The [NiFe]hydrogenase of the photosynthetic bacterium Rhodobacter capsulatus is encoded by the structural hupSLC operon, the expression of which is induced by H2. H2 activation was no longer observable in chromosomal hupR mutants, an indication that HupR is implicated directly in the activation by H2 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 300bp 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-R5-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 H2.

We describe a method for rapid purification of the integration host factor (IHF) homolog of Rhodobacter capsulatus that has allowed us to obtain microgram quantities of highly purified protein. R. capsulatus IHF is an a,3 heterodimer similar to IHF of Escherichia coli. We have cloned and sequenced the hip gene, which encodes the 0i subunit. The deduced amino acid sequence (10.7 kDa) has 46% identity with the I8 subunit of IHF from E. coli. In gel electrophoretic mobility shift DNA binding assays, R. capsulatus IHF was able to form a stable complex in a site-specific manner with a DNA fragment isolated from the promoter of the structural hupSL operon, which contains the IHF-binding site. The mutated IHF protein isolated from the Hup-mutant IR4, which is mutated in the himA gene (coding for the a subunit), gave a shifted band of greater mobility, and DNase I footprinting analysis has shown that the mutated IHF interacts with the DNA fragment from the hupSL promoter region differently from the way that the wild-type IHF does.The purple nonsulfur photosynthetic bacterium Rhodobacter capsulatus possesses a membrane-bound hydrogenase that functions in H2 uptake under physiological conditions (31). This enzyme enables R. capsulatus cells to grow autotrophically with H2 as the source of electrons (17). It is also synthesized under heterotrophic growth conditions, in particular in H2-evolving cultures in which hydrogenase synthesis is stimulated by H2 (9).The level of hydrogenase expression is closely coupled to environmental and growth conditions (9). Regulatory genes necessary for hydrogenase synthesis in R. capsulatus have recently been identified in the cluster of hup and hyp genes (8 (30,33) were grown anaerobically in the light in minimal salts (RCV) medium (15, 32) supplemented with DL-malate (30 mM) and L-glutamate (7 mM) as C and N sources, respectively, as previously described (7). DNA manipulations. Standard recombinant DNA techniques were performed as described previously (25). Plasmids pI,B2 and pI,B3 were constructed by inserting into the polylinker of pUC18 (34) the 5-kbp PstI-PstI and 2.2-kbp HindIll-HindIII fragments, respectively, of genomic R. capsulatus DNA that hybridized with the IHF13 probe. DNA sequencing on both strands was performed by the dideoxy chain termination method (26). DNA sequence analysis and homology searching were performed with LASERGENE programs (DNASTAR, Madison, Wis.). Gel retardation assays were performed as previously described (30), using the 274-bp EcoRI-EcoRI DNA fragment containing the IHF-binding site isolated from the hupS promoter.Purification of IHF. R capsulatus strains were grown anaerobically in 40-liter cultures until the A660 reached 1.2, typically yielding 120 g of wet cells. The cells were harvested by centrifugation and resuspended in 100 mM Tris HCl-20 mM EDTA, and 0.2 mg of lysosyme per ml was added. The cell extract (150 ml), obtained after sonication and ultracentrifugation (150,000 x g, 2 h) and containing 3 g of protein, was applied to a 5-ml Econo-Pac he...

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