US EPA

1227176 

Journal Article 

Genetic and biochemical analysis of methyltransferase gene regulation in Methanosarcina acetivorans C2A 

Bose, A 

2008 

Methanogenesis is biologically mediated by obligate anaerobes called methanogens that belong to the Archaea. They conserve energy and fix carbon by converting a limited number of one and two carbon compounds to methane. Methylotrophic methanogenesis, one of four methanogenic pathways, involves the disproportionation of methylated compounds, such as methanol, to methane and carbon dioxide. Entry of methanol into this pathway is initiated by an enzyme system that has three protein components; MtaB, and MtaC, which form methyltransferase 1 (MT1) and a third protein component, methyltransferase 2 (MtaA). The genes encoding MtaC and MtaB form the operon mtaCB whereas mtaA is monocistronic. Methanosarcina spp. have three mtaCB (mtaCB1, 2 and 3) operons. In order to understand why these organisms maintain genes encoding isozymes we used the three mtaCB operons in M. acetivorans C2A as a model. Mutant analysis established that these operons encode bona fide methanol MT1s, which have distinct roles in methanol utilization. These enzymes might have different biochemical properties and their expression might therefore be regulated in response to varying growth conditions. We addressed the possibility of differential regulation of these operons directly using reporter gene fusions. We established that these operons are differentially regulated by growth substrate and growth phase. We identified cis- and trans-acting elements that mediate the regulation of the three mtaCB operons using sophisticated genetic techniques that have no precedent in Archaea. Five proteins that have weak homology to the bacterial ArsR family regulate the expression of the mtaCB operons. We named these genes methanol specific regulators or msr genes. MsrA and MsrB activate mtaCB1 expression and MsrC, MsrD and MsrE activate mtaCB2 expression. MsrD and MsrE additionally repress expression of mtaCB3 on methanol. MsrD and MsrE emerged as the first archaeal regulators that act as both repressors and activators for different target genes. Electrophoretic mobility shift assays corroborated the genetic experiments and showed that MsrA/B and MsrD/E interact to mediate strong binding to their mtaCB1 and mtaCB2 promoter, respectively. This mode of binding target promoters is rare in prokaryotes and has only one precedent in Bacteria and none in Archaea. MsrD and MsrE, however, bind the mtaCB3 promoter with high affinity individually. We also showed that proteins belonging to the Msr family mediate regulation of other genes, such as three dimethylsulfide methyltransferase isozymes genes in M. acetivorans C2A. In conclusion, we showed that regulatory networks controlling expression of physiologically distinct isozymes might be beneficial to these organisms in Nature and might represent an essential theme. Our data established the Msr proteins as a unique family of archaeal regulators, with over a 100 homologs in Archaea. Unlike the 10 well-characterized archaeal regulators discovered using biochemical tools (7 of which are homologs of bacterial autoregulators), we discovered the Msr proteins using genetic approaches. The novelty of the Msr proteins is obvious from the complexity of methanol regulation, in contrast to mere autoregulation as described for most of the other archaeal regulators. This study thus established powerful genetic methods as unexploited tools to discover unknown regulatory mechanisms in Archaea. 

USA, Illinois; Methanogenesis; Carbon dioxide; Energy; Isoenzymes; Growth; Prokaryotes; Methanogenic bacteria; Homology; Operons; Dimethyl sulfide; Transcription; Reporter gene; Methanosarcina; Models; Methanol; Substrate preferences; Archaea; Gene regulation; Methanosarcina acetivorans; Growth conditions; Promoters; Genes; Biochemical analysis; Data processing; Carbon; Repressors; Gene fusion; Electrophoretic mobility; Enzymes; Methyltransferase; Methane