| Methanogens are anaerobic microorganisms that are able to gain all of their energy for growth by converting a limited number of substrates to methane gas. Methylotrophic methanogenesis involves the disproportionation of methylated compounds, such as methanol, to methane and carbon dioxide. In this study, genetic in vivo analysis was utilized to characterize the C-1 oxidation branch of the methylotrophic pathway. It was found the deletion strains lacking the methyl-H4MPT methyltransferase (mtr ), methylene-H4MPT reductase (mer), methylene-H 4MPT dehydrogenase (mtd), or the formyl-MF:H4 MPT formyltransferase (ftr) were unable to grow on methanol. However, both the mtr and mer deletion strains were still able to oxidize methanol to carbon dioxide in cell suspension. Therefore, a bypass of a portion of the main C-1 oxidation pathway was possible but this bypass was unable to support growth. Surprisingly, the mtr and mer deletion strains were both able to grow on a combination of methanol plus acetate. It was shown that the co-metabolism of methanol and acetate involved the oxidation of acetate in order to produce the reducing equivalents needed to reduce methanol to methane. This unique combination of methanogenic substrates is a novel methanogenic pathway that was previously uncharacterized. Reporter gene fusions to the promoters of each of the genes mentioned above, as well as the methenyl-H 4MPT cyclohydrolase (mch), demonstrated that transcriptional fusions were expressed at high levels on methanol and down-regulated when grown on acetate. Translational fusions to these same promoters, however, where expressed at low levels or not at all. Further studies demonstrated that a novel mechanism of translation initiation may be utilized by the methanogens to express the C-1 oxidation genes. In this work, the use of genetic analysis has allowed for further characterization of the in vivo role, regulation, and expression of the genes involved in the methyl-oxidation branch of methylotrophic methanogenesis. |
