Thiosulfate metabolism in Rhodopseudomonas palustris

The cells of the purple nonsulfur bacterium Rhodopseudomonas palustris, Nakamura strain, are capable of oxidizing thiosulfate and sulfide both under the anaerobic conditions in the light and under the aerobic conditions in the dark. Regardless of the presence of thiosulfate in the medium, the cells contain thiosulfate reductase, rodanase, thiosulfate oxidase, and sulfite oxidase. However, the capability to oxidize thiosulfate and sulfide is induced in Rh. palustris after the cells have been incubated in the presence of thiosulfate for 2--4 hours. The process of induction is related to the synthesis of protein components. Decomposition of thiosulfate in Rh. palustris when its concentration in the medium is low (2--5 mM) is accompanied with the formation of an equimolar quantity of sulfate. When the concentration of thiosulfate is higher (10--20 mM), the products of its oxidation are tetrathionate and sulfate. Therefore, the metabolic pathway of thiosulfate in Rh. palustris depends on its concentration in the medium.     

Chemolithoautotrophic growth on elemental sulfur (S°) and respiratory oxidation of S° by Thiobacillus versutus and another sulfur-oxidizing bacterium

Abstract Thiobacillus versutus was shown to grow chemolithoautotrophically under microaerophilic conditions, with crystalline elemental sulfur (S°) and thiosulfate as sole electron source. The exponential growth rate on S° ( μ = 0.106 h⁻¹) measured in batch culture was similar to the reported maximum growth rate on thiosulfate in chemostat cultures. The rates of thiosulfate, S° and sulfite oxidation were measured respirometrically using an oxygen electrode. During growth under air on thiosulfate, as well as under low oxygen pressure on S° and thiosulfate, a relatively strong sulfuroxidizing activity (SOA) was measured. The induction of the SOA on cells growing with thiosulfate and the similar growth rates on S° and thiosulfate strongly suggest that S° could be an important intermediate during thiosulfate utilization.     

Growth of thermophilic obligately autotrophic hydrogen-oxidizing bacteria on thiosulfate

Thermophilic obligately autotrophic H₂-oxidizing bacteria from Icelandic hot springs were tested for growth on thiosulfate. Ten strains were tested and all grew on thiosulfate but not on sulfite or sulfur. The product of thiosulfate oxidation was sulfate. The growth rate on thiosulfate was slower (μ=0.12 h^-1) than on H₂ (μ=0.34 h^-1). Washed cells which had been grown on thiosulfate could oxidize thiosulfate rapidly but H₂-grown cells oxidized thiosulfate much more slowly and with about a 3 h lag time. The bacteria would not grow on agar medium under H₂ but grew on agar medium containing thiosulfate.     

Effect of Thiosulfate on the Photosynthetic Growth of Rhodopseudomonas palustris

Cell yields of Rhodopseudomonas palustris grown photoheterotrophically in pyruvate-mineral salts medium were increased by the photooxidation of added thiosulfate. However, thiosulfate had no effect on cell yields of cultures grown aerobically in darkness, although thiosulfate was also oxidized. The presence of thiosulfate increased photosynthetic cell yields on a variety of other organic substrates. Growth of cells in thiosulfate-containing medium, or the addition of thiosulfate to cells grown in thiosulfate-free medium, induced the formation of a thiosulfate-oxidizing system which quantitatively photooxidized thiosulfate to sulfate. R. palustris grew photoautotrophically with thiosulfate as an oxidizable substrate. Large amounts of supplemental bicarbonate carbon were incorporated when cells were grown photosynthetically in pyruvate-thiosulfate medium. Cells harvested after photoautotrophic or photoheterotrophic growth in fumarate-thiosulfate medium fixed (14)CO(2) at an 8- to 10-fold greater rate when provided with thiosulfate. The evolution of (14)CO(2) from pyruvate-1-(14)C during photoassimilation by R. palustris was greatly suppressed by the presence of thiosulfate. The increase in photoheterotrophic cell yields of R. palustris caused by the oxidation of thiosulfate may result from assimilation of substrate carbon which is normally evolved as carbon dioxide.     

Are thiosulfate and trithionate intermediates in dissimilatory sulfate reduction?

The fate of 35-S during anaerobic metabolism of [35-S]sulfate, [35-S]thiosulfate, and [35-S]sulfate plus unlabeled thiosulfate by washed cell suspensions of Desulfovibrio spp, and of [35-S]thiosulfate by growing D. desulfuricans was examined. The results appear to be inconsistent with the hypothesis that thiosulfate is an intermediate in sulfate reduction. Since thiosulfate was produced from trithionate, the latter is also unlikely to be an intermediate in the reduction pathway. Extracts of D. desulfuricans catalysed exchange between sulfite and the sulfonate group of thiosulfate.     

Thiosulfate sulfur transferases (Rhodaneses) ofChlorobium vibrioforme f.thiosulfatophilum

Two enzymes containing thiosulfate sulfur transferase activity were purified fromChlorobium vibrioforme f.thiosulfatophilum by ion exchange chromatography, gel filtration and isoelectrofocusing. Enzyme I is a basic protein with an isoelectric point at pH 9.2 and has a molecular weight of 39,000. TheK _m-values for thiosulfate and cyanide of the purified basic protein were 0.25 mM (thiosulfate) and 5 mM (cyanide). Enzyme II is an acidic protein. The enzyme has an isoelectric point at pH 4.6–4.7 and a molecular weight of 34,000. TheK _m-values of the acidic protein were found to be 5 mM for thiosulfate and 125 mM for cyanide.In addition to thiosulfate sulfur transferase activity, cellfree extracts ofChlorobium vibrioforme f.thiosulfatophilum also contained low thiosulfate oxidase activity and negligible thiosulfate reductase activity. The percent distribution of thiosulfate sulfur transferase and thiosulfate oxidase activities in the organism was independent of the offered sulfur compound (thiosulfate, sulfide or both) in the medium.Abbreviations C Chlorobium - SDS sodium dodecylsulfate Dedicated to Prof. Dr. Norbert Pfennig on the occasion of his 60th birthday     

Assimilatory sulfur metabolism in marine microorganisms: a novel sulfate transport system in Alteromonas luteo-violaceus.

The sulfate transport mechanism of a marine bacterium, Alteromonas luteo-violaceus, was unique among microorganisms in its extremely low affinity for the sulfate analog thiosulfate. Distinguishing characteristics included weak inhibition of sulfate transport by thiosulfate, inability to transport thiosulfate effectively, poor growth using thiosulfate as the sole source of sulfur, and a mild effect of the sulfhydryl reagent para-hydroxymercuribenzoate. In contrast, sulfate transport by a marine pseudomonad, Pseudomonas halodurans, was strongly inhibited by thiosulfate, and para-hydroxymercuribenzoate reversibly but completely blocked sulfate transport.     

Thiosulfate oxidation and mixotrophic growth of Methylobacterium oryzae

Thiosulfate oxidation and mixotrophic growth with succinate or methanol plus thiosulfate was examined in nutrient-limited mixotrophic condition for Methylobacterium oryzae CBMB20, which was recently characterized and reported as a novel species isolated from rice. Methylobacterium oryzae was able to utilize thiosulfate in the presence of sulfate. Thiosulfate oxidation increased the protein yield by 25% in mixotrophic medium containing 18.5 mmol.L-1 of sodium succinate and 20 mmol.L-1 of sodium thiosulfate on day 5. The respirometric study revealed that thiosulfate was the most preferable reduced inorganic sulfur source, followed by sulfur and sulfite. Thiosulfate was predominantly oxidized to sulfate and intermediate products of thiosulfate oxidation, such as tetrathionate, trithionate, polythionate, and sulfur, were not detected in spent medium. It indicated that bacterium use the non-S4 intermediate sulfur oxidation pathway for thiosulfate oxidation. Thiosulfate oxidation enzymes, such as rhodanese and sulfite oxidase activities appeared to be constitutively expressed, but activity increased during growth on thiosulfate. No thiosulfate oxidase (tetrathionate synthase) activity was detected.     

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%.     

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.     

A sulfate,sulfite and thiosulfate incorporating system inCandida utilis

Sulfate, sulfite and thiosulfate incorporation in the yeastCandida utilis is inhibited by extracellular sulfate, sulfite and thiosulfate and by sulfate analogues selenate, chromate and molybdate. The three processes are blocked if sulfate, sulfite, thiosulfate, cysteine and homocysteine are allowed to accumulate endogenously. Incorporation of the three inorganic sulfur oxy anions is inactivated by heat at the same rate. Mutants previously shown to be defective in sulfate incorporation are also affected in sulfite and thiosulfate uptake. Revertants of these mutants selected by plating in ethionine-supplemented minimal medium recovered the capacity to incorporate sulfate, sulfite and thiosulfate. These results taken together with previous evidence demonstrate the existence of a common sulfate, sulfite and thiosulfate incorporating system in this yeast.     

Thiosulfate oxidation by nonsulfur purple bacteria

The capability to oxidize thiosulfate was studied in 11 cultures of purple bacteria belonging to Rhodomicrobium vannielii, Rhodopseudmonas viridis, Rh. sphaeroides, Rh. capsulata, and Rhodospirillum rubrum. All the bacteria oxidized thiosulfate under aerobic conditions in the dark. The strains 2R, 8259, A1, A2 and D1 of Rh. sphaeroides oxidized thiosulfate under anaerobic conditions in the light, and the process was coupled with carbon dioxide fixation. All the strains contained thiosulfate reductase, and the majority of them possessed also the activity of thiosulfate oxidase and sulfite oxidase.     

Thiosulfate elimination and permeability in a sulfide-adapted marine invertebrate.

Oxidation of hydrogen sulfide to thiosulfate is one of the best-characterized mechanisms by which animals adapted to sulfide minimize its toxicity, but the mechanism of thiosulfate elimination in these animals has remained unclear. In this study, we examined the accumulation and elimination of thiosulfate in the sulfide-adapted marine worm Urechis caupo. The coelomic fluid of U. caupo exposed to 50-100 micromol L-1 sulfide in hypoxic seawater (Po2 ca. 10 kPa) accumulated (mean+/-SD) 132+/-41 micromol L-1 thiosulfate after 2 h, reaching 227+/-113 micromol L-1 after an additional 4 h in aerated, sulfide-free seawater. In whole-animal thiosulfate clearance studies, the rate of thiosulfate elimination from the coelomic fluid followed a single exponential time course with a half-life of 6 h. The thiosulfate permeability coefficient of isolated preparations mounted in diffusion chambers was 7.6x10-5+/-7. 7x10-5 cm s-1 for the hindgut and 5.5x10-7+/-2.7x10-7 cm s-1 for the body wall. These rates were independent of the direction of net efflux (mucosal-to-serosal or serosal-to-mucosal). Using a simple mathematical model of U. caupo that incorporates the thiosulfate permeability coefficients, the thiosulfate half-life was calculated to be 23 h without hindgut ventilation but less than 1 h with normal hindgut ventilation. Based on this information, we propose that passive thiosulfate diffusion across the hindgut is adequate to explain the observed rates of thiosulfate elimination.     

Metabolism of thiosulfate and tetrathionate by heterotrophic bacteria from soil

Two heterotrophic bacteria that oxidized thiosulfate to tetrathionate were isolated from soil. The enzyme system in one of the isolates (C-3) was constitutive, but in the other isolate (A-50) it was induced by thiosulfate or tetrathionate. The apparent K(m) for oxygen for thiosulfate oxidation by A-50 was about 223 mum, but, for lactate oxidation by A-50 or thiosulfate oxidation by C-3, the apparent K(m) for oxygen was below 2 mm. The oxidation of thiosulfate by A-50 was first order with respect to oxygen from 230 mum. The rate of oxidation was greatest at pH 6.3 to 6.8 and at about 10 mm thiosulfate, and it was strongly inhibited by several metal-binding reagents. Extracts of induced A-50 reduced ferricyanide, endogenous cytochrome c, and mammalian cytochrome c in the presence of thiosulfate. A-50, once induced to oxidize thiosulfate, also reduced tetrathionate to thiosulfate in the presence of an electron donor such as lactate. The optimal pH for this reaction was at 8.5 to 9.5, and the reaction was first order with respect to tetrathionate. There was no correlation between the formation of the thiosulfate-oxidizing enzyme of A-50 and the incorporation of thiosulfate-sulfur into cell sulfur. Thiosulfate did not affect the growth rate or yield of A-50.     

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.     

The role of thiosulfate in sulfur metabolism of Rhodopseudomonas globiformis

Rhodopseudomonas globiformis is able to assimilate both sulfur moieties of thiosulfate. During growth on ³⁵S-labelled thiosulfate the amino acids cysteine, homocysteine and methionine were labelled. The bulk of thiosulfate, however, was oxidized to tetrathionate and accumulated in the medium. A thiosulfate: acceptor oxidoreductase was partially purified and characterized. The enzyme oxidized thiosulfate to tetrathionate in the presence of ferricyanide. A c-type cytochrome isolated from this organism was reduced by this enzyme.     

Thiosulfate oxidation by obligately heterotrophic bacteria

Thiosulfate was oxidized stoichiometrically to tetrathionate during growth on glucose byKlebsiella aerogenes, Bacillus globigii, B. megaterium, Pseudomonas putida, two strains each ofP. fluorescens andP. aeruginosa, and anAeromonas sp. A gram-negative, rod-shaped soil isolate, Pseudomonad H_w, converted thiosulfate to tetrathionate during growth on acetate. None of the organisms could use thiosulfate as sole energy source. The quantitative recovery of all the thiosulfate supplied to heterotrophic cultures either as tetrathionate alone or as tetrathionate and unused thiosulfate demonstrated that no oxidation to sulfate occurred with any of the strains tested. Two strains ofEscherichia coli did not oxidize thiosulfate. Thiosulfate oxidation in batch culture occurred at different stages of the growth cycle for different organisms:P. putida oxidized thiosulfate during lag and early exponential phase,K. aerogenes oxidized thiosulfate at all stages of growth, andB. megaterium andAeromonas oxidized thiosulfate during late exponential phase. The relative rates of oxidation byP. putida andK. aerogenes were apparently determined by different concentrations of thiosulfate oxidizing enzyme. Thiosulfate oxidation byP. aeruginosa grown in chemostat culture was inducible, since organisms pregrown on thiosulfate-containing media oxidized thiosulfate, but those pregrown on glucose only could not oxidize thiosulfate. Steady state growth yield ofP. aeruginosa in glucose-limited chemostat culture increased about 23% in the presence of 5–22 mM thiosulfate, with complete or partial concomitant oxidation to tetrathionate. The reasons for this stimulation are unclear. The results suggest that heterotrophic oxidation of thiosulfate to tetrathionate is widespread across several genera and may even stimulate bacterial growth in some organisms.     

Cloning of the phs genetic locus from Salmonella typhimurium and a role for a phs product in its own induction.

The Salmonella typhimurium phs chromosomal locus essential for the reduction of thiosulfate to hydrogen sulfide was cloned, and some features of its regulation were examined. The phs locus conferred H2S production on Escherichia coli, suggesting that it contains the structural gene for thiosulfate reductase. H2S production by the E. coli host was, as in S. typhimurium, suppressed by nitrate or glucose in the growth medium. The presence of plasmid-borne phs genes in a S. typhimurium chl+ host containing a chromosomal phs::lacZ operon fusion was found to significantly increase the relative induction efficiency of beta-galactosidase by thiosulfate. These results are consistent with a model for phs regulation in which the true inducer is not thiosulfate per se and in which the action of a phs-encoded molybdoprotein, possibly the reductase itself, converts thiosulfate into a compound that resembles the true inducer more closely than does thiosulfate.     

Purification and properties of thiosulfate dehydrogenase from Acidithiobacillus thiooxidans JCM7814

A key enzyme of the thiosulfate oxidation pathway in Acidithiobacillus thiooxidans JCM7814 was investigated. As a result of assaying the enzymatic activities of thiosulfate dehydrogenase, rhodanese, and thiosulfate reductase at 5.5 of intracellular pH, the activity of thiosulfate dehydrogenase was measured as the key enzyme. The thiosulfate dehydrogenase of A. thiooxidans JCM7814 was purified using three chromatographies. The purified sample was electrophoretically homogeneous. The molecular mass of the enzyme was 27.9 kDa and it was a monomer. This enzyme had cytochrome c. The optimum pH and temperature of this enzyme were 3.5 and 35 degrees C. The enzyme was stable in the pH range from 5 to 7, and it was stable up to 45 degrees C. The isoelectric point of the enzyme was 8.9. This enzyme reacted with thiosulfate as a substrate. The Km was 0.81 mM.