Electron Transport in Methanosarcina: Pathway Heterogeneity Within the Genus
Electron transport during methanogenesis in the freshwater methanogen Methanosarcina barkeri is known to utilize hydrogenases to transfer electrons from ferredoxin to methanophenazine. This thesis characterizes electron transport in the marine methanogen Methanosarcina acetivorans. Genomic comparison of hydrogenase gene clusters reveals that M. acetivorans encodes three putative Ni-Fe hydrogenases common to all sequenced Methanosarcina. However, deletion analysis and enzymatic assays indicate M. acetivorans does not produce functional hydrogenase in crude cell extract. This raised the possibility that M. acetivorans contains a unique electron transport chain distinct from that found in M. barkeri. To address the mechanism of M. acetivorans hydrogenase inactivation, reporter gene fusions to the hydrogenase promoters of M. acetivorans and M. barkeri were inserted into the chromosomes of both M. acetivorans and M. barkeri. The M. barkeri promoters were expressed in both organisms, while the M. acetivorans promoters were not expressed in either organism. This suggests that the M. acetivorans hydrogenases have been inactivated via cis-acting mutations in the promoters. Because M. acetivorans cannot use hydrogenases for electron transport, some other pathway must exist. Three putative oxidoreductase gene clusters have been implicated in this electron transport chain in M. acetivorans: rnf, ehr, and MA3739-3743. Deletion analysis of these three gene clusters indicates Rnf is the primary oxidoreductase during growth on acetate. While the Deltaehr and DeltaMA3739-3743 strains have growth phenotypes identical to the parent strains, the Deltarnf strain does not grow on acetate, grows more slowly on methanol, and has a ca. 300 hour lag period before growth on methanol + pyruvate. These data suggest that Rnf is a ferredoxin-dependent oxidoreductase involved in electron transport during methanogenesis in M. acetivorans.
University of Illinois - Urbana-Champaign
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University of Illinois at Urbana-Champaign