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The interaction between hydrogen metabolism, respiration, and photosynthesis was studied in vivo in whole cells of Synechocystis sp. strain PCC 6803 by continuously monitoring the changes in gas concentrations (H2, CO2, and O2) with an online mass spectrometer. The in vivo activity of the bidirectional [NiFe]hydrogenase [H2:NAD(P) oxidoreductase], encoded by the hoxEFUYH genes, was also measured independently by the proton-deuterium (H-D) exchange reaction in the presence of D2. This technique allowed us to demonstrate that the hydrogenase was insensitive to light, was reversibly inactivated by O2, and could be quickly reactivated by NADH or NADPH (+H2). H2 was evolved by cells incubated anaerobically in the dark, after an adaptation period. This dark H2 evolution was enhanced by exogenously added glucose and resulted from the oxidation of NAD(P)H produced by fermentation reactions. Upon illumination, a short (less than 30-s) burst of H2 output was observed, followed by rapid H2 uptake and a concomitant decrease in CO2 concentration in the cyanobacterial cell suspension. Uptake of both H2 and CO2 was linked to photosynthetic electron transport in the thylakoids. In the ndhB mutant M55, which is defective in the type I NADPH-dehydrogenase complex (NDH-1) and produces only low amounts of O2 in the light, H2 uptake was negligible during dark-to-light transitions, allowing several minutes of continuous H2 production. A sustained rate of photoevolution of H2 corresponding to 6 micro mol of H2 mg of chlorophyll(-1) h(-1) or 2 ml of H2 liter(-1) h(-1) was observed over a longer time period in the presence of glucose and was slightly enhanced by the addition of the O2 scavenger glucose oxidase. By the use of the inhibitors DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] and DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone), it was shown that two pathways of electron supply for H2 production operate in M55, namely photolysis of water at the level of photosystem II and carbohydrate-mediated reduction of the plastoquinone pool.

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 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.

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.

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.

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).

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.

The hupT gene, which represses hydrogenase gene expression in the purple photosynthetic bacterium Rhodobacter capsulatus, has been identified and sequenced. The nucleotide sequence of hupT and of the contiguous downstream open reading frame, hupU, is reported. The HupT protein of 456 amino acids (48,414 Da) has sequence similarity with the FixL, DctB, NtrB, and ArcB proteins and is predicted to be a soluble sensor kinase. Insertional inactivation of the hupT gene led to deregulation of transcriptional control, so that the hydrogenase structural operon hupSLC became overexpressed in cells grown anaerobically or aerobically. The HupT- mutants were complemented in trans by a plasmid containing an intact copy of the hupT gene. The hupU open reading frame, capable of encoding a protein of 84,879 Da, shared identity with [NiFe]hydrogenase subunits; the strongest similarity was observed with the periplasmic hydrogenase of Desulfovibrio baculatus.

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 alpha beta heterodimer similar to IHF of Escherichia coli. We have cloned and sequenced the hip gene, which encodes the beta subunit. The deduced amino acid sequence (10.7 kDa) has 46% identity with the beta 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 alpha 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 Escherichia coli beta-galactosidase enzyme was used as a reporter molecule for genetic fusions in Rhodobacter capsulatus. DNA fragments that were from the upstream region of the hydrogenase structural operon hupSLM and contained 5' hupS sequences were fused in frame to a promoterless lacZ gene, yielding fusion proteins comprising the putative signal sequence and the first 22 amino acids of the HupS protein joined to the eight amino acid of beta-galactosidase. We demonstrate the usefulness of the hupS::lacZ fusion in monitoring regulation of hydrogenase gene expression. The activities of plasmid-determined beta-galactosidase and chromosome-encoded hydrogenase changed in parallel in response to various growth conditions (light or dark, aerobiosis or anaerobiosis, and presence or absence of ammonia or of H2), showing that changes in hydrogenase activity were due to changes in enzyme synthesis. Molecular hydrogen stimulated hydrogenase synthesis in dark, aerobic cultures and in illuminated, anaerobic cultures. Analysis of hupS::lacZ expression in various mutants indicated that neither the hydrogenase structural genes nor NifR4 (sigma 54) was essential for hydrogen regulation of hydrogenase synthesis.

The hupR1 gene from Rhodobacter capsulatus was cloned and sequenced. It can encode a protein of 53,843 Da which shares significant similarity with several transcriptional regulators and activates transcription of the structural hupSL genes of [NiFe]hydrogenase, as shown by the use of a translational fusion of lacZ with the hupSL promoter. A Hup- mutant having a point mutation in the hupR1 gene is described.

Translational fusions of the Escherichia coli lacZ gene to Rhodobacter capsulatus nif genes were constructed in order to determine the regulatory circuit of nif gene expression in R. capsulatus, a free-living photosynthetic diazotroph. The expression of nifH, nifA (copies I and II), and nifR4 was measured in different regulatory mutant strains under different physiological conditions. The expression of nifH and nifR4 (the analog of ntrA in Klebsiella pneumoniae) depends on the NIFR1/R2 system (the analog of the ntr system in K. pneumoniae), on NIFA, and on NIFR4. The expression of both copies of nifA is regulated by the NIFR1/R2 system and is modulated by the N source of the medium under anaerobic photosynthetic growth conditions. In the presence of ammonia or oxygen, moderate expression of nifA was detectable, whereas nifH and nifR4 were not expressed under these conditions. The implications for the regulatory circuit of nif gene expression in R. capsulatus are discussed and compared with the situation in K. pneumoniae, another free-living diazotroph.

In vivo genetic engineering by R' plasmid formation was used to isolate an Escherichia coli gene that restored the Ntr+ phenotype to Ntr- mutants of the photosynthetic bacterium Rhodobacter capsulatus (formerly Rhodopseudomonas capsulata; J. F. Imhoff, H. G. Trüper, and N. Pfenning, Int. J. Syst. Bacteriol. 34:340-343, 1984). Nucleotide sequencing of the gene revealed no homology to the ntr genes of Klebsiella pneumoniae. Furthermore, hybridization experiments between the cloned gene and different F' plasmids indicated that the gene is located between 34 and 39 min on the E. coli genetic map and is therefore unlinked to the known ntr genes. The molecular weight of the gene product, deduced from the nucleotide sequence, was 30,563. After the gene was cloned in an expression vector, the gene product was purified. It was shown to have a pI of 5.8 and to behave as a dimer during gel filtration and on sucrose density gradients. Antibodies raised against the purified protein revealed the presence of this protein in R. capsulatus strains containing the E. coli gene, but not in other strains. Moreover, elimination of the plasmid carrying the E. coli gene from complemented strains resulted in the loss of the Ntr+ phenotype. Complementation of the R. capsulatus mutations by the E. coli gene therefore occurs in trans and results from the synthesis of a functional gene product.

Hydrogenase activity was found in cells of Rhodopseudomonas capsulata strain B10 cultured under a variety of growth conditions either anaerobically in the light or aerobically in the dark. The highest activities were found routinely in cells grown in the presence of H2. The hydrogenase of R. capsulata was localized in the particulate fraction of the cells. High hydrogenase activities were usually observed in cells possessing an active nitrogenase. The hydrogen produced by the nitrogenase stimulated the activity of hydrogenase in growing cells. However, the synthesis of hydrogenase was not closely linked to the synthesis of nitrogenase. Hydrogenase was present in dark-grown cultures, whereas nitrogenase synthesis was not significant in the absence of light. Unlike nitrogenase, hydrogenase was present in cultures grown on NH4+. Conditions were established which allowed the synthesis of either nitrogenase or hydrogenase by resting cells. We concluded that hydrogenase can be synthesized independently of nitrogenase.

Nitrogenase proteins were isolated from cultures of the photosynthetic bacterium Rhodopseudomonas capsulata grown on a limiting amount of ammonia. Under these conditions, the nitrogenase N2ase A was active in vivo, and nitrogenase activity in vitro was not dependent upon manganese and the activating factor. The nitrogenase proteins were also isolated from nitrogen-limited cultures in which the in vivo nitrogenase activity had been stopped by an ammonia shock. This nitrogenase activity, N2ase R, showed an in vitro requirement for manganese and the activating factor for maximal activity. The Mo-Fe protein (dinitrogenase) was composed of two dissimilar subunits with molecular weights of 55,000 and 59,500; the Fe protein (dinitrogenase reductase), from either type of culture, was composed of a single subunit (molecular weight), 33,500). The metal and acid labile sulfur contents of both nitrogenase proteins were similar to those found for previously isolated nitrogenases. The Fe proteins from both N2ase A and N2ase R contained phosphate and ribose, 2 mol of each per mol of N2ase R Fe protein and about 1 mol of each per mol of N2ase A Fe protein. The greatest difference between the two types of Fe protein was that the N2ase R Fe protein contained about 1 mol per mol of an adenine-like molecule, whereas the N2ase A Fe protein content of this compound was insignificant. These results are compared with various models previously presented for the short-term regulation of nitrogenase activity in the photosynthetic bacteria.

The metabolic versatility of the purple nonsulfur photosynethetic bacterial permits the expression of either a phototrophic or a dark aerobic mode of growth. These organism also possess nitrogenase activity which may function under semiaerboic conditions. On the basis of these important properties, the light dependence of nitrogenase function and synthesis in Rhodopseudomonas capsulata was investigated. Nitrogenase activity was strictly dependent on light; no activity was observed in the dark, even when energy (ATP) was supplied by oxidative phosphorylation. It was concluded that the low-potential reducing agent required by the nitrogenase-catalyzed reaction could only be generated by a photochemical reaction. Nitrogenase biosynthesis was also largely dependent on light; however, a small amount of synthesis was observed in resting cells incubated in the dark. Resting cells prepared from dark-grown cultures synthesized nitrogenase at high rates upon illumination. The highest stability of nitrogenase in these resting cells was observed when suspensions were exposed to a diurnal pattern of illumination rather than continuous light. Although nitrogenase function and synthesis are closely coupled to photosynthetic activity, the biosyntheses of bacteriochorophyll and nitrogenase are independent of each other and are most probably subject to different regulatory mechanisms by light.

The structural gene (fdxN) encoding ferredoxin I (FdI) in the photosynthetic bacterium Rhodobacter capsulatus was isolated from a cosmid library by using an oligonucleotide probe corresponding to the N-terminal amino acid sequence of FdI. The nucleotide sequences of the gene and of the 3'- and 5'-flanking regions were determined. The gene fdxN codes for a polypeptide of 64 mino acids having a calculated molecular weight of 6,728. Amino acid sequencing of the N- and C-terminal ends of FdI allowed the determination of 86% of the primary structure and confirmed that FdI is the fdxN gene product. Sequence comparisons indicate that FdI shares common structural features with ferredoxins containing two [4Fe-4S] clusters, including eight conserved cysteines. Maximal homology was found with a ferredoxin from Rhodo-pseudomonas palustris. Northern (RNA) hybridization using a 158-base-pair DNA fragment internal to the fdxN coding region revealed the existence of two mRNA transcripts of approximately 330 and 750 nucleotides. Neither of those transcripts was present under nif-repressing growth conditions. The 5' end of the smaller transcript was mapped by S1 nuclease protection and primer extension experiments. On the basis of Southern hybridization experiments, by using probes homologous to fdxN, nifE, and a fragment complementing a nif point mutation, fdxN was localized inside a cluster of nif genes.

Hydrogen evolution and consumption by cell and chromatophore suspensions of the photosynthetic bacterium Rhodopseudomonas capsulata was measured with a sensitive and specific mass spectrometric technique which directly monitors dissolved gases. H2 production by nitrogenase was inhibited by acetylene and restored by carbon monoxide. An H2 evolution activity coupled with HD formation and D2 uptake (H-D exchange) was unaffected by C2H2 and CO. Cultures lacking nitrogenase activity also exhibited H-D exchange activity, which was catalyzed by a membrane-bound hydrogenase present in the chromatophores of R. capsulata. A net hydrogen uptake, mediated by hydrogenase, was observed when electron acceptors such as CO2, O2, or ferricyanide were present in the medium.

By using two-dimensional electrophoresis, five putative soluble nif gene products were identified, and the regulation of nif gene expression in Rhodopseudomonas capsulata was investigated. Expression of nif was repressed by ammonia and atmospheric concentrations of oxygen. Deprivation of molybdenum caused an interesting pattern of partial repression of nif gene expression that was not relieved by tungsten. These results are discussed in relation to the better understood system of nif regulation in Klebsiella pneumoniae.