Showing papers on "Methanosarcina barkeri published in 1990"
TL;DR: Inhibition of the fermentation of acetate to methane and carbon dioxide by acetate was analyzed with an acetate-acclimatized sludge and with Methanosarcina barkeri Fusaro under mesophilic conditions and suggested that the concentration of undissociated acetic acid became a key factor governing the actual threshold acetate concentration for acetate utilization.
Abstract: Inhibition of the fermentation of acetate to methane and carbon dioxide by acetate was analyzed with an acetate-acclimatized sludge and with Methanosarcina barkeri Fusaro under mesophilic conditions. A second-order substrate inhibition model, q ch 4 = q m S /[ K s + S + ( S 2 / K i )], where S was the concentration of undissociated acetic acid, not ionized acetic acid, could be applicable in both cases. The analysis resulted in substrate saturation constants, K s , of 4.0 μM for the acclimatized sludge and 104 μM for M. barkeri. The threshold concentrations of undissociated acetic acid when no further acetate utilization was observed were 0.078 μM (pH 7.50) for the acclimatized sludge and 4.43 μM (pH 7.45) for M. barkeri. These kinetic results suggested that the concentration of undissociated acetic acid became a key factor governing the actual threshold acetate concentration for acetate utilization and that the acclimatized sludge in which Methanothrix spp. appeared dominant could utilize acetate better and survive at a lower concentration of undissociated acetic acid than could M. barkeri. Images
162 citations
TL;DR: Polar ether lipids extracted from 15 methanogenic bacteria, representative of seven genera, were screened by nuclear magnetic resonance and thin layer chromatography for the presence of hydroxyl groups on the C20-phytanyl moieties and confirmed for Methanosaeta concilii.
85 citations
TL;DR: Cell extracts of Methanosarcina barkeri grown on acetate catalyzed the conversion of acetyl‐CoA to CO2 and CH4 at a specific rate of 50 nmol·min−1·mg−1, which indicates that ferredoxin is involved as electron carrier in methanogenesis from acetate.
61 citations
TL;DR: Cell suspensions of methanogenic bacteria reductively dechlorinated 1,2-dichloroethane via two reaction-mechanisms and stimulated methanogenesis caused an increase in the amount of dechlorination products formed, whereas the opposite was found when methane formation was inhibited.
Abstract: 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.
57 citations
TL;DR: Results demonstrate that the molybdenum cofactor isolated from formylmethanofuran dehydrogenase contains the phosphoric anhydride of molybdopterin and 5'‐GMP.
53 citations
TL;DR: The formylmethanofuran dehydrogenase from the archaebacterium Methanosarcina barkeri has been shown to be a novel molybdo-iron-sulfur protein, and it is reported here that the enzyme contains one mol of a bound pterin cofactor/mol molyBdenum, similar but not identical to the molybdopterin of milk xanthine oxidase.
Abstract: Recently formylmethanofuran dehydrogenase from the archaebacterium Methanosarcina barkeri has been shown to be a novel molybdo-iron-sulfur protein. We report here that the enzyme contains one mol of a bound pterin cofactor/mol molybdenum, similar but not identical to the molybdopterin of milk xanthine oxidase. The two pterins, after oxidation with I2 at pH 2.5, showed identical fluorescence spectra and, after oxidation with permanganate at pH 13, yielded pterin 6-carboxylic acid. They differed, however, in their apparent molecular mass: the pterin of formylmethanofuran dehydrogenase was 400 Da larger than that of milk xanthine oxidase, a property also exhibited by the pterin cofactor of eubacterial molybdoenzymes.
A homogeneous formylmethanofuran dehydrogenase preparation was used for these investigations. The enzyme, with a molecular mass of 220 kDa, contained 0.5–0.8 mol molybdenum, 0.6–0.9 mol pterin, 28 ± 2 mol non-heme iron and 28 ± 2 mol acid-labile sulfur/mol based on a protein determination with bicinchoninic acid. The specific activity was 175 μmol · min−1· mg−1 (kcat= 640 s−1) assayed with methylviologen (app. Km= 0.02 mM) as artificial electron acceptor. The apparent Km for formylmethanofuran was 0.02 mM.
48 citations
TL;DR: Cell extracts of acetate-grown Methanosarcina barkeri mediated an isotope exchange between CO2 and the carbonyl group of acetyl-CoA at almost the same specific rate as the above conversion, and both the exchange and the formation of methyl-H4MPT were inhibited by N2O, suggesting that a corrinoid could be the primary methyl group acceptor in the acetyl -CoA C-C-cleavage reaction.
Abstract: From our previous studies on the mechanism of methane formation from acetate it was known that cell extracts of acetate-grown Methanosarcina barkeri (100 000 × g supernatant) catalyze the conversion of acetyl-CoA plus tetrahydromethanopterin (=H4MPT) to methyl-H4MPT, CoA, CO2 and presumably H2. We report here that these extracts, in the absence of H4MPT, mediated an isotope exchange between CO2 ([S]0.5 v=0.2% in the gas phase) and the carbonyl group of acetyl-CoA at almost the same specific rate as the above conversion (10 nmol · min−1 · mg protein−1). Both the exchange and the formation of methyl-H4MPT were inhibited by N2O, suggesting that a corrinoid could be the primary methyl group acceptor in the acetyl-CoA C-C-cleavage reaction. Both activities were dependent on the presence of H2 (E0′=−414 mV). Ti(III)citrate (E0′=−480 mV) was found to substitute for H2, indicating a reductive activation of the system. In the presence of Ti(III)citrate it was shown that the formation of CO2 from the carbonyl group of acetyl-CoA is associated with a 1:1 stoichiometric generation of H2. Free CO, a possible intermediate in CO2 and H2 formation, was not detected.
41 citations
TL;DR: N5N10-Methylenetetrahydromethanopterin reductase was purified 13-fold to apparent homogeneity from methanol grown Methanosarcina barkeri and was found to be similar to CH2=H4MPT reduct enzyme of Methanobacterium thermoautotrophicum which phylogenetically is only distantly related to M. barkeri.
Abstract: 1. Summary N5N10-Methylenetetrahydromethanopterin reductase was purified 13-fold to apparent homogeneity from methanol grown Methanosarcina barkeri. The colourless enzyme was found to be composed of four identical subunits of apparent molecular mass 36 kDa. It catalysed the reduction of methylenetetrahydromethanopterin (Km=15 μM) to methyltetrahydromethanopterin with reduced coenzyme F420 (Km=12 μM) at a specific rate (Vmax) of 2200 μmol min−1 · mg protein−1 (Kcat=1320 s−1). With respect to coenzyme specificity, molecular properties and catalytic mechanism the enzyme was found to be similar to CH2=H4MPT reductase of Methanobacterium thermoautotrophicum which phylogenetically is only distantly related to M. barkeri.
39 citations
TL;DR: Formylmethanofuran: tetrahydromethanopterin formyltransferase was purified from methanol grown Methanosarcina barkeri to apparent homogeneity and characterized with respect to its molecular and kinetic properties.
38 citations
TL;DR: The nitrogenase from strain 227 is similar in overall structure to the eubacterial nitrogenases and shows greatest similarity to alternative nitrogenases.
Abstract: The discovery of nitrogen fixation in the archaebacterium Methanosarcina barkeri 227 raises questions concerning the similarity of archaebacterial nitrogenases to Mo and alternative nitrogenases in eubacteria. A scheme for achieving a 20- to 40-fold partial purification of nitrogenase components from strain 227 was developed by using protamine sulfate precipitation, followed by using a fast protein liquid chromatography apparatus operated inside an anaerobic glove box. As in eubacteria, the nitrogenase activity was resolved into two components. The component 1 analog had a molecular size of approximately 250 kDa, as estimated by gel filtration, and sodium dodecyl sulfate-polyacrylamide gels revealed two predominant bands with molecular sizes near 57 and 62 kDa, consistent with an alpha 2 beta 2 tetramer as in eubacterial component 1 proteins. For the component 2 analog, a molecular size of approximately 120 kDa was estimated by gel filtration, with a subunit molecular size near 31 kDa, indicating that the component 2 protein is a tetramer, in contrast to eubacterial component 2 proteins, which are dimers. Rates of C2H2 reduction by the nearly pure subunits were 1,000 nmol h-1 mg of protein-1, considerably lower than those for conventional Mo nitrogenases but similar to that of the non-Mo non-V nitrogenase from Azotobacter vinelandii. Strain 227 nitrogenase reduced N2 at a higher rate per electron than it reduced C2H2, also resembling the non-Mo non-V nitrogenase of A. vinelandii. Ethane was not produced from C2H2. NH4+ concentrations as low as 10 microM caused a transient inhibition of C2H2 reduction by strain 227 cells. Antiserum against component 2 Rhodospirillum rubrum nitrogenase was found to cross-react with component 2 from strain 227, and Western immunoblots using this antiserum showed no evidence for covalent modification of component 2. Also, extracts of strain 227 cells prepared before and after switch-off had virtually the same level of nitrogenase activity. In conclusion, the nitrogenase from strain 227 is similar in overall structure to the eubacterial nitrogenases and shows greatest similarity to alternative nitrogenases.
36 citations
TL;DR: The 5,10-methenyltetrahydromethanopterin cyclohydrolase from Methanosarcina barkeri was purified 313-fold to a specific activity of 470 mumol min-1 mg-1 at 37 degrees C and pH 7.8 and the monofunctional enzyme was oxygen stable, but the presence of a detergent proved to be essential for its stability.
Abstract: The 5,10-methenyltetrahydromethanopterin cyclohydrolase from Methanosarcina barkeri was purified 313-fold to a specific activity of 470 mumol min-1 mg-1 at 37 degrees C and pH 7.8. At this stage, the enzyme was pure as judged from polyacrylamide gel electrophoresis. The monofunctional enzyme was oxygen stable, but the presence of a detergent proved to be essential for its stability. Like the cyclohydrolase purified from Methanobacterium thermoautotrophicum (A. A. Dimarco, M. I. Donnelly, and R. S. Wolfe, J. Bacteriol. 168:1372-1377, 1986), the protein showed an apparent Mr of 82,000, and it is composed of two identical subunits as was concluded from nondenaturating and denaturating polyacrylamide gel electrophoresis. The enzymes from M. thermoautotrophicum and M. barkeri markedly differ with respect to the hydrolysis product of 5,10-methenyltetrahydromethanopterin: 5-formyl- and 10-formyltetrahydromethanopterin, respectively. The apparent Km for 5,10-methenyltetrahydromethanopterin was 0.57 mM at 37 degrees C and pH 7.8.
TL;DR: Carbon monoxide dehydrogenase, corrinoid, and methylreductase can be observed with EPR spectroscopy during active methanogenesis from acetate in whole cells of Methanosarcina barkeri and cyanide induced formation of two very fast relaxing centers with overlapping rhombic signals in the iron-sulfur region of the spectrum.
TL;DR: In this article, the role of sodium ions in the energy metabolism of methanogenic bacteria was investigated and evidence for a novel mechanism of energy transduction with Na+ as the coupling ion was provided.
Abstract: Recent investigations with Methanosarcina barkeri elucidated the role of sodium ions in the energy metabolism of methanogenic bacteria and provided evidence for a novel mechanism of energy transduction with Na+ as the coupling ion. During methanogenesis from methanol, an eletrochemical sodium gradient generated by a Na+/H+ antiporter is used as the driving force for the thermodynamically unfavourable oxidation of methanol to the formal redox level of formaldehyde. During methanogenesis from H2+ CO2, the reverse reaction, the reduction of formaldehyde to the level of methanol, is accompanied by a primary, electron transport-driven sodium extrusion. Acetogenesis from H2+ CO2 as carried out by Acetobacterium woodii is a sodium-dependent process and is accompanied by the generation of a transmembrane sodium gradient with the reduction of formaldehyde to the level of methanol as the sodium-dependent step.
TL;DR: The concentration of Fe2+ in the methanol minimum medium was found to be a stimulating factor on the formation of cytochromes, the excretion of hemes and the rate of methane formation by Methanosarcino barkeri cells.
Abstract: The concentration of Fe2+ in the methanol minimum medium was found to be a stimulating factor on the formation of cytochromes, the excretion of hemes and the rate of methane formation by Methanosarcino barkeri cells. Overproduced hemes due to the high Fe2+ concentration were released into the medium as free forms. When Fe2+ concentration was employed more than 30 μM for the medium, the contents of cytochrome b and c in the cells seemed to be saturated at 40 nmol/g cell. Under the same condition, not only membrane-bound but also soluble cytochromes were detected in the ultracentrifuged fractions from the cells grown on 35 μM of Fe2+
TL;DR: Protein MC1 has a low amount of alpha-helix but contains antiparallel beta-sheet strands and the larger hydrophobic cluster which contains tryptophan at position 61 appears buried in the protein.
TL;DR: It is reported here that the two enzymes from Methanosarcma barkeri and the formyltransferase from Methanobacterium thermoautotrophicum can also use N‐furfurylfonnamide as a pseudo‐substrate albeit with higher apparent K m and lower apparent V max values.
01 Jan 1990
TL;DR: Among the ATPases so far isolated from archaebacteria, only the halobacterial ATPase has been proven to be the (catalytic) part of ATP synthase.
Abstract: Among the ATPases so far isolated from archaebacteria, only the halobacterial ATPase has been proven to be the (catalytic) part of ATP synthase. This ATPase/synthase is H+-translocating and has unique chracteristics as enzyme/protein.
TL;DR: In an attempt to improve corrinoid production by M. barkeri strain Fusaro, a repeated fed-batch culture coupled with a membrane module on methanol-acetate medium was used.
Abstract: In an attempt to improve corrinoid production byM. barkeri strain Fusaro, a repeated fed-batch culture coupled with a membrane module on methanol-acetate medium was used. Productivity of 22 mg-corrinoid/1. day was obtained during 626 h cultivation with corrinoid and cell mass concentration of 95 mg/l and 25.9 g-dry cell/l, respectively. The minimum value for membrane flux permeation was 18 liter/m2. h for cell mass concentration between 25.9 to 31.0 g-dry cell/l. with rejection of 100 %.
TL;DR: The metabolism of methanol and pyruvate by cells of Methanosarcina barkeri was probed in vivo by NMR taking advantage of the non-invasive characteristics of this technique.
Abstract: The metabolism of methanol and pyruvate by cells of Methanosarcina barkeri was probed in vivo by NMR taking advantage of the non-invasive characteristics of this technique. Upon administration of substrates, the kinetics of substrate consumption, the product formation and the energetic state of the cells was monitored using carbon-13, phosphorus-31 or proton NMR. The effects of several inhibitors and uncouplers were investigated. Cells supplied with pyruvate developed considerable levels of nucleotide triphosphate; methane production was monitored, as well as CO2 and H2 formation. Most of the pyruvate was utilized for the synthesis of valine or intermediates of the valine pathway. The origin of the carbon atom in methane was elucidated using 13C-labelled pyruvate.
01 Oct 1990
TL;DR: In this paper, a second-order substrate inhibition model, q{sub CH{sub 4}} = q{ sub m}S/(k{sub s} + S + (S{sup 2}/K{sub i})), where S was the concentration of undissociated acetic acid, was applied in both cases.
Abstract: Inhibition of the fermentation of acetate to methane and carbon dioxide by acetate was analyzed with an acetate-acclimatized sludge and with Methanosarcina barkeri Fusaro under mesophilic conditions. A second-order substrate inhibition model, q{sub CH{sub 4}} = q{sub m}S/(K{sub s} + S + (S{sup 2}/K{sub i})), where S was the concentration of undissociated acetic acid, not ionized acetic acid, could be applicable in both cases. The analysis resulted in substrate saturation constants, K{sub s}, of 4.0 {mu}M for the acclimatized sludge and 104 {mu}M for M. barkeri. The threshold concentrations of undissociated acetic acid when no further acetate utilization was observed were 0.078 {mu}M (pH 7.50) for the acclimatized sludge and 4.43 {mu}M (pH 7.45) for M. barkeri. These kinetic results suggested that the concentration of undissociated acetic acid became a key factor governing the actual threshold acetate concentration for acetate utilization and that the acclimatized sludge in which Methanothrix spp. appeared dominant could utilize acetate better and survive at a lower concentration of undissociated acetic acid than could M. barkeri.
01 Jan 1990
TL;DR: The F420-reducing hydrogenase (H2ase) of Methanospirillum hungatei was isolated from spheroplast lysates by sedimentation, followed by either sucrose gradients or nickel-affinity chromatography, and gel filtration, and lipid analysis of the H2ase CHCl3/MeOH extracts established that lipid was associated with the enzyme.
Abstract: The F420-reducing hydrogenase (H2ase) of Methanospirillum hungatei was isolated from spheroplast lysates by sedimentation, followed by either sucrose gradients or nickel-affinity chromatography, and gel filtration. Most of the enzyme was free of the cytoplasmic membrane, although isolated membranes retained ca. 15% of the F420-reducing H2ase activity. The brown H2ase protein had an absorption spectrum characteristic of a nonheme iron protein. In electron micrographs it was a coin-shaped, multisubunit protein of 15.9 nm diameter with a central depression on one surface. During chromatography on phenyl Sepharose the H2ase exhibited hydrophobic properties. Labeling with the isoprenoid precursor, [14C]-mevalonate, and lipid analysis of the H2ase CHCl3/MeOH extracts, established that lipid was associated with the enzyme. This association appears to result from membrane contamination of the hydrophobic enzyme. The holoenzyme was about 720 kDa and contained 6–7 Ni2+ atoms. H2-dependent reduction of F420 activity was readily, but transiently, reactivated by anaerobic conditions following exposure of the enzyme to air. Mg2+ or Ca2+ were stimulatory. The holoenzyme was composed of α-subunits of 51 kDa, and 30–31 kDa β and γ subunits of nearly identical mass. The N-terminal amino acid sequences of the first 20–25 residues were very similar in β and γ subunits. Comparisons made to sequences known for other H2ases, established that the M. hungatei H2ase was quite different. Antibody raised against the purified hydrogenase of strain GP1 gave negative reactions with extracts of nine other methanogens, and a reaction of identity with M. hungatei strain JF1 and Methanosarcina barkeri strain MS.
01 Jan 1990
TL;DR: The universal methanogenic reaction from all substates is the reductive demethylation of methyl-CoM by the methyl-coenzyme M methylreductase system according to Gunsalus and Wolfe, 1980 as discussed by the authors.
Abstract: The universal methanogenic reaction from all substates is the reductive demethylation of methyl-CoM by the methyl-coenzyme M methylreductase system according to (Gunsalus and Wolfe, 1980): CH3-S-CoM + H2 → CH4 + HS-CoM This highly exergonic reaction has always been considered as the step coupled with ATP synthesis. Investigations with whole cells of Methanosarcina barkeri led to the conclusion that methanogenesis from methanol plus H2 gives rise to an electrochemical proton gradient, which is subsequently used to synthezise ATP (Blaut and Gottschalk, 1984).
01 Jan 1990
TL;DR: The structural and functional properties of the first ferredoxin isolated from a chemolithotrophic methanogen only growing on C02 and H2 or formate, Methanococcus thermolithotrophus are reported.
Abstract: Ferredoxins have been isolated from organisms belonging to each major group of archaebacteria, including the extreme halophiles, the thermoacidophiles, and the methanogens. So far, two species of methanogenic bacteria were known to contain ferredoxin: Methanosarcina (3,7) and more recently Methanobacterium thermoautotrophicum ΔH (8). In this paper, we report the structural and functional properties of the first ferredoxin isolated from a chemolithotrophic methanogen only growing on C02 and H2 or formate, Methanococcus thermolithotrophicus (4).
01 Jan 1990
TL;DR: The ability of methanogenic archaebacteria to fix N2 was independently reported by four laboratories in 1984–85, indicating that this ability is widespread in methanogens.
Abstract: The ability of methanogenic archaebacteria to fix N2 was independently reported by four laboratories in 1984–85. Murray and Zinder (9) and Belay, Sparling and Daniels (2) published concurrent papers describing diazotrophy in Methanosarcina barkeri strain 227 and Methanococcus thermolithotrophicus respectively, followed soon afterward by the report by Bomar, Knoll and Widdell (3) on diazotrophy in Methanosarcina barkeri strain Fusaro. Sibold et al. (16) found that eubacterial nif probes, especially nifH probes, could hybridize with restriction-enzyme digested methanogen DNA. Since then, diazotrophy has been described in several other methanogens including members of all three major methanogenic orders (1), indicating that this ability is widespread in methanogens. Diazotrophy has not been reported in the other branches of the archaebacteria, the halobacteria and sulfur-dependent thermophiles (20).