A Novel Endo-Hydrogenase Activity Recycles Hydrogen Produced by Nitrogen Fixation

Background: Nitrogen (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 Findings: As 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 b-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