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.

Molecular biology of microbial hydrogenases.

Hydrogenases (H2ases) are metalloproteins. The great majority of them contain iron-sulfur clusters and two metal atoms at their active center, either a Ni and an Fe atom, the [NiFe]-H2ases, or two Fe atoms, the [FeFe]-H2ases. Enzymes of these two classes catalyze the reversible oxidation of hydrogen gas (H2 <--> 2 H+ + 2 e-) and play a central role in microbial energy metabolism; in addition to their role in fermentation and H2 respiration, H2ases may interact with membrane-bound electron transport systems in order to maintain redox poise, particularly in some photosynthetic microorganisms such as cyanobacteria. Recent work has revealed that some H2ases, by acting as H2-sensors, participate in the regulation of gene expression and that H2-evolving H2ases, thought to be involved in purely fermentative processes, play a role in membrane-linked energy conservation through the generation of a protonmotive force. The Hmd hydrogenases of some methanogenic archaea constitute a third class of H2ases, characterized by the absence of Fe-S cluster and the presence of an iron-containing cofactor with catalytic properties different from those of [NiFe]- and [FeFe]-H2ases. In this review, we emphasise recent advances that have greatly increased our knowledge of microbial H2ases, their diversity, the structure of their active site, how the metallocenters are synthesized and assembled, how they function, how the synthesis of these enzymes is controlled by external signals, and their potential use in biological H2 production.

Classification and phylogeny of hydrogenases.

Hydrogenases (H2ases) catalyze the reversible oxidation of molecular hydrogen and play a central role in microbial energy metabolism. Most of these enzymes are found in Archaea and Bacteria, but a few are present in Eucarya as well. They can be distributed into three classes: the [Fe]-H2ases, the [NiFe]-H2ases, and the metal-free H2ases. The vast majority of known H2ases belong to the first two classes, and over 100 of these enzymes have been characterized genetically and/or biochemically. Compelling evidence from sequences and structures indicates that the [NiFe]- and [Fe]-H2ases are phylogenetically distinct classes of proteins. The catalytic core of the [NiFe]-H2ases is a heterodimeric protein, although additional subunits are present in many of these enzymes. Functional classes of [NiFe]-H2ases have been defined, and they are consistent with categories defined by sequence similarity of the catalytic subunits. The catalytic core of the [Fe]-H2ases is a ca. 350-residue domain that accommodates the active site (H-cluster). A few monomeric [Fe]-H2ases are barely larger than the H-cluster domain. Many others are monomeric as well, but possess additional domains that contain redox centers, mostly iron-sulfur. Some [Fe]-H2ases are oligomeric. The modular structure of H2ases is strikingly illustrated in recently unveiled sequences and structures. It is also remarkable that most of the accessory domains and subunits of H2ases have counterparts in other redox complexes, in particular NADH-ubiquinone oxidoreductase (Complex I) of respiratory chains. Microbial genome sequences are bringing forth a significant body of additional H2ase sequence data and contribute to the understanding of H2ase distribution and evolution. Altogether, the available data suggest that [Fe]-H2ases are restricted to Bacteria and Eucarya, while [NiFe]-H2ases, with one possible exception, seem to be present only in Archaea and Bacteria. H2ase processing and maturation involve the products of several genes which have been identified and are currently being characterized in the case of the [NiFe]-H2ases. In contrast, near to nothing is known regarding the maturation of the [Fe]-H2ases. Inspection of the currently available genome sequences suggests that the [NiFe]-H2ase maturation proteins have no similar counterparts in the genomes of organisms possessing [Fe]-H2ases only. This observation, if confirmed, would be consistent with the phylogenetic distinctiveness of the two classes of H2ases. Sequence alignments of catalytic subunits of H2ases have been implemented to construct phylogenetic trees that were found to be consistent, in the main, with trees derived from other data. On the basis of the comparisons performed and discussed here, proposals are made to simplify and rationalize the nomenclature of H2ase-encoding genes.

Characterization of the hydrogen-deuterium exchange activities of the energy-transducing HupSL hydrogenase and H(2)-signaling HupUV hydrogenase in Rhodobacter capsulatus.

Rhodobacter capsulatus synthesizes two homologous protein complexes capable of activating molecular H(2), a membrane-bound [NiFe] hydrogenase (HupSL) linked to the respiratory chain, and an H(2) sensor encoded by the hupUV genes. The activities of hydrogen-deuterium (H-D) exchange catalyzed by the hupSL-encoded and the hupUV-encoded enzymes in the presence of D(2) and H(2)O were studied comparatively. Whereas HupSL is in the membranes, HupUV activity was localized in the soluble cytoplasmic fraction. Since the hydrogenase gene cluster of R. capsulatus contains a gene homologous to hoxH, which encodes the large subunit of NAD-linked tetrameric soluble hydrogenases, the chromosomal hoxH gene was inactivated and hoxH mutants were used to demonstrate the H-D exchange activity of the cytoplasmic HupUV protein complex. The H-D exchange reaction catalyzed by HupSL hydrogenase was maximal at pH 4. 5 and inhibited by acetylene and oxygen, whereas the H-D exchange catalyzed by the HupUV protein complex was insensitive to acetylene and oxygen and did not vary significantly between pH 4 and pH 11. Based on these properties, the product of the accessory hypD gene was shown to be necessary for the synthesis of active HupUV enzyme. The kinetics of HD and H(2) formed in exchange with D(2) by HupUV point to a restricted access of protons and gasses to the active site. Measurement of concentration changes in D(2), HD, and H(2) by mass spectrometry showed that, besides the H-D exchange reaction, HupUV oxidized H(2) with benzyl viologen, produced H(2) with reduced methyl viologen, and demonstrated true hydrogenase activity. Therefore, not only with respect to its H(2) signaling function in the cell, but also to its catalytic properties, the HupUV enzyme represents a distinct class of hydrogenases.

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 (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.

Rhodobacter capsulatus HypF is involved in regulation of hydrogenase synthesis through the HupUV proteins.

The photosynthetic bacterium Rhodobacter capsulatus contains a membrane-bound [NiFe]hydrogenase encoded by the hupSL genes. We show in this study that hypF mutants are devoid of hydrogenase activity and lack the HupL protein. We also observed that, in contrast to the wild-type strain B10, transcription of the hupSL genes was not stimulated by H2 in the hypF mutants RS13 and BSE19. Complementation of the hypF mutants with the plasmid borne hypF gene restored hydrogenase activity to wild-type levels and inducibility by H2. The R. capsulatus hupU and hupV gene products share significant similarities with the small (HupS) and the large (HupL) hydrogenase subunits, respectively. Active HupUV proteins can catalyze the hydrogen-deuterium exchange reaction. In whole cells, this H-D exchange is distinguishable from the H-D exchange catalyzed by the membrane-bound HupSL proteins by its insensitivity to O2 and to acetylene. By measuring the formation of H2 and HD in exchange with D2 uptake, we demonstrated that the hypF mutants have no active HupUV nor HupSL proteins. H-D exchange activity, of both HupUV and HupSL, was restored by hypF gene complementation. These data indicate that the HypF protein participates not only in the maturation of HupSL, but also in the maturation of the HupUV proteins and that the latter are involved in the cellular response to H2.

Unusual organization of the genes coding for HydSL, the stable [NiFe]hydrogenase in the photosynthetic bacterium Thiocapsa roseopersicina BBS.

The characterization of a hyd gene cluster encoding the stable, bidirectional [NiFe]hydrogenase 1 enzyme in Thiocapsa roseopersicina BBS, a purple sulfur photosynthetic bacterium belonging to the family Chromatiaceae, is presented. The heterodimeric hydrogenase 1 had been purified to homogeneity and thoroughly characterized (K. L. Kovacs et al., J. Biol. Chem. 266:947-951, 1991; C. Bagyinka et al., J. Am. Chem. Soc. 115:3567-3585, 1993). As an unusual feature, a 1,979-bp intergenic sequence (IS) separates the structural genes hydS and hydL, which encode the small and the large subunits, respectively. This IS harbors two sequential open reading frames (ORFs) which may code for electron transfer proteins ISP1 and ISP2. ISP1 and ISP2 are homologous to ORF5 and ORF6 in the hmc operon, coding for a transmembrane electron transfer complex in Desulfovibrio vulgaris. Other accessory proteins are not found immediately downstream or upstream of hydSL. A hup gene cluster coding for a typical hydrogen uptake [NiFe]hydrogenase in T. roseopersicina was reported earlier (A. Colbeau et al. Gene 140:25-31, 1994). The deduced amino acid sequences of the two small (hupS and hydS) and large subunit (hupL and hydL) sequences share 46 and 58% identity, respectively. The hup and hyd genes differ in the arrangement of accessory genes, and the genes encoding the two enzymes are located at least 15 kb apart on the chromosome. Both hydrogenases are associated with the photosynthetic membrane. A stable and an unstable hydrogenase activity can be detected in cells grown under nitrogen-fixing conditions; the latter activity is missing in cells supplied with ammonia as the nitrogen source. The apparently constitutive and stable activity corresponds to hydrogenase 1, coded by hydSL, and the inducible and unstable second hydrogenase may be the product of the hup gene cluster.

Cloning, sequence and mutagenesis of the structural gene of Pseudomonas aeruginosa CysB, which can activate algD transcription.

Pseudomonas aeruginosa strains infecting patients with cystic fibrosis (CF) acquire a mucoid phenotype due to overproduction of alginate. The key enzyme in alginate synthesis is AlgD, whose promoter is transcriptionally active in mucoid strains and under the control of several trans-acting factors, including the integration host factor (IHF). The algD promoter (palgD) contains two IHF-binding sites (ihf1 and ihf2). Study of IHF binding to ihf2 of palgD, by electrophoretic mobility-shift assays, led to the discovery of a protein of 36 kDa (p36) able to bind downstream from ihf2, to the 3' region of palgD. The gene encoding p36 was isolated from the mucoid strain CHA of P. aeruginosa and sequenced. It can encode a 324-amino-acid protein, which shares a high degree of sequence identity (63%) with CysB from Escherichia coli and from Salmonella typhimurium, a transcriptional factor of the LysR superfamily. Furthermore, both p36 and S. typhimurium CysB bind the same site of palgD; p36 was therefore termed CysB and its structural gene was called cysB. Next to cysB, on the opposite DNA strand, cysH was capable of encoding a protein sharing 26% identity with CysH (PAPS reductase) of E. coli and an even greater identity (54%) with the nucleotide-deduced protein from Arabidopsis. A CysB-deficient mutant of CHA, constructed by insertional inactivation of cysB, was a cysteine auxotroph and was unable to form a specific complex with palgD in vitro. Activity of palgD in the cysB mutant, in CHA and in the non-mucoid strain PAO was assessed by the use of a transcriptional algD-xylE fusion. Cells of PAO and of the cysB mutant grown in minimal media in the presence of 0.3 M NaCl exhibited a palgD activity, which was 10% or less that of the mucoid strain CHA. Thus, P. aeruginosa CysB can act as an activator of algD expression.

Purification and in vitro phosphorylation of HupT, a regulatory protein controlling hydrogenase gene expression in Rhodobacter capsulatus.

The HupT protein of Rhodobacter capsulatus, involved in negative regulation of hydrogenase gene expression, is predicted to be a histidine kinase on the basis of sequence comparisons. The protein was overproduced in Escherichia coli, purified to homogeneity, and demonstrated to autophosphorylate in vitro in the presence of [gamma-32P]ATP. An H217N hupt mutant was constructed, and the mutant protein was shown to have lost kinase activity. This result, and the fact that the phosphoryl group in phosphorylated HupT appeared to be bound to an N atom, support the suggestion from sequence comparisons that HupT is a histidine kinase, which can autophosphorylate on the His217 residue.

HupUV proteins of Rhodobacter capsulatus can bind H2: evidence from the H-D exchange reaction.

The H-D exchange reaction has been measured with the D2-H2O system, for Rhodobacter capsulatus JP91, which lacks the hupSL-encoded hydrogenase, and R. capsulatus BSE16, which lacks the HupUV proteins. The hupUV gene products, expressed from plasmid pAC206, are shown to catalyze an H-D exchange reaction distinguishable from the H-D exchange due to the membrane-bound, hupSL-encoded hydrogenase. In the presence of O2, the uptake hydrogenase of BSE16 cells catalyzed a rapid uptake and oxidation of H2, D2, and HD present in the system, and its activity (H-D exchange, H2 evolution in presence of reduced methyl viologen [MV+]) depended on the external pH, while the H-D exchange due to HupUV remained insensitive to external pH and O2. These data suggest that the HupSL dimer is periplasmically oriented, while the HupUV proteins are in the cytoplasmic compartment.

Structural study of the response regulator HupR from Rhodobacter capsulatus. Electron microscopy of two-dimensional crystals on a nickel-chelating lipid.

Two-dimensional crystals of the histidine-tagged-HupR protein, a transcriptional regulator from the photosynthetic bacterium Rhodobacter capsulatus, were obtained upon specific interaction with a Ni2+-chelated lipid monolayer. HupR is a response regulator of the NtrC family; it activates the transcription of the structural genes, hupSLC, of the [NiFe]hydrogenase. The lipid (Ni-NTA-DOGA) uses the metal chelator nitrilotriacetic group as the hydrophilic headgroup and contains unsaturated oleyl tails to provide the fluidity necessary for two-dimensional protein crystallization. A projection map of the full-length protein at 18 A resolution was generated by analysing electron microscopy micrographs of negatively stained crystals. The HupR protein appeared to be dimeric and revealed a characteristic "propeller-like" motif. Each monomer forms an L-shaped structure.

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 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.

Inhibition by iodoacetamide and acetylene of the H-D-exchange reaction catalyzed by Thiocapsa roseopersicina hydrogenase.

The kinetics of H-D isotope exchange catalyzed by the thermostable hydrogenase from Thiocapsa roseopersicina have been studied by analysis of the exchange between D2 and H2O. The pH dependence of the exchange reaction was examined between pH 2.5 and pH 11. Over the whole pH range, HD was produced at a higher initial velocity than H2, with a marked optimum at pH 5.5; a second peak in the pH profile was observed at around pH 8.5. The rapid formation of H2 with respect to HD in the D2/H2O system is consistent with a heterolytic cleavage of D2 into D+ and an enzyme hydride that can both exchange with the solvent. The H-D-exchange activity was lower in the H2/D2O system than in the D2/H2O system. The other reactions catalyzed by the hydrogenase, H2 oxidation and H2 evolution, are pH dependent; the optimal pH were 9.5 for H2 uptake and 4.0 for H2 production. Treatment of the active form of hydrogenase by iodoacetamide led to a slow and irreversible inhibition of the H-D exchange. When iodo[1-14C]acetamide was incubated with hydrogenase, the radioactive labeling of the large subunit was higher for the enzyme activated under H2 than for the inactive oxidized form. Cysteine residues were identified as the alkylated derivative by amino acid analysis. Acetylene, which inhibits H-D exchange and abolishes the Ni-C EPR signal, protected the enzyme from irreversible inhibition by iodoacetamide. These data indicate that iodoacetamide can reach the active site of the H2-activated hydrogenase from T. roseopersicina. This was not found to be the case with the seleno hydrogenase from Desulfovibrio baculatus (now Desulfomicrobium baculatus). Cysteine modification by iodoacetamide upon activation of the enzyme concomitant with loss of H-D exchange indicates that reductive activation makes at least one Cys residue of the active site available for alkylation.

Isolation of an IHF-deficient mutant of a Pseudomonas aeruginosa mucoid isolate and evaluation of the role of IHF in algD gene expression.

The role of integration host factor (IHF) in the regulation of alginate synthesis was investigated in a mucoid strain of Pseudomonas aeruginosa (strain CHA) isolated from a cystic fibrosis patient. Escherichia coli strain BL21(DE3) was made IHF-deficient by inactivation of its chromosomal IHF genes, himA and himD, then used as host strain to overproduce P. aeruginosa IHF. The purified recombinant IHF protein was used to determine the affinity of IHF for the two IHF binding sites in the algD promoter. The Kd values were determined to be 130 nM for algD IHF site 2 and about 2 microM for algD IHF site 1. Two IHF-deficient mutants of P. aeruginosa strain CHA were constructed by insertional inactivation of the himA gene, and the activity of the algD promoter was determined using transcriptional fusion with xylE as reporter gene. The expression of algD, the structural gene for GDP-mannose dehydrogenase, was decreased three- to fourfold in the himA mutants under conditions of high salinity and nitrogen limitation. Assays of alginate production by cultures grown on agar plates indicated that the IHF-deficient mutants synthesized 50% less polymer than the mucoid parental strain. These results demonstrate clearly that although IHF is dispensable for alginate production, himA expression is required for full activation of algD expression.

The Pseudomonas aeruginosa fumc and soda genes belong to an iron-responsive operon.

Pseudomonas aeruginosa contains two superoxide dismutases (SOD), a Mn-containing SOD (Mn-SOD) and a Fe-SOD, which are encoded by sodA and sodB, respectively. We have cloned and sequenced a DNA fragment from P. aeruginosa, strain CHA, which contains the sodA gene and three other open reading frames (ORF). We report here that one of the ORFs upstream from sodA is fumC, which encodes the O2.- resistant isoform of fumarase (or fumarate hydratase). It is shown that fumC and sodA belong to the same operon. By primer extension experiments, the transcription initiation site has been located at -413 from the ATG codon of the fumC gene. The fumC-sodA operon was found to be negatively regulated in presence of iron and the E. coli FUR protein was shown to bind to the 19-bp FUR consensus sequence present at the transcription start site of the operon.

The hupTUV operon is involved in negative control of hydrogenase synthesis in Rhodobacter capsulatus.

The hupT, hupU, and hupV genes, which are located upstream from the hupSLC and hypF genes in the chromosome of Rhodobacter capsulatus, form the hupTUV operon expressed from the hupT promoter. The hupU and hupV genes, previously thought to belong to a single open reading frame, encode HupU, of 34.5 kDa (332 amino acids), and HupV, of 50.4 kDa (476 amino acids), which are >/= 50% identical to the homologous Bradyrhizobium japonicum HupU and HupV proteins and Rhodobacter sphaeroides HupU1 and HupU2 proteins, respectively; they also have 20 and 29% similarity with the small subunit (HupS) and the large subunit (HupL), respectively, of R. capsulatus [NiFe]hydrogenase. HupU lacks the signal peptide of HupS and HupV lacks the C-terminal sequence of HupL, which are cleaved during hydrogenase processing. Inactivation of hupV by insertional mutagenesis or of hupUV by in-frame deletion led to HupV- and Hup(UV)- mutants derepressed for hydrogenase synthesis, particularly in the presence of oxygen. These mutants were complemented in trans by plasmid-borne hupTUV but not by hupT or by hupUV, except when expressed from the inducible fru promoter. Complementation of the HupV- and Hup(UV)- mutants brought about a decrease in hydrogenase activity up to 10-fold, to the level of the wild-type strain B10, indicating that HupU and HupV participate in negative regulation of hydrogenase expression in concert with HupT, a sensor histidine kinase involved in the repression process. Plasmid-borne gene fusions used to monitor hupTUV expression indicated that the operon is expressed at a low level (50- to 100-fold lower than hupS).

Role of manganese superoxide dismutase in a mucoid isolate of Pseudomonas aeruginosa: adaptation to oxidative stress.

Chronic infection by alginate-producing (mucoid) Pseudomonas aeruginosa is a leading cause of morbidity among cystic fibrosis (CF) patients. In the lungs of CF patients, the bacteria are exposed to activated oxygen species produced by the phagocytes of the host or resulting from the metabolism of oxygen. Two isoforms of superoxide dismutase are synthesized by P. aeruginosa; they differ by the metal present at their active site, which is either iron or manganese. To evaluate the role of manganese-containing superoxide dismutase (MnSOD), encoded by sodA, we have isolated a sodA mutant of the mucoid P. aeruginosa strain CHA isolated from the bronchopulmonary tract of a CF patient. The sodA mutant exhibited an increased sensitivity to oxidative stress generated by paraquat and was less resistant to oxidative stress in the stationary phase of growth compared with its parental strain. It was observed that MnSOD was expressed in the parental strain solely during the stationary phase of growth and that cells of the sodA mutant taken at the stationary phase resumed growth with a longer delay than the sodA+ cells when reinoculated in a new medium, especially in the presence of paraquat. These results suggest that MnSOD may participate in the adaptation of mucoid strains of P. aeruginosa to the stationary phase of growth in the lungs of CF patients.

Characterisation of the mcpA and mcpB genes capable of encoding methyl-accepting type chemoreceptors in Rhodobacter capsulatus.

Two contiguous mcp genes, mcpA and mcpB, transcribed from the same DNA strand and capable of encoding methyl-accepting chemotaxis proteins (Mcp) have been isolated from Rhodobacter capsulatus (Rc), sequences and overexpressed in Escherichia coli (Ec). The deduced proteins (McpA, 69 171 Da; McpB, 81 629 Da) show a structure similar to that of Ec Mcp. The products of mcpA and mcpB, overproduced in Ec, were recognized by anti-Ec Mcp (Trg) antibodies.

Isolation of Rhodobacter capsulatus transketolase: cloning and sequencing of its structural tktA gene.

Rhodobacter capsulatus transketolase (Tkt) protein has been isolated from strain B10 by heparin affinity chromatography. Oligodeoxyribonucleotides (oligo) constructed as based on the amino-acid sequences were used for polymerase chain reaction (PCR) amplification on total genomic DNA. Southern hybridization with the PCR product as a probe allowed the isolation of a 5-kb PstI DNA fragment containing the structural Tkt-encoding gene (tktA) which was cloned and sequenced. The deduced tktA product of 671 aa (72815 Da) shares 59% identity with Rhodobacter sphaeroides Tkt.

Cloning and sequence analyses of the genes coding for the integration host factor (IHF) and HU proteins of Pseudomonas aeruginosa.

Histone-like proteins, such as HU and the integration host factor (IHF), are small, dimeric, DNA-bending proteins which play a role in maintaining constrained DNA structures and hence in regulating gene expression. Two different strategies were used to isolate the genes coding for Pseudomonas aeruginosa (Pa) HU and IHF, two proteins that we have previously isolated from a mucoid strain. By use of a PCR-based technique with oligodeoxyribonucleotides (oligos) designed from the N-terminal amino acid (aa) sequences of HU and the beta-subunit of IHF, and Southern blot analyses, hupB and himD, encoding HU and IHF beta, respectively, have been cloned. The himA gene of Pa, encoding the alpha-subunit of IHF, was isolated using himA of Escherichia coli (Ec) as a probe in Southern blot analyses. The deduced hupB product (90 aa, 9 kDa) is 79% identical to HU beta and 61% to HU alpha of Ec. The predicted products of himA (100 aa, 11.5 kDa) and of himD (94 aa, 10.6 kDa) share 77 and 70% identity with IHF alpha and IHF beta of Ec, respectively. The promoter region of himD contains an IHF consensus sequence, as is the case for Ec himD.