BackgroundNitrogen (N2) fixation also yields hydrogen (H2) at 1∶1 stoichiometric amounts. In aerobic diazotrophic (able to grow on N2 as sole N-source) bacteria, orthodox respiratory hupSL-encoded hydrogenase activity, associated with the cell membrane but facing the periplasm (exo-hydrogenase), has nevertheless been presumed responsible for recycling such endogenous hydrogen.Methods and FindingsAs shown here, for Azorhizobium caulinodans diazotrophic cultures open to the atmosphere, exo-hydrogenase activity is of no consequence to hydrogen recycling. In a bioinformatic analysis, a novel seven-gene A. caulinodans hyq cluster encoding an integral-membrane, group-4, Ni,Fe-hydrogenase with homology to respiratory complex I (NADH : quinone dehydrogenase) was identified. By analogy, Hyq hydrogenase is also integral to the cell membrane, but its active site faces the cytoplasm (endo-hydrogenase). An A. caulinodans in-frame hyq operon deletion mutant, constructed by “crossover PCR”, showed markedly decreased growth rates in diazotrophic cultures; normal growth was restored with added ammonium—as expected of an H2-recycling mutant phenotype. Using A. caulinodans hyq merodiploid strains expressing β-glucuronidase as promoter-reporter, the hyq operon proved strongly and specifically induced in diazotrophic culture; as well, hyq operon induction required the NIFA transcriptional activator. Therefore, the hyq operon is constituent of the nif regulon.ConclusionsRepresentative of aerobic N2-fixing and H2-recycling α-proteobacteria, A. caulinodans possesses two respiratory Ni,Fe-hydrogenases: HupSL exo-hydrogenase activity drives exogenous H2 respiration, and Hyq endo-hydrogenase activity recycles endogenous H2, specifically that produced by N2 fixation. To benefit human civilization, H2 has generated considerable interest as potential renewable energy source as its makings are ubiquitous and its combustion yields no greenhouse gases. As such, the reversible, group-4 Ni,Fe-hydrogenases, such as the A. caulinodans Hyq endo-hydrogenase, offer promise as biocatalytic agents for H2 production and/or consumption.