Methanosarcina acetivorans is the primary model to understand the physiology of methanogens that do not use hydrogenase to consume or produce hydrogen (H2) during methanogenesis. The genome of M. acetivorans encodes putative methanophenazine-reducing hydrogenases (Vht and Vhx), F420-reducing hydrogenase (Frh), and hydrogenase maturation machinery (Hyp), yet cells lack significant hydrogenase activity under all growth conditions tested to date. Thus, the importance of hydrogenase to the physiology of M. acetivorans has remained a mystery. M. acetivorans can fix dinitrogen (N2) using nitrogenase that is documented in bacteria to produce H2 during the reduction of N2 to ammonia. Therefore, we hypothesized that M. acetivorans uses hydrogenase to recycle H2 produced by nitrogenase during N2 fixation. Results demonstrate that hydrogenase expression and activity is higher in N2-grown cells compared to cells grown with fixed nitrogen (NH4Cl). To test the importance of each hydrogenase and the maturation machinery, the CRISPRi-dCas9 system was used to generate separate M. acetivorans strains where transcription of the vht, frh, vhx, or hyp operons is repressed. Repression of vhx and frh does not alter growth with either NH4Cl or N2 and has no effect on H2 metabolism. However, repression of vht or hyp results in impaired growth with N2 but not NH4Cl. Importantly, H2 produced endogenously by nitrogenase is detected in the headspace of culture tubes containing the vht or hyp repression strains. Overall, the results reveal that Vht hydrogenase recycles H2 produced by nitrogenase that is required for optimal growth of M. acetivorans during N2 fixation.
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