Showing papers on "Methanosarcina barkeri published in 1991"

Characterization of Methanosarcina barkeri MST and 227, Methanosarcina mazei S-6T, and Methanosarcina vacuolata Z-761T

Abstract: Members of the genus Methanosarcina are recognized as major aceticlastic methanogens, and several species which thrive in low-salt, pH-neutral culture medium at mesophilic temperatures have been described. However, the environmental conditions which support the fastest growth of these species (Methanosarcina barkeri MST [T = type strain] and 227, Methanosarcina mazei S-6T, and Methanosarcina vacuolata Z-761T) have not been reported previously. Although the members of the genus Methanosarcina are widely assumed to grow best at pH values near neutrality, we found that some strains prefer acidic pH values. M. vacuolata and the two strains of M. barkeri which we tested were acidophilic when they were grown on H2 plus methanol, growing most rapidly at pH 5 and growing at pH values as low as 4.3. M. mazei grew best at pH values near neutrality. We found that all of the strains tested grew most rapidly at 37 to 42°C on all of the growth substrates which we tested. None of the strains was strongly halophilic, although the growth of some strains was slightly stimulated by small amounts of added NaCl. The catabolic substrates which supported most rapid growth were H2 plus methanol; this combination sometimes allowed growth of a strain under extreme environmental conditions which prevented growth on other substrates. The cell morphology of all strains was affected by growth conditions.

Effect of pH on Anaerobic Mild Steel Corrosion by Methanogenic Bacteria.

Abstract: Methanogens can use H2 produced by cathodic depolarization-mediated oxidation of elemental iron to produce methane. Thermodynamic consideration of the cathodic depolarization mechanism predicts more oxidation of Fe0 at lower pH. Methanogenic responses to pH by Methanococcus deltae, Methanococcus thermolithotrophicus, and Methanosarcina barkeri were examined. When grown on H2-CO2, these bacteria had pH optima from 6.2 to 7.0, but when all H2 was supplied from Fe0, methanogenic pH optima were lower, 5.4 to 6.5. Corrosion was monitored with and without cultures and at various pHs; more corrosion occurred when cultures were present, biologically induced corrosion was greatest at the pH optima for methanogenesis from Fe0, and corrosion without cultures increased with a drop in pH.

Activities of formylmethanofuran dehydrogenase methylenetetrahydromethanopterin dehydrogenase, methylenetetrahydromethanopterin reductase, and heterodisulfide reductase in methanogenic bacteria

Abstract: The activities of formylmethanofuran dehydrogenase, methylenetetrahydromethanopterin dehydrogenase, methylenetetrahydromethanopterin reductase, and heterodisulfide reductase were tested in cell extracts of 10 different methanogenic bacteria grown on H2/CO2 or on other methanogenic substrates. The four activities were found in all the organisms investigated: Methanobacterium thermoautotrophicum,M. wolfei, Methanobrevibacter arboriphilus, Methanosphaera stadtmanae, Methanosarcina barkeri (strains Fusaro and MS), Methanothrix soehngenii, Methanospirillum hungatei, Methanogenium organophilum, and Methanococcus voltae. Cell extracts of H2/CO2 grown M. barkeri and of methanol grown M. barkeri showed the same specific activities suggesting that the four enzymes are of equal importance in CO2 reduction to methane and in methanol disproportionation to CO2 and CH4. In contrast, cell extracts of acetate grown M. barkeri exhibited much lower activities of formylmethanofuran dehydrogenase and methylenetetrahydromethanopterin dehydrogenase suggesting that these two enzymes are not involved in methanogenesis from acetate. In M. stadtmanae, which grows on H2 and methanol, only heterodisulfide reductase was detected in activities sufficient to account for the in vivo methane formation rate. This finding is consistent with the view that the three other oxidoreductases are not required for methanol reduction to methane with H2.

Archaebacteria: Lipids, Membrane Structures, and Adaptation to Environmental Stresses

Abstract: In the past few years a revolution has occurred in the taxonomy of living organisms. In fact, on the basis of genetic studies and on the acquisition of other general biochemical features, organisms are no longer merely gathered into two groups of eubacteria and eukaryotes, but may be considered to belong to a third line, the archaebacteria (Woese 1987).

Purification and properties of N5,N10-methylenetetrahydromethanopterin reductase (coenzyme F420-dependent) from the extreme thermophile Methanopyrus kandleri.

Abstract: Methanopyrus kandleri belongs to a novel group of abyssal methanogenic archaebacteria that can grow at 110°C on H2 and CO2 and that shows no close phylogenetic relationship to any methanogens known so far. N 5 N 10 -Methylenetetrahydromethanopterin reductase, an enzyme involved in methanogenesis from CO2, was purified from this hyperthermophile. The apparent molecular mass of the native enzyme was found to be 300 kDa. Sodium dodecylsulfate/polyacrylamide gel electrophoresis revealed the presence of only one polypeptide of apparent molecular mass 38 kDa. The ultraviolet/visible spectrum of the enzyme was almost identical to that of albumin indicating the absence of a chromophoric prosthetic group. The reductase was specific for reduced coenzyme F420 as electron donor; NADH, NADPH or reduced dyes could not substitute for the 5-deazaflavin. The catalytic mechanism was found to be of the ternary complex type as deduced from initial velocity plots. V max at 65°C and pH 6.8 was 435 U/mg (kcat=275 s-1) and the K m for methylenetetrahydro-methanopterin and for reduced F420 were 6 μM and 4 μM, respectively. From Arrhenius plots an activation energy of 34 kJ/mol was determined. The Q 10 between 40°C and 90°C was 1.5. The reductase activity was found to be stimulated over 100-fold by sulfate and by phosphate. Maximal stimulation (100-fold) was observed at a sulfate concentration of 2.2 M and at a phosphate concentration of 2.5 M. Sodium-, potassium-, and ammonium salts of these anions were equally effective. Chloride, however, could not substitute for sulfate or phosphate in stimulating the enzyme activity. The thermostability of the reductase was found to be very low in the absence of salts. In their presence, however, the reductase was highly thermostable. Salt concentrations between 0.1 M and 1.5 M were required for maximal stability. Potassium salts proved more effective than ammonium salts, and the latter more effective than sodium salts in stabilizing the enzyme activity. The anion was of less importance. The N-terminal amino acid sequence of the reductase from M. kandleri was determined and compared with that of the enzyme from Methanobacterium thermoautotrophicum and Methanosarcina barkeri. Significant similarity was found.

N 5 , N 10 -Methylenetetrahydromethanopterin reductase (coenzyme F 420 -dependent) and formylmethanofuran dehydrogenase from the hyperthermophile Archaeoglobus fulgidus

Abstract: Methylene-H4MPT reductase was found to be present in Archaeoglobus fulgidus in a specific activity of 1 U/mg. The reductase was purified 410-fold. The native enzyme showed an apparent molecular mass of approximately 200 kDa. Sodium dodecylsulfate/polyacrylamide gel electrophoresis revealed the presence of only 1 polypeptide of apparent molecular mass 35 kDa. The ultraviolet/visible spectrum of the reductase was almost identical to that of albumin indicating the absence of a chromophoric prosthetic group. The reductase was dependent on reduced coenzyme F420 as electron donor. Neither NADH, NADPH, nor reduced viologen dyes could substitute for the reduced deazaflavin. From reciprocal plots, which showed an intersecting patter, a Km for methylene-H4MPT of 16 μM, a Km for F420H2 of 4 μM, and a Vmax of 450 U/mg (Kcat=265 s-1) were obtained. The enzyme was found to be rapidly inactivated when incubated at 80°C in 100 mM Tris/HCl pH 7. The rate of inactivation, however, decreased to essentially zero in the presence of either F420 (0.2 mM), methylene-H4MPT (0.2 mM), albumin (1 mg/ml), or KCl (0.5 M). The N-terminal amino acid sequence was determined and found to be similar to that of methylene-H4MPT reductase (F420-dependent) from the methanogens Methanobacterium thermoautotrophicum, Methanosarcina barkeri, and Methanopyrus kandleri. The purification and some properties of formylmethanofuran dehydrogenase from A. fulgidus are also described.

Acetate-dependent methylation of two corrinoid proteins in extracts of Methanosarcina barkeri.

Abstract: Corrinoid proteins have been implicated as methyl carriers in methane formation from acetate, yet specific corrinoid proteins methylated by acetate-derived intermediates have not been identified. In the presence of ATP, H2, and bromoethanesulfonic acid, label from 3H- or 2-14C-labeled acetate was incorporated into the protein fraction of cell extracts of Methanosarcina barkeri. Incorporated label was susceptible to photolysis, yielding labeled methane as the anaerobic photolysis product. Size exclusion high-pressure liquid chromatography (HPLC) demonstrated the presence of at least three labeled proteins with native molecular sizes of 480, 200, and 29 kDa, while electrophoresis indicated that four major labeled proteins were present. Dual-label experiments demonstrated that these four proteins were methylated rather than acetylated. Two of the proteins (480 and 29 kDa) contained the majority of radiolabel and were stably methylated. After labeling with [2-14C]acetate, the stable 14CH3-proteins were partially purified, and 14CH3-cofactors were isolated from each protein. UV-visible spectroscopy and HPLC demonstrated these to be methylated corrinoids. When the 480-kDa corrinoid protein was purified to 70% homogeneity, the preparation was found to have subunits of 40 and 30 kDa. The 480-kDa protein but not the 29-kDa protein was methylated during in vitro methanogenesis from acetate and demethylated as methanogenesis ceased, consistent with the involvement of this protein in methane formation.

Pattern of organotin inhibition of methanogenic bacteria.

Abstract: Seven organotin compounds and tin chloride were tested for their effects on the methanogenic bacteria Methanococcus thermolithotrophicus, Methanococcus deltae delta LH, and Methanosarcina barkeri 227. The methanogens were strongly inhibited by triethyltin, tripropyltin, and monophenyltin compounds, generally at concentrations below 0.05 mM. Less inhibition by tributyltin and diphenyltin was observed at levels below 0.1 mM, but complete inhibition was observed at a 1 mM concentration. Tin chloride inhibited all methanogens, with nearly complete inhibition at a 1 mM concentration. There was no inhibition by tetra-n-butyltin and triphenyltin compounds even at 2 mM, the highest concentration tested. The 50 and 100% inhibitory concentrations of all compounds were estimated; these values varied with both the compound tested and the bacterium tested. The 50% inhibitory concentration estimate generally decreased (i.e., giving a higher toxicity) as the total surface area of the alkyltin molecules decreased. These results differ considerably from those reported previously for aerobic microorganisms (G. Eng, E. J. Tierney, J. M. Bellama, and F. E. Brinckman, Appl. Organometallic Chem. 2:171-175, 1988), where a clear correlation between increasing total molecular surface area and increasing toxicity was documented with a variety of organisms. Using the same procedures as for the methanogens, we examined the effects of organotin compounds on Escherichia coli growing aerobically or anaerobically. The E. coli inhibition pattern clearly resembled that seen in the data of Eng et al., under both aerobic and anaerobic conditions.

Coenzyme F420 dependent N5, N10-methylenetetrahydromethanopterin dehydrogenase in methanol grown Methanosarcina barkeri

Abstract: The dehydrogenation of N 5,N 10-methylenetetrahydromethanopterin (CH2=H4MPT) to N 5,N 10-methenyltetrahydromethanopterin (CH≡H4MPT+) is an intermediate step in the oxidation of methanol to CO2 in Methanosarcina barkeri. The reaction is catalyzed by CH2=H4MPT dehydrogenase, which was found to be specific for coenzyme F420 as electron acceptor; neither NAD, NADP nor viologen dyes could substitute for the 5-deazaflavin. The dehydrogenase was anaerobically purified almost 90-fold to apparent homogeneity in a 32% yield by anion exchange chromatography on DEAE Sepharose and Mono Q HR, and by affinity chromatography on Blue Sepharose. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed only one protein band with an apparent mass of 31 kDa. The apparent molecular mass of the native enzyme determined by polyacrylamide gradient gel electrophoresis was 240 kDa. The ultraviolet/visible spectrum of the purified enzyme was almost identical to that of albumin suggesting the absence of a chromophoric prosthetic group. Reciprocal plots of the enzyme activity versus the substrate concentrations were linear: the apparent K m for CH2=H4MPT and for coenzyme F420 were found to be 6 μM and 25 μM, respectively. Vmax was 4,000 μmol min-1·mg-1 protein (kcat=2,066 s-1) at pH 6 (the pH optimum) and 37°C. The Arrhenius activation energy was 40 kJ/mol. The N-terminal amino acid sequence was found to be 50% identical with that of the F420-dependent CH2=H4MPT dehydrogenase isolated from H2/CO2 grown Methanobacterium thermoautotrophicum.

Immunocytochemical localization of the coenzyme F420-reducing hydrogenase in Methanosarcina barkeri Fusaro.

Abstract: The cytological localization of the 8-hydroxy-5-deazaflavin (coenzyme F420)-reducing hydrogenase of Methanosarcina barkeri Fusaro was determined by immunoelectron microscopy, using a specific polyclonal rabbit antiserum raised against the homogeneous deazaflavin-dependent enzyme. In Western blot (immunoblot) experiments this antiserum reacted specifically with the native coenzyme F420-reducing hydrogenase, but did not cross-react with the coenzyme F420-nonreducing hydrogenase activity also detectable in crude extracts prepared from methanol-grown Methanosarcina cells. Immunogold labelling of ultrathin sections of anaerobically fixed methanol-grown cells from the exponential growth phase revealed that the coenzyme F420-reducing hydrogenase was predominantly located in the vicinity of the cytoplasmic membrane. From this result we concluded that the deazaflavin-dependent hydrogenase is associated with the cytoplasmic membrane in intact cells of M. barkeri during growth on methanol as the sole methanogenic substrate, and a possible role of this enzyme in the generation of the electrochemical proton gradient is discussed.

Nucleotide-activated oligosaccharides are intermediates of the cell wall polysaccharide of Methanosarcina barkeri.

Abstract: The cell wall of Methanosarcina barkeri consists of a heteropolysaccharide (methanochondroitin), which resembles the eukaryotic chondroitin. From cell extracts of Methanosarcina barkeri four uridine diphosphate and one undecaprenyl pyrophosphate-activated intermediate(s) of the methanochondroitin were isolated. In contrast to the known biosynthetic pathways of polysaccharides from other prokaryotes and eukaryotes, nucleotide activated oligosaccharide precursors are involved in the case of the methanochondroitin. Usually, oligosaccharides are synthesized at the lipid stage.

Reductive activation of the corrinoid-containing enzyme involved in methyl group transfer between methyl-tetrahydromethanopterin and coenzyme M in Methanosarcina barkeri.

Abstract: The conversion of methyl-tetrahydromethanopterin to methylcoenzyme M inMethanosarcina barkeri is catalyzed by two enzymes: an enzyme with a bound corrinoid, which becomes methylated during the reaction and an enzyme which tranfers the methyl group from this corrinoid to coenzyme M. As in the similar methyltransfer reaction inMethanobacterium thermoautotrophicum the corrinoid enzyme inM. barkeri needs to be activated by H2 and ATP. ATP can be replaced by Ti(III)citrate or CO.

Formation of factor 390 by cell extracts of Methanosarcina barkeri.

Abstract: Cell extracts of Methanosarcina barkeri converted coenzyme F420 in an ATP-dependent reaction to the adenylylated derivative factor 390. Although it was reported previously (L. M. Gloss and R. P. Hausinger, BioFactors 1:237-240, 1988) that whole cells were unable to perform this conversion, we observed the conversion in 7 of 11 extracts, all of which were prepared from different batches of cells.

Ammonia assimilation and glutamate incorporation in coenzyme F420 derivatives of Methanosarcina barkeri.

Abstract: Methanosarcina barkeri was able to grow on L-alanine and L-glutamate as sole nitrogen sources. Cell yields were 0.5 g/l and 0.7 g/l (wet wt), respectively. The mechanism of ammonia assimilation in Methanosarcina barkeri strain MS was studied by analysis of enzyme activities. Activity levels of nitrogen-assimilating enzymes in extracts of cells grown on different nitrogen sources (ammonia, 0.05-100 mM; L-alanine, 10 mM; L-glutamate, 10 mM) were compared. Activities of glutamate dehydrogenase, glutamate synthase, glutamine synthetase, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase could be measured in cells grown on these three nitrogen sources. Alanine dehydrogenase was not detected under the growth conditions used. None of the measured enzyme activities varied significantly in response to the NH4+ concentration. The length of the poly-gamma-glutamyl side chain of F420 derivatives turned out to be independent of the concentration of ammonia in the culture medium.

Anaerobic degradation of sulphite evaporator condensate in a fixed-bed loop reactor by a defined bacterial consortium

Abstract: After elucidating the composition of an anaerobic bacterial enrichment culture treating sulphite evaporator condensate (SEC), an effluent in the pulp and paper industry, we built up stepwise a defined mixed culture to convert the organic constituents of SEC (acetate, methanol, furfural) to methane and CO2 In batch cultures Desulfovibrio furfuralis and Methanobacterium bryantii degraded furfural in the absence of sulphate via inter-species H2 transfer yielding 042 mol methane and 187 mol acetate/mol furfural degraded When Methanosarcina barkeri was added to this diculture, acetate was also transformed to methane yielding 093 mol methane/mol acetate converted This consortium (D furfuralis, Methanobacterium bryantii and Methanosarcina barkeri) degraded furfural in continuous culture (fixed-bed loop reactor) to 92%, but the conversion of acetate was only 67% The conversion of acetate could be further improved to 86% by adding 10 mm sulphate to the medium This resulted in a space time yield of 109 g chemical oxygen demand (COD)/1 per day for the overall conversion With a consortium consisting of M barkeri, Methanobrevibacter arboriphilus, Methanosaeta concilii and D furfuralis, a synthetic SEC could be degraded at a space time yield of 1335 g COD/1 per day This defined culture degraded all the constituents of SEC at an efficiency of almost 90% compared to an enrichment culture under identical conditions

The effects of pesticides on anaerobic digestion processes

Abstract: The effects of the fungicide Mancozeb, the herbicide Ametryne, and the molluscicide Niclosamide, on the anaerobic digestion of glucose by a mixed bacterial population were evaluated. Effects on activity of a pure culture of the methanogen, Methanosarcina barkeri was also tested. Pronounced inhibition of methanogenesis from glucose was observed for Mancozeb at a concentration of 100 mg.l‐1. In contrast, methanogenesis by Methanosarcina barkeri was not affected by Ametryne and Mancozeb, although Niclosamide delayed the onset of methanogenesis. Long‐term incubations (up to 60 days) indicated relief from Niclosamide inhibition.

Dna sequence coding principal subunit of atp synthetase originated from methane bacterial

Abstract: PURPOSE:To enable the synthesis of ATP in high efficiency by using a DNA sequence coding a principal subunit of ATP synthetase originated from methane bacteria. CONSTITUTION:Methanosarcina barkeri (a kind of methane bacteria) is cultured in a medium containing yeast extract, NH4Cl, etc., and the obtained bacterial cell is extracted to obtain a chromosome DNA (A). A fragment produced by the restriction enzyme treatment of the component A is linked with a fragment produced by the restriction enzyme treatment of a plasmid vector to obtain a recombinant DNA plasmid (B). The component B is introduced into E.coli, etc., and a gene bank (C) of methane bacteria is produced from the transformed E.coli. A transformant (D) containing a recombinant DNA coding ATP synthetase is selected from the component C using P as a label. The strain D is cultured on an agar medium, etc., and the obtained colony is extracted and purified to collect a DNA sequence coding principal subunits of ATP synthetase and containing an alpha-subunit exhibiting an amino acid sequence of formula in the principal subunits.