-S-CoM) isreductively demethylated tomethane byCH3-S-CoM methylreductase (4, 8,10,14,15,17). CH3-S-CoM istheonly substrate known tobereductively demethylated toformmethane. These results support CH3-S-CoM asacandidate fortheproposed universal intermediate X-CH3.However. ithasnotbeen previously determined whether CH3-S-CoM isalso anintermediate inmethanogenesis fromacetate. Studies onacetate metabolism havelagged behind studies onmethanogenesis fromother substrates because ofthe difficulties inculturing these oxygen-sensitive andslow-growingorganisms onacetate (4). In1956, itwasdemonstrated thatCD3COO ismetabolized toCD3Hinacetate enrichmentcultures (12). Morerecently, Methanosarcina barkeri wasshowntometabolize CD3COO to75%CD3Hand25% CD2H2(7). Wenowreport that Methanosarcina strain TM-1 produces CD3-S-CoM andCD2H-S-CoM during theconversion ofCD3COO toCD3HandCD2H2. Theseresults, along withthedemonstration ofcatabolically significant CH3-SCoMmethylreductase activity inthis organism (11), indicate thatCH3-S-CoM isanintermediate inthepathway for methanogenesis fromacetate andsupport Barker's (5)hypothesis ofaunifying mechanism fortheterminal stepin methanogenesis fromallknownsubstrates.
Patent•
14 Aug 1984
TL;DR: New anaerobic microorganism called Clostridium proteolyticum sp. nov. as mentioned in this paper was used for the production of proteases and collagenases and, on the other hand, of proline.
Abstract: New anaerobic microorganism called Clostridium proteolyticum sp. nov. and a process for isolating it from sludge or similar materials. The use of this microorganism on gelatine or a similar material for the production, on the one hand, of proteases and collagenases and, on the other hand, of proline. The use of this microorganism in combination with Methanosarcina barkeri for the substantially complete degradation of complex proteins and in particular of gelatine, into methane and into carbonic anhydride; process for preparing the C. proteolyticum and M. barkeri co-culture used in the methanisation process and the culture thus obtained.