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.     

Purification and properties of hydrogenase from phototrophic bacterium Thiocapsa roseopersicina]

The isolation method and some peoperties of purple sulphur bacteria (Thiocapsa roseopersicina strain BBS) hydrogenase are described Hydrogenase molecular weight is found to be 66000; it contains 3.7 moles of S2- and 3.9 moles of Fe2+ per one mole of the enzyme;pI=4.2. The enzyme absorption spectrum has the maximum at 400-412 nm which is characteristic of proteins containing non-haem iron. Hydrogenase is suggested to consist pf 4 subunits of two types: with molar weight 27000 and 6000. Unlike other hydrogenases, this enzyme is rather resistant to O2 and is more thermostable: the inactivation of the enzyme was observed at the temperature above 80 degrees C; Hydrogenase preparation catalyses D2-H2O exchange reaction, H2 evolution from the reduced methyl viologene (MV) and H2 absorption in the presense of MV or benzylviologene but not in the presense of NAD(P), FAD, FMN, azocarmine, methylene blue and ferricyanide.     

Growth of Thiocapsa roseopersicina purple sulfur bacteria in darkness under anaerobic conditions]

The phototrophic sulphur bacterium. Thiocapsa roseopersicina, strain BBS, was grown under anaerobic conditions in the darkness on the medium containing glucose and thiosulphate or molecular sulphur. The assimilation of glucose is accompanied by the accumulation of small amounts of pyruvate in the medium, and the uptake of thiosulphate or molecular sulphur leads to the formation of sulphates and hydrogen sulphide.     

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.     

Growth and oxidation of sulfur compounds by Thiocapsa roseopersicina in darkness]

The purple sulphur bacterium Thiocapsa roseopersicina, strain BBS, grown in the darkness in aerobic autotrophic conditions, oxidized sulphides to free sulphur and then to sulphates. This was accompanied with the fixation of carbon dioxide by the cells. Addition of glucose to the mineral medium increased the biomass yield; the cells oxidized thiosulphate still at a high rate. These results prove the possibility of switching T. roseopersicina from photosynthesis to a dark chemolithautotrophic way of life.     

Comparative study of NADP-reductase properties in two species of purple bacteria]

Unlike Rhodospirillum rubrum, the highly purified preparations of NADP-reductase Thiocapsa roseopersicina are capable of reduction of cytochrome c though they do not catalyse diaphorase reaction in the presence of methyl viologen or benzyl viologen and NADH. T. roseopersicina reductase has more high temperature optimum (50-65 degrees) and more high thermal stability (65 degrees) and it is capable to catalyse diaphorase and menadione-reductase reactions under more high pH values (11.0-12.0) than NADP-reductase of R. rubrum. NADP-reductase of T. roseopersicina is more stable under storing than the enzyme from R. rubrum: the semi-inactivation period of the enzyme when storing in Ar or the air is about 10 and 4 days, respectively, and it takes about three days for R. rubrum.     

Low potential c-type cytochrome of Thiocapsa roseopersicina

The low potential c-type cytochrome from the phototrophic purple sulphur bacterium Thiocapsa roseopersicina, strain BBS was isolated in electrophoretically homogeneous state. The bulk of the cytochrome (approximately 90%) after disruption of the cells remained in the membrane fraction. The absorption spectrum of the cytochrome was characterized by the maxima at 420, 523 and 552 nm in the reduced state and at 408 nm in the oxidized one. The cytochrome interacted with CO in the reduced state. The molecular weight of the cytochrome is 50 000. The cytochrome contains great amounts of phenylalanine, leucine, valine, aspartic and glutamic acids and can be reduced by dithionite but not by cysteine, sulfide or ascorbate. Besides, the cytochrome can also be reduced by NAD(P)H in the presence of NAD(P)-reductases of T. roseopersicina, when ferredoxin of Spirulina platensis or benzyl viologen are added to the reaction mixture. The cytochrome can act as an electron donor (acceptor) for T. roseopersicina hydrogenase.     

Macromolecular subunits on the surfaces of the cell wall of Thiocapsa roseopersicina]

A layer of fungiform macromolecular subunits was found on the surface of the cell wall of Thiocapsa roseopersicina, a purple sulphur bacterium, strain BBS. The cap of a particle has a diameter of 40 to 60 A; the stalk is 80 to 100 A long and 20 to 30 A thick. Under the conditions of nitrogen fixation and a low content of vitamin B12 (0.1 mcg/litre) in the cultural broth, a second layer of similar particles is formed over the first layer.     

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.     

Purification and Properties of Adenylyl Sulfate Reductase from the Phototrophic Sulfur Bacterium, Thiocapsa roseopersicina

Adenylyl sulfate reductase was purified from Thiocapsa roseopersicina 60- to 80- fold, and the properties were studied. The molecular weight is 180,000. The enzyme contains, per molecule; one flavine group, two heme groups of cytochrome c character, four atoms of nonheme iron, and six labile sulfide groups. Cytochrome c and ferricyanide serve as electron acceptors. With ferricyanide as the electron acceptor, the pH optimum of the enzyme is at 8.0; with cytochrome c, the pH optimum is at 9.0. Of the nucleotides studied, adenosine 5'-monophosphate is most effective. The influence of substrate concentrations on the activity of the enzyme was studied, and the K(m) values for sulfite, adenosine 5'-monophosphate, ferricyanide, and cytochrome c were determined. The properties of the enzyme are compared with those of adenylyl sulfate reductases purified from sulfate-reducing bacteria and thiobacilli.     

Photo-autotrophic growth of Thiocapsa roseopersicina on dimethyl sulfide

Abstract: The purple sulfur bacterium Thiocapsa roseopersicina was examined for photo-autotrophic growth on dimethyl sulfide (DMS). The maximum specific growth rate μ _max (0.068 h⁻¹), saturation constant K _s (38 μm l⁻¹), and yield (5.24 mg protein mmol⁻¹ DMS) were determined in chemostat experiments. Dimethyl sulfoxide was the only product of DMS oxidation. Batch experiments revealed the simultaneous oxidation of DMS and hydrogen sulfide.     

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.     

Heterologous expression of Alteromonas macleodii and Thiocapsa roseopersicina [NiFe] hydrogenases in Synechococcus elongatus

Oxygen-tolerant [NiFe] hydrogenases may be used in future photobiological hydrogen production systems once the enzymes can be heterologously expressed in host organisms of interest. To achieve heterologous expression of [NiFe] hydrogenases in cyanobacteria, the two hydrogenase structural genes from Alteromonas macleodii Deep ecotype (AltDE), hynS and hynL, along with the surrounding genes in the gene operon of HynSL were cloned in a vector with an IPTG-inducible promoter and introduced into Synechococcus elongatus PCC7942. The hydrogenase protein was expressed at the correct size upon induction with IPTG. The heterologously-expressed HynSL hydrogenase was active when tested by in vitro H(2) evolution assay, indicating the correct assembly of the catalytic center in the cyanobacterial host. Using a similar expression system, the hydrogenase structural genes from Thiocapsa roseopersicina (hynSL) and the entire set of known accessory genes were transferred to S. elongatus. A protein of the correct size was expressed but had no activity. However, when the 11 accessory genes from AltDE were co-expressed with hynSL, the T. roseopersicina hydrogenase was found to be active by in vitro assay. This is the first report of active, heterologously-expressed [NiFe] hydrogenases in cyanobacteria.     

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.     

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.     

Reconstruction of HydSL Hydrogenase from Thiocapsa roseopersicina after Cyanide Inhibition

Applied Biochemistry and Microbiology - It is shown that the catalytic center of Thiocapsa roseopersicina remains active after prolonged treatment with cyanide. It was found that the incubation of...     

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.     

Hydrogenase activity in Thiocapsa roseopersicina according to the D2--H20 metabolic reaction]

Extracts of Thiocapsa roseopersicina cells show hydrogenase activity, measured by evolution of H2 from reduced methylviologene (MV) and by D2-H2O exchange reaction. According to these reactions the most part of hydrogenases is found to be in the soluble fraction. Hydrogenase activity measured in the exchange reaction is completely inhibited by p-chloromercurybenzoate (5-10- minus 3 M), iodacetate (1-10- minus 2 M) and 26% inhibited by KCN and o-phenanthroline (5-10- minus 3 M). Evolution of H2 from reduced MV was not inhibited by o-phenanthroline, KCN and iodacetate and was inhibited by 66% only with p-chloromercurybenzoate. Light and ATP stimulated hydrogenase activity of chromatophores did not affect on its activity in the soluble fraction. The results obtained show that there are certain differences in hydrogenase systems responsible for the exchange reaction and evolution of H2.