The complete 1,751,377-bp sequence of the genome of the thermophilic archaeon Methanobacterium thermoautotrophicum deltaH has been determined by a whole-genome shotgun sequencing approach. A total of 1,855 open reading frames (ORFs) have been identified that appear to encode polypeptides, 844 (46%) of which have been assigned putative functions based on their similarities to database sequences with assigned functions. A total of 514 (28%) of the ORF-encoded polypeptides are related to sequences with unknown functions, and 496 (27%) have little or no homology to sequences in public databases. Comparisons with Eucarya-, Bacteria-, and Archaea-specific databases reveal that 1,013 of the putative gene products (54%) are most similar to polypeptide sequences described previously for other organisms in the domain Archaea. Comparisons with the Methanococcus jannaschii genome data underline the extensive divergence that has occurred between these two methanogens; only 352 (19%) of M. thermoautotrophicum ORFs encode sequences that are >50% identical to M. jannaschii polypeptides, and there is little conservation in the relative locations of orthologous genes. When the M. thermoautotrophicum ORFs are compared to sequences from only the eucaryal and bacterial domains, 786 (42%) are more similar to bacterial sequences and 241 (13%) are more similar to eucaryal sequences. The bacterial domain-like gene products include the majority of those predicted to be involved in cofactor and small molecule biosyntheses, intermediary metabolism, transport, nitrogen fixation, regulatory functions, and interactions with the environment. Most proteins predicted to be involved in DNA metabolism, transcription, and translation are more similar to eucaryal sequences. Gene structure and organization have features that are typical of the Bacteria, including genes that encode polypeptides closely related to eucaryal proteins. There are 24 polypeptides that could form two-component sensor kinase-response regulator systems and homologs of the bacterial Hsp70-response proteins DnaK and DnaJ, which are notably absent in M. jannaschii. DNA replication initiation and chromosome packaging in M. thermoautotrophicum are predicted to have eucaryal features, based on the presence of two Cdc6 homologs and three histones; however, the presence of an ftsZ gene indicates a bacterial type of cell division initiation. The DNA polymerases include an X-family repair type and an unusual archaeal B type formed by two separate polypeptides. The DNA-dependent RNA polymerase (RNAP) subunits A', A", B', B" and H are encoded in a typical archaeal RNAP operon, although a second A' subunit-encoding gene is present at a remote location. There are two rRNA operons, and 39 tRNA genes are dispersed around the genome, although most of these occur in clusters. Three of the tRNA genes have introns, including the tRNAPro (GGG) gene, which contains a second intron at an unprecedented location. There is no selenocysteinyl-tRNA gene nor evidence for classically organized IS elements, prophages, or plasmids. The genome contains one intein and two extended repeats (3.6 and 8.6 kb) that are members of a family with 18 representatives in the M. jannaschii genome.

Complete genome sequence of Methanobacterium thermoautotrophicum deltaH: functional analysis and comparative genomics.

Max-Planck-Institut fur terrestrische Mikrobiologie, Karl-von-Frisch-Strase, D-35043 Marburg, and Laboratorium fur Mikrobiologie, Fachbereich Biologie, Philipps-Universitat, Karl-von-Frisch-Strase, D-35032 Marburg, Germany
In 1933, Stephenson & Stickland (1933a) published that they had isolated from river mud, by the single cell technique, a methanogenic organism capable of growth in an inorganic medium with formate as the sole carbon source.

Biochemistry of methanogenesis: a tribute to Marjory Stephenson:1998 Marjory Stephenson Prize Lecture

Inferred amino acid sequences of the methyl coenzyme-M reductase (mcrA) gene from five different methanogen species were aligned and two regions with a high degree of homology flanking a more variable region were identified. Analysis of the DNA sequences from the conserved regions yielded two degenerate sequences from which a forward primer, a 32-mer, and a reverse primer, a 23-mer, could be derived for use in the specific PCR-based detection of methanogens. The primers were successfully evaluated against 23 species of methanogen representing all five recognized orders of this group of Archaea, generating a PCR product between 464 and 491 bp. Comparisons between the mcrA and 16S small subunit rRNA gene sequences using PHYLIP demonstrated that the tree topologies were strikingly similar. Methods were developed to enable the analysis of methanogen populations in landfill using the mcrA gene as the target. Two landfill sites were examined and 63 clones from a site in Mucking, Essex, and 102 from a site in Odcombe, Somerset, were analysed. Analysis revealed a far greater diversity in the methanogen population within landfill material than has been seen previously.

The mcrA gene as an alternative to 16S rRNA in the phylogenetic analysis of methanogen populations in landfill

Dissimilatory Sulfate- and Sulfur-Reducing Prokaryotes

Methyl–coenzyme M reductase (MCR), the enzyme responsible for the microbial formation of methane, is a 300-kilodalton protein organized as a hexamer in an α 2 β 2 γ 2 arrangement. The crystal structure of the enzyme from Methanobacterium thermoautotrophicum , determined at 1.45 angstrom resolution for the inactive enzyme state MCR ox1-silent , reveals that two molecules of the nickel porphinoid coenzyme F 430 are embedded between the subunits α, α′, β, and γ and α′, α, β′, and γ′, forming two identical active sites. Each site is accessible for the substrate methyl–coenzyme M through a narrow channel locked after binding of the second substrate coenzyme B. Together with a second structurally characterized enzyme state (MCR silent ) containing the heterodisulfide of coenzymes M and B, a reaction mechanism is proposed that uses a radical intermediate and a nickel organic compound.

https://science.sciencemag.org/content/sci/278/5342/1457.full.pdf

Crystal structure of methyl-coenzyme M reductase: the key enzyme of biological methane formation.

1.

Growth of Vibrio succinogenes with nitrate as terminal electron acceptor was found to be a function of the following two catabolic reactions: 

$$HCO _2^ - + NO _3^ - + H^ + \to CO_2 + NO _2^ - + H_2 O$$

(a)



$$3HCO _2^ - + NO _2^ - + 5H^ + \to 3CO_2 + NH _4^ + + 2H_2 O.$$

(b)



The latter reaction (b) was responsible for growth with nitrite.




2.

Either succinate or fumarate could serve as sole carbon source during growth with nitrate or nitrite. Biosynthesis from succinate proceeded via fumarate. The ATP requirement for cell synthesis from succinate was equal to that calculated earlier for growth with fumarate as carbon source and electron acceptor (Brounder et al. 1982).




3.

The cell yield at infinite dilution rate (Ymax) as obtained with chemostat cultures was 8.5g dry cells/mol formate with either nitrate or nitrite as acceptor. This value amounts to 60% of that measured earlier with fumarate as acceptor (Mell et al. 1982).




4.

Membrane vesicles prepared from V. succinogenes catalyzed electron transport from H2 to nitrate. The reaction was dependent on the menaquinone present in the membrane.




5.

Electron transport with H2 and nitrite was coupled to the phosphorylation of ADP. The P/H2 ratio with nitrite was 40% of that measured with fumarate as acceptor using the same preparation. The phosphorylation but not the electron transport was abolished by an uncoupling agent.

Energy metabolism and biosynthesis of Vibrio succinogenes growing with nitrate or nitrite as terminal electron acceptor

The genes coding for methyl coenzyme M reductase were cloned from a genomic library of Methanobacterium thermoautotrophicum Marburg into Escherichia coli by using plasmid expression vectors When introduced into E coli, the reductase genes were expressed, yielding polypeptides identical in size to the three known subunits of the isolated enzyme, alpha, beta, and gamma The polypeptides also reacted with the antibodies raised against the respective enzyme subunits In M thermoautotrophicum, the subunits are encoded by a gene cluster whose transcript boundaries were mapped Sequence analysis revealed two more open reading frames of unknown function located between two of the methyl coenzyme M reductase genes

Cloning and characterization of the methyl coenzyme M reductase genes from Methanobacterium thermoautotrophicum.

Methyl-coenzyme-M reductase from Methanobacterium thermoautotrophicum (strain Marburg) was purified to a stage where, besides the α, β and γ subunits, no additional polypeptides were detectable in the preparation. Under appropriate conditions the enzyme was found to catalyze the reduction of methyl-CoM with 7-mercaptoheptanoylthreonine phosphate (H-S-HTP) to CH4 at a specific rate of 2.5 μmol · min−1· mg protein−1. This finding contradicts a recent report that methyl-CoM reductase is only active when some contaminating proteins are present.



The two polypeptides encoded by the open reading frames ORF1 and ORF2 of the methyl-CoM reductase transcription unit did not co-purify with the α, β and γ subunits. They were neither required nor did they stimulate the activity under the assay conditions.



3-Bromopropanesulfonate (apparent Ki= 0.05 μM) and 2-azidoethanesulfonate (apparent Ki= 1 μM) were found to be two new competitive inhibitors of methyl-CoM reductase. Both inhibitors were considerably more effective than the “classical” 2-bromoethanesulfonate (apparent Ki= 4 μM).

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1989.tb14990.x

Methyl-coenzyme-M reductase from Methanobacterium thermoautotrophicum (strain Marburg). Purity, activity and novel inhibitors.

Nucleotide sequence of the methyl coenzyme M reductase gene cluster from Methanosarcina barkeri

The sequence of the gene cluster encoding the methyl coenzyme M reductase (MCR) in Methanococcus voltae was determined. It contains five open reading frames (ORF), three of which encode the known enzyme subunits. Putative ribosome binding sites were found in front of all ORFs. They differ in their degrees of complementarity to the 3′ end of the 16 S rRNA, which is discussed in terms of different translation efficiencies of the respective genes. The codon usage bias is different in the subunit encoding genes compared with the two other ORFs in the cluster and two other known genes of Mc. voltae. This is interpreted in terms of increased translational accuracy of the highly expressed MCR subunit genes. The derived polypeptide sequences encoded by the five ORFs of the MCR cluster were compared to those of the respective genes in Methanobacterium thermoautotrophicum Marburg and Methanosarcina barkeri. Conserved regions were detected in the enzyme subunits, which are candidates for factor binding domains. Conserved hydrophobic sequences found in the α and β subunits are discussed with respect to the membrane association of the enzyme.

Martin Bokranz

Papers

Energy metabolism and biosynthesis of Vibrio succinogenes growing with nitrate or nitrite as terminal electron acceptor

Cloning and characterization of the methyl coenzyme M reductase genes from Methanobacterium thermoautotrophicum.

Methyl-coenzyme-M reductase from Methanobacterium thermoautotrophicum (strain Marburg). Purity, activity and novel inhibitors.

Nucleotide sequence of the methyl coenzyme M reductase gene cluster from Methanosarcina barkeri

Comparative analysis of genes encoding methyl coenzyme M reductase in methanogenic bacteria.