THE AZOTOBACTER-CHROOCOCCUM HYDROGENASE GENE-CLUSTER - SEQUENCES AND GENETIC-ANALYSIS OF 4 ACCESSORY GENES, HUPA, HUPB, HUPY AND HUPC

The Azotobacter chroococcum chromosome contains a region spanning abou t 14 kb associated with hydrogen-uptake (Hup) activity. The small and large subunits of the hydrogenase are encoded by the structural genes hupS and hupL. Two other genes, hupD and hupE, are located 8.9 kb down stream from hupL and are required for the formation of a catalytically active hydrogenase. In this study, we determined the nucleotide seque nce of a 3.8-kb region immediately upstream from hupD. This revealed f our additional closely linked ORFs which we designated hupA, hupB, hup Y and hupC; these genes potentially encode polypeptides with predicted masses of 12.6, 33.3, 80.4 and 9.0 kDa, respectively. This cluster of genes was shown to be essential for hydrogenase activity by insertion mutagenesis using antibiotic-resistance gene cassettes and a Tn5 deri vative carrying a promoterless lacZ gene. A 10.5-kb fragment of DNA be ginning 3.4 kb downstream from hupL, and including the sequenced regio n, was able to complement hupA and hupY mutants, supporting earlier ev idence for a promoter downstream from hupSL. The deduced amino acid se quences of hupA, hupB and hupC are homologous to the Escherichia coli hypA, hypB and hypC gene products, respectively. Of particular interes t is the fact that there is no homologue of the hupY gene product in t he E. coli hyp operon. Mutations in hupY or hupB had little effect on beta-galactosidase activity in a strain also carrying a hupL::lacZ fus ion, showing that hupY and hupB are not major factors in regulating th e transcription of the hydrogenase structural genes.