Transcriptional regulation of the uptake [NiFe]hydrogenase genes in Rhodobacter capsulatus

Transcription of the hupSL genes, which encode the uptake [NiFe]hydrogenase of Rhodobacter capsulatus, is specifically activated by H-2. Three proteins are involved, namely the H-2-sensor HupUV, the histidine kinase HupT and the transcriptional activator HupR. hupT and hupUV mutants have the same phenotype, i.e. an increased level of hupSL expression (assayed by phupS::lacZ fusion) in the absence of H-2; they negatively control hupSL gene expression. HupT can autophosphorylate its conserved His(217), and in vitro phosphotransfer to Asp(54) of its cognate response regulator, HupR, was demonstrated. The non-phosphorylated form of HupR binds to an enhancer site (5'-TTG-N-5-CAA) of phupS localized at -162/-152 nt and requires integration host factor to activate fully hupSL transcription. HupUV is an O-2-insensitive [NiFe]hydrogenase, which interacts with HupT to regulate the phosphorylation state of HupT in response to H-2 availability. The N-terminal domain of HupT, encompassing the PAS domain, is required for interaction with HupUV. This interaction with HupT, leading to the formation of a (HupT)(2)-(HupUV)(2) complex, is weakened in the presence of H-2, but incubation of HupUV with H-2 has no effect on the stability of the heterodimer/tetramer, HupUV-(HupUV)(2), equilibrium. HupSL biosynthesis is also under the control of the global two-component regulatory system RegB/RegA, which controls gene expression in response to redox. RegA binds to a site close to the -35 promoter recognition site and to a site overlapping the integration host factor DNA-binding site (5'-TCACACACCATTG, centred at -87 nt) and acts as a repressor.