H2 metabolism in the photosynthetic bacterium Rhodopseudomonas capsulata: H2 production by growing cultures.

Purple photosynthetic bacteria produce H2 from organic compounds by an anaerobic light-dependent electron transfer process in which nitrogenase functions as the terminal catalyst. It has been established that the H2-evolving function of nitrogenase is inhibited by N2 and ammonium salts, and is maximally expressed in cells growing photoheterotrophically with certain amino acids as sources of nitrogen. In the present studies with Rhodopseudomonas capsulata, nutritional factors affecting the rate and magnitude of H2 photoproduction in cultures growing with amino acid nitrogen sources were examined. The highest H2 yields and rates of formation were observed with the organic acids: lactate, pyruvate, malate, and succinate in media containing glutamate as the N source; under optimal conditions with excess lactate, H2 was produced at rates of ca. 130 ml/h per g(dry weight) of cells. Hydrogen production is significantly influenced by the N/C ratio in the growth substrates; when this ratio exceeds a critical value, free ammonia appears in the medium and H2 is not evolved. In the "standard" lactate + glutamate system, both H2 production and growth are "saturated" at a light intesity of ca. 600 ft-c (6,500 lux). Evolution of H2, however, occurs during growth at lithe intensities as low as 50 to 100 ft-c (540 to 1,080 lux), i.e., under conditions of energy limitation. In circumstances in which energy conversion rate and supplies of reducing power exceed the capacity of the biosynthetic machinery, energy-dependent H2 production presumably represents a regulatory device that facilitates "energy-idling." It appears that even when light intensity (energy) is limiting, a significant fraction of the available reducing power and adenosine 5'-triphosphate is diverted to nitrogenase, resulting in H2 formation and a bioenergetic burden to the cell.     

Inorganic nitrogen assimilation by the photosynthetic bacterium Rhodopseudomonas capsulata.

The photosynthetic bacterium Rhodopseudomonas capsulata lacks glutamate dehydrogenase and normally uses the glutamine synthetase/glutamate synthase sequence of reactions for assimilation of N2 and ammonia. The glutamine synthetase in cell-free extracts of the organism is completely sedimented by centrifugation at 140,000 X g for 2 h, is inhibited by L-alanine but not by adenosine 5'-monophosphate, and exhibits two apparent Km values for ammonia (ca. 13 muM and 1 mM).     

Growth of the photosynthetic bacterium Rhodopseudomonas capsulata chemoautotrophically in darkness with H2 as the energy source.

The phototrophic bacterium Rhodopseudomonas capsulata was found to be capable of growing chemoautotrophically under aerobic conditions in darkness. Growth was strictly dependent on the presence of H2 as the source of energy and reducing power, O2 as the terminal electron acceptor for energy transduction, and CO2 as the sole carbon source; under optimal conditions the generation time was about 6 h. Chemoautotrophically grown cells showed a relatively high content of bacteriochlorophyll a and intracytoplasmic membranes (chromatophores). Experiments with various mutants of R. capsulata, affected in electron transport, indicate that either of the two terminal oxidases of this bacterium can participate in the energy-yielding oxidation of H2. The ability of R. capsulata to multiply in at least five different physiological growth modes suggests that it is one of the most metabolically versatile procaryotes known.     

Nucleotide exchange in membrane vesicles from the photosynthetic bacterium Rhodopseudomonas capsulata

Membrane vesicles (heavy chromatophores) prepared from the photosynthetic bacteria Rhodopseudomonas capsulata catalyze photophosphorylation of exogenous ADP and also take up [³H]ADP from the external medium. The rate of uptake depends on the concentration of external ADP reaching half-maximal velocity at 2.7 mm. The rate increases also with the increase in the concentration of internal ADP. Vesicles, preloaded with [³H]ADP release the radioactive nucleotide when ADP is included in the external medium. Regular chromatophores, which are inside-out membrane vesicles also take up [³H]ADP from the external medium when preloaded with ADP. These results are interpreted to indicate the existence of nucleotide transport across the cytoplasmic membrane of these bacteria which is catalyzed by an ADP exchange carrier.     

Regulation of nitrogenase in the photosynthetic bacterium Rhodopseudomonas capsulata as studied by two-dimensional gel electrophoresis.

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.     

Purification and characterization of a catalase-peroxidase from the photosynthetic bacterium Rhodopseudomonas capsulata

Catalase-peroxidase was isolated from aerobically grown Rhodopseudomonas capsulata. The enzyme resembles typical catalases in some of its physicochemical properties. It has an apparent molecular weight of 236,000 and is composed of four identical subunits. It shows a typical high spin ferric heme spectrum with absorption maxima at 403 and 635 nm and shoulders at 503 and 535 nm. Upon binding of cyanide, the enzyme is converted to the low spin state, as shown by the shift of the Soret maximum to 418 nm and the band at 532 nm. It has an isoelectric point at pH 4.5. The enzyme differs from typical catalases in also having a strong peroxidatic activity with dianisidine, pyrogallol, and diaminobenzidine as electron donors. Both the catalatic and the peroxidatic activities are similarly inactivated by treatment with 1 mM H2O2, heating to 50 degrees C, exposure to ethanol/chloroform, and photooxidative conditions. In contrast to typical catalases, but similarly to peroxidases, the enzyme is reduced by sodium dithionite. The pH optimum of the peroxidatic activity is 5-5.3 (in contrast to 6-6.5 of the catalatic activity). 50% of the apparent maximal activities are reached at 0.3 and 4.2 mM H2O2 for the peroxidatic and catalatic activities, respectively. Both enzymic activities are equally inhibited by cyanide, 50% inhibition being achieved with 2.2 X 10(-5) M KCN. Contrarily, the two activities differ in their response to hydroxylamine and azide. 50% inhibition of the catalatic activity is obtained with 1.5 X 10(-4) M azide or 2.15 X 10(-6) M hydroxylamine; 50% inhibition of the peroxidatic activity requires 7.3 X 10(-4) M azide or 7.8 X 10(-5) M hydroxylamine. The activation energies of the catalatic and the peroxidatic activities are 1.9 and 1.7 kcal/mol, respectively.     

The size and number of intramembrane particles in cells of the photosynthetic bacterium Rhodopseudomonas capsulata studied by freeze-fracture electron microscopy.

By freeze-fracture electron microscopy, particles have been observed on the protoplasmic leaflet (PF face) of cytoplasmic and intracytoplasmic membranes of the photosynthetic bacterium Rhodopseudomonas capsulata. The particles are present under all culture conditions of chemotrophically and phototrophically grown cells. However, the number of particles per microM2 increased significantly when the formation of the photosynthetic apparatus in the membrane is induced. Intracytoplasmic membranes, where the bulk of photosynthetic activity is localized, always have a higher density of particles than cytoplasmic membranes. Under all conditions particles with a diameter of 9.5 nm dominate. The frequency of particles with diameters greater or smaller than 9.5 nm changed with culture conditions. A comparison of biochemical and electron microscopic data have lead us to the conclusion that the particles, formed under conditions which allow the synthesis of the photosynthetic apparatus, are composed of photochemical reaction centers and antenna light-harvesting bacteriochlorophyll I (B 875)-protein complexes. The total molecular weight of these particles is calculated to be 500,000.     

Glycerol utilization by a mutant of Rhodopseudomonas capsulata.

The glycerol-catabolizing enzymes of a mutant of Rhodopseudomonas capsulata were found to be constitutive and modulated coordinately, although apparently not functional in the presence of malate. No difference in glycerol permeation was found between the mutant and wild type.     

Continuous monitoring, by mass spectrometry, of H2 production and recycling in Rhodopseudomonas capsulata.

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.     

Hydrogen production by Rhodopseudomonas capsulata cells entrapped in carrageenan beads

Summary The photosynthetic bacteria Rhodopseudomonas capsulata strain B10 were immobilized in agar or in carrageenan beads (Ø = 1–3 mm). Beads containing 5.8 mg cell dry weight/mL of gel produced hydrogen from lactate at a rate of 54 mL/h.g dry weight; the efficiency of H₂ production by immobilized cells was comparable to that of free cells and was 60 to 65% that of the theoretical maximum from lactate. Carrageenan-entrapped cells produced H₂ steadily over a 16-day period.     

Anaerobic and aerobic metabolism of diverse aromatic compounds by the photosynthetic bacterium Rhodopseudomonas palustris.

The purple nonsulfur photosynthetic bacterium Rhodopseudomonas palustris used diverse aromatic compounds for growth under anaerobic and aerobic conditions. Many phenolic, dihydroxylated, and methoxylated aromatic acids, as well as aromatic aldehydes and hydroaromatic acids, supported growth of strain CGA001 in both the presence and absence of oxygen. Some compounds were metabolized under only aerobic or under only anaerobic conditions. Two other strains, CGC023 and CGD052, had similar anaerobic substrate utilization patterns, but CGD052 was able to use a slightly larger number of compounds for growth. These results show that R. palustris is far more versatile in terms of aromatic degradation than had been previously demonstrated. A mutant (CGA033) blocked in aerobic aromatic metabolism remained wild type with respect to anaerobic degradative abilities, indicating that separate metabolic pathways mediate aerobic and anaerobic breakdown of diverse aromatics. Another mutant (CGA047) was unable to grow anaerobically on either benzoate or 4-hydroxybenzoate, and these compounds accumulated in growth media when cells were grown on more complex aromatic compounds. This indicates that R. palustris has two major anaerobic routes for aromatic ring fission, one that passes through benzoate and one that passes through 4-hydroxybenzoate.