Detoxication of cyanide in the chicken by conversion to thiocyanate, as influenced by the availability of transferable sulphur

The urinary excretion of thiocyanate by hens after dosage with cyanide (30 mumol) has been studied in a series of acute experiments involving 6 hr urine collection periods. More than half of the dose could be recovered as thiocyanate when cyanide was given by intravenous infusion and the rate of excretion closely paralleled plasma thiocyanate concentration. Little cyanide was excreted directly. The excretion of thiosulphate fell by an amount that suggested that availability of sulphane sulphur might limit the extent of conversion. However, neither thiosulphate nor sulphur amino acids enhanced thiocyanate excretion when they were infused together with cyanide; indeed, thiocyanate excretion decreased as the level of sulphur compound given was increased. Both nitrite and sulphite depressed thiocyanate excretion also but they differed in their effects on plasma thiocyanate levels and the pattern of urinary excretion. Comparison of excretion from both sides of the kidneys separately emphasised the importance of the first pass of cyanide in its conversion to thiocyanate. The results suggest that although sulphur availability may be limited the in vivo production of sulphite also restricts cyanide detoxication.     

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.     

Thiosulphate oxidation in the phototrophic sulphur bacterium Allochromatium vinosum

Two different pathways for thiosulphate oxidation are present in the purple sulphur bacterium Allochromatium vinosum: oxidation to tetrathionate and complete oxidation to sulphate with obligatory formation of sulphur globules as intermediates. The tetrathionate:sulphate ratio is strongly pH-dependent with tetrathionate formation being preferred under acidic conditions. Thiosulphate dehydrogenase, a constitutively expressed monomeric 30 kDa c-type cytochrome with a pH optimum at pH 4.2 catalyses tetrathionate formation. A periplasmic thiosulphate-oxidizing multienzyme complex (Sox) has been described to be responsible for formation of sulphate from thiosulphate in chemotrophic and phototrophic sulphur oxidizers that do not form sulphur deposits. In the sulphur-storing A. vinosum we identified five sox genes in two independent loci (soxBXA and soxYZ). For SoxA a thiosulphate-dependent induction of expression, above a low constitutive level, was observed. Three sox-encoded proteins were purified: the heterodimeric c-type cytochrome SoxXA, the monomeric SoxB and the heterodimeric SoxYZ. Gene inactivation and complementation experiments proved these proteins to be indispensable for thiosulphate oxidation to sulphate. The intermediary formation of sulphur globules in A. vinosum appears to be related to the lack of soxCD genes, the products of which are proposed to oxidize SoxY-bound sulphane sulphur. In their absence the latter is instead transferred to growing sulphur globules.     

Alternative sulphur donors for detoxification of cyanide in the chicken.

1. Urinary excretion of thiocyanate by hens after dosage with cyanide was studied over 3 hr periods during which various sulphur sources were infused. 2. With 20 mumoles cyanide, endogenous sulphur supplies appeared to be almost sufficient. 3. With 45 mumoles cyanide, thiocyanate excretion was doubled with 90 mumoles of sulphur donor. Higher doses of mercaptopyruvate were also effective but not rhodanese substrates (thiosulphate or methanethiosulphonate): they interfered with thiocyanate excretion and may also have suppressed its formation. 4. Mercaptopyruvate and rhodanese substrates also differed in their effects on blood cyanide concentration and on the excretion of isotope from radiolabelled cyanide.     

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.     

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.     

The effect of cAMP and some sulphur compounds upon the activity of mercaptopyruvate sulphurtransferase and rhodanese in mouse liver.

The aim of the experiments was to evaluate the effect of administration of cysteine, methionine, thiocystine, and thiosulphate upon the activity of mercaptopyruvate sulphurtransferase (MPST) and rhodanese in mouse liver. It was found that rhodanese activity increased following thiocystine and methionine administration and showed a smaller increase after cysteine and thiosulphate. The MPST activity significantly increased after cysteine and to a lesser extent after thiocystine and thiosulphate. On the other hand, methionine seemed to exert no effect upon the enzymatic activity. The results suggested that methionine metabolic cycle in mouse liver proceeded from cysteine to sulphane sulphur as thiocystine and, therefore, these three compounds increased rhodanese activity. Thiosulphate seemed rather to be involved in metabolic changes related to maintaining the stability of the physiological thiosulphate level and activated both the enzymes.     

Isolation of some new sulphur bacteria from activated sludge

E mtiazi , G., H abibi , M.H. & S etareh , M. 1990. Isolation of some new sulphur bacteria from activated sludge. Journal of Applied Bacteriology 69 , 864–870.
During studies on bulking of activated sludge some new sulphur micro-organisms, which were able to grow aerobically and anaerobically on reduced sulphur compounds, were isolated on thiosulphate agar. These were capable of autotrophic and heterotrophic growth on a wide range of substrates. In view of their ability to oxidize reduced sulphur compounds, and because one of them was an oxidase- and catalase-positive, Gram-negative, motile coccus (0.36–0.48 μm) it was named Thiosphaera persica . The second one was an oxidase- and catalase-positive, Gram-negative, motile rod (1.32–1.80 μm) and was named Thiobacillus persica . The third one was oxidase-negative, catalase-positive, motile, Gram-negative and polymorphic and was named Sulphobacter polymorpha .     

Isolation and physiological characterisation of Thiobacillus thyasiris sp. nov., a novel marine facultative autotroph and the putative symbiont of Thyasira flexuosa

A novel facultatively chemolithoautotropic Thiobacillus, isolated from the gill tissue of the marine bivalve Thyasira flexuosa, is described. It is believed to be the symbiont from this animal, providing the animal with carbon fixed by the Calvin cycle. The organism grows lithoautotrophically on thiosulphate, tetrathionate and elemental sulphur, which are oxidised to sulphate. It oxidizes sulphide, thiosulphate, trithionate, tetrathionate and hexathionate, but not thiocyanate. Kinetic constants for these substrates are presented. In autotrophic batch culture it produces yields that are among the lowest reported for thiosulphate or tetrathionate as energy substrates (1.25 and 2.5 g cell-carbon per mol substrate, respectively). Autotrophic cultures contain ribulose bisphosphate carboxylase and excreted 20% of their fixed carbon into the medium during growth. Mixotrophic growth on acetate and thiosulphate resulted in partial repression of the carboxylase. The organism is slightly halophilic and markedly halotolerant, showing optimum growth at about pH 7.5 and maximum growth rate at 37° C. It contains ubiquinone Q-10 and its DNA contains 52 mol % G+C. These characteristics distinguish it from any other Thiobacillus or Thiomicrospira species previously described. The organism is formally described and named as Thiobacillus thyasiris.     

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.     

Observations on the source of acid in empoldered mangrove soils

Summary Formation of elemental sulphur as an intermediate in polysulphide oxidation in mangrove soils has been demonstrated both in laboratory experiments and in the field. The oxidation of soil polysulphides occurred in the absence of more reactive sulphur compounds and it is suggested that these are not essential for the initiation of sulphur oxidation in high sulphur soils. Field experiments contained some evidence of instability of soil polysulphides at pH values near neutrality, but laboratory experiments suggested that early decomposition of polysulphides may be accelerated by non-biological oxidations which lower the soil pH value.Maximum population ofThiobacillus thio-oxidans was reached much earlier than maximum oxidative activity, although an early peak in oxidative activity could be obtained by adding elemental sulphur to soil. It is suggested that while conditions in the early stages of the oxidation may be suitable for multiplication of the organisms, maximum oxidative activity is limited by the rate of release of sulphur from the decomposing polysulphide.     

The occurrence of denitrifying colourless sulphur-oxidising bacteria in marine waters and sediments as shown by the agar shake technique

Abstract The agar shake technique has been tested for the enumeration and isolation of bacteria involved in the anaerobic oxidation of reduced sulphur compounds. High numbers of colony forming units were observed from regions rich in sulphide, and the numbers of these forms were sometimes significantly correlated with the number of sulphate-reducing bacteria. The isolates could oxidise not only thiosulphate but also sulphide in liquid medium at the expense of nitrate. Addition of 1 mM glucose to the medium enhanced the rate and amount of thiosulphate oxidised by many of the isolates. Hence the use of the agar shake technique is recommended for the study of these little known facultatively or even obligately chemolithotrophic bacteria involved in the anaerobic oxidation of reduced inorganic sulphur compounds in the marine and estuarine environment.     

THE BIOLOGICAL OXIDATION OF SPENT GAS LIQUOR

SUMMARY: Mixed cultures of bacteria grown in spent gas liquor readily oxidized phenol, o -, m - and p -cresol, catechol, 3-methyl catechol, 4-methyl catechol, resorcinol, 2-methyl resorcinol, and 4-methyl resorcinol. Quinol, pyrogallol and phloroglucinol were more resistant. The optimum temperature was 30° and the best pH range 6·5–7·8. Yeast extract and sterile sewage sludge both increased the rate of growth of organisms in liquor when the inoculum was small. Five phenol oxidizing organisms were isolated in pure culture. Copper in concentrations greater than 1 p/m inhibited both growth and phenol oxidation by one of these.
Mixed cultures grown in an ammonium thiocyanate medium originally inoculated with Thiobacillus thiocyanoxidans oxidized potassium thiocyanate and sodium thiosulphate. Chloride inhibited thiocyanate oxidation in concentrations above 5,000 p/m, although adaptation to 15,000 p/m was possible. Phenol inhibited thiocyanate oxidation in concentrations of 300 p/m or more. Mixed cultures grown on sodium thiosulphate oxidized sodium trithionate and tetrathionate, potassium pentathionate and hexa-thionate, and potassium and ammonium thiocyanate
Manometric determinations of the 5 day biological oxygen demand of effluents after treatment showed good agreement with the values obtained by the conventional method, the manometric values being usually somewhat higher.     

Studies on the in situ physiology of Thiothrix spp. present in activated sludge

The in situ physiology of the filamentous sulphur bacterium Thiothrix spp. was investigated in an industrial wastewater treatment plant with severe bulking problems as a result of overgrowth of Thiothrix. Identification and enumeration using fluorescence in situ hybridization (FISH) with species-specific 16S and 23S rRNA probes revealed that 5–10% of the bacteria in the activated sludge were Thiothrix spp. By using a combination of FISH and microautoradiography it was possible to study the in situ physiology of probe-defined Thiothrix filaments under different environmental conditions. The Thiothrix filaments were very versatile and showed incorporation of radiolabelled acetate and/or bicarbonate under heterotrophic, mixotrophic and chemolithoautotrophic conditions. The Thiothrix filaments were active under anaerobic conditions (with or without nitrate) in which intracellular sulphur globules were formed from thiosulphate and acetate was taken up. Thiothrix -specific substrate uptake rates and growth rates in activated sludge samples were determined under different conditions. Doubling times of 6–9 h under mixotrophic conditions and 15–30 h under autotrophic conditions were estimated. The key properties that Thiothrix might be employing to outcompete other microorganisms in activated sludge were probably related to the mixotrophic growth potential with strong stimulation of acetate uptake by thiosulphate, as well as stimulation of bicarbonate incorporation by acetate in the presence of thiosulphate.     

Identification of a major facilitator protein from Escherichia coli involved in efflux of metabolites of the cysteine pathway

A chromosomal fragment has been identified in a gene bank from Escherichia coli, which augmented the yield of cysteine in an industrial production strain. Subcloning and genetic analysis showed that an open reading frame coding for a product of 299 amino acids (Orf299) was responsible. Orf299 was synthesized in the T7 polymerase/promoter system and exhibited the properties of an integral membrane protein. Mutational interruption of orf299 did not cause a distinct phenotype; however, transformants overexpressing orf299 had lost the ability to grow in minimal medium unless it was supplemented with a source of reduced sulphur compounds, and they excreted considerable amounts of cysteine and O-acetyl-L-serine, especially in the presence of thiosulphate. Most of the cysteine was found to be masked in 2-methyl-2,4-thiazolidinedicarboxylic acid. N-acetyl-L-serine was also present in the medium, but it is open to question whether it represents a primary excretion product. Measurement of the induction status of the cysteine regulon by means of a cysK'-'lacZ gene fusion demonstrated that the regulon is not induced upon growth in the presence of a poor sulphur source and that the introduction of a constitutive cysB allele alleviates this deficiency. The results indicate that orf299 codes for an export pump for different metabolites of the cysteine pathway. Its relation to other efflux systems and the physiological role are discussed.     

Identification of two outer membrane proteins involved in the oxidation of sulphur compounds in Thiobacillus ferrooxidans

Abstract: Shift of three Thiobacillus ferrooxidans strains from Fe(II) to S⁰ or thiosulphate liquid medium caused distinctive changes in the outer membrane protein profile. In addition to a new 55-kDa protein which was synthesized only in the presence of sulphur compounds, a higher expression of a 47-kDa protein was observed. This latter protein appeared to be constitutively synthesized, since it was detectable in small amounts even in tile presence of ferrous iron as sole energy source, but its expression was greatly enhanced when elemental sulphur or thiosulphate were present in the growth medium.     

Chemolithotrophic potential of a Hyphomicrobium species,capable of growth on methylated sulphur compounds

The yield of Hyphomicrobium EG on dimethyl sulphoxide, dimethyl sulphide and methylamine, considering the metabolic pathways of these compounds, suggested that the organism gained energy from the oxidation of the sulphur moiety of the former compounds. Indeed, a comparison of chemostat cultures of Hyphomicrobium EG grown on methylamine in the presence and absence of sulphide or thiosulphate proved this obligate methylotroph to be a chemolithoheterotroph. The apparent Y_sulphide and Y_thiosulphate were comparable, being 8–10 g dry weight/mol. In batch cultures thiosulphate concentrations up to 10 mM had a stimulatory effect on the growth rate of Hyphomicrobium EG, whereas higher concentrations increased the organisms doubling time.Enzyme- and respiration data showed that the organism had constitutive enzymes for the breakdown of dimethyl sulphoxide although they were clearly regulated to need. Addition of sulphide or thiosulphate to methylamine-limited chemostat cultures of Hyphomicrobium EG not only resulted in the induction of enzymes necessary for their breakdown, but also caused the enzymes for dimethyl sulphoxide metabolism, especially methyl mercaptan oxidase, to be induced. The formation of H₂O₂, a product of the latter enzyme, was reflected in the relatively high catalase activities during growth on dimethyl sulphoxide and in the organisms inability to grow on this compound in the presence of a catalase inhibitor.Abbreviations DMSO dimethyl sulphoxide - DMS dimethyl sulphide - MM methyl mercaptan - TMAO trimethylamine N-oxide - D dilution rate - GSH redticed glutathione - DCPIP 2,6-dichlorophenolindophenol - PMS phenazine methosulphate - PES phenazine ethosulphate - RubPCase ribulose 1,5-bisphosphate carboxylase - PEPCase phosphoenol pyruvate carboxylase - Wurster's blue (TMPD) N,N,N,N-tetramethyl-p-phenylenediamine     

Soil heterotrophic bacteria in transformations of inorganic sulphur

The occurrence of biochemical activities of the sulphur cycle was followed in isolates of heterotrophic bacteria from the fermentative horizon of a spruce stand, a grass-covered withered spruce stand and of mountain ash and birch stand in the area strongly influenced by sulphur immissions. The occurrence of bacteria capable of reducing S⁰ to S²⁻, oxidizing S⁰ and S₂O₃ ²⁻ to SO₄ ²⁻ and solubilizing S⁰ increased in the above order. The occurrence of producers of thiosulphate sulphurtransferase (rhodanese), thiosulphate oxidase and sulphite oxidase increased and the level of the production of these enzymes increased as well. Heterotrophic bacteria (mostly pseudomonads) from the grass-covered stands exhibit more activities of the sulphur cycle than bacteria from the spruce stand without ground vegetation.     

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.     

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.