Showing papers on "Methanosarcina barkeri published in 1995"

Substrate and accessory protein requirements and thermodynamics of acetyl-CoA synthesis and cleavage in Methanosarcina barkeri.

Abstract: Enzymological studies on the multienzyme acetyl-CoA decarbonylase synthase (ACDS) complex from Methanosarcina barkeri have been conducted in order to identify and characterize physiologically relevant substrates and reactions in acetyl-CoA synthesis and decomposition in methanogens. Whereas previous investigations employed carbon monoxide as substrate and reducing agent for acetyl-CoA synthesis, we discovered that bicarbonate (or CO2) acts as a highly efficient carbonyl group precursor substrate in the presence of either hydrogen or Ti3+.EDTA as reducing agent. In reactions with Ti3+.EDTA, synthesis of acetyl-CoA was strongly dependent on ferredoxin, and in reactions with H2, dependence on ferredoxin was absolute. Two major hydrogenases were resolved from the enzyme complex preparation by HPLC gel filtration. One of these hydrogenases was shown to be active in reconstitution of acetyl-CoA synthesis in CO2-containing reactions with H2 as reducing agent. The hydrogenase active in reconstitution was capable of reducing ferredoxin, but was unreactive toward the 8-hydroxy-5-deazaflavin derivative coenzyme F420. In contrast, the hydrogenase that did not reconstitute acetyl-CoA synthesis was reactive with F420 but was unable to reduce ferredoxin. Further experiments were performed in which the value of the equilibrium constant (Keq) was determined for the reaction: H2 + CO2 + CH3-H4SPt + CoASH acetyl-CoA + H4SPt + H2O, where CH3-H4SPt and H4SPt stand for N5-methyl-tetrahydrosarcinapterin and tetrahydrosarcinapterin, respectively. Keq for this reaction was found to be 2.09 x 10(6) M-1ATMH2-1 at 37 degrees C. Calculations of thermodynamic values for additional, related reactions were made and are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)

Chloroform degradation in methanogenic methanol enrichment cultures and by Methanosarcina barkeri 227.

Abstract: The effects of methanol addition and consumption on chloroform degradation rate and product distribution in methanogenic methanol enrichment cultures and in cultures of Methanosarcina barkeri 227 were investigated. Degradation of chloroform with initial concentrations up to 27.3 microM in enrichment cultures and 4.8 microM in pure cultures was stimulated by the addition of methanol. However, methanol consumption was inhibited by as little as 2.5 microM chloroform in enrichment cultures and 0.8 microM chloroform in pure cultures, suggesting that the presence of methanol, not its exact concentration or consumption rate, was the most significant variable affecting chloroform degradation rate. Methanol addition also significantly increased the number of moles of dichloromethane produced per mole of chloroform consumed. In enrichment cultures, the number of moles of dichloromethane produced per mole of chloroform consumed ranged from 0.7 (methanol consumption essentially uninhibited) to 0.35 (methanol consumption significantly inhibited) to less than 0.2 (methanol not added to the culture). In pure cultures, the number of moles of dichloromethane produced per mole of chloroform consumed was 0.47 when methanol was added and 0.24 when no methanol was added. Studies with [14C]chloroform in both enrichment and pure cultures confirmed that methanol metabolism stimulated dichloromethane production compared with CO2 production. The results indicate that while the addition of methanol significantly stimulated chloroform degradation in both methanogenic methanol enrichment cultures and cultures of M. barkeri 227, the prospects for use of methanol as a growth substrate for anaerobic chloroform-degrading systems may be limited unless the increased production of undesirable chloroform degradation products and the inhibition of methanol consumption can be mitigated.

Biochemical characterization of the 8-hydroxy-5-deazaflavin-reactive hydrogenase from Methanosarcina barkeri Fusaro.

Abstract: The membrane-associated coenzyme F420-reactive hydrogenase of the anaerobic methanogenic archaeon Methanosarcina barkeri Fusaro has been purified 95-fold to apparent homogeneity. A new purification procedure and altered storage conditions gave substantially higher yield (13.4% versus 4.3%) and specific coenzyme F420-reducing activity (82.8 μmol · min−1· mg protein−1 versus 11.5 μmol · min−1· mg protein−1) than reported previously [Fiebig, K. & Friedrich, B. (1989) Eur. J. Biochem. 184, 79–88]. The predominant coenzyme F420-reactive form of the hydrogenase has an apparent molecular mass of 198 kDa and is composed of three non-identical subunits with apparent molecular masses of 48 (α), 33 (β), and 30 kDa (γ), apparently in a stoichiometry of α2β2γ1. This minimal coenzyme F420-reducing hydrogenase formed aggregates with apparent molecular masses of approximately 845 kDa. 1 mol of the 198-kDa form of hydrogenase contained 2 mol FAD, 2 mol nickel, 28–32 mol non-heme iron, and 34 mol acid-labile sulfur; in addition, 0.2 mol selenium was detected. The isoelectric point was 5.30. The amino acid sequence PXXRXEGH, where X is any amino acid, was found to be conserved in the N-termini of the putative nickel-binding subunits of most [NiFe]- and [NiFe]Sehydrogenases of methanogenic Archaea and Bacteria. However, this motif was not detected in the protein sequences of [Fe]hydrogenases. Maximal coenzyme F420-reducing activity was obtained with reductively reactivated enzyme at 55°C in the pH range 6.5–7.25. The Km, values of the purified enzyme for H2 with coenzyme F420 or methylviologen as electron acceptor were extremely low, namely 3μM and 4μM. The catalytic efficiency coefficients (kcat/Km) for H2 with both reducible cosubstrates were high: 2.5×107M−1· s−1 with coenzyme F420 and 6.9×107 M−1· S−1 with methylviologen.

Growth of Methanosarcina barkeri (Fusaro) under nonmethanogenic conditions by the fermentation of pyruvate to acetate: ATP synthesis via the mechanism of substrate level phosphorylation.

Abstract: A mutant of Methanosarcina barkeri (Fusaro) is able to grow on pyruvate as the sole carbon and energy source. During growth, pyruvate is converted to CH4 and CO2, and about 1.5 mol of ATP per mol of CH4 is formed (A.-K. Bock, A. Prieger-Kraft, and P. Schonheit, Arch. Microbiol. 161:33-46, 1994). The pyruvate-utilizing mutant of M. barkeri could also grow on pyruvate when methanogenesis was completely inhibited by bromoethanesulfonate (BES). The mutant grew on pyruvate (80 mM) in the presence of 2 mM BES with a doubling time of about 30 h up to cell densities of about 400 mg (dry weight) of cells per liter. During growth on pyruvate, the major fermentation products were acetate and CO2 (about 0.9 mol each per mol of pyruvate). Small amounts of acetoin, acetolactate, alanine, leucine, isoleucine, and valine were also detected. CH4 was not formed. The molar growth yield (Yacetate) was about 9 g of cells (dry weight) per mol of acetate, indicating an ATP yield of about 1 mol/mol of acetate formed. Growth on pyruvate in the presence of BES was limited; after six to eight generations, the doubling times increased and the final cell densities decreased. After 9 to 11 generations, growth stopped completely. In the presence of BES, suspensions of pyruvate-grown cells fermented pyruvate to acetate, CO2, and H2. CH4 was not formed. Conversion of pyruvate to acetate, in the complete absence of methanogenesis, was coupled to ATP synthesis. Dicyclohexylcarbodiimide, an inhibitor of H(+)-translocating ATP synthase, did not inhibit ATP formation. In the presence of dicyclohexylcarbodiimide, stoichiometries of up to 0.9 mol of ATP per mol of acetate were observed. The uncoupler arsenate completely inhibited ATP synthesis, while the rates of acetate, CO2, and H2 formation were stimulated up to fourfold. Cell extracts of M. barkeri grown on pyruvate under nonmethenogenic conditions contained pyruvate: ferredoxin oxidoreductase (0.5 U/mg), phosphate acetyltransferase (12 U/mg), and acetate kinase (12 U/mg). From these data it is concluded that ATP was synthesized by substrate level phosphorylation during growth of the M. barkeri mutant on pyruvate in the absence of methanogenesis. This is the first report of growth of a methanogen under nonmethanogenic conditions at the expense of a fermentative energy metabolism.

Immunological characterization of hydrogenases in the nitrogen-fixing cyanobacterium nostoc sp strain pcc-73102

Abstract: N2-fixing Nostoc sp strain PCC 73102 was examined for the presence of hydrogenases Native-PAGE/immunoblots demonstrated that two proteins with molecular masses of approximately 200 kDa and 215 kDa are immunologically related to hydrogenases purified from Bradyrhizobium japonicum, Azotobacter vinelandii, Methanosarcina barkeri, and Thiocapsa roseopersicina SDS-PAGE/immunoblots showed that one polypeptide, with a molecular mass of about 58 kDa, is immunologically related to the hydrogenases purified from all the microorganisms mentioned above In addition, two polypeptides, with molecular masses of approximately 34 and 70 kDa, are immunologically related to the hydrogenases purified from T roseopersicina and M barkeri respectively Immunogold/transmission electron microscopy showed that the hydrogenase proteins are present in both the heterocysts and the vegetative cells

Purification and characterization of 5-aminolevulinic acid dehydratase from Methanosarcina barken

Abstract: 5-Aminolevulinic acid dehydratase from the archaebacterium Methanosarcina barken resembles the mammalian and yeast enzymes in its activation by Zn2+, whereas its activation by K+ resembles the characteristic of bacterial enzymes. This enzyme is activated with Ni2+ which is a component of F430, a cofactor present mainly in methanogens. The Mr of 280000 for the native enzyme and 30 000 ± 2000 for the individual subunit suggest that the enzyme is composed of eight apparently indentical subunits similar to mammalian and yeast enzymes. The enzyme has two pH optima, at 8.5 and 9.4. Higher levels of 5-aminolevulinic acid dehydratase in acetate-grown cells suggest the possibility that regulation and control of this enzyme could be different on various growth substrates.