Organic sulphur sources for the growth of the dermatophyte microsporum gypseum

The suitability of 30 organic compounds (of them 19 sulphur-containing amino acids) at a concentration of 1mm as sulphur sources for the growth of the dermatophyteMicrosporum gypseum was investigated. The dry mass of the mycelium after an 11-d growth served as a measure of utilizability. Of sulphur amino acids cystine, cysteine, reduced and oxidized glutathione, cysteic and cysteinesulphinic acids, S-sulphocysteine, lanthionine, taurine and serine sulphate were the best sources. Methionine and methionine-sulphone were utilized slightly less effectively. Other compounds were medium to poor sources and only S-carboxymethylcysteine was not utilized at all. All organic compounds that are not of amino acid type were poor sulphur sources or were utilized at all. Sodium dodecyl sulphate inhibited germination and growth completely.     

Utilization ofL- andDL-cystine by the fungusMicrosporum gypseum

Growth of the fungusMicrosporum gypseum and utilization of cystine during this growth was studied in a glucose-arginine medium containing either sodium sulphate, andL-cystine orDL-cystine. Replacement of sulphate withL-cystine brought about no significant changes in the growth of the microorganism. Utilization ofL-cystine as a source of carbon and nitrogen was rapid and complete and excess sulphur was excreted into the medium in the form of sulphate. Similarly excreted were also minute amounts of sulphite which immediately reacted with the remaining cystine to formS. sulphocysteine. Growth ofM. gypseum in a medium withDL-cystine was slow. Although this substance was not utilized as readily asL-cystine, its utilization was still complete and excess sulphur was similarly excreted in the form of sulphate and sulphite. The initial step in the utilization of theD-isomer is probably its extracellular deamination.     

Cysteine and S-sulphocysteine biosynthesis in bacteria

Summary Four components of O-acetylserine (OAS) sulphhydrylase with different molecular weights have been detected in extracts of Pseudomonas aeruginosa. This bacterium also contained an enzyme, S-sulphocysteine synthase, which catalysed the formation of S-sulphocysteine from OAS and thiosulphate. The latter enzyme was possibly associated with a low molecular weight form of OAS sulphhydrylase. Some properties of OAS sulphhydrylase and S-sulphocysteine synthase in P. aeruginosa are reported.The distribution of OAS sulphhydrylase, serine transacetylase and S-sulphocysteine synthase in a number of bacteria including sulphur photoautotrophs, sulphur chemoautotrophs and dissimilatory sulphate reducers was examined.All organisms contained the first two enzymes but only about half had S-sulphocysteine synthase. There was no correlation between the presence of S-sulphocysteine synthase and other aspects of sulphur metabolism in the organisms studied or the source of sulphur in the growth medium. No enzymic degradation of S-sulphocysteine was detected in P. aeruginosa. Strong repression of OAS sulphhydrylase synthesis by cysteine occurred in Escherichia coli and Rhodopseudomonas spheroides but in P. aeruginosa, Bacillus megaterium and Desulfotomaculum nigrificans the levels of the enzyme did not correlate with the source of sulphur for growth.     

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.     

The role of microorganisms in transformations of sulphite in spruce forest soil

Transformations of sulphite and the participation of microorganisms were investigated in samples of fermentative (A₀₂) and humus (A₀₃) horizons from a spruce forest. About 80% of sulphite were almost immediately oxidized abiotically to sulphate, less than 1% persisted in the soil for several days and almost 20% were converted to a form not demonstrable as sulphite or sulphate. Microorganisms accelerated the disappearance of less than 10% of the added sulphite.     

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.     

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.     

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.     

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.     

The control of sulphate reduction in Escherichia coli by O-acetyl-l-serine

1. Extracts of Escherichia coli A.T.C.C. 9723 and K(12)703 contain serine transacetylase and O-acetylserine sulphhydrase. Synthesis of the latter enzyme is repressed by growth on l-cyst(e)ine and other sulphur compounds. 2. O-Acetyl-l-serine added to cells growing on glutathione or sulphate as source of sulphur induces the enzymes that catalyse (a) the activation of sulphate to adenosine 3'-phosphate 5'-sulphatophosphate (EC 2.7.7.4 and 2.7.1.25), (b) the reduction of adenosine 3'-phosphate 5'-sulphatophosphate to sulphite and (c) the reduction of sulphite to sulphide (EC 1.8.1.2). Hydrogen sulphide is liberated from cultures growing on sulphate as source of sulphur and in the presence of O-acetylserine. 3. The cysE mutants of E. coli K(12) lack serine transacetylase. Addition of O-acetylserine permits growth on sulphate as source of sulphur; at the same time the enzymes of sulphate reduction, previously absent, are synthesized. Such mutants have no detectable intracellular cyst(e)ine when starved of sulphur. 4. These results suggest that O-acetylserine is necessary for synthesizing the enzymes of sulphate reduction in E. coli. Its action does not appear to be by interference with the repressive control exerted over these enzymes by cyst(e)ine.     

Control of 5-aminolaevulinate synthetase activity in Rhodopseudomonas spheroides. The involvement of sulphur metabolism

1. The ;initial' 5-aminolaevulinate synthetase activity, that is the activity observed immediately after cell disruption, in extracts prepared from unharvested semianaerobically grown Rhodopseudomonas spheroides, was twice that observed under the same assay conditions in extracts prepared from harvested cells. 2. The effect of oxygenation of a culture on the ;maximum' aminolaevulinate synthetase activity, that is the activity observed 1h after disruption of harvested cells, is markedly influenced by the contents of the growth medium. Oxygenation of organisms for 1h in the medium in which they have grown produces an 80-90% decrease in maximum activity, whereas similar treatment of organisms resuspended in fresh medium produces less than a 40% decrease. 3. This protective effect of fresh medium is absolutely dependent on the presence of sulphate. When cells are suspended in sulphate-deficient fresh medium, the maximum activity falls by 65-75% even without oxygenation. A high maximum activity is regenerated when sulphate is resupplied. 4. When organisms are oxygenated in the medium in which they have grown, the cellular contents of GSH+GSSG and cysteine+cystine fall very markedly and homolanthionine is formed. Both the fall in aminolaevulinate synthetase activity and the changes in sulphur metabolism are largely prevented by the addition of compounds which stimulate synthesis of cysteine de novo or inhibit the conversion of cysteine S into homocysteine S. 5. The maximum aminolaevulinate synthetase activity was directly proportional to the GSH+GSSG content of all cell preparations. In glutathione-depleted extracts the ;low'-activity enzyme could be re-activated in vitro by the addition of GSH, GSSG, cysteine or cystine, whereas in extracts with a high glutathione content the ;high'-activity enzyme was unaffected by these sulphur compounds. 6. The activation of low-activity enzyme with exogenous sulphur compounds was prevented by excluding air or by adding NADH. Studies with purified enzyme indicate that sulphur compounds do not interact directly with the enzyme, but that their effect is mediated by a number of other endogenous factors.     

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.     

Sulphur oxidation by a Streptomyces sp. growing in a carbon-deficient medium and autoclaved soil

Streptomyces colonies, apparently all of the same species, were isolated from a range of soils using a polysulphide medium lacking an organic carbon source. Growth on this medium, and clearing of the otherwise white, opaque overlay, suggested that the organisms were capable of growing autotrophically. However, investigation of one of these isolates showed that it was unable to fix ¹⁴CO₂ and did not possess the enzyme ribulose bisphosphate carboxylase, showing that it was incapable of autotrophic growth. The isolate oxidized elemental sulphur, thiosulphate and tetrathionate to sulphate in vitro in carbon-deficient medium, and also oxidized elemental sulphur to sulphate when inoculated into autoclaved soil supplemented with sulphur. It also oxidized polysulphide when growing on Czapek Dox and plate count agars. The isolate can therefore grow heterotrophically in both carbon-rich media and in media lacking organic carbon — presumably by scavenging organic carbon from the laboratory atmosphere. The possible role of these organisms in sulphur oxidation in soils is commented upon.     

The formation of exchangeable sulphite from adenosine 3''-phosphate 5''-sulphatophosphate in yeast.

A new low-molecular-weight bound sulphite was found in yeast enzyme reaction systems which convert the sulphur of 35S-labelled adenosine 3'-phosphate 5'-sulphatophosphate into exchangeable radioactive sulphite. This bound sulphite was separated from other components by paper electrophoresis and Sephadex G-25 chromatography, and shown to be a peptide with multiple thiol groups and an estimated mol.wt. of 1400. The labelled sulphur in this peptide is highly exchangeable with unlabelled sulphite, but exchangeability decreases with time and freeze-drying. The low-molecular-weight acceptor is tightly bound to enzyme B of the yeast system and, apparently, accepts the sulpho group of adenosine 3'-phosphate 5'-sulphatophosphate and is released as bound sulphite only in the presence of enzymically or chemically reduced fraction C. It is proposed that the low-molecular-weight acceptor is a carrier peptide which, after release of the reduced sulphur, becomes re-oxidized and returns to enzyme B. Fraction C appears to function as an obligatory reductant of the oxidized acceptor before it can accept another-SO-3-moiety from adenosine 3'-phosphate 5'-sulphatophosphate. These findings are consistent with mechanisms proposed for sulphate reduction in spinach and Chlorella, and suggest that fraction C is the natural thiol required in these systems. An improved column technique for the preparation of adenosine 3'-phosphate 5'-sulphatophosphate is described.     

Sulphate transport inCandida utilis

Sulphate uptake byCandida utilis follows Michaelis-Menten type kinetics characterized by a K_m of 1.43 mM for sulphate. The process is unidirectional, pH, temperature and energy dependent. Molybdate, selenate, thiosulphate, chromate and sulphite are competitive inhibitors. Dithionite is a mixed-type inhibitor of sulphate uptake. If cells are pre-incubated with sulphate, sulphite, thiosulphate, dithionite or sulphide, sulphate uptake is severely blocked. Inhibition by endogenous sulphate, sulphite and thiosulphate was specific for sulphate uptake. Thus, incorporation of extracellular sulphate seems to be under the control of a heterogeneous pool of sulphur compounds. These results are discussed in connection with the regulation of sulphur ammo acid biosynthesis inC.utilis.     

Utilization of cystine by dermatophytes on a gelatin medium

All 16 strains of dermatophytes investigated utilized cystine (added to the gelatin medium) as a source of sulfur and also of carbon and nitrogen. Excess sulfur oxidized and excreted to the medium, primarily as inorganic sulfate. Six strains used up all cystine and excreted more than 90% stoichiometric amount of sulfur. Cystine utilization proceeded in parallel with the development of the culture and was terminated during the stationary phase or as late as in the autolytic phase. Other strains did not use up cystine completely and excreted 17-70% sulfur in the oxidized form. In addition to sulfate, sulfite was always produced during the initial growth phases and in poorly growing strains. Free sulfite was only rarely detected; it usually reacted with the residual cystine yielding S-sulfocysteine that was also used up later. Specific features of cystine metabolism (known from Microsporum gypseum) are generally valid in dermatophytes.     

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.     

Activation of proteases in an anaerobic sulphidogenic bioreactor

Activities of proteases were stimulated by specific sulphur metabolites during the enhanced hydrolysis of complex polymeric organic carbon in an anaerobic sulphidogenic environment. While sulphate at 1000 mg l(-1) inhibited proteases by 50%, there was a 2.5-fold increase in activity of proteases by added sulphite and a 3.6-fold increase from added sulphide. Two hypothetical models are proposed. First the sulphur species, sulphite (HSO3-) and sulphide (HS-), liberated at different times during the sulphate reduction process, directly activate the proteases, which are associated with the organic particulate matter, leading to a subsequent enhancement of hydrolysis of polymeric material. Second, they indirectly activate the proteases by neutralising the cations on the floc surface disrupting the integrity of the organic particulate floc therebye releasing further entrapped enzymes from the organic particulate matter.     

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.     

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.