Showing papers on "Methanosarcina barkeri published in 1994"

Carbon isotope effects associated with aceticlastic methanogenesis.

Abstract: The carbon isotope effects associated with synthesis of methane from acetate have been determined for Methanosarcina barkeri 227 and for methanogenic archaea in sediments of Wintergreen Lake, Michigan. At 37 degrees C, the 13C isotope effect for the reaction acetate (methyl carbon) --> methane, as measured in replicate experiments with M. barkeri, was - 21.3% +/- 0.3%. The isotope effect at the carboxyl portion of acetate was essentially equal, indicating participation of both positions in the rate-determining step, as expected for reactions catalyzed by carbon monoxide dehydrogenase. A similar isotope effect, - 19.2% +/- 0.3% was found for this reaction in the natural community (temperature = 20 degrees C). Given these observations, it has been possible to model the flow of carbon to methane within lake sediment communities and to account for carbon isotope compositions of evolving methane. Extension of the model allows interpretation of seasonal fluctuations in 13C contents of methane in other systems.

Cloning, DNA sequencing, and characterization of a nifD-homologous gene from the archaeon Methanosarcina barkeri 227 which resembles nifD1 from the eubacterium Clostridium pasteurianum.

Abstract: L. Sibold, M. Henriquet, O. Possot, and J.-P. Aubert (Res. Microbiol. 142:5-12, 1991) cloned and sequenced two nifH-homologous open reading frames (ORFs) from Methanosarcina barkeri 227. Phylogenetic analysis of the deduced amino acid sequences of the nifH ORFs from M. barkeri showed that nifH1 clusters with nifH genes from alternative nitrogenases, while nifH2 clusters with nifH1 from the gram-positive eubacterium Clostridium pasteurianum. The N-terminal sequence of the purified nitrogenase component 2 (the nifH gene product) from M. barkeri was identical with that predicted for nifH2, and dot blot analysis of RNA transcripts indicated that nifH2 (and nifDK2) was expressed in M. barkeri when grown diazotrophically in Mo-containing medium. To obtain nifD2 from M. barkeri, a 4.7-kbp BamHI fragment of M. barkeri DNA was cloned which contained at least five ORFs, including nifH2, ORF105, and ORF125 (previously described by Sibold et al.), as well as nifD2 and part of nifK2. ORFnifD2 is 1,596 bp long and encodes 532 amino acid residues, while the nifK2 fragment is 135 bp long. The deduced amino acid sequences for nifD2 and the nifK2 fragment from M. barkeri cluster most closely with the corresponding nifDK1 gene products from C. pasteurianum. The predicted M. barkeri nifD2 product contains a 50-amino acid insert near the C terminus which has previously been found only in the clostridial nifD1 product. Previous biochemical and sequencing evidence indicates that the C. pasteurianum nitrogenase is the most divergent of known eubacterial Mo-nitrogenases, most likely representing a distinct nif gene family, which now also contains M. barkeri as a member. The similarity between the methanogen and clostridial nif sequences is especially intriguing in light of the recent findings of sequence similarities between gene products from archaea and from low-G+C gram-positive eubacteria for glutamate dehydrogenase, glutamine synthetase I, and heat shock protein 70. It is not clear whether this similarity is due to horizontal gene transfer or to the resemblance of the M. barkeri and C. pasteurianum nitrogenase sequences to an ancestral nitrogenase.

Purification of a two-subunit cytochrome-b-containing heterodisulfide reductase from methanol-grown Methanosarcina barkeri.

Abstract: Heterodisulfide reductase catalyzes the terminal step in the energy-conserving electron-transport chain in methanogenic Archaea. The heterodisulfide reductase activity of the membrane fraction of methanol-grown Methanosarcina barkeri was solubilized by Chaps. Chromatography on Q-Sepharose and Superdex-200 yielded a high-molecular-mass fraction (> 700 kDa) which was dissociated by dodecyl β-D-maltoside. After chromatography on Q-Sepharose, an active heterodisulfide reductase preparation was obtained which was composed of only two different subunits of apparent molecular masses 46 kDa and 23 kDa. For each 69 kDa, the enzyme contained 0.6 mol cytochrome b, 0.2 mol FAD, 20 mol non-heme iron and 20 mol acid-labile sulfur. The 23-kDa subunit possessed heme-derived peroxidase activity, showing that this polypeptide is the cytochrome b. The purified enzyme contained the cytochrome b in the reduced form. Upon addition of the heterodisulfide of coenzyme M and N-7-mercaptoheptanoylthreonine phosphate the cytochrome was instantaneously oxidized, indicating that the cytochrome b served as electron donor for heterodisulfide reduction.

Decrease of the hydraulic conductivity of sand columns by Methanosarcina barkeri

Abstract: The extent to which a methanogen can clog sand columns was examined: two permeameters packed with clean quartz sand were sterilized, saturated with water, inoculated with Methanosarcina barkeri and percolated under upward flow conditions. After approx. 5 months, the hydraulic conductivity of the sand had decreased to 3% and 25% of the highest values measured earlier. At that point, gas-filled regions in the sand were clearly visible through the transparent walls of the permeameters, and methane bubbles were continuously released from the columns into the effluent. Scanning electron microscopy observations and biomass assays indicated that cell mass accumulation did not contribute significantly to the observed decrease of the hydraulic conductivity. This decrease was therefore attributed to pore blocking due to the entrapment of methane bubbles.

Syntrophic Acetate Oxidation and “Reversible Acetogenesis”

Abstract: Acetate is an important CH4 precursor in nature, accounting for two-thirds of the CH4 produced in many natural habitats and in anaerobic bioreactors. Although microbial methanogenesis from acetate was first described in the early 1900s, the mechanism of methanogenesis from acetate was controversial until 1978, when it was demonstrated that a pure culture of Methanosarcina barkeri could grow on acetate (Mah et al., 1978; Smith and Mah, 1978; Weimer and Zeikus, 1978) and convert acetate to CH4 by a decarboxylation mechanism sometimes called the aceticlastic reaction. With the description of a similar mechanism for Methanothrix soehngenii in 1980 (Zehnder et al., 1980), it appeared that acetate decarboxylation was “the” mechanism for methanogenesis from acetate.

Formylmethanofuran dehydrogenases from methanogenic Archaea. Substrate specificity, EPR properties and reversible inactivation by cyanide of the molybdenum or tungsten iron-sulfur proteins.

Abstract: Formylmethanofuran dehydrogenases, which are found in methanogenic Archaea, are molybdenum or tungsten iron-sulfur proteins containing a pterin cofactor. We report here on differences in substrate specificity, EPR properties and susceptibility towards cyanide inactivation of the enzymes from Methanosarcina barkeri, Methanobacterium thermoautotrophicum and Methanobacterium wolfei. The molybdenum enzyme from M. barkeri (relative activity with N-formylmethanofuran = 100%) was found to catalyze, albeit at considerably reduced apparent Vmax, the dehydrogenation of N-furfurylformamide (11%), N-methylformamide (0.2%), formamide (0.1%) and formate (1%). The molybdenum enzyme from M. Wolfei could only use N-furfurylformamide (1%) and formate (3%) as pseudosubstrates. The molybdenum enzyme from M. thermoautotrophicum and the tungsten enzymes from M. thermoautotrophicum and M. wolfei were specific for N-formylmethanofuran. The molybdenum formylmethanofuran dehydrogenases exhibited at 77 K two rhombic EPR signals, designated FMDred and FMDox, both derived from Mo as shown by isotopic substitution with 97Mo. The FMDred signal was only displayed by the active enzyme in the reduced form and was lost upon enzyme oxidation; the FMDox signal was displayed by an inactive form and was not quenched by O2. The tungsten isoenzymes were EPR silent. The molybdenum formylmethanofuran dehydrogenases were found to be inactivated by cyanide whereas the tungsten isoenzymes, under the same conditions, were not inactivated. Inactivation was associated with a characteristic change in the molybdenum-derived EPR signal. Reactivation was possible in the presence of sulfide.

Pyruvate : a novel substrate for growth and methane formation in Methanosarcina barkeri

Abstract: Methanosarcina barkeri strain Fusaro was found to grow on pyruvate as sole carbon and energy source after an incubation period of 10–12 weeks in the presence of high pyruvate concentrations (100 mM). Growth studies, cell suspension experiments and enzymatic investigations were performed with pyruvate-utilizing M. barkeri. For comparison acetate-adapted cells of M. barkeri were analyzed. 1. Pyruvate-utilizing M. barkeri grew on pyruvate (100 mM) with an initial doubling time of about 25 h (37 °C, pH 6.5) up to cell densities of about 0.8 g cell dry weight/l. The specific growth rate was linearily dependent on the pyruvate concentration up to 100 mM indicating that pyruvate was taken up by passive diffusion. Only CO2 and CH4 were detected as fermentation products. As calculated from fermentation balances pyruvate was converted to CH4 and CO2 according to following equation: Pyruvate-+H++0.5 H2O » 1.25 CH4+1.75 CO2. The molar growth yield (Ych4) was about 14 g dry weight cells/mol CH4. In contrast the growth yield (Ych4) of M. barkeri during growth on acctate (Acetate-+H+ » CH4+CO2) was about 3 g/mol CH4. 2. Cell suspensions of pyruvate-grown M. barkeri catalyzed the conversion of pyruvate to CH4, CO2 and H2 (5–15 nmol pyruvate consumed/min x mg protein). At low cell concentrations (0.5 mg protein/ml) 1 mol pyruvate was converted to 1 mol CH4, 2 mol CO2 and 1 mol H2. At higher cell concentration less H2 and CO2 and more CH4 were formed due to CH4 formation from H2/CO2. The rate of pyruvate conversion was linearily dependent on the pyruvate concentration up to about 30 mM. Cell suspensions of acetate-grown M. barkeri also catalyzed the conversion of 1 mol pyruvate to 1 mol CH4, 2 mol CO2 and 1 mol H2 at similar rates and with similar affinity for pyruvate as pyruvate-grown cells. 3. Cell extracts of both pyruvate-grown and acetate-grown M. barkeri contained pyruvate: ferredoxin oxidoreductase. The specific activity in pyruvate-grown cells (0.8 U/mg) was 8-fold higher than in acetate-grown cells (0.1 U/mg). Coenzyme F420 was excluded as primary electron acceptor of pyruvate oxidoreductase. Cell extracts of pyruvate-grown M. barkeri contained carbon monoxide dehydrogenase activity and hydrogenase activity catalyzing the reduction by carbon monoxide and hydrogen of both methylviologen and ferredoxin (from Clostridium).

Transformation of nitroaromatic compounds by a methanogenic bacterium, Methanococcus sp. (strain B)

Abstract: The transformation of several nitroaromatic compounds by a newly isolated methanogenic bacterium, Methanococcus sp. (strain B) was studied. The presence of nitroaromatic compounds (0.5 mM) viz., nitrobenzene, 2,4-dinitrobenzene, 2,4,6-trinitrobenzene, 2,4-dinitrophenol, 2,4-dinitrobenzene, and 2,6-dinitrotoluene in the culture medium did not inhibit growth of the isolate. The bacteria grew rapidly and reached stationary phase within seven days of incubation. All the nitroaromatic compounds tested were 80 to 100% transformed by the bacterium to amino compounds by a reduction process. The isolate did not use the nitroaromatic compounds as the sole source of carbon or nitrogen. The transformation of nitroaromatic compounds by this isolate was compared to that of other methanogenic bacteria. Out of five methanogens studied, only Methanococcus deltae and Methanococcus thermolithotrophicus could transform the nitroaromatic compounds; however, the transformation rates were significantly less than that of the new isolate Methanococcus sp. (strain B). The nitroaromatic compounds were not transformed by Methanosarcina barkeri, Methanobacterium thermoautotrophicum, and Methanobrevibacter ruminantium.

Purification and characterization of membrane-bound hydrogenase from Methanosarcina barkeri MS

Abstract: Hydrogenase was solubilized from the membrane of acetate-grown Methanosarcina barkeri MS and purification was carried out under aerobic conditions. The enzyme was reactivated under reducing conditions in the presence of H2. The enzyme showed a maximal activity of 120±40 μmol H2 oxidized · min−1 · min−1 with methyl viologen as an electron acceptor, a maximal hydrogen production rate of 45±4 μmol H2 · min−1 · mg−1 with methyl viologen as electron donor, and an apparent K m for hydrogen oxidation of 5.6±1.7 μM. The molecular weight estimated by gel filtration was 98,000. SDS-PAGE showed the enzyme to consist of two polypeptides of 57,000 and 35,000 present in a 1:1 ratio. The native protein contained 8±2 mol Fe, 8±2 mol S2−, and 0.5 mol Ni/mol enzyme. Cytochrome b was reduced by hydrogen in a solubilized membrane preparation. The hydrogenase did not couple with autologous F420 or ferredoxin, nor with FAD, FMN, or NAD(P)+. The physiological function of the membrane-bound hydrogenase in hydrogen consumption is discussed.

Purine and pyrimidine biosynthesis in methanogenic bacteria

Abstract: Following long-term labeling with [1-13C]acetate, [2-13C]acetate, 13CO2, H13COOH, or 13CH3OH, NMR spectroscopy was used to determine the labeling patterns of the purified ribonucleosides of Methanospirillum hungatei, Methanococcus voltae, Methanobrevibacter smithii, Methanosphaera stadtmanae, Methanosarcina barkeri and Methanobacterium bryantii. Major differences were observed among the methanogens studied, specifically at carbon positions 2 and 8 of the purines, positions at which one-carbon carriers are involved during synthesis. In Methanospirillum hungatei and Methanosarcina barkeri, the labcl at both positions came from carbon atom C-2 of acetate, as predicted from known eubacterial pathways, whereas in Methanococcus voltae and Methanobacterium bryantii both originated from CO2. In Methanosphaera stadtmanae grown in the presence of formate, the C-2 of purines originated exclusively from formate and the C-8 was labeled by the C-2 of acetate. When grown in media devoid of formate, the C-2 of the purine ring originated mainly from the C-2 of acetate and in part from CH3OH. In Methanobrevibacter smithii grown in the presence of formate, C-2 and C-8 of purines were derived from CO2 and/or formate. The labeling patterns obtained for pyrimidines are consistent with the biosynthetic pathways common to eubacteria and eucaryotes.

Tungstate does not support synthesis of active formylmethanofuran dehydrogenase in Methanosarcina barkeri

Abstract: Cell extracts of Methanosarcina barkeri grown on methanol in media supplemented with molybdate exhibited a specific activity of formylmethanofuran dehydrogenase of approximately 1 U (1 μmol/min)/mg protein. When the growth medium was supplemented with tungstate rather than with molybdate, the specific activity was only 0.04 U/mg. Despite this reduction in specific activity growth on methanol was not inhibited. An inhibition of both growth and synthesis of active formylmethanofuran dehydrogenase was observed, however, when H2 and CO2 were the energy substrates. The results indicate that, in contrast to Methanobacterium wolfei and Methanobacterium thermoautotrophicum, M. barkeri possesses only a molybdenum containing formylmethanofuran dehydrogenase and not in addition a tungsten isoenzyme.

Ribose biosynthesis in methanogenic bacteria

Abstract: NMR spectroscopy was used to determine the labeling patterns of the ribose moieties of ribonucleosides purified from Methanospirillum hungatei, Methanococcus voltae, Methanobrevibacter smithii, Methanosphaera stadtmanae, Methanosarcina barkeri and Methanobacterium bryantii labeled with 13C-precursors. In most methanogens tested ribose was labeled in a manner consistent with the operation of the oxidative branch of the pentose phosphate pathway. In contrast, transaldolase and transketolase reactions typical of a partial nonoxidative pentose phosphate pathway are hypothesized to explain the different labeling patterns and enrichments of carbon atoms observed in the ribose moiety of Methanococcus voltae. The source of erythrose 4-phosphate needed for the transaldolase reaction proposed in Methanococcus voltae, and for biosynthesis of aromatic amino acids in methanogenic bacteria in general, was assessed. Phenylalanine carbon atom C-7 was labeled by [1-13C]pyruvate in Methanospirillum hungatei, Methanococcus voltae, and Methanococcus jannaschii, the only methanogens which incorporated sufficient label from pyruvate for testing. Reductive carboxylation of a triose precursor (derived from pyruvate) to synthesize erythrose 4-phosphate is consistent with the labeling patterns observed in phenylalanine and ribose.

Refolding of denatured restriction endonucleases with ribosomal preparations from Methanosarcina barkeri.

Abstract: Two restriction endonucleases, EcoRI and HindIII were denatured by guanidine HC1 or by storing at room temperature denatured by guanidine HC1 or by storing at room temperature (28 degrees C) for several days. The activity of these enzymes could be restored by incubating with ribosomal preparations from an archaebacterium Methanosarcina barkeri. These results hint at a possible role of ribosomal preparations in folding polypeptides and could be useful in working with restriction enzymes in experiments on genetic engineering and molecular biology.

Inhibition of methanogens by n- and iso-volatile fatty acids.

Abstract: n-Butyrate, n-valerate and n-caproate were more inhibitory towards Methanobacterium byrantii, Methanobacterium formicicum and Methanosarcina barkeri than the corresponding iso-acids. Butyrate caused maximum inhibition irrespective of isomer. Methanobacterium bryantii was more sensitive to inhibition than Methanobacterium formicicum.

Selectivity of food bacteria for the growth of anaerobic ciliate Trimyema compressum

Abstract: The anaerobic ciliate Trimyema compressum was cultivated on various food bacteria. Significant growth was observed when Lactobacillus sp., Escherichia coli, Enterobacter aerogenes, Desulfovibrio vulgaris, Methanoculleus bourgense, or Pelobacter propionicus cells were fed to the ciliates. The highest cell yield which we obtained was ca. 9,000 cells/ml when feeding D. vulgaris. However, no growth of the ciliates was observed on the culture with Clostridium novyi, Propionibacterium sp., Desulfobulbus propionicus, Methanobrevibacter arboriphilicus, Methanobacterium sp., Methanosarcina barkeri, or Methanothrix soehngenii cells. The ciliates produced acetate and methane as major end products in any cultures and small amounts of propionate, butyrate and hydrogen were also detected in some cultures. Physiological studies on the food bacteria which we tested indicated that the growth of T. compressum depended on the bacterial species, but there was no apparent correlation between the digestibility and the basic properties of those bacteria (i.e. size of the bacteria, gram-staining properties, susceptibility to the known lytic enzymes, Archaea or Bacteria).

Pyruvate as a substrate for growth and methanogenesis forMethanosarcina barkeri

Abstract: Methanosarcina barkeri strains (227, MS, and UBS) were tested for their ability to utilize pyruvate for growth and methanogenesis. All three strains grown on methanol required 4–5 weeks of adaptation for growth on pyruvate, whereas they required only 2–3 weeks of adaptation for growth on acetate. The adapted cells had a lag of 3–4 days for growth on acetate and 5–10 days for growth on pyruvate. Equimolar amounts of methane were produced from acetate, whereas 0.6–0.7 mol of methane was produced per mol of pyruvate. The optimal concentration of pyruvate for growth and methanogenesis for all three strains was 100 mM, and doubling times were in the range of 135–170 h.

Low-spin sulfite reductases

Abstract: Publisher Summary This chapter focuses on low-spin sulfite reductases, which are isolated from Desulfovibrio vulgaris (Hildenborough) (DvH), Methanosarcina barkeri ( Ms. Barkeri ), and Desulforomonas acetoxidans ( Drm. Acetoxidans ). Sulfite reductase catalyzes the six-electron reduction of SO 3 2- to S 2- .This enzyme contains an iron tetrahydroporphyrin prosthetic group, termed “siroheme,” in addition to nonheme iron. On the basis of physiological function, two types of sulfite reductases can be defined (1) the assimilatory type, which is involved in the synthesis of sulfur-containing compounds, and (2) the dissimilatory one, which participates in the respiratory pathway for sulfate-reducing bacteria (SRB). Under certain assay conditions of dissimilatory sulfite reductases, in addition to sulfide, trithionate and thiosulfate are irreversibly produced. All purification procedures of sulfite reductases are carried out at 4 and all buffers are adjusted to pH 7.6. The purified enzyme is stable for six months in 0.05 M Tris-HCl buffer at –80. The purified sulfite reductase has a molecular mass of 27.2 kDa, determined by amino-acid composition. The iron content is consistent with the existence of a single [4Fe-4S] cluster and a siroheme.

Regulation and function of ferredoxin-linked versus cytochrome b-linked hydrogenase in electron transfer and energy metabolism of Methanosarcina barkeri MS

Abstract: Acetate-grown cells of Methanosarcina barkeri MS were found to form methane from H2:CO2 at the same rate as hydrogen-grown cells. Cells grown on acetate had similar levels of soluble F420-reactive hydrogenase I, and higher levels of cytochrome-linked hydrogenase II compared to hydrogen-grown cells. The hydrogenase I and II activities in the crude extract of acetate-grown cells were separated by differential binding properties to an immobilized Cu2+ column. Hydrogenase II did not react with ferredoxin or F420, whereas hydrogenase I coupled to both ferredoxin and F420. A reconstituted soluble protein system composed of purified CO dehydrogenase, F420-reactive hydrogenase I fraction, and ferredoxin produced H2 from CO oxidation at a rate of 2.5 nmol/min · mg protein. Membrane-bound hydrogenase II coupled H2 consumption to the reduction of CoM-S-S-HTP and the synthesis of ATP. The differential function of hydrogenase I and II is ascribed to ferredoxin-linked hydrogen production from CO and cytochrome b-linked H2 consumption coupled to methanogenesis and ATP synthesis, respectively.

Differential in vitro methylation and synthesis of the 480-kilodalton corrinoid protein in Methanosarcina barkeri grown on different substrates.

Abstract: The 480-kDa corrinoid protein was significantly methylated in extracts of acetate- but not methanol-grown cells incubated with 14CH3OH, in part because of its decreased synthesis in cells grown on substrates other than acetate. In addition, a 200-kDa corrinoid protein was methylated in extracts of methanol- but not acetate-grown cells. Images

Corrinoid-Dependent Mechanism of Acetogenesis from Methanol

Abstract: More than 13 reactions are catalyzed by corrinoid-containing enzymes in prokaryotes and eukaryotes. These reactions were reviewed previously (Stadtman, 1971; Halpern, 1985) but some additional corrinoid-dependent reactions have been discovered since then. For example, the methanol conversion into methane proceeds via a corrinoid-dependent methyltransferase (van der Meijden et al., 1984a). The enzyme from Methanosarcina barkeri provides the methyl group from methanol to the specific methanogenic cofactor 2-mercaptoethanesulfonic acid (HS-CoM) after the protein is reductively activated by H2 and ATP. That enzyme revealed an α2β structure and it contained 3–4 mol loosely bound corrinoid per mole of protein. In vitro studies with mixed cell-free extracts of Methanosarcina barkeri and Eubacterium limosum indicated that the acetogenic bacterium also possesses a methylated corrinoid enzyme. This enzyme was demethylated in the presence of methylcobalamin: coenzyme M methyltransferase from the methanogenic bacterium with concomitant formation of methyl-coenzyme M (van der Meijden et al., 1984b).

Cytochromebc purified from the methanogenMethanosarcina barkeri

Abstract: Cytochromebc was partially purified from the methanogen,Methanosarcina barkeri. The cytochrome was composed of three subunits with molecular masses of 23.4, 20.9, and 9.1 kDa, respectively, and the 23.4 kDa subunit contained haemc. The absorption spectrum of cytochromebc showed a peak at 411 nm in the oxidized form, and peaks at 554, 524, and 422 nm in the reduced form. The cytochrome reacted with CO, and its low temperature absorption spectrum showed the α peak at 552 nm with a shoulder at 557 nm.