In Azotobacter chroococcum the hydrogenase gene (hup) cluster spans about 14 kb of DNA. In this study about 12 kb of the hup region beginning immediately downstream of the structural genes (hupSL) were sequenced. This revealed 14 additional open reading frames (ORFs) which we designated hupZMNOQRTVABYCDE. All of them are transcribed from the same strand as hupSL and are closely linked. The polypeptides predicted from all these genes are homologous to products of the gene clusters of membrane-bound (NiFe) hydrogenases from other bacteria, including Azotobacter vinelandii, Alcaligenes eutrophus, Rhodobacter capsulatus, Rhizobium leguminosarum and Escherichia coli. The products of hupR and hupZ may be involved in hydrogenase-linked electron transport since they are similar to rubredoxins and b-type cytochromes, respectively. / Site-directed mutagenesis of hupB, hupY, hupD and hupE abolished Hup activity with either O$ sb2$ or methylene blue as the electron acceptor whereas two insertions downstream of the hupE gene had no effect on Hup activity. A 10.5 kb fragment of DNA beginning in hupR was able to complement hupD and hupE mutants, supporting earlier evidence for a promoter downstream of hupSL. / Mutations in hupB, hupY and hupD had little effect on $ beta$-galactosidase activity in a strain also carrying a hupL-lacZ fusion, indicating that hupB, hupY and hupD are probably not involved in regulating the transcription of hupSL. / Adding nickel to the medium restored wild-type Hup activity to a hupB mutant and about half of the activity in a hupA mutant, indicating that the hupB and hupA gene products may be involved in Ni metabolism.

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