Annette Colbeau

The synthesis of Rhodobacter capsulatus HupSL hydrogenase is regulated by the two-component HupT/HupR system.

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 (phupSu200a) was studied using gel retardation and footprinting assays. HupR protected a 50u2003bp 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/−152u2003nt. 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 σ70‐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.

Enlarging the gas access channel to the active site renders the regulatory hydrogenase HupUV of Rhodobacter capsulatus O2 sensitive without affecting its transductory activity

In the photosynthetic bacterium Rhodobacter capsulatus, the synthesis of the energy‐producing hydrogenase, HupSL, is regulated by the substrate H2, which is detected by a regulatory hydrogenase, HupUV. The HupUV protein exhibits typical features of [NiFe] hydrogenases but, interestingly, is resistant to inactivation by O2. Understanding the O2 resistance of HupUV will help in the design of hydrogenases with high potential for biotechnological applications. To test whether this property results from O2 inaccessibility to the active site, we introduced two mutations in order to enlarge the gas access channel in the HupUV protein. We showed that such mutations (Ile65→Val and Phe113→Leu in HupV) rendered HupUV sensitive to O2 inactivation. Also, in contrast with the wild‐type protein, the mutated protein exhibited an increase in hydrogenase activity after reductive activation in the presence of reduced methyl viologen (up to 30% of the activity of the wild‐type). The H2‐sensing HupUV protein is the first component of the H2‐transduction cascade, which, together with the two‐component system HupT/HupR, regulates HupSL synthesis in response to H2 availability. In vitro, the purified mutant HupUV protein was able to interact with the histidine kinase HupT. In vivo, the mutant protein exhibited the same hydrogenase activity as the wild‐type enzyme and was equally able to repress HupSL synthesis in the absence of H2.

Genetic and physical mapping of an hydrogenase gene cluster from Rhodobacter capsulatus

SummaryAn hydrogenase-deficient (Hup−) mutant of Rhodobacter capsulatus was obtained by adventitious insertion of IS21 DNA into an hydrogenase structural gene (hup) of the wild-type strain 1310. The resulting Hup− mutant, strain JP91, selected by its inability to grow autotrophically (Aut− phenotype) together with other Hup− mutant strains obtained by classical ethyl methane sulphonate mutagenesis were used in R plasmid-mediated conjugation experiments to map the hup/aut loci on the chromosome of R. capsulatus. The hup genes tested in this study were found to cluster on the chromosome in the proximity of the his-1 marker. A cluster of hup genes comprising the structural genes was isolated from a gene bank constructed in the cosmid vector pHC79 with 40 kb insert DNA. The clustered hup genes, characterized by hybridization studies and complementation analyses of the R. capsulatus Hup− mutants, span 15–20 kb of DNA.

Cloning and sequencing the genes encoding uptake-hydrogenase subunits of Rhodocyclus gelatinosus

SummaryRhodocyclus gelatinosus grew photosynthetically in the light and consumed H2 at a rate of about 665 nmol/min per mg protein. The uptake-hydrogenase (H2ase) was found to be membrane bound and insensitive to inhibition by CO. The structural genes of R. gelatinosus uptake-H2ase were isolated from a 40 kb cosmid gene library of R. gelatinosus DNA by hybridization with the structural genes of uptake-H2ase of Bradyrhizobium japonicum and Rhodobacter capsulatus. The R. gelatinosus genes were localized on two overlapping DNA restriction fragments subcloned into pUC18. Two open reading frames (ORF1 and ORF2) were observed. ORF1 contained 1080 nucleotides and encoded a 39.4 kDa protein. ORF2 had 1854 nucleotides and encoded a 68.5 kDa protein. Amino acid sequence analysis suggested that ORF1 and ORF2 corresponded to the small (HupS) and large (HupL) subunits, respectively, of R. gelatinosus uptake-H2ase. ORF1 was approximately 80% homologous with the small, and ORF2 was maximally 68% homologous with the large subunit of typical membrane-bound uptake-H2ases.

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 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 300u2003bp 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.

Molecular biology studies of the uptake hydrogenase of Rhodobacter capsulatus and Rhodocyclus gelatinosus

In the photosynthetic bacteria, as in other N2-fixing bacteria, two main enzymes are involved in H2 metabolism: nitrogenase, which catalyses the photoproduction of H2, and a membrane-bound (NiFe) hydrogenase, which functions as an H2-uptake enzyme. The structural genes for Rhodobacter capsulatus and Rhodocyclus gelatinosus uptake hydrogenases were isolated and sequenced. They present the same organization, with the gene encoding the small subunit (hupS) (molecular masses 34.2 and 34.6 kDa, respectively) preceding the gene encoding the large one (hupL) (molecular masses 65.8 and 68.5 kDa, respectively). The two hupSL genes apparently belong to the same operon. The deduced protein sequences of the small and of the large subunits share nearly 80% and maximally 70% identity, respectively, with their counterparts in uptake hydrogenases found in N2-fixing bacteria. However, unlike in Bradyrhizobium japonicum, R. gelatinosus or Azotobacter chroococcum, another open reading frame (ORFX) was found downstream and contiguous to the R. capsulatus hupSL whose transcription seemed to depend on the same hup promoter as hupSL. ORFX contained 786 nucleotides capable of encoding a hydrophobic polypeptide of 262 amino acids (30.2 kDa).

Phosphorylation coupled to H2 oxidation by chromatophores from Rhodopseudomonas capsulata.

The phototrophic bacterium, Rhodopseudomonas capsulata can use Hz as electron donor for either photoautotrophic or chemoautotrophic growth [ 1,2]. When grown in the presence of Ha, cells contain a relatively high hydrogenase activity of the Hz -uptake type [3]. This hydrogenase is located in the membrane [3], which also contains the redox components of the respiratory chain. Cells of Rps. capsulata grown anaerobically in the light have been shown to have respiratory activities [4] similar to those found in aerobic cells [5]. The purpose of the experiments reported here was to show that hydrogenase formed in the membranes of photosynthetically grown cells can feed electrons to the respiratory chain and that Hz oxidation is linked to ATP synthesis.

Chapter 11: Hydrogen Respiration

Hydrogen respiration can be considered either as (i) the oxidation of H2 to H+, with the electrons released being channeled into a membrane-bound, respiratory electron transport chain or (ii) as the reduction of H+ to H2 in the terminal reaction of an anaerobic, low-potential electron transport system. In both cases, the redox reaction involving H2 is catalyzed by a hydrogenase enzyme (H2ase) and electron transport to or from H2 is coupled to the vectorial translocation of H+ across a membrane, leading to the conservation of energy in the form of a protonmotive force.

ORGANIZATION OF THE GENES ENCODING UPTAKE HYDROGENASE IN THE PHOTOSYNTHETIC BACTERIUM RHODOBACTER CAPSULATUS

Mutants of Rhodobacter capsulatus deficient in hydrogenase activity (Hup- mutants) were used for genetic mapping. The hup markers were found to cluster on the chromosome in the proximity of the his-1 marker. A cluster of hup genes comprising the structural genes was isolated from a cosmid library of R capsulatus B10 containing recombinant DNA with 40 kbp insert DNA and was sequenced. Downstream of the structural genes (hupSL) 15 open reading frames were identified, the function of many of them has not been identified yet. The product of the hupR1 gene shares sequence and domain similarities with the transcriptional activators NifA and NtrC from R. capsulatus and Klebsiella pneumoniae, resp.. The hupSL promoter was fused to the lacZ gene and plasmid pAC142 carrying the fusion was introduced into the Hup- mutants and into the wild type strain B10 to study comparatively the β-galactosidase activity and the chromosomally-encoded hydrogenase activity in response to environmental stimuli. In a mutant carrying a mutation in hupR1, both the β-galactosidase activity and the hydrogenase activity were reduced to 6% of that found in the wild type strain, a result consistent with the assumption that HupR1 protein acts as a transcriptional activator.

ORGANIZATION OF GENES ENCODING HYDROGENASE ACTIVITY IN THE PHOTOSYNTHETIC BACTERIUM RHODOBACTER CAPSULATUS

Autotrophic bacteria, including the phototrophs, can use hydrogen gas as an energy source by means of H2-uptake hydrogenase. Uptake hydrogenases (Hup) are intrinsic membrane proteins and transfer H2 electrons either to the respiratory chain, under aerobic conditions, or, under anaerobic conditions, to redox carrier(s), still unidentified for the reduction of CO2.