Showing papers on "Methanosarcina barkeri published in 1998"

An escherichia coli hydrogenase-3-type hydrogenase in methanogenic archaea

Abstract: Methanogenic archaea are known to contain two types of [NiFe] hydrogenases designated F420-reducing hydrogenase and F420-non-reducing hydrogenase. We report here that they additionally contain Escherichia coli hydrogenase-3-type [NiFe] hydrogenases. The evidence is based on biochemical studies and analysis of the subunit primary structure of this hydrogenase (designated Ech) purified from membranes of acetate-grown cells of Methanosarcina barkeri. The subunits EchE and EchC of the EchABCDEF complex showed 34% and 45% sequence identity to the nickel-containing large subunit HycE and to the iron-sulfur cluster containing small subunit HycG, respectively, of the hydrogenase in the formate hydrogen lyase complex from E. coli. Analysis of the totally sequenced genomes of Methanococcus jannaschii and Methanobacterium thermoautotrophicum strain deltaH revealed that these organisms contain similar open reading frames, indicating the presence of an E. coli hydrogenase-3-type hydrogenase also in these methanogenic archaea.

Clustered Genes Encoding the Methyltransferases of Methanogenesis from Monomethylamine

Abstract: Coenzyme M (CoM) is methylated during methanogenesis from monomethyamine in a reaction catalyzed by three proteins. Using monomethylamine, a 52-kDa polypeptide termed monomethylamine methyltransferase (MMAMT) methylates the corrinoid cofactor bound to a second polypeptide, monomethylamine corrinoid protein (MMCP). Methylated MMCP then serves as a substrate for MT2-A, which methylates CoM. The genes for these proteins are clustered on 6.8 kb of DNA in Methanosarcina barkeri MS. The gene encoding MMCP (mtmC) is located directly upstream of the gene encoding MMAMT (mtmB). The gene encoding MT2-A (mtbA) was found 1.1 kb upstream of mtmC, but no obvious open reading frame was found in the intergenic region between mtbA and mtmC. A single monocistronic transcript was found for mtbA that initiated 76 bp from the translational start. Separate transcripts of 2.4 and 4.7 kb were detected, both of which carried mtmCB. The larger transcript also encoded mtmP, which is homologous to the APC family of cationic amine permeases and may therefore encode a methylamine permease. A single transcriptional start site was found 447 bp upstream of the translational start of mtmC. MtmC possesses the corrinoid binding motif found in corrinoid proteins involved in dimethylsulfide- and methanol-dependent methanogenesis, as well as in methionine synthase. The open reading frame of mtmB was interrupted by a single in-frame, midframe, UAG codon which was also found in mtmB from M. barkeri NIH. A mechanism that circumvents UAG-directed termination of translation must operate during expression of mtmB in this methanogen.

Enhanced Dechlorination of Carbon Tetrachloride and Chloroform in the Presence of Elemental Iron and Methanosarcina barkeri, Methanosarcina thermophila, or Methanosaeta concillii

Abstract: Previous experiments in our laboratory have demonstrated that the rate and extent of carbon tetrachloride (CT) and chloroform (CF) dechlorination were enhanced when a methanogenic enrichment culture and iron (Fe0) were incubated together. Batch experiments with three pure cultures of methanogens, Methanosarcina barkeri, Methanosarcina thermophila, and Methanosaeta concillii were performed to determine how this enhanced transformation occurred. When hydrogen (H2) was added as an electron donor, degradation of CT for all organisms and CF for M. thermophila was more rapid. H2 was produced from the oxidation of iron, which therefore served as an H2 source for the organisms, enhancing the transformation of CT and CF. Experiments with M. thermophila and M. concillii, which could not grow on H2−CO2 under the conditions tested, showed that H2 could serve as an electron donor for dechlorination of CT and CF with these organisms as well. Experiments with supernatants from M. thermophila grown with and without iron ...

Methanol:coenzyme M methyltransferase from Methanosarcina barkeri--identification of the active-site histidine in the corrinoid-harboring subunit MtaC by site-directed mutagenesis.

Abstract: The enzyme system catalyzing the formation of methyl-coenzyme M from methanol and coenzyme M in Methanosarcina barkeri is composed of the three different polypeptides MtaA, MtaB and MtaC of which MtaC harbors a corrinoid prosthetic group. The heterologous expression of mtaA and mtaB in Escherichia coli has been described previously. We report here on the overproduction of the apoprotein of MtaC in E. coli, on its reconstitution to the active holoprotein with either cob(II)alamin or methyl-cob(III)alamin, and on the properties of the reconstituted corrinoid protein. Reconstituted MtaC was found to contain 1 mol bound cobamide/mol. EPR spectroscopic evidence is presented for a His residue as an axial ligand to Co2+ of the bound corrinoid. This active-site His was identified by site-directed mutagenesis as His136 in the MtaC sequence that contains four His residues. The reconstituted MtaC, in the cob(I)amide oxidation state, was methylated with methanol in the presence of MtaB and demethylated with coenzyme M in the presence of MtaA. In the presence of both MtaB and MtaA, methyl-coenzyme M was formed from methanol and coenzyme M at specific rates comparable to those determined for the enzyme system purified from M. barkeri. M. barkeri contains an isoenzyme of MtaA designated MtbA. The isoenzyme reacted with MtaC with only 2.5% of the activity of MtaA.

Purification and Properties of the Heme- and Iron−Sulfur-Containing Heterodisulfide Reductase from Methanosarcina thermophila†

Abstract: The heterodisulfide reductase (HDR) from Methanosarcina thermophila was purified to homogeneity from acetate-grown cells. In the absence of detergents, HDR consisted of an eight-protein complex with hydrogenase activity. However, when HDR was purified in the presence of 0.6% Triton X-100, a two-subunit (53 and 27 kDa) high specific activity (∼200 units mg-1) enzyme was obtained that lacked hydrogenase activity. Sedimentation equilibrium experiments demonstrated that HDR has a molecular mass of 206 kDa and a high partial specific volume (0.9 cm3/g), indicating that the purified protein contains a significant amount of bound lipid. Like the HDR from Methanosarcina barkeri [Kunkel, A., Vaupel, M., Heim, S., Thauer, R. K., and Hedderich, R. (1997) Eur. J. Biochem. 244, 226−234], it was found to contain two discrete b-type hemes in the small subunit and two distinct [Fe4S4]2+/1+ clusters in the large subunit. One heme is high-spin and has a high midpoint potential (−23 mV), whereas the other heme apparently is...

Thiol:fumarate reductase (Tfr) from Methanobacterium thermoautotrophicum--identification of the catalytic sites for fumarate reduction and thiol oxidation.

Abstract: Most methanogenic Archaea contain an unusual cytoplasmic fumarate reductase which catalyzes the reduction of fumarate with coenzyme M (CoM-S-H) and coenzyme B (CoB-S-H) as electron donors forming succinate and CoM-S-S-CoB as products. We report here on the purification and characterization of this thiol :fumarate reductase (Tfr) from Methanobacterium thermoautotrophicum (strain Marburg). The purified enzyme, which was composed of two different subunits with apparent molecular masses of 58 kDa (TfrA) and 50 kDa (TfrB), was found to catalyze the following reactions : (a) the reduction of fumarate with CoM-S-H and CoB-S-H (150 U/mg); (b) the reduction of fumarate with reduced benzyl viologen (620 U/mg); (c) the oxidation of CoM-S-H and CoB-S-H to CoM-S-S-CoB with methylene blue (95 U/mg); and (d) the reduction of CoM-S-S-CoB with reduced benzyl viologen (250 U/mg). The flavoprotein contained 12 mol non-heme iron and approximately the same amount of acid-labile sulfur/mol heterodimer. The genes encoding TfrA and TfrB were cloned and sequenced. Sequence comparisons with fumarate reductases and succinate dehydrogenases from Bacteria and Eucarya and with heterodisulfide reductases from M. thermoautotrophicum and Methanosarcina barkeri revealed that TfrA harbors FAD-binding motifs and the catalytic site for fumarate reduction and that TfrB harbors one [2Fe-2S] cluster and two [4Fe-4S] clusters and the catalytic site for CoM-S-H and CoB-S-H oxidation.

Redox-dependent acetyl transfer partial reaction of the acetyl-CoA decarbonylase/synthase complex: kinetics and mechanism.

Abstract: Acetyl-CoA decarbonylase/synthase (ACDS) is a multienzyme complex that plays a central role in energy metabolism in Methanosarcina barkeri grown on acetate. The ACDS complex carries out an unusual reaction involving net cleavage of the acetyl C-C and thioester bonds of acetyl-CoA. The overall reaction is composed of several partial reactions, one of which involves catalysis of acetyl group transfer. To gain insight into the overall reaction, a study was carried out on the kinetics and mechanism of the acetyltransferase partial reaction. Analysis by HPLC was used to quantify rates of acetyl transfer from acetyl-CoA both to 3'-dephospho-CoA and, by isotope exchange, to 14C-labeled CoA. Acetyl transfer activity was observed only under strongly reducing conditions, and was half-maximal at -486 mV at pH 6.5. The midpoint activation potential became increasingly more negative as the pH was increased, indicating the involvement of a protonation step. Cooperative dependence on acetyl-CoA concentration was exhibited in reactions that contained incompletely reduced enzyme; however, under redox conditions supporting maximum activity, hyperbolic kinetics were found. A ping-pong steady state kinetic mechanism was established, consistent with formation of an acetyl-enzyme intermediate. Analysis of the inhibitory effects of CoA on acetyl transfer to 3'-dephospho-CoA provided values for KiCoA of 6.8 microM and for Kiacetyl-CoA of 45 microM; isotope exchange analyses yielded values of 32 and 120 microM, respectively. Two separate measures of stability yielded values for the free energy of hydrolysis of the acetyl-enzyme intermediate of -9.6 and -9.3 kcal/mol, an indication of a high-energy bonding interaction in the acetyl-enzyme species. Implications for the mechanism of C-C bond cleavage are discussed.

Tetramethylammonium:coenzyme M methyltransferase system from methanococcoides sp

Abstract: A methanogen (strain NaT1) that belongs to the family of Methanosarcinaceae and that can grow on tetramethylammonium as the sole energy source has recently been isolated. We report here that cell extracts of the archaeon catalyze the formation of methyl-coenzyme M from coenzyme M and tetramethylammonium. The activity was dependent on the presence of Ti(III) citrate and ATP, and was rapidly lost under oxic conditions. Anoxic chromatography on DEAE-Sepharose revealed that two fractions, fractions 3 and 4, were required for activity. A 50-kDa protein that together with fraction 3 catalyzed methyl-coenzyme M formation from tetramethylammonium and coenzyme M was purified from fraction 4. From fraction 3, a 22-kDa corrinoid protein and a 40-kDa protein exhibiting methylcobalamin:coenzyme M methyltransferase (MT2) activity were purified. The N-terminal amino acid sequences of these purified proteins were determined. The 40-kDa protein showed sequence similarity to MT2 isoenzymes from Methanosarcina barkeri. Cell extract of strain NaT1 grown on trimethylamine rather than on tetramethylammonium did not exhibit tetramethylammonium:coenzyme M methyltransferase activity. The strain was identified as belonging to the genus of Methanococcoides, its closest relative being Methanococcoides methylutens.

Two F420-reducing hydrogenases in Methanosarcina barkeri

Abstract: F420-reducing hydrogenases are nickel iron-sulfur flavoproteins involved in CO2 reduction with H2 to methane in methanogenic archaea. Evidence is presented that Methanosarcina barkeri contains two isoenzymes for which the encoding genes have been cloned and sequenced. The genes are organized in two operons, frhADGB and freAEGB, each comprising four open reading frames. Transcription analysis revealed that both operons are transcribed during growth of Ms. barkeri on H2/CO2, on methanol, and on trimethylamine, but not during growth on acetate.

Purification and characterization of dimethylamine : 5-hydroxybenzimidazolyl-cobamide methyltransferase from methanosarcina barkeri fusaro

Abstract: Dimethylamine : 5-hydroxybenzimidazolylcobamide methyltransferase (DMA-MT) was purified from cells of Methanosarcina barkeri Fusaro grown on trimethylamine. In the presence of methylcobalamine :coenzyme M methyltransferase isoenzyme II [MT2(II)] the enzyme quite specifically catalyzed the stoichiometric conversion of dimethylamine (apparent Km = 0.45 mM) and 2-mercaptoethanesulfonate (coenzyme M) to monomethylamine and methyl-coenzyme M. Monomethylamine was a competitive inhibitor of the reaction (Ki = 4.5 mM). The apparent molecular mass of DMA-MT was 100 kDa and the enzyme was found to be a dimer, composed of identical 50-kDa subunits. A corrinoid content of 0.9 ± 0.1 mol B12/mol holoenzyme was calculated from HPLC analysis. The as-isolated methyltransferase was inactive, but it could be reductively reactivated. Activation required the presence of methyltransferase-activating protein, ATP and dimethylamine. Incubation with these compounds resulted in the methylation of the corrinoid prosthetic group.

Interactions between the promoter regions of nitrogenase structural genes (nifHDK2) and DNA-binding proteins from N2- and ammonium-grown cells of the archaeon Methanosarcina barkeri 227.

Abstract: Transcription initiation in Archaea (archaebacteria) resembles the eucaryotic process, having been shown to involve TATA box-like promoter regions as well as TATA-binding protein and TFIIB homologs. However, little is known about transcription regulation in archaea. We have previously demonstrated that transcripts of nifHDK2 genes, encoding Methanosarcina barkeri nitrogenase, are present in N2-grown cells but not in ammonium-grown cells, indicating that nif transcription is regulated by the nitrogen source. In this study, we detected proteins in M. barkeri cell extracts that bind specifically to DNA containing the putative promoter region of nifHDK2. No binding was found when the promoter region was deleted from the DNA. A competition assay showed that the methyl coenzyme M reductase (mcr) promoter region DNA and the nifH2 promoter region DNA competed for a common factor(s). There was no binding to the nifH2 promoter region by extracts of ammonium-grown cells, but there was binding by these extracts to promoter regions for mcr genes, which are presumably constitutively expressed. Interestingly, extracts of ammonium-grown cells inhibited binding to the nif promoter region by extracts of N2-grown cells. Fractionation of extracts of ammonium-grown cells with a heparin-Sepharose column resolved them into a fraction eluting at 0 M NaCl, which inhibited binding by extracts of N2-grown cells, and a fraction eluting at 0.5 to 0.75 M NaCl, which showed binding to the promoter region. These results are congruent with a model for regulation of nif gene expression in M. barkeri in which a substance present in ammonium-grown cells inhibits DNA binding by a transcription-associated protein or proteins.

Overproduction and one-step purification of the N5,N10-methenyltetrahydromethanopterin cyclohydrolase (Mch) from the hyperthermophilic Methanopyrus kandleri.

Abstract: 5,N 10-Methenyltetrahydromethanopterin cyclohydrolase (Mch) is an enzyme involved in methanogenesis from CO2 and H2 which represents the energy metabolism of Methanopyrus kandleri, a methanogenic Archaeon growing at a temperature optimum of 98°C. The gene mch from M. kandleri was cloned, sequenced, and expressed in Escherichia coli. The overproduced enzyme could be purified in yields above 90% in one step by chromatography on phenyl Sepharose in 80% ammonium sulfate. From 3.5 g cells (250 mg protein), approximately 18 mg cyclohydrolase was obtained. The purified enzyme showed essentially the same catalytic properties as the enzyme purified from M. kandleri cells. The primary structure and properties of the cyclohydrolase are compared with those of the enzyme from Methanococcus jannaschii (growth temperature optimum 85°C), from Methanobacterium thermoautotrophicum (65°C), and from Methanosarcina barkeri (37°C). Of the four enzymes, that from M. kandleri has the lowest isoelectric point (3.8) and the lowest hydrophobicity of amino acid composition. Besides, it has the highest relative content of glutamate, leucine, and valine and the lowest relative content of isoleucine, serine, and lysine. Some of these properties are unusual for enzymes from hyperthermophilic organisms. They may reflect the observation that the cyclohydrolase from M. kandleri is not only adapted to hyperthermophilic conditions but also to the high intracellular concentrations of lyotrophic salts prevailing in this organism.

Interspecific, intraspecific and interoperonic variability in the 16S rRNA gene of methanogens revealed by length and single-strand conformation polymorphism analysis

Abstract: Thirty-seven strains of mesophilic and thermophilic methanogenic Archaea, belonging to 30 species, were analyzed by length polymorphism (LP) and single-strand conformation polymorphism (SSCP) of an amplified 300-bp fragment of the 16S rRNA gene (Escherichia coli positions 9–331) including the variable regions V1 and V2. LPs and SSCPs were detected between species and between strains of the same species (Methanobacterium formicicum). LPs were found in Mb. formicicum DSMZ 3637, Mb. ivanovii DSMZ 2611, Mb. wolfei DSMZ 2970, Methanosarcina barkeri DSMZ 800, and Methanosaeta concilii DSMZ 3671, suggesting the presence of polymorphic 16S rRNA genes in the genome. We propose that LP and SSCP analysis of the 16S rRNA gene could be of practical help for strain identification.

Investigation of serine hydroxymethyltransferase in methanogens.

Abstract: The cofactor specificity of serine hydroxymethyltransferase (SHMT) activities was tested in extracts of several methanogens using tetrahydromethanopterin (H4MPt) from Methanobacterium thermoautotrophicum Marburg, tetrahydrosarcinapterin (H4SPt) from Methanosarcina barkeri, and tetrahydrofolate (H4folate) as the potential C1 carrier. In Methanosphaera stadtmanae and Methanococcus thermolithotrophicus, the activities were H4MPt dependent. In Methanospirillum hungatei GP1, Methanosaeta concilii, Methanolobus tindarius, and Methanosarcina barkeri Fusaro, the activities were strictly H4folate dependent. H4SPt was reactive with the SHMT of Methanosphaera stadtmanae but not with that of Methanosarcina barkeri. In both Methanosarcina barkeri and Methanospirillum hungatei, pyridoxal phosphate stimulated SHMT activity. The apparent Km values for H4folate and L-serine were 0.086 and 0.29 mM in Methanosarcina barkeri and 0.065 and 0.31 mM in Methanospirillum hungatei, respectively. Key words: tetrahydromethanopterin,...

Activation and reaction kinetics of the dimethylamine/coenzyme M methyltransfer in Methanosarcina barkeri strain Fusaro

Abstract: Dimethylamine/5-hydroxybenzimidazolylcobamide methyltransferase (DMA-MT) from Methanosarcina barkeri Fusaro catalyzes (Vmax = 4700 nmol min−1 mg−1 protein; kcat = 7.8 s−1) the transfer of a methyl group from dimethylamine (apparent Km = 0.45 mM) to its corrinoid prosthetic group to yield monomethylamine (MMA) and the methylated enzyme. The product, MMA, is a competitive inhibitor of the reaction (apparent Ki = 5.5 mM). The methyl group bound to the corrinoid prosthetic group of DMA-MT is subsequently transferred to coenzyme M in a reaction mediated by methylcobalamin/coenzyme M methyltransferase isoenzyme II [MT2(II)], which binds with high affinity to DMA-MT (apparent Km = 0.22 μM). As isolated, DMA-MT is inactive, but it can enzymically be reactivated by methyltransferase activating protein (MAP), ATP, and hydrogenase. Apart from the established role in corrinoid activation, ATP was found to act as a powerful allosteric effector on the methyltransferase reaction. The results of kinetic studies, supported by the resolution of as-yet partially purified auxiliary protein fractions, demonstrate that DMA-MT, MT2(II), MAP, and hydrogenase are the only enzymic components involved in the dimethylamine/coenzyme M methyltransfer in M. barkeri Fusaro.

How many rumen methanogens are there

Abstract: For nearly 70 years in ruminant nutrition the loss as methane of gross energy intake by ruminants has been measured, but surprisingly little is known about the populations of methane-producing microorganisms (methanogens) in the rumen. Numbers of methanogens in the cattle rumen are ca. 10/mL rumen contents, and differ little with time after feeding or with the roughage content of the diet (Leedle and Greening 1982). Methanogens that have been isolated from the rumen and from faeces of ruminants belong to the genera Methanobrevibacter, Methanomicrobium and Methanosarcina (Miller et al. 1986, Wolin and Miller 1997). Methanobrevibacter spp. and Methanomicrobium spp. are present in high numbers in the rumen (ca. 10/mL), but the sarcina are present in low numbers (ca. 10/mL) except in animals fed unusual diets where rumen dilution rates are very low. Interestingly, few of the type species are rumen isolates (Sowers and Schreier 1995). Here we report observations of the size and composition of the populations of methanogens in sheep and cattle from several studies. An agar medium (Miller and Wolin 1982) was used for enumeration and isolation of methanogens. Methanogens were identified by their autofluorescence at 420nm and production of methane in culture. Methane was determined by gas chromatography with flame ionisation or thermal conductivity detectors. Serum antibodies raised in rabbits were used as ‘probes’ to indicate possible phylogenetic relatedness of isolates, or of cells in rumen contents, observed using immunofluorescence microscopy (see Conway de Macario et al. (1982) for more detail). In rumen inocula from 12 sheep fed oaten hay, lupin grain and a mineral mix (88:10:2) the size of the methanogen population, estimated from colony counts in the agar medium, varied between sheep from less than 10 up to 10/mL rumen inoculum. In the culture dilution series, the final dilutions at which methane in the head-space was detectable also varied, from dilutions of 10 to more than 10/mL rumen inoculum. From two cattle fed either hay or hay silage, methanogens were isolated from dilutions of 10 to 10/mL rumen contents. The isolates were cocci, coccobacilli or rods and when they were tested against antisera raised to various methanogens they bound most strongly antibodies to either Methanobrevibacter ruminantium M1 or Methanobacterium formicicum MF. There was little or no cross-reactivity with antibodies to Methanobrevibacter smithii B181 or Methanobrevibacter arboriphilicus DC. From a sheep fed silage the morphology of methanogens that were isolated (in the laboratory of M.J. Wolin, New York State Department of Health) resembled Methanobrevibacter spp. Antisera (in the laboratory of A. J. L. Macario, New York State Department of Health) raised against Mb. ruminantium M1, Mb. smithii PS, ALI, Mb. arboriphilicus DH1, AZ, DC, Methanobacterium bryantii M.o.H.G, M. formicicum MF, Methanomicrobium mobile BP, Methanosarcina mazei S6 and Methanosarcina barkeri MS were used to probe methanogens in rumen fluid samples. Coccobacilli that morphologically resembled Methanobrevibacter spp. and strongly bound antibodies to Mb. smithii PS were most numerous followed by cocobacilli that strongly bound antibodies to Mb. arboriphilicus DH1 and DC. Low numbers of large cocci strongly bound antibodies to Ms. mazei S6 and Ms. barkeri MS. Since antibody probes revealed a greater number and diversity of methanogens in the rumen population than did culture techniques, ecological studies of rumen methanogens using culture techniques also should employ complementary techniques such as the use of antibody or oligonucleotide probes.