| Genetic analysis of the methylotrophic methanogenesis in Methanosarcina acetivorans has revealed a complex system for the utilization of methanol. M. acetivorans contains three isozymes of mtaCB and two of mtaA, encoding methyltransferase 1 (MT1) and methyltransferase 2 (MT2), respectively, which activate methanol to methyl-CoM. To study these genes, a markerless exchange method was developed and deletion mutants were constructed which lacked one or more mtaCB or mtaA isozymes. None of the mtaCB or mtaA deletion mutants was affected in trimethylamine and acetate metabolism indicating they have no role in the utilization of these substrates. Single and double mtaCB deletion mutants grew on methanol, whereas deletion of all three mtaCB isozymes abolished the ability to grow on methanol showing that each isozyme is a bona fide methanol methyltransferase. Strains with mtaCB1 or mtaCB2 alone had similar generation times, rates of methane production, and cell yield to wild type, but exhibited longer methanol-switching times, while strains with mtaCB3 alone displayed a lower capacity to utilize methanol. The methanol:CoM methyltransferase activity of mtaCB1 was similar to wild type and two-fold higher than mtaCB2 and four-fold higher than mtaCB3. In contrast, deletion of mtaA1 alone caused a methanol-minus phenotype indicating that mtaA2 cannot substitute for mtaA1 in methanol metabolism. Deletion of mtaA2 led to a generation time, methanol-switching time, cell yield, and methanol:CoM methyltransferase activity similar to wild type indicating it does not affect the ability to grow on methanol, however, this strain did have a decreased rate of methane production relative to wild type. These data suggest that each mtaCB isozyme is differentially regulated due to dissimilar mtaC promoter regions and to increasing methanol-switching times which could result from gene expression differences. To study the regulation of these multiple isozymes with regard to methanol utilization, the gene uidA encoding beta-glucuronidase from E. coli was demonstrated as a suitable reporter gene for Methanosarcina . Additionally, the E. coli proC gene was developed as a positive selectable marker further increasing the capacity to use genetics in the analysis of methanogenesis. |
