Energy Conservation via Hydrogen Cycling in the Methanogenic Archaeon Methanosarcina barkeri

Energy conservation via hydrogen cycling, which generates proton motive force by intracellular H₂ production coupled to extracellular consumption, has been controversial since it was first proposed in 1981. It was hypothesized that the methanogenic archaeon Methanosarcina barkeri is capable of energy conservation via H₂ cycling, based on genetic data that suggest that H₂ is a preferred, but nonessential, intermediate in the electron transport chain of this organism. Here, we characterize a series of hydrogenase mutants to provide direct evidence of H₂ cycling. M. barkeri produces H₂ during growth on methanol, a phenotype that is lost upon mutation of the cytoplasmic hydrogenase encoded by frhADGB, although low levels of H₂, attributable to the Ech hydrogenase, accumulate during stationary phase. In contrast, mutations that conditionally inactivate the extracellular Vht hydrogenase are lethal when expression of the vhtGACD operon is repressed. Under these conditions, H₂ accumulates, with concomitant cessation of methane production and subsequent cell lysis, suggesting that the inability to recapture extracellular H₂ is responsible for the lethal phenotype. Consistent with this interpretation, double mutants that lack both Vht and Frh are viable. Thus, when intracellular hydrogen production is abrogated, loss of extracellular H₂ consumption is no longer lethal. The common occurrence of both intracellular and extracellular hydrogenases in anaerobic microorganisms suggests that this unusual mechanism of energy conservation may be widespread in nature.IMPORTANCE ATP is required by all living organisms to facilitate essential endergonic reactions required for growth and maintenance. Although synthesis of ATP by substrate-level phosphorylation is widespread and significant, most ATP is made via the enzyme ATP synthase, which is energized by transmembrane chemiosmotic gradients. Therefore, establishing this gradient across the membrane is of central importance to sustaining life. Experimental validation of H₂ cycling adds to a short list of mechanisms for generating a transmembrane electrochemical gradient that is likely to be widespread, especially among anaerobic microorganisms.