Analysis of the periplasmic [NiFe] hydrogenase transcription unit from Desulfovibrio fructosovorans.

Two genes, hynA and hynB, encode the two subunits of the periplasmic [NiFe] hydrogenase in Desulfovibrio fructosovorans. Sequencing downstream from hynB revealed a third open reading frame (hynC) that has the potential for encoding a polypeptide showing 21% identity with the HyaD, HoxM, and HupD proteins, belonging to putative operons encoding Escherichia coli hydrogenase 1, Alcaligenes eutrophus H16 membrane-bound hydrogenase, and Rhizobium leguminosarum uptake hydrogenase, respectively. Northern (RNA) blotting with a structural gene probe revealed the existence of a major transcript of 2.9 kb, which is the appropriate length to contain the two hydrogenase subunits only. In addition, two minor 4.4- and 5.8-kb transcripts that could contain hynABC and additional genes were found. The 5' end of the most abundant [NiFe] hydrogenase mRNA was found 170 bp upstream from the translational start site of hynA. The sequences at -10 and -35 relative to the transcriptional starting site showed 55% homology with the consensus sequences of the Escherichia coli sigma 70-type promoter. The cloning of that particular region as a promoter to control transcription of the lacZ gene in E. coli DH5 alpha or the hynA, hynB, and hynC genes in D. fructosovorans MR400 led to strong expression in both systems.