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In this work, we report the complete genome sequence of an obligate aceticlastic methanogen,  Methanosaeta harundinacea  6Ac. Genome comparison indicated that the three cultured  Methanosaeta  spp.,  M. thermophila ,  M. concilii  and  M. harundinacea  6Ac, each carry an entire suite of genes encoding the proteins involved in the methyl-group oxidation pathway, a pathway whose function is not well documented in the obligately aceticlastic methanogens. Phylogenetic analysis showed that the methyl-group oxidation-involving proteins, Fwd, Mtd, Mch, and Mer from  Methanosaeta  strains cluster with the methylotrophic methanogens, and were not closely related to those from the hydrogenotrophic methanogens. Quantitative PCR detected the expression of all genes for this pathway, albeit ten times lower than the genes for aceticlastic methanogenesis in strain 6Ac. Western blots also revealed the expression of  fwd  and  mch , genes involved in methyl-group oxidation. Moreover,  13 C-labeling experiments suggested that the  Methanosaeta  strains might use the pathway as a methyl oxidation shunt during the aceticlastic metabolism. Because the  mch  mutants of  Methanosarcina barkeri  or  M. acetivorans  failed to grow on acetate, we suggest that  Methanosaeta  may use methyl-group oxidation pathway to generate reducing equivalents, possibly for biomass synthesis. An  fpo  operon, which encodes an electron transport complex for the reduction of CoM-CoB heterodisulfide, was found in the three genomes of the  Methanosaeta  strains. However, an incomplete protein complex lacking the FpoF subunit was predicted, as the gene for this protein was absent. Thus, F 420 H 2  was predicted not to serve as the electron donor. In addition, two gene clusters encoding the two types of heterodisulfide reductase (Hdr),  hdrABC , and  hdrED , respectively, were found in the three  Methanosaeta  genomes. Quantitative PCR determined that the expression of  hdrED  was about ten times higher than  hdrABC , suggesting that  hdrED  plays a major role in aceticlastic methanogenesis.

The Genome Characteristics and Predicted Function of Methyl-Group Oxidation Pathway in the Obligate Aceticlastic Methanogens, Methanosaeta spp