Methanogenic archaea belonging to the Order Methanosarcinales conserve energy using an electron transport chain (ETC). In the genetically tractable strain Methanosarcina acetivorans, ferredoxin donates electrons to the ETC via the Rnf (Rhodobacter nitrogen fixation) complex. The Rnf complex in M. acetivorans, unlike its counterpart in Bacteria, contains a multiheme c-type cytochrome (MHC) subunit called MmcA. Early studies hypothesized MmcA is a critical component of Rnf, however recent work posits that the primary role of MmcA is facilitating extracellular electron transport. To explore the physiological role of MmcA, we characterized M. acetivorans mutants lacking either the entire Rnf complex (∆mmcA-rnf) or just the MmcA subunit (∆mmcA). Our data show that MmcA is essential for growth during acetoclastic methanogenesis but neither Rnf nor MmcA is required for methanogenic growth on methylated compounds. On methylated compounds, the absence of MmcA alone leads to a more severe growth defect compared to a Rnf deletion likely due to different strategies for ferredoxin oxidation that arise in each strain. Transcriptomic data suggest that the ∆mmcA mutant might oxidize ferredoxin by upregulating the cytosolic Wood-Ljundahl pathway for acetyl-CoA synthesis, whereas the ∆mmcA-rnf mutant may repurpose the F 420 dehydrogenase complex (Fpo) to oxidize ferredoxin coupled to proton translocation. Beyond energy conservation, the deletion of rnf or mmcA leads to global transcriptional changes of genes involved in methanogenesis, carbon assimilation and regulation. Overall, our study provides systems-level insights into the non-overlapping roles of the Rnf bioenergetic complex and the associated MHC, MmcA.

Methanogenic archaea belonging to the Order Methanosarcinales conserve energy using an electron transport chain (ETC). In the genetically tractable strain Methanosarcina acetivorans, ferredoxin donates electrons to the ETC via the Rnf (Rhodobacter nitrogen fixation) complex. The Rnf complex in M. acetivorans, unlike its counterpart in Bacteria, contains a multiheme c-type cytochrome (MHC) subunit called MmcA. Early studies hypothesized MmcA is a critical component of Rnf, however recent work posits that the primary role of MmcA is facilitating extracellular electron transport. To explore the physiological role of MmcA, we characterized M. acetivorans mutants lacking either the entire Rnf complex (Δrnf) or just the MmcA subunit (ΔmmcA). Our data show that MmcA is essential for growth during acetoclastic methanogenesis but neither Rnf nor MmcA are required for methanogenic growth on methylated compounds. On methylated compounds, the absence of MmcA alone leads to a more severe growth defect compared to a Rnf deletion likely due to different strategies for ferredoxin regeneration that arise in each strain. Transcriptomic data suggest that the ΔmmcA mutant might regenerate ferredoxin by upregulating the cytosolic Wood-Ljundahl pathway for acetyl-CoA synthesis, whereas the Δrnf mutant may repurpose the F420 dehydrogenase complex (Fpo) to regenerate ferredoxin coupled to proton translocation. Beyond energy conservation, the deletion of Rnf or MmcA leads to some shared and some unique transcriptional changes in methyltransferase genes and regulatory proteins. Overall, our study provides systems-level insights into the non-overlapping roles of the Rnf bioenergetic complex and the associated MHC, MmcA.ImportanceMethane is a greenhouse gas that is ten times more potent than carbon dioxide and efforts to curb emissions are crucial to meet climate goals. Methane emissions primarily stem from the metabolic activity of microorganisms called methanogenic archaea (methanogens). The electron transport chain (ETC) in methanogens that belong to the Order Methanosarcinales has been the focus of many in vitro studies to date, but the endogenous functions of the bioenergetic complexes that comprise the ETC have rarely been investigated. In this study, we use genetic techniques to functionally characterize the Rnf bioenergetic complex and the associated multi-heme c-type cytochrome MmcA in the model methanogen, Methanosarcina acetivorans. Our results show that MmcA and Rnf have shared and unique roles in the cell, and that, contrary to current knowledge, M. acetivorans has the capacity to induce at least two alternative pathways for ferredoxin regeneration in the absence of a functional Rnf complex.