The synthesis of the membrane-bound [NiFe]hydrogenase of Rhodobacter capsulatus (HupSL) is regulated negatively by the protein histidine kinase, HupT, and positively by the response regulator, HupR. It is demonstrated in this work that HupT and HupR are partners in a two-component signal transduction system. The binding of HupR protein to the hupS promoter regulatory region (phupS ) was studied using gel retardation and footprinting assays. HupR protected a 50 bp region localized upstream from the binding site of the histone-like integration host factor (IHF) regulator. HupR, which belongs to the NtrC subfamily, binds to an enhancer site (TTG-N5-CAA) localized at -162/-152 nt. However, the enhancer-binding HupR protein does not require the RpoN sigma factor for transcriptional activation, as is the case for NtrC from enteric bacteria, but functions with sigma70-RNA polymerase, as is the case for R. capsulatus NtrC. Besides, unlike NtrC from Escherichia coli, HupR activates transcription in the unphosphorylated form and becomes inactive by phosphorylation. This was demonstrated by replacing the putative phosphorylation site (D54) of the HupR protein with various amino acids or by deleting it using site-directed mutagenesis. Strains expressing mutated hupR genes showed high hydrogenase activities even in the absence of H2, indicating that hupSL transcription is activated by the binding of unphosphorylated HupR protein. Strains producing mutated HupRD54 proteins were derepressed for hupSL expression as were HupT- mutants. It is shown that the phosphorylated form of HupT was able to transfer phosphate to wild-type HupR protein but not to mutated D54 HupR proteins. Thus, it is concluded that HupT and HupR are the partners of a two-component regulatory system that regulates hupSL gene transcription.

Purple photosynthetic bacteria are capable of generating cellular energy from several sources, including photosynthesis, respiration, and H 2 oxidation. Under nutrient-limiting conditions, cellular energy can be used to assimilate carbon and nitrogen. This study provides the first evidence of a molecular link for the coregulation of nitrogenase and hydrogenase biosynthesis in an anoxygenic photosynthetic bacterium. We demonstrated that molybdenum nitrogenase biosynthesis is under the control of the RegB-RegA two-component regulatory system in Rhodobacter capsulatus. Footprint analyses and in vivo transcription studies showed that RegA indirectly activates nitrogenase synthesis by binding to and activating the expression of nifA2, which encodes one of the two functional copies of the nif-specific transcriptional activator, NifA. Expression of nifA2 but not nifA1 is reduced in the reg mutants up to eightfold under derepressing conditions and is also reduced under repressing conditions. Thus, although NtrC is absolutely required for nifA2 expression, RegA acts as a coactivator of nifA2. We also demonstrated that in reg mutants, [NiFe]hydrogenase synthesis and activity are increased up to sixfold. RegA binds to the promoter of the hydrogenase gene operon and therefore directly represses its expression. Thus, the RegB-RegA system controls such diverse processes as energy-generating photosynthesis and H 2 oxidation, as well as the energy-demanding processes of N 2 fixation and CO 2 assimilation.The purple nonsulfur photosynthetic bacterium Rhodobacter capsulatus exhibits remarkable metabolic diversity (30). This bacterium is capable of generating energy from light via photosynthesis as well as from dark aerobic and anaerobic respiration. Another feature is the capacity to grow heterotrophically as well as autotrophically. When growing autotrophically, the cells are also capable of generating cellular energy and reducing power by the oxidation of H 2 , which occurs at a membrane-bound [NiFe]hydrogenase complex. Several decades ago, Gest and colleagues described the presence of redox-related interrelationships among carbon assimilation, N 2 fixation, and photophosphorylation (26; reviewed in reference 27). However, the nature and even the existence of specific molecular mechanisms for balancing the use of reducing equivalents have remained unclear.Recent studies have suggested that balancing different metabolic processes could result, at least partially, from the activity of a global two-component regulatory system that regulates the synthesis of the enzymes involved in different energetic processes. Indeed, the three fundamental biological processes catalyzed by photosynthetic bacteria, i.e., photosynthesis, CO 2 fixation, and N 2 assimilation, are affected by the RegB-RegA global two-component transduction signal system in Rhodobacter sphaeroides (32). In R. capsulatus, the RegB-RegA system functions as a classic two-component system, with RegB being a membrane-spanning histidine kinase capable of autophosphorylating ...