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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↗
PubMed2006

MTH187 from Methanobacterium thermoautotrophicum has three HEAT-like repeats.

With the completion of genome sequencing projects, there are a large number of proteins for which we have little or no functional information. Since protein function is closely related to three-dimensional conformation, structural proteomics is one avenue where the role of proteins with unknown function can be investigated. In the present structural project, the structure of MTH187 has been determined by solution-state NMR spectroscopy. This protein of 12.4 kDa is one of the 424 non-membrane proteins that were cloned and purified for the structural proteomic project of Methanobacterium thermoautotrophicum [Christendat, D., Yee, A., Dharamsi, A., Kluger, Y., Gerstein, M., Arrowsmith, C.H. and Edwards, A.M. (2000) Prog. Biophys. Mol. Biol., 73, 339-345]. Methanobacterium thermoautotrophicum is a thermophilic archaeon that grows optimally at 65 degrees C. A particular characteristic of this microorganism is its ability to generate methane from carbon dioxide and hydrogen [Smith, D.R., Doucette-Stamm, L.A., Deloughery, C., Lee, H., Dubois, J., Aldredge, T., Bashirzadeh, R., Blakely, D., Cook, R., Gilbert, K., Harrison, D., Hoang, L., Keagle, P., Lumm, W., Pothier, B., Qiu, D., Spadafora, R., Vicaire, R., Wang, Y., Wierzbowski, J., Gibson, R., Jiwani, N., Caruso, A., Bush, D., Reeve, J. N. et al. (1997) J. Bacteriol., 179, 7135-7155].

Archaeal Proteins↗
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↗
PubMed2001

Methanobacterium congolense sp. nov., from a methanogenic fermentation of cassava peel.

Strain CT, a non-motile, mesophilic, hydrogenotrophic, methanogenic bacterium, was isolated from an anaerobic digester used for the treatment of raw cassava-peel waste in Congo. The cells were rods, 0.4-0.5 x 2-10 microm in size, and stained Gram-positive. Hydrogen and carbon dioxide were the only substrates that supported growth and methane production. Methane production, but not growth, occurred with CO2 in the presence of either 2-propanol, 2-butanol or cyclopentanol as hydrogen donors. The temperature range for growth was 25-50 degrees C, the optimum being between 37 and 42 degrees C. The optimum pH for growth was 7.2; consistent growth and methane production were not observed below pH 5.9 or above pH 8.2. The doubling time under optimal growth conditions was 7.5 h. The DNA base composition was 39.5 mol% G+C. On the basis of 16S rRNA gene sequence analysis and phenotypic characteristics, the isolate is proposed as a new species of the genus Methanobacterium, namely Methanobacterium congolense sp. nov. The type strain is strain CT (= DSM 7095T = OCM 779T).

Bioreactors↗
PubMed2005

Methanobacterium beijingense sp. nov., a novel methanogen isolated from anaerobic digesters.

Two methanogenic strains, 8-2T and 4-1, with rod-shaped (0.4-0.5 x 3-5 microm), non-motile cells, sometimes observed in chains, were isolated from two anaerobic digesters in Beijing, China. The two strains used H2/CO2 and formate for growth and produced methane. The temperature range for growth was 25-50 degrees C, with fastest growth at 37 degrees C. The pH ranges for growth and methane production were 6.5-8.0 for strain 8-2T and 6.8-8.6 for strain 4-1, with the fastest growth at pH 7.2 for strain 8-2T and pH 7.5-7.7 for strain 4-1. The G+C content of genomic DNA for strain 8-2T was 38.9 mol%. The similarity levels of the 16S rRNA sequence of strain 8-2T with other species of the genus Methanobacterium ranged from 93.8 to 96.0 %. Based on the phylogenetic analysis and phenotypic characteristics, the novel species Methanobacterium beijingense sp. nov. is proposed, with the type strain 8-2T (=DSM 15999T=CGMCC 1.5011T).

Anaerobiosis↗
PubMed1992

Differential expression of the two methyl-coenzyme M reductases in Methanobacterium thermoautotrophicum as determined immunochemically via isoenzyme-specific antisera.

Methanobacterium thermoautotrophicum contains two isoenzymes of methyl-coenzyme M reductase (MCR), MCR I and MCR II, which catalyze the methane-forming step and which together represent more than 10% of the cellular protein. We describe here the preparation of isoenzyme-specific antisera against the two MCR isoenzymes and their use in the quantitative immunochemical determination of the two isoenzymes in the methanogen. The relative and absolute cellular concentration of the two proteins is shown to be strongly affected by growth conditions such as the temperature, pH, and substrate concentration. Conditions were found yielding cells which contained essentially only MCR I or MCR II. Using antisera against MCR I and MCR II, MCR from other methanogens were immunochemically compared. Evidence is presented that Methanobacterium wolfei also contains two isoenzymes of MCR.

Immune Sera↗
PubMed1992

Physical map of the Methanobacterium thermoautotrophicum Marburg chromosome.

A physical map of the Methanobacterium thermoautotrophicum Marburg chromosome was constructed by using pulsed-field gel electrophoresis of restriction fragments generated by NotI, PmeI, and NheI. The order of the fragments was deduced from Southern blot hybridization of NotI fragment probes to various restriction digests and from partial digests. The derived map is circular, and the genome size was estimated to be 1,623 kb. Several cloned genes were hybridized to restriction fragments to locate their positions on the map. Genes coding for proteins involved in the methanogenic pathway were located on the same segment of the circular chromosome. In addition, the genomes of a variety of thermophilic Methanobacterium strains were treated with restriction enzymes and analyzed by pulsed-field gel electrophoresis. The sums of the fragment sizes varied from 1,600 to 1,728 kb among the strains, and widely different macrorestriction patterns were observed.

Chromosome Mapping↗
PubMed1995

Purification of the copper response extracellular proteins secreted by the copper-resistant methanogen Methanobacterium bryantii BKYH and cloning, sequencing, and transcription of the gene encoding these proteins.

When the copper-resistant methanogen Methanobacterium bryantii BKYH was exposed to 1 mM Cu(II), it secreted approximately fourfold increased levels of three proteins, copper response extracellular (CRX) proteins. The members of the CRX protein trio had apparent molecular masses of 40.8, 42.3, and 42.9 kDa and were purified together from the culture supernatant and separated from each other by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequences of the three proteins were essentially identical, and antibodies raised against one of the trio reacted with all three proteins and with three other intracellular proteins with slightly higher molecular weights. The N-terminal amino acid sequence of one of these larger proteins was different from that of the secreted CRX proteins. The gene crx, which encodes the CRX proteins, was cloned and sequenced, and crx transcription was characterized. The crx sequence predicts that the encoded polypeptide is synthesized as a precursor with an N-terminal leader peptide, containing 28 amino acid residues, that is removed during the extracellular secretion of the CRX proteins. Transcription was initiated 274 bp upstream from the crx gene, producing an approximately 1.4-kb monocistronic transcript that was present in M. bryantii BKYH cells under all growth conditions but that increased approximately fourfold in vivo in response to Cu addition. The CRX proteins appear to be glycosylated, since they react with concanavalin A and neuraminidase, and to be the products of one gene that have different levels of posttranslational glycosylation. This is supported by very similar chromatographic and electrophoretic properties, identical N-terminal amino acid sequences, immunological cross-reactivities, and the detection of only one crx-related sequence by Southern blotting. Western blots (immunoblots) showed no evidence for CRX proteins in cell lysates of several other Methanobacterium strains.

Amino Acid Sequence↗
PubMed1997

Growth- and substrate-dependent transcription of the formate dehydrogenase (fdhCAB) operon in Methanobacterium thermoformicicum Z-245.

The formate dehydrogenase-encoding fdhCAB operon and flanking genes have been cloned and sequenced from Methanobacterium thermoformicicum Z-245. fdh transcription was shown to be initiated 21 bp upstream from fdhC, although most fdh transcripts terminated or were processed between fdhC and fdhA. The resulting fdhC, fdhAB, and fdhCAB transcripts were present at all growth stages in cells growing on formate but were barely detectable during early exponential growth on H2 plus CO2. The levels of the fdh transcripts did, however, increase dramatically in cells growing on H2 plus CO2, coincident with the decrease in the growth rate and the onset of constant methanogenesis that occurred when culture densities reached an optical density at 600 nm of approximately 0.5. The mth transcript that encodes the H2-dependent methenyl-H4 MPT reductase (MTH) and the frh and mvh transcripts that encode the coenzyme F420-reducing (FRH) and nonreducing (MVH) hydrogenases, respectively, were also present in cells growing on formate, consistent with the synthesis of three hydrogenases, MTH, FRH, and MVH, in the absence of exogenously supplied H2. Reducing the H2 supply to M. thermoformicicum cells growing on H2 plus CO2 reduced the growth rate and CH4 production but increased frh and fdh transcription and also increased transcription of the mtd, mer, and mcr genes that encode enzymes that catalyze steps 4, 5, and 7, respectively, in the pathway of CO2 reduction to CH4. Reducing the H2 supply to a level insufficient for growth resulted in the disappearance of all methane gene transcripts except the mcr transcript, which increased. Regions flanking the fdhCAB operon in M. thermoformicicum Z-245 were used as probes to clone the homologous region from the Methanobacterium thermoautotrophicum deltaH genome. Sequencing revealed the presence of very similar genes except that the genome of M. thermoautotrophicum, a methanogen incapable of growth on formate, lacked the fdhCAB operon.

Bacteriological Techniques↗
PubMed2001

[Characterization and phylogenetics of a new species of genus Methanobacterium].

A strain of methanogenic bacterium Px1 was isolated from an anaerobic digester in the department of environmental sciences, TsingHua University, which treated the wastewater from Beijing Beer Factory. The isolate is curved rod, light-yellow colony, uses only H2 + CO2 as the substrate for CH4 formation. On the basis of physiology, morphology, structural characteristics and phylogenetic analysis, it is turned out that strain Px1 is a new species of the genus Methanobacterium, the name Methanobacterium curvum sp. nov. is proposed.

DNA, Bacterial↗
PubMed1980

Incorporation of 8 succinate per mol nickel into factors F430 by Methanobacterium thermoautotrophicum.

Factors F430 from methanogenic bacteria have recently been shown to contain nickel and it has been speculated that they may have a nickel tetrapyrrole structure. This assumption was tested by determining whether succinate is incorporated by growing Methanobacterium thermoautotrophicum into three factors F430. Succinate is assimilated by Methanobacterium thermoautotrophicum into the amino acids glutamate, arginine and proline and into tetrapyrroles rather than other cell components. It was found that per mol nickel 8--9 mol of succinate were incorporated into the three factors F430 which is the amount predicted for a tetrapyrrole structure. Since the three factors F430 only contained significant amounts of glutamate rather than arginine or proline, the incorporation data suggest that factors F430 are nickel tetrapyrrole compounds. Spectral properties of the three factors F430, apparent molecular weights, and the absence of phosphor in these compounds are also described.

Amino Acids↗
PubMed1988

Reversible conversion of coenzyme F420 to the 8-OH-AMP and 8-OH-GMP esters, F390-A and F390-G, on oxygen exposure and reestablishment of anaerobiosis in Methanobacterium thermoautotrophicum.

Intracellular levels of F390 (AMP and GMP adducts of the 5-deazaflavin cofactor F420) in Methanobacterium thermoautotrophicum were analysed after gasing fermenter cultures with several consecutive cycles of substrate gas and gas mixtures containing 5% oxygen. No F390 was detected in growing cells, hydrogen starved cells and CO2 starved cells prior to O2 contamination. Also, no F390 was found in hydrogen depleted cells after O2 treatment. Exposure of exponentially growing cells and CO2 starved cells to oxygen lead to the formation of F390 species; the increase in the detected amount of F390 was coupled to a decrease of the F420 level. As soon as anaerobiosis was reestablished F390 cofactors were degraded and growth proceeded. Independent of the physiological condition of Methanobacterium thermoautotrophicum methanopterin was formed upon O2 exposure. After normal growth conditions were restored the level of detected methanopterin decreased again.

Anaerobiosis↗
PubMed1983

A plasmid in the archaebacterium Methanobacterium thermoautotrophicum.

The archaebacterium Methanobacterium thermoautotrophicum Marburg (DSM 2133) was found to contain a plasmid (pME2001) in covalently closed circular form. It was isolated by CsCl gradient centrifugation of total DNA in the presence of ethidium bromide. Multimers up to the hexamer were observed upon agarose gel electrophoresis and electron microscopy of a purified plasmid preparation. A restriction map was constructed. The length of plasmid pME2001 was determined to be approximately 4,500 bp. Southern hybridization of plasmid DNA to DNA extracted from Methanobacterium thermoautotrophicum delta H (DSM1053) revealed the presence of a plasmid with homologous sequences in the delta H strain.

DNA Restriction Enzymes↗
PubMed1987

Autotrophic synthesis of activated acetic acid from CO2 in Methanobacterium thermoautotrophicum. Synthesis from tetrahydromethanopterin-bound C1 units and carbon monoxide.

The synthesis of acetyl-CoA from CO2, H2, and various C1 compounds was studied in vitro with extracts and with protein fractions of Methanobacterium thermoautotrophicum. Acetyl-CoA synthesis from CO2 and H2 by extracts required CO2 reduction to CH4 to proceed. Both processes were highly stimulated by formaldehyde which served as the carbon precursor of both CH4 and the CH3 group of acetate. Carbon monoxide in combination with formaldehyde dramatically stimulated the acetyl-CoA synthesis up to 150-fold. In this system, which did not require CO2 reduction to the formaldehyde and CO level, acetyl-CoA synthesis was no longer dependent on CH4 formation. The soluble (100,000 X g supernatant) cell protein was resolved into a protein fraction [45-60% (NH4)2SO4-fraction] which catalyzed acetyl-CoA synthesis at a specific rate of 15 nmol X min-1 X (equivalent of mg cell protein)-1 (60 degrees C). This oxygen-sensitive enzyme reaction required dithioerythritol for activity and was strictly dependent on coenzyme A, CO, and N5,N10-methylene tetrahydromethanopterin, N5-methyl tetrahydromethanopterin or formaldehyde plus tetrahydromethanopterin. The incorporation of formaldehyde is explained by the spontaneous formation of methylene tetrahydromethanopterin. The product of the reaction, acetyl-CoA, was quantitatively derived from CO (carboxyl of acetate) and a C1 derivative of tetrahydromethanopterin (methyl of acetate). The C1 derivative of tetrahydromethanopterin could not be replaced by a C1 derivative of tetrahydrofolate or by methyl-coenzyme M; ATP was not required. The active protein fraction contained CO dehydrogenase and at least on corrinoid protein. These results provide strong biochemical arguments for the proposed mechanism of autotrophic acetyl-CoA synthesis in Methanobacterium.

Acetyl Coenzyme A↗
PubMed1977

Fermentation of cellulose by Ruminococcus flavefaciens in the presence and absence of Methanobacterium ruminantium.

The anaerobic cellulolytic rumen bacterium Ruminococcus flavefaciens normally produces succinic acid as a major fermentation product together with acetic and formic acids, H2, and CO2. When grown on cellulose and in the presence of the methanogenic rumen bacterium Methanobacterium ruminantium, acetate was the major fermentation product; succinate was formed in small amounts; little formate was detected; H2 did not accumulate; and large amounts of CH4 were formed. M. ruminantium depends for growth on the reduction of CO2 to CH4 by H2, which it can obtain directly or by producing H2 and CO2 from formate. In mixed culture, the methanobacterium utilized the H2 and possibly the formate produced by the ruminococcus and in so doing stimulated the flow of electrons generated during glycolysis by the ruminococcus toward H2 formation and away from formation of succinate. This type of interaction may be of significance in determining the flow of cellulose carbon to the normal rumen fermentation products.

Acetates↗