Energy conservation in methanogenic archaea associated with the human gut

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The possible involvement of human microbiome-associated methanoarchaea in disease and health warrants further investigation to understand their physiology and metabolism. Therefore, the major aim of this proposal is to elucidate the energy conservation mechanism in methanogenic organisms found in the human gut which exclusively use H2 and methanol or methylamines as substrates. The experiments will be based on the model organism Methanomassiliicoccus (M.) luminyensis. This organism does not fit into the current scheme of how methanogens gain energy from their substrates. M. luminyensis contains the typical hydrogenase/ heterodisulfide oxidoreductase complex (HdrABC/MvhADG) as found in hydrogenotrophic methanogens but the energy conserving methyl-H4MPT coenzyme M methyltransferase complex is absent. So the question arises how energy is conserved in this organism. In the previous funding period we could show that the soluble HdrABC/MvhADG complex was highly active in the cytoplasmic compartment of M. luminyensis. In the course of the reaction ferredoxin functions as electron acceptor. Furthermore, we could show that reduced ferredoxin (Fdred) is used by washed cytoplasmic membranes to reduce the heterodisulfide CoM-S-S-CoB in the presence of recombinant HdrD. Our current hypothesis is that Fdred is reoxidized by a truncated form of the F420H2 dehydrogenase (headless Fpo complex) which misses the F420H2-oxidizing subunit FpoF. Accordingly, electrons are transferred to HdrD that directly interacts with the headless Fpo complex. In turn, HdrD catalyzes the reduction of CoM-S-S-CoB. The overall process could be coupled to ion translocation (H+ or Na+) and the generation of an electrochemical ion gradient for the synthesis of ATP via a A1A0 type ATP synthase. To verify this hypothesis, it is planned to analyze the interaction of HdrD and Fdred with the headless Fpo complex. The approach includes the partial purification of the Fpo complex or Fpo subcomplexes to reconstitute the anaerobic electron transport chain. Also protein: protein interaction will be analyzed to elucidate the route of electrons and to identify the subunit(s) which are involved in the electron transfer from Fdred to CoM-S-S-CoB as final electron acceptor of the proposed novel energy-conserving electron transport chain in M. luminyensis.