The Azotobacter chroococcum hydrogenase gene cluster: sequences and genetic analysis of four accessory genes, hupA, hupB, hupY and hupC

Abstract

The Azotobacter chroococcum chromosome contains a region spanning about 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 downstream from hupL and are required for the formation of a catalytically active hydrogenase. In this study, we determined the nucleotide sequence of a 3.8-kb region immediately upstream from hupD. This revealed four additional closely linked ORFs which we designated hupA, hupB, hupY 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 derivative carrying a promoterless lacZ gene. A 10.5-kb fragment of DNA beginning 3.4 kb downstream from hupL, and including the sequenced region, was able to complement hupA and hupY mutants, supporting earlier evidence for a promoter downstream from hupSL. The deduced amino acid sequences of hupA, hupB and hupC are homologous to the Escherichia coli hypA, hypB and hypC gene products, respectively. Of particular interest is the fact that there is no homologue of the hupY gene product in the E. coli hyp operon. Mutations in hupY or hupB had little effect on beta-galactosidase activity in a strain also carrying a hupL::lacZ fusion, showing that hupY and hupB are not major factors in regulating the transcription of the hydrogenase structural genes.