Oxidation of reduced sulphur compounds by intact cells of Thiobacillus acidophilus

Oxidation of reduced sulphur compounds by Thiobacillus acidophilus was studied with cell suspensions from heterotrophic and mixotrophic chemostat cultures. Maximum substrate-dependent oxygen uptake rates and affinities observed with cell suspensions from mixotrophic cultures were higher than with heterotrophically grown cells. ph Optima for oxidation of sulphur compounds fell within the pH range for growth (pH 2–5), except for sulphite oxidation (optimum at pH 5.5). During oxidation of sulphide by cell suspensions, intermediary sulphur was formed. Tetrathionate was formed as an intermediate during aerobic incubation with thiosulphate and trithionate. Whether or not sulphite is an inter-mediate during sulphur compound oxidation by T. acidophilus remains unclear. Experiments with anaerobic cell suspensions of T. acidophilus revealed that trithionate metabolism was initiated by a hydrolytic cleavage yielding thiosulphate and sulphate. A hydrolytic cleavage was also implicated in the metabolism of tetrathionate. After anaerobic incubation of T. acidophilus with tetrathionate, the substrate was completely converted to equimolar amounts of thiosulphate, sulphur and sulphate. Sulphide- and sulphite oxidation were partly inhibited by the protonophore uncouplers 2,4-dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) and by the sulfhydryl-binding agent N-ethylmaleimide (NEM). Oxidation of elemental sulphur was completely inhibited by these compounds. Oxidation of thiosulphate, tetrathionate and trithionate was only slightly affected. The possible localization of the different enzyme systems involved in sulphur compound oxidation by T. acidophilus is discussed.     

Isolation of sulphate transport defective mutants ofCandida utilis: Further evidence for a common transport system for sulphate,sulphite and thiosulphate

Selenate-resistant mutants ofCandida utilis were isolated. They did not take up sulphate while incorporation of an organic sulphur source, such asl-methionine, was similar to the wild-type strain. They grew poorly on sulphate, sulphite and thiosulphate and, as expected, grew well on methionine. Sulphite reductase activities of the mutants were similar to the wild type strain. The properties of these mutants support the view of a common transport system for sulphate, sulphite and thiosulphate.     

Physiological analysis of mutants of Saccharomyces cerevisiae impaired in sulphate assimilation.

The assimilation of sulphate in Saccharomyces cerevisiae, comprising the reduction of sulphate to sulphide and the incorporation of the sulphur atom into a four-carbon chain, requires the integrity of 13 different genes. To date, the functions of nine of these genes are still not clearly established. A set of strains, each bearing a mutation in one MET gene, was studied. Phenotypic studies and enzyme determinations showed that the products of at least five genes are needed for the synthesis of an enzymically active sulphite reductase. These genes are MET1, MET5, MET8, MET10 and MET20. Wild-type strains of S. cerevisiae can use organic metabolites such as homocysteine, cysteine, methionine and S-adenosylmethionine as sulphur sources. They are also able to use inorganic sulphur sources such as sulphate, sulphite, sulphide or thiosulphate. Here we show that both of the two sulphur atoms of thiosulphate are used by S. cerevisiae. Thiosulphate is cleaved into sulphite and sulphide prior to utilization by the sulphate assimilation pathway, as the metabolism of one sulphur atom from thiosulphate requires the presence of an active sulphite reductase.     

Transport du sulfate à travers la double membrane limitante, ou enveloppe, des chloroplastes d'épinard

Evidence is presented for low rates of carriermediated uptake of sulphate, thiosulphate and sulphite into the stroma of the C3 plant Spinacia oleracea. Uptake of sulphate in the dark was followed using two techniques (1) uptake of sulphate [³⁵S] as determined by silicon oil centrifugal filtration and (2) uptake as indicated by inhibition of CO₂-dependant O₂ evolution rates after addition of sulphate.Sulphate, thiosulphate and sulphite were transported across the envelope leading to an accumulation in the chloroplasts. Sulphate transport had saturation kinetics of the Michaelis-Menten type (Vmax : 25 μmoles . mg⁻¹ chl . h⁻¹ at 22°C ; Km : 2.5 mM). The rate of transport for sulphate was not influenced either by illumination or pH change in the external medium. Phosphate was a competitive inhibitor of sulphate uptake by chloroplasts (Ki : 0.7 mM, fig. 1). The rate of transport for phosphate appeared to be much higher than for sulphate. When the chloroplasts were pre-loaded with labelled sulphate, radioactivity was rapidly released after addition of phosphate into the external medium. Consequently, the transport of sulphate occurs by a strict counter-exchange : for each molecule of sulphate entering the chloroplast, one molecule of phosphate leaves the stroma, and vice-versa.The uptake of sulphate by isolated intact chloroplasts exchanging for internal free phosphate induced a lower rate of photophosphorylation, which in turn inhibited CO₂-dependent O₂ evolution.The presence, on the inner membrane of the chloroplast envelope, of a specific sulphate carrier, distinct from the phosphate translocator, is discussed.     

Sulphur acquisition by Neisseria meningitidis

Group B Neisseria meningitidis (SD1C) was grown on defined medium supplemented with each of a variety of sulphur compounds as the sole source of sulphur. The organism grew on sulphate, sulphite, bisulphite, thiosulphate, dithionite, hydrosulphide, thiocyanate, L-cysteine, L-cystine, reduced glutathione, methionine, mercaptosuccinate, and lanthionine, but not on dithionate unless previously sulphur starved. Good growth was seen on concentrations of sulphate or thiosulphate as low as 10 microM. When pregrown on and subsequently starved for sulphate, the meningococcus showed enhanced transport capacity for this ion. Optimal conditions for assessing sulphur transport by active sulphur-limited cells were determined. The maximal sulphate uptake velocity was 9.3 nmol sulphate X mg protein-1 X min-1, and the apparent Km was 1.4 microM, far below human nasopharyngeal or serum sulphate levels.     

Characterization of Sulphate Uptake in Anacystis nidulans

Sulphate uptake in the blue-green alga Anacystis nidulans appears based upon an active mechanism with a Km of 0.75 μM and V_max of 0.7 pmol/min × 10⁶ cells. Sulphate uptake is competitively inhibited by thiosulphate and sulphite. The sulphate uptake has a pH optimum at 8 and a temperature optimum at 40°C. By increasing the extracellular sulphate concentration from 0.1 to 10 μM the sulphate pool in Anacystis was altered from 8.3, 10^?5M to 5.9, 10^?4M.     

Sulphate metabolism of selenate-resistant Schizosaccharomyces pombe mutants

Selenate-resistant mutants were obtained from several strains of Schizosaccharomyces pombe. The obtained mutants all belonged to the same genetic complementation group. They were low in sulphate uptake activity and in ATP sulphurylase activity. They grew on medium containing sulphite, thiosulphate, cysteine or glutathione but not methionine as the sole source of sulphur. From these results, the mutants were concluded to carry mutations in the ATP sulphurylase gene. Inability of the mutants to utilize methionine as a sulphur source is rationalized by the absence of the reverse transsulphurylation pathway in this organism; wild type strains must utilize methionine as a sulphur source after it is degraded to give rise to sulphate.     

Effect of some sulphur compounds on soil microflora of spruce rhizosphere

The effect of a long-term application of sulphite, thiosulphate and sodium sulphate on the soil microflora and spruce seedlings was investigated in a pot experiment. Sulphur compounds decreased the concentration of bacteria, including thiobacilli, increased the concentration of microscopic fungi and sulphate-reducing bacteria; they inhibited respiration, nitrification and oxidation of thiosulphate, stimulated ammonification and oxidation of elemental sulphur. In certain cases the spruce rhizosphere exhibited just the opposite effect. In the rhizosphere the sulphate-reducing bacteria were suppressed together with thiobacilli, whose unit oxidative activity increased substantially. Growth of seedlings was inhibited by sulphite and stimulated by thiosulphate and sulphate. Sulphite, the effects of which were similar to those of sulphur dioxide immissions, was the most effective compound. In regions influenced by immissions the soil is apparently intoxicated by the absorbed sulphite.     

Transport of H+ in mitochondria induced by the uptake of sulfite, sulfate and thiosulfate

The addition of sulphite to rat-liver mitochondria (RLM) causes an uptake of H+ that is unaffected by NEM and butylmalonate. The uptake of H+ induced by sulphate or thiosulphate is abolished by NEM and butylmalonate in freshly isolated RLM, whereas it is inhibited only by butylmalonate in sulphite-pretreated mitochondria. The data suggest that sulphite is cotransported with H+, whereas the movement of H+ associated to the uptake of sulphate or thiosulphate by RLM is mediated by either phosphate or sulphite.     

DsrL mediates electron transfer between NADH and rDsrAB in Allochromatium vinosum

Dissimilatory sulphite reductase DsrAB occurs in sulphate/sulphite-reducing prokaryotes, in sulphur disproportionators and also in sulphur oxidizers, where it functions in reverse. Predictions of physiological traits in metagenomic studies relying on the presence of dsrAB, other dsr genes or combinations thereof suffer from the lack of information on crucial Dsr proteins. The iron–sulphur flavoprotein DsrL is an example of this group. It has a documented essential function during sulphur oxidation and was recently also found in some metagenomes of probable sulphate and sulphite reducers. Here, we show that DsrL and reverse acting rDsrAB can form a complex and are copurified from the phototrophic sulphur oxidizer Allochromatium vinosum. Recombinant DsrL exhibits NAD(P)H:acceptor oxidoreductase activity with a strong preference for NADH over NADPH. In vitro, the rDsrABL complex effectively catalyses NADH-dependent sulphite reduction, which is strongly enhanced by the sulphur-binding protein DsrC. Our work reveals NAD⁺ as suitable in vivo electron acceptor for sulphur oxidation in organisms operating the rDsr pathway and points to reduced nicotinamide adenine dinucleotides as electron donors for sulphite reduction in sulphate/sulphite-reducing prokaryotes that contain DsrL. In addition, dsrL cannot be used as a marker distinguishing sulphate/sulphite reducers and sulphur oxidizers in metagenomic studies without further analysis.     

Selection of sulphur sources for the growth of Butyribacterium methylotrophicum and Acetobacterium woodii

Sulphide and cysteine inhibited growth of batch cultures of Butyribacterium methylotrophicum at moderate concentrations (above 0.5 mM) during growth on glucose (10 mM). The ability of several sulphur sources to replace sulphide was tested in cultures of B. methylotrophicum or Acetobacterium woodii. With sulphite (1 mM), thiosulphate (0.5 mM), elemental sulphur, and dithionite (1 mM), but not sulphate (1 mM), cultures of both organisms grew and produced some sulphide. With elemental sulphur as the sulphur source, toxic levels of sulphide accumulated. Optimal levels for the cultivation of B. methylotrophicum with sulphite were 0.5–2.0 mM, but at higher concentrations the growth rate decreased rapidly, while with dithionite up to 4.0 mM the growth rate was relatively unaffected. In chemostat cultures of B. methylotrophicum with dithionite (1 mM) as the sulphur source and glucose as the limiting substrate, dilution rates up to 0.40 h^–1 were obtained. Thiosulphate could only be used in batch cultures in combination with the reductant titanium(III)nitriloacetate, but in continuous cultures the addition of the reductant to the reservoir was not necessary, because once growth had started enough sulphide was produced to keep the fermentor reduced. The maximum growth rate of B. methylotrophicum with thiosulphate in batch and continuous culture was 0.26 h^–1. Both thiosulphate and dithionite are more convenient sulphur sources than sulphide, but dithionite is more versatile because of its reductive properties and the faster growth it allows.Offprint requests to: T. A. Hansen     

Sulphur metabolism in Thiorhodaceae I. Quantitative measurements on growing cells ofChromatium okenii

Growth experiments and short term experiments in a stirred cuvette showed thatChromatium okenii strain Ostrau is not able to oxidize any reduced sulphur compounds except sulphide and elementary sulphur; thiosulphate, sulphite, and thioglycolate can not be utilized as reducing agents for photosynthesis. The cells are not able to use H₂; hydrogenase could not be demonstrated. In the dark, sulphide is formed from intracellular sulphur and the carbon content of the cells decreases. Growth and turnover of sulphur compounds was followed in the light in the presence and absence of acetate as a second carbon source. Sulphide oxidation depends on the presence of CO₂ and on light intensity, i.e. sulphur metabolism is governed by the photosynthetic activity of the cells.     

Dynamics of oxidation of inorganic sulphur compounds in upper soil horizons of spruce forests

Dynamics of oxidation of inorganic sulphur compounds to sulphate by the soil of spruce forests was investigated. Sulphide, sulphite and thiosulphate are oxidized to sulphate at a maximal rate at the beginning of the reaction, oxidation of elemental sulphur exhibits a lag phase. Linear relationships between the amounts of the produced sulphate and concentrations of substrates in the soil could be detected. On the basis of this finding a method for comparison of the oxidative activity of various soils was proposed.     

Sulphur requirements of certain isolates ofAlternaria tenuis Auct

The sulphur nutrition of three isolates ofAlternaria tenuis Auct., isolated from the diseased leaves ofMangifera indica L.,Musa paradisiaca L. andPsidium guajava L., was studied. They were grown on the medium devoid of sulphur as well as on media containing various sources of sulphur viz., ammonium sulphate, sodium hyposulphite, sodium thiosulphate, magnesium sulphate, potassium sulphate, potassium metabisulphite, zinc sulphate and thiourea. Sodium hyposulphite, sodium thiosulphate, magnesium sulphate, potassium sulphate and zinc sulphate were generally found to be satisfactory sources for the growth of all the isolates under study. Poor growth of the different isolates was observed on the medium devoid of sulphur.     

Studies on sulphur-selenium antagonism in blue-green algae

Summary Anacystis nidulans and Anabaena variabilis contain sufficient sulphur reserves to enable them to perform only one round of growth cycle in the non-sulphur growth medium. Sulphate, sulphite, l-methionine and d-methionine, each can act as a suitable sulphur source, but they differ in respect of their growth promoting action; sulphate uptake seems to be a light driven phenomenon and the sulphate metabolizing enzymes are inducible in nature. Methionine appears to act as a repressor of sulphate-metabolizing enzymes.     

Utilization of hydrogen and formate by Campylobacter spec. under aerobic and anaerobic conditions

A free-living aspartate-fermenting Campylobacter spec. was shown to utilize hydrogen produced in mixed culture by Clostridium cochlearium from glutamate. Resting cells of Campylobacter were shown to reduce aspartate, fumarate and malate as well as nitrate, nitrite, hydroxylamine, sulphite, thiosulphate and elemental sulphur with molecular hydrogen. Growth of Campylobacter spec. was demonstrated with formate as electron donor and nitrate, thiosulphate, elemental sulphur or oxygen as electron acceptor in the presence of acetate as carbon source.     

Tetrathionate Disproportionation by Thiomonas intermedia K12

Thiomonas intermedia K12, a moderately acidophilic bacterium, which oxidises sulphur compounds, – exhibited the capability to use tetrathionate under oxic and anoxic conditions. Whereas under oxic conditions, the reduced sulphur tetrathionate compound was oxidised, under anoxic conditions, the organism disproportionated the compound. In both cases, trithionate and sulphate were produced but in different amounts. The results of the tetrathionate degradation experiments under oxic conditions pointed towards a cyclic degradation process with a transient formation of trithionate and sulphate as the final products, similar to the mechanism described for acidophilic sulphur compound oxidising bacteria. The results of the tetrathionate degradation experiments under anoxic conditions hinted to a partial reduction of tetrathionate to thiosulphate and a fractional oxidation to trithionate and sulphate. 4 M tetrathionate were converted to 6 M thiosulphate, 1 M trithionate, 1 M sulphate, and 8 M protons. The ΔG₀' of this reaction was found to be –16.1 kJ per mol tetrathionate degraded. Additionally, Thiomonas intermedia K12 grew under anoxic conditions with tetrathionate as the sole energy source. The cell numbers increased from 10⁵ as the start value to 10⁷/mL at the end. Organic compounds, excluding traces of yeast extract, did not enhance growth. Therefore, it is proposed that tetrathionate disproportionation is a novel lithotrophic metabolism, which allowed Thiomonas intermedia K12 to survive changing conditions of oxygen supply in sulphur‐compound‐rich environments and even to grow during this reaction. The extensive sulphur compound analysis was carried out by ion‐pair chromatography.     

Sulphur metabolism in Thiorhodaceae. III. Storage and turnover of thiosulphate sulphur inThiocapsa floridana andChromatium species

Thiocapsa floridana strain 1711 andChromatium strains 1211 and 1611 utilize sulphide, thiosulphate, and elementary sulphur as electron donors for growth; sulphite can be used only byChromatium strain 1611. In contrast to the other strains, thiosulphate utilization inChromatium strain 1211 is inducible and not constitutive: thiosulphate is consumed only after an induction period of about 20 hours. The turnover rate of different sulphur compounds is controlled by the CO₂ fixation rate. Using differently labeled³⁵S thiosulphates in short term experiments in a special stirred cuvette, it was shown that the maximum amount of stored intracellular sulphur depends on the strain as well as on the experimental conditions like pH and thiosulphate concentration. WhileChromatium strain 1211 showed a maximum storage of only 10% from sulphane-labeled thiosulphate at pH 6.7, and of 25.7% at pH 6.2,Thiocapsa floridana accumulated 75–90% of the radioactivity into the cells at pH 6.7. While in theChromatium strains the labeling of the cells remained at a constant level until all thiosulphate was consumed, inThiocapsa floridana a defined peak of radioactivity storage was obtained, followed by a steady but 3–4 times slower rate of excretion. With sulphonelabeled thiosulphate no significant accumulation of radioactivity occurred in the cells. During dark-incubation ofThiocapsa floridana (free of intracellular sulphur) in phosphate buffer, pH 6.5, with thiosulphate a production of sulphide could be measured while sulphite was not detected; no sulphide was produced by disrupted cells under the same conditions. The results obtained withThiocapsa floridana strongly support the concept of an initial cleavage of thiosulphate. The present observations do not allow a decision concerning the enzymatic mechanism of the cleavage itself.     

Inorganic sulphur sources for the growth of the dermatophyte microsporum gypseum

Suitability of 10 inorganic compounds at a concentration of 1mm as sulphur sources for the growth of the dermatophyteMicrosporum gypseum was investigated. Dry mass of the mycelium after a 11-d growth served as indicator. Sodium sulphate, sulphite and also disulphite, peroxodisulphate and dithionite were the best sources. Growth in the presence of sodium thiosulphate and tetrathionate was slightly worse. Sulphide inhibited the growth, which began only after a longer adaptation. Sodium thiocyanate and amidosulphate were not utilizable as sulphur sources.     

Isolation and characterization of alkaliphilic, chemolithoautotrophic, sulphur-oxidizing bacteria

Alkaliphilic sulphur-oxidizing bacteria were isolated from samples from alkaline environments including soda soil and soda lakes. Two isolates, currently known as strains AL 2 and AL 3, were characterized. They grew over a pH range 8.0–10.4 with an optimum at 9.5–9.8. Both strains could oxidize thiosulphate, sulphide, polysulphide, elemental sulphur and tetrathionate. Strain AL 3 more actively oxidized thiosulphate and sulphide, while isolate AL 2 had higher activity with elemental sulphur and tetrathionate. Isolate AL 2 was also able to oxidize trithionate. The pH optimum for thiosulphate and sulphide oxidation was between 9–10. Some activity remained at pH 11, but was negligible at pH 7. Metabolism of tetrathionate by isolate AL 2 involved initial anaerobic hydrolysis to form sulphur, thiosulphate and sulphate in a sequence similar to that in other colourless sulphur-oxidizing bacteria. Sulphate was produced by both strains. During batch growth on thiosulphate, elemental sulphur and sulphite transiently accumulated in cultures of isolates AL 2 and AL 3, respectively. At lower pH values, both strains accumulated sulphur during sulphide and thiosulphate oxidation. Both strains contained ribulose bisphosphate carboxylase. Thiosulphate oxidation in isolate AL 3 appeared to be sodium ion-dependent. Isolate AL 2 differed from AL 3 by its high GC mol % value (65.5 and 49.5, respectively), sulphur deposition in its periplasm, the absence of carboxysomes, lower sulphur-oxidizing capacity, growth kinetics (lower growth rate and higher growth yield) and cytochrome composition.