Gene Products of the hupGHIJ Operon Are Involved in Maturation of the Iron-Sulfur Subunit of the [NiFe] Hydrogenase from Rhizobium leguminosarum bv. viciae

In the present study, we investigate the functions of thehupGHIJ operon in the synthesis of an active [NiFe] hydrogenase in the legume endosymbiontRhizobium leguminosarum bv. viciae. These genes are clustered with 14 other genes including the hydrogenase structural geneshupSL . A set of isogenic mutants with in-frame deletions (ΔhupG , ΔhupH , ΔhupI , and ΔhupJ ) was generated and tested for hydrogenase activity in cultures grown at different oxygen concentrations (0.2 to 2.0%) and in symbiosis with peas. In free-living cultures, deletions in these genes severely reduced hydrogenase activity. The ΔhupH mutant was totally devoid of hydrogenase activity at any of the O2 concentration tested, whereas the requirement ofhupGIJ for hydrogenase activity varied with the O2 concentration, being more crucial at higher pO2 . Pea bacteroids from the mutant strains affected inhupH, hupI , andhupJ exhibited reduced (20 to 50%) rates of hydrogenase activity compared to the wild type, whereas rates were not affected in theΔhupG mutant. Immunoblot experiments with HupL- and HupS-specific antisera showed that free-living cultures fromΔhupH, ΔhupI , andΔhupJ mutants synthesized a fully processed mature HupL protein and accumulated an unprocessed form of HupS (pre-HupS). Both the mature HupL and the pre-HupS forms were located in the cytoplasmic fraction of cultures from theΔhupH mutant. Affinity chromatography experiments revealed that cytoplasmic pre-HupS binds to the HupH protein before the pre-HupS-HupL complex is formed. From these results we propose thathupGHIJ gene products are involved in the maturation of the HupS hydrogenase subunit.