Purification and properties of phototrophic bacteria Thiocapsa roseopersicina hydrogenase bound with chromatophores]

The method of solution and puridication of hydrogenase from chromatophores of purpur sulphur bacteria Thiocapsa roseopersicina strain BBS are described. Hydrogenase molecular weight is 73000. It contains 4,4 mole S2- and 3.1 mole Fe2+ per mole of protein; pI 4.15. The enzyme absorption spectrum has the maximun et 400-410 nm, which is characteristic of proteins containing non-haem iron. Membrane--linked enzyme as well as soluble hydrogenase of that microorganism is characterized by high thermal stability: inactivation occurs at the temperature above 78 degrees C when the optimal temperature for that enzyme is 70 degrees C. Homogenous enzyme catalyses D2--H2O exchange reaction, reversible redox reaction of methyl viologene and benzyl viologene.     

Chemolithotrophic growth of the phototrophic sulfur bacterium Thiocapsa roseopersicina

Chemotrophic growth capacities of the purple sulfur bacterium Thiocapsa roseopersicina strain M1 were studied in continuous culture under thiosulfate limitation.Pigment synthesis was completely inhibited upon a shift from anaerobic to semi-aerobic conditions (52 μM O₂) in the light, but no active breakdown occurred. During the transient state, the cells grew in a mixed photo- and chemolithotrophic mode; the specific respiration rate gradually increased with a concomitant drop in the bacteriochlorophyll a content. Photolithotrophically grown cells have the ability to respire. It was concluded that photosynthesis and respiration compete for electrons, but that photosynthesis is preferred under electron donor-limiting conditions, when the cells still contain large amounts of pigments. Eventually, a fully chemolithotrophic steady state was attained.The chemolithotropic growth of T. roseopersicina was studied in the dark under semiaerobic conditions at various dilution rates. The maximum specific growth rate was 68% of the maximum attainable growth rate under photolithotrophic conditions. The growth affinity for thiosulfate was high (K_m = 1.5 μM). The yield on thiosulfate under chemolithotrophic conditions exceeded that of thiobacilli. Oxygen uptake was studied in short-term experiments. It was shown that respiration in T. roseopersicina has a K_m of approx. 1 μM O₂. the ecological importance for T. roseopersicina of chemolithotrophic growth and pigment content is discussed with respect to the occurrence of T. roseopersicina in laminated microbial ecosystems and its possible competition with colorless sulfur bacteria.     

The capacity of phototrophic sulfur bacterium Thiocapsa roseopersicina for chemosynthesis

Purple sulfur bacterium Thiocapsa roseopersicina strain BBS requiring vitamin B₁₂ may grow in the dark in media containing no other organic compounds. Under such conditions the cells oxidize sulfide and thiosulfate with the use of O₂ and assimilate carbon dioxide. After 10–30 s assimilation of NaH¹⁴CO₃ about 60% of radioactivity is found in phosphorylated compounds characteristic for the reductive pentose phosphate cycle. The possibility of the function of this cycle in the dark in the presence of O₂ is confirmed by the capacity of cells grown under such conditions to synthesize ribulose-1,5-diphosphate carboxylase. All this evidence suggests the ability of T. roseopersicina to change from phototrophy to aerobic chemolithoautotrophy.     

Purification and properties of cytochrome c552 from purple sulfur bacterium Thiocapsa roseopersicina

The method of purification up to electrophoretical homogeneity of cytochrome c552 from the phototrophic bacterium Thiocapsa roseopersicina, strain BBS is described. For the cytochrome absorption spectrum the maxima at 417, 523 and 552 nm are characteristic for the reduced state and at 409 nm--for the oxidized state. The molecular weight is equal to 62000. The cytochrome contains two hemes per molecule and consists of two subunits. pI is 4.1; E0' is about 10 mV. Cytochrome c552 is a flavoprotein according to its fluorescence spectrum and subunit structure. T. roseopersicina cytochrome c552 is able to be reduced with sulphide, cysteine and ascorbate as well as with H2 in the presence of hydrogenase from the same bacterium. These data suggest that cytochrome c552 from T. roseopersicina functions in vivo at the initial stage of electron transport from hydrogen and sulphide.     

Localization of hydrogenase in the purple sulphur bacterium Thiocapsa roseopersicina

The localization of hydrogenase in the phototrophic bacterium Thiocapsa roseopersicina was investigated by subcellular fractionations, and transmission electron microscopic immunocytochemistry. By using sonicated cells and measuring in vitro hydrogenase activities in soluble and membrane fractions, respectively, a weak hydrophobic interaction between the hydrogenase enzyme and the T. roseopersicina membranes was observed. Polyclonal antisera directed against the purified hydrogenase were raised in rabbits and exhibited one band in native-PAGE/Western immunoblot analysis. Native-PAGE/activity stain confirmed the identity of this band as being hydrogenase. Immunocytolocalization experiments using ultrathin sections showed an internal localization of the hydrogenase enzyme. A higher specific labeling was associated with chromatophores, indicating a possible coupling of hydrogenase with the photosynthetic membranes in the T. roseopersicina cells.     

Influence of metal ions on hydrogenase from the purple sulfur bacterium Thiocapsa roseopersicina

The effects of some metal ions on the activity and activation of Thiocapsa roseopersicina hydrogenase have been studied. Inhibitory effects of Ni2+ and Cd2+ on the catalytic activity of the enzyme were reversible and competitive with respect to methyl viologen (MV) in the reaction of hydrogen oxidation. The affinity of these metal ions to the enzyme increased significantly with increasing pH, suggesting that their interactions are determined by electrostatic forces. Cu2+ and Hg2+ irreversibly inhibited the hydrogenase activity. A decrease in absorption of hydrogenase at 400 nm in the presence of these metal ions is indicative of the destruction of the FeS cluster in the enzyme.     

Aerobic turnover of dimethyl sulfide by the anoxygenic phototrophic bacterium Thiocapsa roseopersicina

This is the first report describing the complete oxidation of dimethyl sulfide (DMS) to sulfate by an anoxygenic, phototrophic purple sulfur bacterium. Complete DMS oxidation was observed in cultures of Thiocapsa roseopersicina M11 incubated under oxic/light conditions, resulting in a yield of 30.1 mg protein mmol^–1. No oxidation of DMS occurred under anoxic/light conditions. Chloroform, methyl butyl ether, and 3-amino-1,2,4-triazole, which are specific inhibitors of aerobic DMS oxidation in thiobacilli and hyphomicrobia, did not affect DMS oxidation in strain M11. This could be due to limited transport of the inhibitors through the cell membrane. The growth yield on sulfide as sole electron donor was 22.2 mg protein mmol^–1 under anoxic/light conditions. Since aerobic respiration of sulfide would have resulted in yields lower than 22 mg protein mmol^–1, the higher yield on DMS under oxic/light conditions suggests that the methyl groups of DMS have served as an additional carbon source or as an electron donor in addition to the sulfide moiety. The kinetic parameters V _max and K _m for DMS oxidation under oxic/light conditions were 12.4 ± 1.3 nmol (mg protein)^–1 min^–1 and 2 μM, respectively. T. roseopersicina M11 also produced DMS by cleavage of dimethylsulfoniopropionate (DMSP). Specific DMSP cleavage rates increased with increasing initial substrate concentrations, suggesting that DMSP lyase was only partly induced at lower initial DMSP concentrations. A comparison of T. roseopersicina strains revealed that only strain M11 was able to oxidize DMS and cleave DMSP. Both strain M11 and strain 5811 accumulated DMSP intracellularly during growth, while strain 1711 showed neither of these characteristics. Phylogenetic comparison based on 16S rRNA gene sequence revealed a similarity of 99.0% between strain M11 and strain 5811, and 97.6% between strain M11 and strain 1711. DMS and DMSP utilization thus appear to be strain-specific. Received: 26 March 1999 / Accepted: 18 June 1999     

Purification and properties of NADP-reductase of phototropic bacteria Thiocapsa roseopersicina]

The method of purification up to homogenous states and properties of NADP-reductase of purple bacteria Thiocapsa roseopersicina, strain BBS, are described. The molecular weight of NADP-reductase is about 47 000; it is flavoprotein consisting of two subunits. Atebrim and chloromercury bensoate inhibit the activity of NADP-reductase (34% and 33--60%, respectively). The enzyme is specific to NADPH; it catalyzes menadion-reductase reaction, diaphorase reaction of benzyl viologen reduction, oxidation of reduced benzyl viologen in the presence of NADP, reduction of ferredoxin and cytochrome c in the presence of NADPH, but it is not capable to catalyze transhydrogenase reaction.     

Three-dimensional structure of the nickel-containing hydrogenase from Thiocapsa roseopersicina.

The three-dimensional structure of the nickel-containing hydrogenase from Thiocapsa roseopersicina has been determined at a resolution of 2 nm in the plane and 4 nm in the vertical direction by electron microscopy and computerized image processing on microcrystals of the enzyme. The enzyme forms a large ring-shaped complex containing six each of the large (62-kDa) and small (26-kDa) subunits. The complex is very open, with six well-separated dumbbell-shaped masses surrounding a large cylindrical hole. Each dumbbell is interpreted as consisting of one large and one small subunit.     

Simultaneous phototrophic and chemotrophic growth in the purple sulfur bacterium Thiocapsa roseopersicina M1

Abstract The anoxygenic phototrophic purple sulfur bacterium Thiocapsa roseopersicina was grown in illuminated continuous cultures with thiosulfate as growth limiting substrate. Aeration resulted in completely colorless cells growing chemotrophically, whereafter the conditions were changed to a 23 h oxic/1 h anoxic regime. After 11 volume changes at a dilution rate of 0.031 h⁻¹ (35% of μ_max) a time dependent equilibrium was established. During the 23 h oxic periods bacteriochlorophyll a synthesis (BChl a ) was not observed, whereas during the 1 h anoxic periods synthesis was maximal (i.e. 1.1 μg (mg protein)⁻¹ h⁻¹). As a result the BChl a concentration gradually increased from zero to an average value over 24 h of 1.9 μg (mg protein)⁻¹. Concomitantly, the protein concentration increased from 13.9 mg 1⁻¹ during continuous oxic conditions to 28.8 mg 1⁻¹. For comparison, the protein concentration during fully phototrophic growth at an identical thiosulfate concentration in the inflowing medium was 53.7 mg 1⁻¹. The specific respiration rate was 8 μmol O₂ (mg protein)⁻¹ h⁻¹ during full chemotrophic growth and gradually decreased to 3.5 μmol O₂ (mg protein)⁻¹ h⁻¹ after 11 volume changes at the regime employed. These data show that T. rosepersicina is able to simultaneously utilize light and aerobic respiration of thiosulfate as sources of energy. The ecological relevance of the data is discussed.     

Localization of hydrogenase in Thiocapsa roseopersicina photosynthetic membrane

The photosynthetic cell membrane is impermeable to the oxidized redox dyes Methyl Viologen and Benzyl Viologen, whereas the reduced forms easily penetrate into the cells. By exploiting this permeability difference, the orientation of the membrane-bound hydrogenase has been determined.     

Interaction of HydSL hydrogenase from the purple sulfur bacterium Thiocapsa roseopersicina BBS with methyl viologen and positively charged polypeptides

The effect of polypeptides having different charge on the activity of Thiocapsa roseopersicina HydSL hydrogenase was studied. Strong inhibition was shown for poly-L-lysine bearing positive charge. The inhibition was reversible and competitive to methyl viologen, an electron acceptor, in the reaction of hydrogen oxidation catalyzed by the hydrogenase. Peptides carrying less positive charge had weaker inhibiting effect, while neutral and negatively charged peptides did not inhibit the hydrogenase. Molecular docking of poly-L-lysine to T. roseopersicina hydrogenase showed strong affinity of this polypeptide to the acceptor-binding site of the enzyme. The calculated binding constant is close to the experimentally measured value (K _i = 2.1 μM).     

Structural rearrangements in active and inactive forms of hydrogenase from Thiocapsa roseopersicina.

The electrophoretic behavior of Thiocapsa roseopersicina hydrogenase on sodium dodecyl sulfate gels demonstrates that the protein exists in two active forms, A1 and A2, which may be interconverted. Each of these forms has a characteristic electrophoretic mobility and differs in its sensitivity to O2. Form A1 is O2-labile and converts to A2 under O2. Form A2 is less sensitive to O2 and may be converted into A1 under H2 atmosphere. Both active forms are present in aerobically isolated samples. Because the proteins are still active on 15% sodium dodecyl sulfate gels, they are not completely denatured, and the apparent molecular masses do not necessarily represent the true molecular masses of the enzymes. A1 has an Rf = 0.19, corresponding to an apparent molecular mass of 90 kDa, and A2 has an Rf = 0.35, corresponding to an apparent molecular mass of 49 kDa. A sedimentation equilibrium centrifugation study of the active enzyme shows that the holoenzyme has a molecular mass of 98 kDa. Form A2 may be separated into two subunits of molecular mass of 64 kDa and 34 kDa, respectively. Thus, form A2 represents the holoenzyme with a true molecular mass of 98 kDa. Amino acid compositions and N-terminal amino acid sequences of the A2 protein and these subunits are consistent with a heterodimeric holoenzyme. The relationship between the conformational changes detected in this study and a three-state scheme proposed on the basis of EPR spectroscopic studies of the metal-containing cofactors present in the enzyme is also discussed.     

Protein structural changes associated with active and inactive states of hydrogenase from Thiocapsa roseopersicina

Electrophoretic and isoelectrofocusing behavior of the hydrogenase from Thiocapsa roseopersicina under various conditions revealed remarkable properties of this enzyme: there are two active forms which differ in their molecular masses as well as in oxygen sensitivity; the apparent molecular masses of the active hydrogenase forms (90 and 49 kDa) differ considerably from those in the inactive state (64, 34, and 15 kDa); the active forms and some of the inactivated ones can be transformed into each other reversibly; urea can unfold the 64 and 34 kDa proteins but not the 49 kDa form at room temperature; the pI of these proteins are different in the presence of urea. The results suggest large rearrangements in the hydrogenase protein structure which are associated with the enzymatically active and inactive states. It is concluded that reversible formation of disulfide bonds cannot be the major cause for maintaining the enzyme conformation. Strong hydrophobic interactions are suggested to be primarily responsible for the structural stability and for the rearrangements.     

Metal composition analysis of hydrogenase from Thiocapsa roseopersicina by proton induced X-ray emission spectroscopy

Polyacrylamide gel electrophoresis combined with proton induced X-ray emission spectroscopy is suitable to identify and to determine the relative amounts of protein bound metals in situ. An analysis of the hydrogenase from Thiocapsa roseopersicina has shown the feasibility of the technique and provides new insight into the relative amount as well as the intramolecular location of Fe and Ni metal atoms in this enzyme.     

Spectroscopic characterization of the nickel and iron-sulphur clusters of hydrogenase from the purple photosynthetic bacterium Thiocapsa roseopersicina. 2. Electron spin-echo spectroscopy

Pulsed electron-spin-resonance techniques were applied to the hydrogenase of the purple photosynthetic bacterium Thiocapsa roseopersicina, an enzyme which contains nickel and iron-sulphur clusters but no flavin. The linear electric field effect profile of the spectrum in the region of g = 2.01 indicated that the strong ESR signal in the oxidized protein is due to a [3Fe-4S] cluster. The electron spin-echo envelope of this spectrum was modulated by hyperfine interactions with 1H and 14N nuclei, probably from the polypeptide chain. The ESR spectrum of this species shows a complex pattern arising from spin-spin interaction with another paramagnet. When the protein was partially reduced by ascorbate plus phenazine methosulphate, the complexity of the spectrum was abolished but the form of the electron spin-echo envelope modulation (ESEEM) pattern was unchanged. This indicates that the reversible disappearance of the spin-spin interaction pattern on partial reduction is not due to cluster interconversion to a [4Fe-4S] cluster. In the ESR spectrum of nickel(III), weak hyperfine interactions with 1H and 14N were also observed by ESEEM. The nature of the interacting nuclei is discussed.     

Enhanced aniline degradation by Desulfatiglans anilini in a synthetic microbial community with the phototrophic purple sulfur bacterium Thiocapsa roseopersicina

Desulfatiglans anilini is a sulfate-reducing bacterium (SRB) capable of oxidizing aniline, although growth and aniline turnover rates are slow, making it difficult to analyze the metabolism of the strain. Therefore, this study was designed to investigate the effect of sulfide on growth of D. anilini cultures, in order to improve its growth and aniline turnover rates, and study the biochemical mechanisms of sulfide inhibition. Hydrogen sulfide was found to inhibit growth of D. anilini, regardless of whether the strain was grown with aniline or phenol, and complete inhibition was observed at 20 mM hydrogen sulfide. For improving the growth of D. anilini with aniline, the sulfide-consuming phototrophic bacterium Thiocapsa roseopersicina was co-cultured in a synthetic microbial community with D. anilini using a co-cultivation device that continuously removed hydrogen sulfide from the culture. The doubling time of D. anilini with aniline was 15 days in the co-cultivation device, compared to 26 days in the absence of a sulfide-oxidizing partner. Moreover, the aniline degradation rate was significantly increased by a factor of 2.66 during co-cultivation of D. anilini with T. roseopersicina. The initial carboxylation reaction during aniline degradation was measured in cell-free extracts of D. anilini with carbon dioxide (CO₂) as a co-substrate in the presence of aniline and ATP. The effects of hydrogen sulfide on this aniline carboxylating system and on phenylphosphate synthase activity for phenol activation were studied, and it was concluded that hydrogen sulfide severely inhibited these enzyme activities.     

The role of Hox hydrogenase in the H2 metabolism of Thiocapsa roseopersicina

The purple sulfur phototrophic bacterium Thiocapsa roseopersicina BBS synthesizes at least three NiFe hydrogenases (Hox, Hup, Hyn). We characterized the physiological H(2) consumption/evolution reactions in mutants having deletions of the structural genes of two hydrogenases in various combinations. This made possible the separation of the functionally distinct roles of the three hydrogenases. Data showed that Hox hydrogenase (unlike the Hup and Hyn hydrogenases) catalyzed the dark fermentative H(2) evolution and the light-dependent H(2) production in the presence of thiosulfate. Both Hox(+) and Hup(+) mutants demonstrated light-dependent H(2) uptake stimulated by CO(2) but only the Hup(+) mutant was able to mediate O(2)-dependent H(2) consumption in the dark. The ability of the Hox(+) mutant to evolve or consume hydrogen was found to depend on a number of interplaying factors including both growth and reaction conditions (availability of glucose, sulfur compounds, CO(2), H(2), light). The study of the redox properties of Hox hydrogenase supported the reversibility of its action. Based on the results a scheme is suggested to describe the role of Hox hydrogenase in light-dependent and dark hydrogen metabolism in T. roseopersicina BBS.     

Induced mutagenesis by bleomycin in the purple sulfur bacterium Thiocapsa roseopersicina

Cell death and mutagenesis in bleomycin-treated cells of Thiocapsa roseopersicina (a purple sulfur bacterium) was studied by cultivation in a semisolid medium (agar-shake technique). This technique has also proven useful in assessing the frequency of antibiotic mutations by detecting and counting individual colonies of Thiocapsa roseopersicina. The frequencies of spontaneous mutants resistant to ampicillin, rifampicin, cloramphenicol, tetracycline, kanamycin, streptomycin, and neomycin were also studied: they ranged between 2×10^-9 and 9×10^-8. Bleomycin (4 g/ml) sharply increased the frequency of ampicillin-resistant mutants, from 10^-8 (spontaneous) to 4×10^-4 (induced), in 17 h. An inducible, error-prone mechanisms of DNA synthesis seems to be responsible for this enhancement of the mutagenic effect. This is the first report on the sensitivity to several antibiotics, and capacity of lethality and mutagenesis by bleomycin has been studied in a purple sulfur bacterium.