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At least 19 recordsLinked to original sources

PubMed1992

DNA relatedness among some thermophilic members of the genus Methanobacterium: emendation of the species Methanobacterium thermoautotrophicum and rejection of Methanobacterium thermoformicicum as a synonym of Methanobacterium thermoautotrophicum.

DNA reassociation was used to determine levels of relatedness among four thermophilic Methanobacterium strains that are able to use formate and between these organisms and two representative strains of Methanobacterium thermoautotrophicum, strain delta HT (= DSM 1053T = ATCC 29096T) (T = type strain) and strain Marburg (= DSM 2133). Three homology groups were delineated, and these groups coincided with the clusters identified by antigenic fingerprinting. The first group, which had levels of cross hybridization that ranged from 73 to 99%, included M. thermoautotrophicum delta HT, Methanobacterium thermoformicicum Z-245, Methanobacterium sp. strain THF, and Methanobacterium sp. strain FTF. The second and third groups were each represented by only one strain, Methanobacterium sp. strain CB-12 and M. thermoautotrophicum Marburg, respectively (cross-hybridization levels, 13 to 30 and 29 to 33%, respectively). Our results indicate that the name M. thermoformicicum should be rejected as it is a synonym of M. thermoautotrophicum. The taxonomic positions of strains Marburg and CB-12 need further investigation.

Antigens, Bacterial↗

PubMed1998

The formylmethanofuran dehydrogenase isoenzymes in Methanobacterium wolfei and Methanobacterium thermoautotrophicum: induction of the molybdenum isoenzyme by molybdate and constitutive synthesis of the tungsten isoenzyme.

Formylmethanofuran dehydrogenase catalyzes the first step in methane formation from CO2 in methanogenic archaea. Methanobacterium wolfei and Methanobacterium thermoautotrophicum have been shown to contain two isoenzymes, a tungsten-containing isoenzyme (Fwd) and a molybdenum-containing isoenzyme (Fmd). We report here that in both thermophilic organisms the encoding genes are organized in a highly conserved fwdHFGDACB tungsten operon and in an fmdECB molybdenum operon. In both organisms, the tungsten isoenzyme was found to be constitutively transcribed, whereas the transcription of the molybdenum operon was induced by molybdate. Induction by molybdate was not significantly affected by tungstate.

Aldehyde Oxidoreductases↗

PubMed1989

Nucleotide sequence of the glyceraldehyde-3-phosphate dehydrogenase gene from the mesophilic methanogenic archaebacteria Methanobacterium bryantii and Methanobacterium formicicum. Comparison with the respective gene structure of the closely related extreme thermophile Methanothermus fervidus.

The genes for glyceraldehyde-3-phosphate dehydrogenase (gap genes) from the mesophilic methanogenic archaebacteria Methanobacterium formicicum and Methanobacterium bryantii were cloned and sequenced. The deduced amino acid sequences show 95% identity to each other and about 70% identity to the glyceraldehyde-3-phosphate dehydrogenase from the thermophilic methanogenic archaebacterium Methanothermus fervidus. Although the sequence similarity between the archaebacterial glyceraldehyde-3-phosphate dehydrogenase and the homologous enzyme of eubacteria and eukaryotes is low, an equivalent secondary-structural arrangement can be deduced from the profiles of the physical parameters hydropathy, chain flexibility and amphipathy. In order to find possible thermophile-specific structural features of the enzyme from M. fervidus, a comparative primary-sequence analysis was performed. Amino acid exchanges leading, to a stabilization of the main-chain conformation, could be found throughout the sequence of the thermophile enzyme. Striking features of the thermophile sequence are the preference for isoleucine, especially in beta-sheets, and a low arginine/lysine ratio of 0.54.

Amino Acids↗

PubMed1987

Purification and use of Methanobacterium wolfei pseudomurein endopeptidase for lysis of Methanobacterium thermoautotrophicum.

The pseudomurein-degrading enzyme from autolysates of Methanobacterium wolfei was purified approximately 500-fold to electrophoretic homogeneity by ion-exchange chromatography under anaerobic conditions. Analysis of the soluble cell wall fragments produced by the pure enzyme from a cell wall preparation of M. thermoautotrophicum indicated that it is a peptidase hydrolyzing the epsilon-Ala-Lys bond of pseudomurein. A partially purified preparation of pseudomurein endopeptidase was free of nuclease activity and thus proved useful for the preparation in high yields of undegraded chromosomal and plasmid DNA from M. thermoautotrophicum. The partially purified enzyme was also used for the preparation of protoplasts, which were stabilized by 0.8 M sucrose. Under growth conditions the protoplasts produced methane and increased up to 100-fold in size, but failed to regenerate a cell wall.

Endopeptidases↗

PubMed1994

Aspartate aminotransferase from a thermophilic formate-utilizing methanogen, Methanobacterium thermoformicicum strain SF-4: relation to serine and phosphoserine aminotransferases, but not to the aspartate aminotransferase family.

The primary structure of the aspartate aminotransferase (AspAT) of an archaebacterium, Methanobacterium thermoformicicum strain SF-4, has been determined by cloning and sequencing of the gene for the enzyme. The gene had a consensus promoter and a ribosome binding sequence of methanogens in the 5' untranslated region, followed by an open reading frame starting with ATG and terminating with TGA. The deduced amino acid sequence was identical with the partial amino acid sequences of the enzyme including the N-terminal sequence, and the deduced molecular weight of 41,684 was virtually identical to that reported earlier for this enzyme [Tanaka, T., Yamamoto, S., Taniguchi, M., Hayashi, H., Kuramitsu, S., Kagamiyama, H., & Oi, S. (1992) J. Biochem. 112, 811-815]. The gene was expressed in Escherichia coli by inserting it into an expression vector just downstream of the lacZ promoter, and this verified that the cloned gene really encodes the Methanobacterium AspAT. The primary structure of the Methanobacterium AspAT showed extremely low homology, 5%, with AspATs of eubacteria, eukaryotes, and a thermoacidophilic arachaebacterium, Sulfolobus solfataricus. On the other hand, the Methanobacterium AspAT showed remarkable amino acid sequence homology, 31.5%, with rat serine:pyruvate aminotransferase and, 13.5%, with E. coli phosphoserine aminotransferase. Thus, the Methanobacterium AspAT apparently belongs to subgroup IV of the aminotransferases [Mehta, P.K., Hale, T.I., & Christen, P. (1993) Eur. J. Biochem. 214, 549-561], but not to subgroup I, in which all the AspATs known so far are included.

Amino Acid Sequence↗

PubMed1988

Cloning and physical mapping of RNA polymerase genes from Methanobacterium thermoautotrophicum and comparison of homologies and gene orders with those of RNA polymerase genes from other methanogenic archaebacteria.

The structural genes encoding the four largest subunits of RNA polymerase, A, B', B", and C, were physically mapped in Methanobacterium thermoautotrophicum Winter. The genes formed a cluster in the order B", B', A, C and had a common orientation. DNA hybridization experiments yielded different degrees of homology between RNA polymerase gene sequences of different species of Methanobacterium and Methanococcus voltae. No homology was detectable between Methanobacterium thermoautotrophicum and Methanosarcina barkeri. From Southern hybridization experiments in which probes of the four genes from Methanobacterium thermoautotrophicum Winter and restriction digests of the genomic DNAs of the different methanogens were used, a common gene order of the RNA polymerase genes could be deduced.

Archaea↗

PubMed1994

Na(+)-driven ATP synthesis in Methanobacterium thermoautotrophicum and its differentiation from H(+)-driven ATP synthesis by rhodamine 6G.

Rhodamine 6G (3 microM) effectively inhibited delta pH-driven ATP synthesis in Methanobacterium thermoautotrophicum while delta pNa-driven ATP synthesis was not affected by it. Rhodamine 6G inhibited Mg(2+)-stimulated ATPase activity of membrane vesicles prepared from these cells but the ATPase catalytic sector detached from the membrane was insensitive to this inhibitor. Methanogenesis-driven ATP synthesis at pH 6.8 of cells grown in the presence of 50 mM NaCl was inhibited by rhodamine 6G both in the presence of 5 mM and 50 mM NaCl. On the other hand, the methanogenesis-driven ATP synthesis at pH 8.0 of cells grown in the presence of 50 mM NaCl was slightly inhibited by rhodamine 6G in the presence of 5 mM NaCl and was not inhibited at all in the presence of 50 mM NaCl. The growth experiments have shown that cells of Methanobacterium thermoautotrophicum can grow under alkaline conditions even in the presence of rhodamine 6G and of high NaCl concentration when the growth media were inoculated with the cells which had been grown in the presence of 50 mM NaCl. These results indicate that sodium-motive force-driven ATP synthase in Methanobacterium thermoautotrophicum operates effectively in alkaline conditions and it might be the sole ATP synthesizing system when the proton-motive force-supported ATP synthesis is inhibited by rhodamine 6G.

Adenosine Triphosphatases↗

PubMed1994

Na(+)-driven ATP synthesis in Methanobacterium thermoautotrophicum and its differentiation from H(+)-driven ATP synthesis by rhodamine 6G.

Rhodamine 6G (3 microM) effectively inhibited delta pH-driven ATP synthesis in Methanobacterium thermoautotrophicum while delta pNA-driven ATP synthesis was not affected by it. Rhodamine 6G inhibited Mg(2+)-stimulated ATPase activity of membrane vesicles prepared from these cells but the ATPase catalytic sector detached from the membrane was insensitive to this inhibitor. Methanogenesis-driven ATP synthesis at pH 6.8 of the cells grown in the presence of 50 mM NaCl was inhibited by rhodamine 6G both in the presence of 5 mM and 50 mM NaCl. On the other hand, the methanogenesis-driven ATP synthesis at pH 8.0 of cells grown in the presence of 50 mM NaCl was slightly inhibited by rhodamine 6G in the presence of 5 mM NaCl and was not inhibited at all in the presence of 50 mM NaCl. The growth experiments have shown that cells of Methanobacterium thermoautotrophicum can grow under alkaline conditions even in the presence of rhodamine 6G and of high NaCl concentration when the growth media were inoculated with the cells which had been grown in the presence of 50 mM NaCl. These results indicate that sodium-motive force-driven ATP synthase in Methanobacterium thermoautotrophicum operates effectively at alkaline conditions and it might be the sole ATP synthesizing system when the proton motive force-supported ATP synthesis is inhibited by rhodamine 6G.

Adenosine Triphosphatases↗

PubMed1995

The presence of H+ and Na(+)-translocating ATPases in Methanobacterium thermoautotrophicum and their possible function under alkaline conditions.

Two ATPases with different apparent molecular masses of approx. 500 kDa and 400 kDa were identified in the EDTA extract of the cell membranes of Methanobacterium thermoautotrophicum. Western blotting with polyclonal antiserum reactive with beta-subunit of mitochondrial ATPase from rat liver and yeast was used for further analysis of these ATPases. A strong crossreactivity with a single protein band with an apparent molecular weight of about 53 kDa (similar to beta-subunit of F-type ATPase from other sources) was found in protein extracts of whole cells of Methanobacterium thermoautotrophicum strains delta H and Marburg, as well as of Methanospirillum hungatei. This indicates the presence of F-type ATPase in methanogens. ATP synthesis driven by membrane potential which was generated by artificially-imposed delta pH in the presence of protonophorous uncoupler and sodium ions was stimulated by bafilomycin A1, an inhibitor of V- and A-type ATPases, as well as by harmaline, an inhibitor of Na+/H+ antiporter. These results indicate that cells of Methanobacterium thermoautotrophicum strain delta H contain the F-type ATP synthase which is Na(+)-translocating in addition to V- or A-type ATP synthase which is H(+)-translocating.

Adenosine Triphosphate↗

PubMed2000

Phylogenetic analysis of 18 thermophilic Methanobacterium isolates supports the proposals to create a new genus, Methanothermobacter gen. nov., and to reclassify several isolates in three species, Methanothermobacter thermautotrophicus comb. nov., Methanothermobacter wolfeii comb. nov., and Methanothermobacter marburgensis sp. nov.

Using a combination of 16S rRNA analysis and antigenic fingerprinting consisting of new and published data, the phylogenetic position of 18 thermophilic isolates currently classified as Methanobacterium species was reinvestigated. The results were verified by independent methods, including, where applicable, plasmid and phage typing. Comparative analysis of 16S rRNA data for 30 strains belonging to the order Methanobacteriales strongly suggested that mesophilic and thermophilic Methanobacterium isolates are distantly related and should be assigned to separate genera. For the thermophilic strains the genus Methanothermobacter was initially proposed by Boone, Whitman and Rouvière. Furthermore, the results support a reclassification of 15 isolates in three species within the proposed genus: (i) Methanothermobacter thermautotrophicus comb. nov., containing eight isolates, six of which are able to utilize formate (type strain deltaHT); (ii) Methanothermobacter wolfeii comb. nov., containing four formate-utilizing isolates (type strain DSM 2970T); (iii) Methanothermobacter marburgensis sp. nov., containing three obligately autotrophic isolates (type strain MarburgT). Of the nine isolates formerly referred to as Methanobacterium thermoformicicum, six were reclassified as Methanothermobacter thermautotrophicus and three as Methanothermobacter wolfeii.

Antigens, Archaeal↗

PubMed1993

Cloning, sequencing and immunological characterization of the corrinoid-containing subunit of the N5-methyltetrahydromethanopterin: coenzyme-M methyltransferase from Methanobacterium thermoautotrophicum.

A 3.5-kb EcoRI fragment of the Methanobacterium thermoautotrophicum chromosome contains five open reading frames, mtrA to mtrE. The deduced N-terminal amino acid sequence of mtrA is identical with 26 N-terminal amino acids of a corrinoid-containing membrane protein from Methanobacterium. Computer-aided analyses of mtrA predicts 237 amino acids with a molecular mass of 25,603 Da for its gene product. A hydropathy plot of this amino acid sequence indicates one hydrophobic helical conformation near the N-terminus of the peptide which represents a tentative membrane-spanning region. The main part of the protein, however, shows hydrophilic domains, suggesting a location outside the cytoplasmic membrane. These domains are probably accessible by monospecific polyclonal antibodies raised previously against the corrinoid-containing membrane protein. The immunogold-labeling technique revealed that the corrinoid-dependent membrane protein was detectable at the cytoplasmic face of the membranes and of vesicle preparations. No significant identity of the deduced amino acid sequence was found with sequences of several corrinoid-containing enzymes. In contrast to the hydrophilic gene product of mtrA, four other gene products from the gene cluster encode extremely hydrophobic proteins. The N-terminal sequences of mtrC and mtrD are identical with two peptides of the N5-methyltetrahydromethanopterin:coenzyme-M methyltransferase complex from Methanobacterium, indicating that the mtr genes encode this membrane protein.

Amino Acid Sequence↗

PubMed1995

Evidence for a defective prophage on the chromosome of Methanobacterium wolfei.

Evidence shows the presence on the chromosome of Methanobacterium wolfei of a defective prophage which, by DNA-DNA hybridization, is closely related to the virulent archaeophage psi M1 of Methanobacterium thermoautotrophicum Marburg. Partial sequencing of a M. wolfei 16S rRNA gene and phylogenetic analysis indicated that this organism is more closely related to other representatives of the genus Methanobacterium than to M. thermoautotrophicum Marburg. The chromosomal region of M. wolfei encoding the putative prophage was found to be deleted for two non-contiguous segments of the phage psi M1 genome and thus encompassed only 80 to 90% of the psi M1 DNA. The prophage region was mapped to a 30 kb restriction fragment on the physical map of the M. wolfei chromosome. A randomly chosen DNA fragment was cloned from phage psi M1 DNA, as was its homologous counterpart from the chromosome of M. wolfei. The 126-bp region present in both clones exhibited 100% sequence identity.

Bacteriophages↗

PubMed1972

Methanobacterium thermoautotrophicus sp. n., an anaerobic, autotrophic, extreme thermophile.

The isolation of a new methanogenic bacterium, Methanobacterium thermoautotrophicus sp. n., is described. Successful isolation required a medium containing inorganic salts, an atmosphere consisting of an 80:20 mixture of hydrogen-carbon dioxide, and incubation temperatures of 65 to 70 C. Isolates of M. thermoautotrophicus were gram-positive, nonmotile, irregularly curved rods which frequently formed long filaments. The organism was found to be an autotroph and a strict anaerobe, and to have a pH optimum of 7.2 to 7.6. The optimal temperature for growth was 65 to 70 C, the maximum being 75 C and the minimum about 40 C. The generation time at the optimum was about 5 hr. The deoxyribonucleic acid of M. thermoautotrophicus had a guanine plus cytosine (GC) content of 52 moles per cent, whereas Methanobacterium sp. strain M.O.H. had a GC content of 38%. When heated, intact ribosomes of Methanobacterium sp. strain M.O.H. were stable up to 55 C and had a T(m) of 73 C. In contrast, ribosomes of M. thermoautotrophicus were stable up to 75 C and had a T(m) of 82 C. Upon complete thermal denaturation, ribosomes of strain M.O.H. underwent a 59% hyperchromic shift, whereas those of the thermophile showed only a 20% increase in hyperchromicity. Methane formation in cell-free extracts of M. thermoautotrophicus was temperature-dependent and required hydrogen and carbon dioxide; methyl cobalamin served as a methyl donor, and addition of coenzyme M stimulated methanogenesis.

Anaerobiosis↗

PubMed1989

Aerobic purification of N5,N10-methylenetetrahydromethanopterin dehydrogenase, separated from N5,N10-methylenetetrahydromethanopterin cyclohydrolase, from Methanobacterium thermoautotrophicum strain Marburg.

The N5,N10-methylenetetrahydromethanopterin dehydrogenase from Methanobacterium thermoautotrophicum strain Marburg has been purified with reasonable yield and much higher specific activity than previously reported. For the first time it has been shown that both N5,N10-methylenetetrahydromethanopterin dehydrogenase and N5,N10-methenyltetrahydromethanopterin cyclohydrolase activities were stable under air and could be purified using aerobic operations. The dehydrogenase activity from Methanobacterium thermoautotrophicum Marburg was stable in phosphate buffer with or without glycerol or ammonium sulfate under both aerobic and anaerobic conditions. However, the presence of either 2-mercaptoethanol or dithiothreitol in the enzyme solution destroyed the enzyme activity during both aerobic and anaerobic incubations. Dehydrogenase was purified 62-fold using Phenyl-Sepharose and DEAE-Sephadex chromatography in succession under air. Both of these chromatographic methods separated dehydrogenase activity from N5,N10-methenyltetrahydromethanopterin cyclohydrolase; DEAE-Sephadex provided the best separation. Phenyl-Sepharose chromatography of the supernatant of cell extracts containing ammonium sulfate at 60% of saturation provided a 4.7-fold purification and 98% recovery of cyclohydrolase; this result established the air stability of N5,N10-methenyltetrahydromethanopterin cyclohydrolase from Methanobacterium thermoautotrophicum Marburg.

Aerobiosis↗

PubMed1993

Distribution and characterization of plasmid-related sequences in the chromosomal DNA of different thermophilic Methanobacterium strains.

The genomes of several thermophilic members of the genus Methanobacterium were analyzed for homology to the related restriction-modification plasmids pFV1 and pFZ1 from M. thermoformicicum strains THF and Z-245, respectively. Two plasmid regions, designated FR-I and FR-II, could be identified with chromosomal counterparts in six Methanobacterium strains. Multiple copies of the pFV1-specific element FR-I were detected in the M. thermoformicicum strains CSM3, FF1, FF3 and M. thermoautotrophicum delta H. Sequence analysis showed that one FR-I element had been integrated in almost identical sequence contexts into the chromosomes of the strains CSM3 and delta H. Comparison of the FR-I elements from these strains with that from pFV1 revealed that they consisted of two subfragments, boxI (1118 bp) and boxII (383 bp), the order of which is variable. Each subfragment was identical on the sequence level with the corresponding plasmid-borne element and was flanked by terminal direct repeats with the consensus sequence A(A/T)ATTT. These results suggest that FR-I represents a mobile element. FR-II was located on both plasmids pFV1 and pFZ1, and on the chromosome of M. thermoformicicum strains THF, CSM3 and HN4. Comparison of the nucleotide sequences of the two plasmid FR-II copies and that from the chromosome of strain CSM3 showed that the FR-II segments were approximately 2.5-3.0 kb in size and contained large open reading frames (ORFs) that may encode highly related proteins with an as yet unknown function.

Amino Acid Sequence↗

PubMed1992

Spectroscopic characterization of the alternate form of S-methylcoenzyme M reductase from Methanobacterium thermoautotrophicum (strain delta H).

Two forms (MR1 and MR2) of S-methylcoenzyme M reductase were purified from Methanobacterium thermoautotrophicum (strain delta H) as recently described (Rospert, S., Linder, D., Ellerman, J. and Thauer, R.K. (1990) Eur. J. Biochem. 194, 871-877). MR2 was at least 50-fold more active than MR1, independent of assay conditions. The two forms are spectroscopically similar, but not identical, by UV-visible, magnetic circular dichroism and resonance Raman spectroscopies. MR2 exhibited an EPR signal corresponding to 20% of the enzyme-bound nickel. Strong EPR signals similar to those previously assigned to Ni(I)F430 bound to methylreductase in Methanobacterium thermoautotrophicum (strain Marburg) (Albracht, S.P.J., Ankel-Fuchs, D., Bocher, R., Ellerman, J., Moll, J., Van der Zwann, J.W. and Thauer, R.K. (1988) Biochim. Biophys. Acta 955, 86-102) were observed in MR2-rich, log-phase, as well as in MR1-rich, slow-growing bacteria. Log-phase cells had dramatically different EPR spectra depending on whether they were removed from the fermenter (under gas flow) before or after cooling to 10 degrees C. EPR spectra of slow-growing cells were insensitive to harvesting conditions. The possible biological significance of the alternate form of methylreductase is discussed.

Circular Dichroism↗

PubMed1998

Methanobacterium subterraneum sp. nov., a new alkaliphilic, eurythermic and halotolerant methanogen isolated from deep granitic groundwater.

Deep subterranean granitic aquifers have not been explored regarding methanogens until now. Three autotrophic methane-producing Archaea were isolated from deep granitic groundwater at depths of 68, 409 and 420 m. These organisms were non-motile, small, thin rods, 0.1-0.15 micron in diameter, and they could use hydrogen and carbon dioxide or formate as substrates for growth and methanogenesis. One of the isolates, denoted A8p, was studied in detail. It grew with a doubling time of 2.5 h under optimal conditions (20-40 degrees C, pH 7.8-8.8 and 0.2-1.2 M NaCl). Strain A8p is eurythermic as it grew between 3.6 and 45 degrees C. It was resistant to up to 20 mg bacitracin l-1. The G + C content was 54.5 mol%, as determined by thermal denaturation. Phylogenetic studies based upon 16S rRNA gene sequence comparisons placed the isolate A8p in the genus Methanobacterium. Phenotypic and phylogenetic characters indicate that the alkaliphilic, halotolerant strain A8p represents a new species. We propose the name Methanobacterium subterraneum for this species, and strain A8p (= DSM 11074T) is the type strain.

Anti-Bacterial Agents↗

PubMed2000

Methanobacterium oryzae sp. nov., a novel methanogenic rod isolated from a Philippines ricefield.

A rod (0.3-0.4 micron x 3-10 microns) to filamentous (up to 40 microns) non-motile methanogenic bacterium, designated strain FPiT (T = type strain), was isolated from ricefield soil in the Philippines. The strain uses H2 + CO2 or formate for growth and produces CH4. Optimum growth temperature is 40 degrees C; no growth is observed at 15 degrees C or 45 degrees C. Optimum pH for growth is 7; no growth is observed at pH 5.5 or 9.0. Strain FPiT is halotolerant and grows at NaCl concentrations of 0-25 g l-1. The G + C content of its DNA is 31 mol%. Based on 16S rRNA gene sequence analysis, the isolate was identified as a new species of the genus Methanobacterium: Methanobacterium oryzae sp. nov. The type strain is FPiT (= DSM 11106T).

Agriculture↗