We report complete genome sequence of a mesophilic hydrogenotrophic methanogen Methanocella paludicola, the first cultured representative of the order Methanocellales once recognized as an uncultured key archaeal group for methane emission in rice fields. The genome sequence of M. paludicola consists of a single circular chromosome of 2,957,635 bp containing 3004 protein-coding sequences (CDS). Genes for most of the functions known in the methanogenic archaea were identified, e.g. a full complement of hydrogenases and methanogenesis enzymes. The mixotrophic growth of M. paludicola was clarified by the genomic characterization and re-examined by the subsequent growth experiments. Comparative genome analysis with the previously reported genome sequence of RC-I(MRE50), which was metagenomically reconstructed, demonstrated that about 70% of M. paludicola CDSs were genetically related with RC-I(MRE50) CDSs. These CDSs included the genes involved in hydrogenotrophic methane production, incomplete TCA cycle, assimilatory sulfate reduction and so on. However, the genetic components for the carbon and nitrogen fixation and antioxidant system were different between the two Methanocellales genomes. The difference is likely associated with the physiological variability between M. paludicola and RC-I(MRE50), further suggesting the genomic and physiological diversity of the Methanocellales methanogens. Comparative genome analysis among the previously determined methanogen genomes points to the genome-wide relatedness of the Methanocellales methanogens to the orders Methanosarcinales and Methanomicrobiales methanogens in terms of the genetic repertoire. Meanwhile, the unique evolutionary history of the Methanocellales methanogens is also traced in an aspect by the comparative genome analysis among the methanogens.
Obligate anaerobic bacteria fermenting volatile fatty acids in syntrophic association with methanogenic archaea share the intermediate bottleneck step in organic-matter decomposition. These organisms (called syntrophs) are biologically significant in terms of their growth at the thermodynamic limit and are considered to be the ideal model to address bioenergetic concepts. We conducted genomic and proteomic analyses of the thermophilic propionate-oxidizing syntroph Pelotomaculum thermopropionicum to obtain the genetic basis for its central catabolic pathway. Draft sequencing and subsequent targeted gap closing identified all genes necessary for reconstructing its propionate-oxidizing pathway (i.e., methylmalonyl coenzyme A pathway). Characteristics of this pathway include the following. (i) The initial two steps are linked to later steps via transferases. (ii) Each of the last three steps can be catalyzed by two different types of enzymes. It was also revealed that many genes for the propionate-oxidizing pathway, except for those for propionate coenzyme A transferase and succinate dehydrogenase, were present in an operon-like cluster and accompanied by multiple promoter sequences and a putative gene for a transcriptional regulator. Proteomic analysis showed that enzymes in this pathway were up-regulated when grown on propionate; of these enzymes, regulation of fumarase was the most stringent. We discuss this tendency of expression regulation based on the genetic organization of the open reading frame cluster. Results suggest that fumarase is the central metabolic switch controlling the metabolic flow and energy conservation in this syntroph.
