The Rhodobacter capsulatus hupSLC promoter:: identification of cis-regulatory elements and of trans-activating factors involved in H2 activation of hupSLC transcription

The [NiFe]hydrogenase of the photosynthetic bacterium Rhodobacter capsulatus is encoded by the structural hupSLC operon, the expression of which is induced by H-2. H-2 activation was no longer observable in chromosomal hupR mutants, an indication that HupR is implicated directly in the activation by H-2 of hupS gene expression. The transcriptional start site of the hupS promoter, determined by primer extension mapping, was located 55 nucleotides upstream from the translational start codon of the hupS gene. Regulatory sequences were identified by serial 5' deletions of the 300 bp hupS promoter-regulatory region (phupS) and phupS-lacZ translational fusions, Cis-regulatory sequences capable of interacting with two transcription factors, IHF and HupR, a response regulator of the NtrC subfamily, were studied by electrophoretic mobility shift assays (EMSAs). The R. capsulatus IHF and HupR proteins were overexpressed in Escherichia coli and purified by affinity chromatography, IHF binds to a site, 5'-TCACACACCATTG, centred at -87 nt from the transcription start site, The HupR protein binds to one site within the -162 to -152 nt region, which contains the palindromic sequence 5'-TTG-R-5-CAA. By the use of 5' deletions and site-directed mutagenesis of the -162/-152 region, this palindrome was shown to be required for in vivo hupS transcriptional activation by H-2.