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

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, 13C-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, F420H2 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

Cloning and characterization of two groESL operons of Rhodobacter sphaeroides: transcriptional regulation of the heat-induced groESL operon.

The nonsulfur purple bacterium Rhodobacter sphaeroides was found to contain two groESL operons. The groESL1 heat shock operon was cloned from a genomic library, and a 2.8-kb DNA fragment was sequenced and found to contain the groES and groEL genes. The deduced amino acid sequences of GroEL1 (cpn60) and GroES1 (cpn10) were in agreement with N-terminal sequences previously obtained for the isolated proteins (K. C. Terlesky and F. R. Tabita, Biochemistry 30:8181-8186, 1991). These sequences show a high degree of similarity to groESL genes isolated from other bacteria. Northern analysis indicated that the groESL1 genes were expressed as part of a 2.2-kb polycistronic transcript that is induced 13-fold after heat shock. Transcript size was not affected by heat shock; however, the amount of transcript was induced to its greatest extent 15 to 30 min after a 40 degrees C heat shock, from an initial temperature of 28 degrees C, and remained elevated up to 120 min. The R. sphaeroides groESL1 operon contains a putative hairpin loop at the start of the transcript that is present in other bacterial heat shock genes. Primer extension of the message showed that the transcription start site is at the start of this conserved hairpin loop. In this region were also found putative -35 and -10 sequences that are conserved upstream from other bacterial heat shock genes. Transcription of the groESL1 genes was unexpectedly low under photoautotrophic growth conditions. Thus far, it has not been possible to construct a groESL1 deletion strain, perhaps indicating that these genes are essential for growth. A second operon (groESL2) was also cloned from R. sphaeroides, using a groEL1 gene fragment as a probe; however, no transcript was observed for this operon under several different growth conditions. A groESL2 deletion strain was constructed, but there was no detectable change in the phenotype of this strain compared to the parental strain

Membrane-Bound Electron Transport in Methanosaeta thermophila▿

The obligate aceticlastic methanogen Methanosaeta thermophila uses a membrane-bound ferredoxin:heterodisulfide oxidoreductase system for energy conservation. We propose that the system is composed of a truncated form of the F420H2 dehydrogenase, methanophenazine, and the heterodisulfide reductase. Hence, the electron transport chain is distinct from those of well-studied Methanosarcina species