Purification and properties of the sulfur oxygenase/reductase from the acidothermophilic archaeon, <Emphasis Type="Italic">Acidianus </Emphasis>strain S5

The sulfur oxygenase/reductase (SOR) of Acidianus strain S5 was purified and characterized after expressing the SOR gene in a recombinant strain of Escherichia coli. The N-terminal sequence of the purified SOR protein was the same as the deduced amino acid sequence from previously cloned SOR genes. Enzymatic studies indicated that the SOR catalyzed the conversion of elemental sulfur (S(o)) to sulfite, thiosulfate, and sulfide. The optimal pH and temperature were 5.0 and 70 degrees C, respectively. Comparison of this SOR and that of A. ambivalens revealed several differences between these two SORs. The most striking difference is that the SOR of Acidianus S5 had maximal activity at acidic pH. By application of anti-SOR serum and the Western blot technique, it was found that SOR proteins existed in A. brierleyi and in Acidianus S5 cells cultivated with thiosulfate as the sole energy source, indicating that SOR may also play a role in thiosulfate metabolism.     

The phs gene and hydrogen sulfide production by Salmonella typhimurium.

Salmonella typhimurium produces H2S from thiosulfate or sulfite. The respective pathways for the two reductions must be distinct as mutants carrying motations in phs, chlA, and menB reduced sulfite, but not thiosulfate, to H2S, and glucose repressed the production of H2S from thiosulfate while it stimulated its production from sulfite. The phs and chlA mutants also lacked a methyl viologen-linked thiosulfate reductase activity present in anaerobically grown wild-type cultures. A number of hydroxylamine, transposon Tn10 insertion, and Mu d1(Apr lac) operon fusion mutants defective in phs were characterized. One of the hydroxylamine mutants was an amber mutant, as indicated by suppression of its mutation in a supD background. The temperature-sensitive phs mutants produced H2S and methyl viologen-linked thiosulfate reductase at 30 degrees C but not at 42 degrees C. The reductases in all such mutants grown at 30 degrees C were as thermostable as the wild-type enzyme and did not differ in electrophoretic relative mobility, suggesting that phs is not the structural gene for thiosulfate reductase. Expression of beta-galactosidase in phs::Mu d1(Apr lac) mutants was dependent on anaerobiosis and the presence of reduced sulfur. It was also strongly influenced by carbon source and growth stage. The results are consistent with a model in which the phs gene encodes a regulatory protein essential for the reduction of thiosulfate to hydrogen sulfide.     

Purification and properties of thiosulfate reductase from Desulfovibrio gigas.

Thiosulfate reductase of the dissimilatory sulfate-reducing bacterium Desulfovibrio gigas has been purified 415-fold and its properties investigated. The enzyme was unstable during the different steps of purification as well as during storage at - 15 degrees C. The molecular weight of thiosulfate reductase estimated from the chromatographic behaviour of the enzyme on Sephadex G-200 was close to 220000. The absorption spectrum of the purified enzyme exhibited a protein peak at 278 nm without characteristic features in the visible region. Thiosulfate reductase catalyzed the stoichiometric production of hydrogen sulfide and sulfite from thiosulfate, and exhibited tetrathionate reductase activity. It did not show sulfite reductase activity. The optimum pH of thiosulfate reduction occurred between pH 7.4 and 8.0 and its Km value for thiosulfate was calculated to be 5 - 10(-4)M. The sensitivity of thiosulfate reductase to sulfhydryl reagent and the reversal of the inhibition by cysteine indicated that one or more sulfhydryl groups were involved in the catalytic activity. The study of electron transport between hydrogenase and thiosulfate reductase showed that the most efficient coupling was obtained with a system containing cytochromes c3 (Mr = 13000) and c3 (Mr = 26000).     

Sulfite-oxido-reductase is involved in the oxidation of sulfite in Desulfocapsa sulfoexigens during disproportionation of thiosulfate and elemental sulfur

The enzymatic pathways of elemental sulfur and thiosulfate disproportionation were investigated using cell-free extract of Desulfocapsa sulfoexigens. Sulfite was observed to be an intermediate in the metabolism of both compounds. Two distinct pathways for the oxidation of sulfite have been identified. One pathway involves APS reductase and ATP sulfurylase and can be described as the reversion of the initial steps of the dissimilatory sulfate reduction pathway. The second pathway is the direct oxidation of sulfite to sulfate by sulfite oxidoreductase. This enzyme has not been reported from sulfate reducers before. Thiosulfate reductase, which cleaves thiosulfate into sulfite and sulfide, was only present in cell-free extract from thiosulfate disproportionating cultures. We propose that this enzyme catalyzes the first step in thiosulfate disproportionation. The initial step in sulfur disproportionation was not identified. Dissimilatory sulfite reductase was present in sulfur and thiosulfate disproportionating cultures. The metabolic function of this enzyme in relation to elemental sulfur or thiosulfate disproportionation was not identified. The presence of the uncouplers HQNO and CCCP in growing cultures had negative effects on both thiosulfate and sulfur disproportionation. CCCP totally inhibited sulfur disproportionation and reduced thiosulfate disproportionation by 80% compared to an unamended control. HQNO reduced thiosulfate disproportionation by 80% and sulfur disproportionation by 90%.     

Purification and properties of thiosulfate reductase from Desulfovibrio vulgaris, Miyazaki F

Thiosulfate reductase was purified to an almost homogeneous state from Desulfovibrio vulgaris, strain Miyazaki F, by ammonium sulfate precipitation, chromatography on DEAE-Toyopearl, Ultrogel AcA 34, and hydroxylapatite, and disc electrophoresis. The specific activity was increased 580-fold over the crude extract. The molecular weight was determined by gel filtration to be 85,000-89,000, differing from those reported for thiosulfate reductases from other Desulfovibrio strains. The enzyme had no subunit structure. When coupled with hydrogenase and methyl viologen, it stoichiometrically reduced thiosulfate to sulfite and sulfide with consumption of hydrogen. It did not reduce sulfite or trithionate. Cytochrome c3 was active as an electron donor. More than 0.75 mM thiosulfate inhibited the enzyme activity. o-Phenanthroline and 2,2'-bipyridine inhibited the enzyme and ferrous ion stimulated the reaction.     

The sulfur oxygenase reductase from the mesophilic bacterium Halothiobacillus neapolitanus is a highly active thermozyme

A biochemical, biophysical, and phylogenetic study of the sulfur oxygenase reductase (SOR) from the mesophilic gammaproteobacterium Halothiobacillus neapolitanus (HnSOR) was performed in order to determine the structural and biochemical properties of the enzyme. SOR proteins from 14 predominantly chemolithoautotrophic bacterial and archaeal species are currently available in public databases. Sequence alignment and phylogenetic analysis showed that they form a coherent protein family. The HnSOR purified from Escherichia coli after heterologous gene expression had a temperature range of activity of 10 to 99°C with an optimum at 80°C (42 U/mg protein). Sulfite, thiosulfate, and hydrogen sulfide were formed at various stoichiometries in a range between pH 5.4 and 11 (optimum pH 8.4). Circular dichroism (CD) spectroscopy and dynamic light scattering showed that the HnSOR adopts secondary and quaternary structures similar to those of the 24-subunit enzyme from the hyperthermophile Acidianus ambivalens (AaSOR). The melting point of the HnSOR was ≈20°C lower than that of the AaSOR, when analyzed with CD-monitored thermal unfolding. Homology modeling showed that the secondary structure elements of single subunits are conserved. Subtle changes in the pores of the outer shell and increased flexibility might contribute to activity at low temperature. We concluded that the thermostability was the result of a rigid protein core together with the stabilizing effect of the 24-subunit hollow sphere.     

Nutritional studies with Pseudomonas aeruginosa grown on inorganic sulfur sources.

Pseudomonas aeruginosa was grown on a succinate-basal salts medium supplemented with various inorganic sulfur compounds as its sole source of sulfur. The organism was able to grow on the sodium salts of sulfide, thiosulfate, tetrathionate, dithionite, metabisulfite, sulfite, or sulfate, but not on those of dithionate. Analyses of the culture media after 24 h of growth indicated accumulation of sulfate from each inorganic sulfur source except sulfate. Manometric studies with resting cells obtained by growth on each of these sulfur sources yielded net oxygen uptake for all substrates except sulfite and dithionate. Similar results were obtained with extracts from these cells by spectrophotometric techniques. Thiosulfate oxidase activity appeared to be induced by growth on sulfide, thiosulfate, or tetrathionate, with little or no activity observed when cells were grown on inorganic sulfur sources of higher oxidative states. Metabisulfite oxidase appeared to be associated with growth on all inorganic sulfur compounds. Rhodanese activity appeared to be constitutively present, and its activity, observed only in soluble fraction, seemed independent of the growth medium employed. Thiosulfate and tetrathionate oxidase activities were studied in greater detail than some of the other sulfur oxidases, and both were found to be distributed between particulate and soluble fractions.     

Anaerobic oxidation of thiosulfate and elemental sulfur in Thiobacillus denitrificans

Thiobacillus denitrificans strain RT could be grown anaerobically in batch culture on thiosulfate but not on other reduced sulfur compounds like sulfide, elemental sulfur, thiocyanate, polythionates or sulfite. During growth on thiosulfate the assimilated cell sulfur was derived totally from the outer or sulfane sulfur. Thiosulfate oxidation started with a rhodanese type cleavage between sulfane and sulfone sulfur leading to elemental sulfur and sulfite. As long as thiosulfate was present elemental sulfur was transiently accumulated within the cells in a form that could be shown to be more reactive than elemental sulfur present in a hydrophilic sulfur sol, however, less reactive than sulfane sulfur of polythionates or organic and inorganic polysulfides. When thiosulfate had been completely consumed, intracellular elemental sulfur was rapidly oxidized to sulfate with a specific rate of 45 natom S°/min·mg protein. Extracellularly offered elemental sulfur was not oxidized under anaerobic conditions.     

Reduced sulfur compound oxidation by Thiobacillus caldus.

The oxidation of reduced inorganic sulfur compounds was studied by using resting cells of the moderate thermophile Thiobacillus caldus strain KU. The oxygen consumption rate and total oxygen consumed were determined for the reduced sulfur compounds thiosulfate, tetrathionate, sulfur, sulfide, and sulfite in the absence and in the presence of inhibitors and uncouplers. The uncouplers 2,4-dinitrophenol and carbonyl cyanide m-chlorophenyl-hydrazone had no affect on the oxidation of thiosulfate, suggesting that thiosulfate is metabolized periplasmically. In contrast, the uncouplers completely inhibited the oxidation of tetrathionate, sulfide, sulfur, and sulfite, indicating that these compounds are metabolized in the cytoplasm of T. caldus KU. N-Ethylmaleimide inhibited the oxidation of tetrathionate and thiosulfate at the stage of elemental sulfur, while 2-heptyl-4-hydroxyquinoline-N-oxide stopped the oxidation of thiosulfate, tetrathionate, and elemental sulfur at the stage of sulfite. The following intermediates in the oxidation of the sulfur compounds were found by using uncouplers and inhibitors: thiosulfate was oxidized to tetrathionate, elemental sulfur was formed during the oxidation of tetrathionate and sulfide, and sulfite was found as an intermediate of tetrathionate and sulfur metabolism. On the basis of these data we propose a model for the metabolism of the reduced inorganic sulfur compounds by T. caldus KU.     

Purification and some properties of sulfur:ferric ion oxidoreductase from Thiobacillus ferrooxidans.

A sulfur:ferric ion oxidoreductase that utilizes ferric ion (Fe3+) as an electron acceptor of elemental sulfur was purified from iron-grown Thiobacillus ferrooxidans to an electrophoretically homogeneous state. Under anaerobic conditions in the presence of Fe3+, the enzyme reduced 4 mol of Fe3+ with 1 mol of elemental sulfur to give 4 mol of Fe2+ and 1 mol of sulfite, indicating that it corresponds to a ferric ion-reducing system (T. Sugio, C. Domatsu, O. Munakata, T. Tano, and K. Imai, Appl. Environ. Microbiol. 49:1401-1406, 1985). Under aerobic conditions, sulfite, but not Fe2+, was produced during the oxidation of elemental sulfur by this enzyme because the Fe2+ produced was rapidly reoxidized chemically by molecular oxygen. The possibility that Fe3+ serves as an electron acceptor under aerobic conditions was ascertained by adding o-phenanthroline, which chelates Fe2+, to the reaction mixture. Sulfur:ferric ion oxidoreductase had an apparent molecular weight of 46,000, and it is composed of two identical subunits (Mr = 23,000) as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sulfur oxidation by this enzyme was absolutely dependent on the presence of reduced glutathione. The enzyme had an isoelectric point and a pH optimum at pH 4.6 and 6.5, respectively. Almost all the activity of sulfur:ferric ion oxidoreductase was observed in the osmotic shock fluid of the cells, suggesting that it was localized in the periplasmic space of the cells.     

Stable sulfur isotope fractionation during the reduction of thiosulfate by Dethiosulfovibrio russensis

Stable sulfur isotope fractionation was investigated during reduction of thiosulfate by growing batch cultures of Dethiosulfovibrio russensis at a cell-specific reduction rate of 2.4 +/- 0.72 fmol cell(-1) d(-1) (28 degrees C). Citrate was used as carbon and energy source. The hydrogen sulfide produced by this sulfur- and thiosulfate-reducing bacterium was depleted in 34S by 11% compared to total thiosulfate sulfur, in agreement with previous results observed for sulfate-reducing bacteria. This indicates the operation of a similar pathway for thiosulfate reduction in these phylogenetically different bacteria.     

Key role of cysteine residues in catalysis and subcellular localization of sulfur oxygenase-reductase of Acidianus tengchongensis

Analysis of known sulfur oxygenase-reductases (SORs) and the SOR-like sequences identified from public databases indicated that they all possess three cysteine residues within two conserved motifs (V-G-P-K-V-C(31) and C(101)-X-X-C(104); numbering according to the Acidianus tengchongensis numbering system). The thio-modifying reagent N-ethylmaleimide and Zn(2+) strongly inhibited the activities of the SORs of A. tengchongensis, suggesting that cysteine residues are important. Site-directed mutagenesis was used to construct four mutant SORs with cysteines replaced by serine or alanine. The purified mutant proteins were investigated in parallel with the wild-type SOR. Replacement of any cysteine reduced SOR activity by 98.4 to 100%, indicating that all the cysteine residues are crucial to SOR activities. Circular-dichroism and fluorescence spectrum analyses revealed that the wild-type and mutant SORs have similar structures and that none of them form any disulfide bond. Thus, it is proposed that three cysteine residues, C(31) and C(101)-X-X-C(104), in the conserved domains constitute the putative binding and catalytic sites of SOR. Furthermore, enzymatic activity assays of the subcellular fractions and immune electron microscopy indicated that SOR is not only present in the cytoplasm but also associated with the cytoplasmic membrane of A. tengchongensis. The membrane-associated SOR activity was colocalized with the activities of sulfite:acceptor oxidoreductase and thiosulfate:acceptor oxidoreductase. We tentatively propose that these enzymes are located in close proximity on the membrane to catalyze sulfur oxidation in A. tengchongensis.     

A combined pathway of sulfur compound disproportionation in Desulfovibrio desulfuricans

The fates of the two different sulfur atoms of the thiosulfate molecule during anaerobic disproportionation by the sulfate-reducing bacterium Desulfovibrio desulfuricans were followed by isotope mass spectrometry. During disproportionation, ³²S-thiosulfate was preferentially metabolized, and the residual thiosulfate became enriched in ³⁴S. The sulfate formed was isotopically heavier than the inner sulfur of the consumed thiosulfate. Vice versa, the sulfide formed was isotopically lighter than the outer sulfur of the consumed thiosulfate. These results indicate that thiosulfate is cleaved to intermediates that undergo further disproportionation to sulfate and sulfide in a second step. These intermediates are probably elemental sulfur and sulfite. It is concluded that disproportionation of thiosulfate, sulfite and elemental sulfur includes a combined pathway.     

Reduced inorganic sulfur oxidation supports autotrophic and mixotrophic growth of Magnetospirillum strain J10 and Magnetospirillum gryphiswaldense

Magnetotactic bacteria are present at the oxic–anoxic transition zone where opposing gradients of oxygen and reduced sulfur and iron exist. Growth of non‐magnetotactic lithoautotrophic Magnetospirillum strain J10 and its close relative magnetotactic Magnetospirillum gryphiswaldense was characterized in microaerobic continuous culture. Both strains were able to grow in mixotrophic (acetate + sulfide) and autotrophic (sulfide or thiosulfate) conditions. Autotrophically growing cells completely converted sulfide or thiosulfate to sulfate and produced 7.5 g dry weight per mol substrate at a maximum observed growth rate of 0.09 h^?1 for strain J10 and 0.07 h^?1 for M. gryphiswaldense. The respiratory activity for acetate was repressed in autotrophic and also in mixotrophic cultures, suggesting acetate was used as C‐source in the latter. We have estimated the proportions of substrate used for assimilatory processes and evaluated the biomass yields per mol dissimilated substrate. The yield for lithoheterotrophic growth using acetate as the C‐source was approximately twice the autotrophic growth yield and very similar to the heterotrophic yield, showing the importance of reduced sulfur compounds for growth. In the draft genome sequence of M. gryphiswaldense homologues of genes encoding a partial sulfur‐oxidizing (Sox) enzyme system and reverse dissimilatory sulfite reductase (Dsr) were identified, which may be involved in the oxidation of sulfide and thiosulfate. Magnetospirillum gryphiswaldense is the first freshwater magnetotactic species for which autotrophic growth is shown.     

Crystal structure studies on sulfur oxygenase reductase from Acidianus tengchongensis

Sulfur oxygenase reductase (SOR) simultaneously catalyzes oxidation and reduction of elemental sulfur to produce sulfite, thiosulfate, and sulfide in the presence of molecular oxygen. In this study, crystal structures of wild type and mutants of SOR from Acidianus tengchongensis (SOR-AT) in two different crystal forms were determined and it was observed that 24 identical SOR monomers form a hollow sphere. Within the icosatetramer sphere, the tetramer and trimer channels were proposed as the paths for the substrate and products, respectively. Moreover, a comparison of SOR-AT with SOR-AA (SOR from Acidianus ambivalens) structures showed that significant differences existed at the active site. Firstly, Cys31 is not persulfurated in SOR-AT structures. Secondly, the iron atom is five-coordinated rather than six-coordinated, since one of the water molecules ligated to the iron atom in the SOR-AA structure is lost. Consequently, the binding sites of substrates and a hypothetical catalytic process of SOR were proposed.     

Growth of thermophilic, obligatorily chemolithoautotrophic hydrogen-oxidizing bacteria related to Hydrogenobacter with thiosulfate and elemental sulfur as electron and energy source

Abstract Strains related to Hydrogenobacter , a genus of thermophilic, obligatorily chemolithoautotrophic bacteria, were able to utilize elemental sulfur or thiosulfate, as well as molecular hydrogen, as sole electron and energy source. Extracellular elemental sulfur was produced as an intermediate during oxidation of thiosulfate. Growth with thiosulfate alone was strongly microaerophilic, whereas no hydrogenase activity was detected. Mixolithotrophic growth with both hydrogen and thiosulfate was faster than with hydrogen alone, and the cells harbored a hydrogenase activity comparable to that of cells grown under hydrogen without thiosulfate.     

Utilization of different sulfur sources by propionic acid bacteria

The object of this work was to study the ability of propionic bacteria to utilize sulfur compounds having various degrees of oxidation. Propionibacterium shermanii was found to utilize sulfite, thiosulfate, sulfide and elemental sulfur, apart from sulfate, as a sulfur source. When the culture grew in a medium with elemental sulfur, sulfide was produced. The utilization of sulfate by P. shermanii had a peculiar character. In the process of the culture growth, the utilization of sulfate alternated with its release into the medium.     

Characterization of a novel thiosulfate dehydrogenase from a marine acidophilic sulfur-oxidizing bacterium,Acidithiobacillus thiooxidans strain SH

A marine acidophilic sulfur-oxidizing bacterium, Acidithiobacillus thiooxidans strain SH, was isolated to develop a bioleaching process for NaCl-containing sulfide minerals. Because the sulfur moiety of sulfide minerals is metabolized to sulfate via thiosulfate as an intermediate, we purified and characterized the thiosulfate dehydrogenase (TSD) from strain SH. The enzyme had an apparent molecular mass of 44 kDa and was purified 71-fold from the solubilized membrane fraction. Tetrathionate was the product of the TSD-oxidized thiosulfate and ferricyanide or ubiquinone was the electron acceptor. Maximum enzyme activity was observed at pH 4.0, 40 °C, and 200 mM NaCl. To our knowledge, this is the first report of NaCl-stimulated TSD activity. TSD was structurally different from the previously reported thiosulfate-oxidizing enzymes. In addition, TSD activity was strongly inhibited by 2-heptyl-4-hydroxy-quinoline N-oxide, suggesting that the TSD is a novel thiosulfate:quinone reductase.     

Thiosulfate Reductase of Desulfovibrio vulgaris

The thiosulfate reductase of Desulfovibrio vulgaris has been purified and some of its properties have been determined. Only one protein component was detected when the purified enzyme was subjected to polyacrylamide gel electrophoresis at pH values of 8.9, 8.0, and 7.6. In the presence of H(2), the enzyme, when coupled to hydrogenase and with methyl viologen as an electron carrier, catalyzed the reduction of thiosulfate to hydogen sulfide. The use of specifically labeled (35)S-thiosulfate revealed that the outer sulfur atom was reduced to sulfide and the inner sulfur atom was released as sulfite. Thus, the enzyme catalyzes the reductive dismutation of thiosulfate to sulfide and sulfite. The molecular weight of the enzyme was determined by sedimentation equilibrium (16,300) and amino acid analysis (15,500). The enzyme sedimented as a single, symmetrical component with a calculated sedimentation coefficient of 2.21S. Amino acid analysis revealed the presence of two half-cystine residues per mole of enzyme and a total of 128 amino acid residues. Carbohydrate and organic phosphorus analyses revealed the presence of 9.2 moles of carbohydrate and 4.8 moles of phosphate per mole of enzyme. The substrate specificity of the enzyme was studied.     

Quantitative proteomics of Chlorobaculum tepidum: insights into the sulfur metabolism of a phototrophic green sulfur bacterium

Chlorobaculum (Cba.) tepidum is a green sulfur bacterium that oxidizes sulfide, elemental sulfur, and thiosulfate for photosynthetic growth. To gain insight into the sulfur metabolism, the proteome of Cba. tepidum cells sampled under different growth conditions has been quantified using a rapid gel-free, filter-aided sample preparation (FASP) protocol with an in-solution isotopic labeling strategy. Among the 2245 proteins predicted from the Cba. tepidum genome, approximately 970 proteins were detected in unlabeled samples, whereas approximately 630-640 proteins were detected in labeled samples comparing two different growth conditions. Wild-type cells growing on thiosulfate had an increased abundance of periplasmic cytochrome c-555 and proteins of the periplasmic thiosulfate-oxidizing SOX enzyme system when compared with cells growing on sulfide. A dsrM mutant of Cba. tepidum, which lacks the dissimilatory sulfite reductase DsrM protein and therefore is unable to oxidize sulfur globules to sulfite, was also investigated. When compared with wild type, the dsrM cells exhibited an increased abundance of DSR enzymes involved in the initial steps of sulfur globule oxidation (DsrABCL) and a decreased abundance of enzymes putatively involved in sulfite oxidation (Sat-AprAB-QmoABC). The results show that Cba. tepidum regulates the cellular levels of enzymes involved in sulfur metabolism and other electron-transferring processes in response to the availability of reduced sulfur compounds.