Microbial Thiocyanate Utilization under Highly Alkaline Conditions

Three kinds of alkaliphilic bacteria able to utilize thiocyanate (CNS⁻) at pH 10 were found in highly alkaline soda lake sediments and soda soils. The first group included obligate heterotrophs that utilized thiocyanate as a nitrogen source while growing at pH 10 with acetate as carbon and energy sources. Most of the heterotrophic strains were able to oxidize sulfide and thiosulfate to tetrathionate. The second group included obligately autotrophic sulfur-oxidizing alkaliphiles which utilized thiocyanate nitrogen during growth with thiosulfate as the energy source. Genetic analysis demonstrated that both the heterotrophic and autotrophic alkaliphiles that utilized thiocyanate as a nitrogen source were related to the previously described sulfur-oxidizing alkaliphiles belonging to the gamma subdivision of the division Proteobacteria (the Halomonas group for the heterotrophs and the genus Thioalkalivibrio for autotrophs). The third group included obligately autotrophic sulfur-oxidizing alkaliphilic bacteria able to utilize thiocyanate as a sole source of energy. These bacteria could be enriched on mineral medium with thiocyanate at pH 10. Growth with thiocyanate was usually much slower than growth with thiosulfate, although the biomass yield on thiocyanate was higher. Of the four strains isolated, the three vibrio-shaped strains were genetically closely related to the previously described sulfur-oxidizing alkaliphiles belonging to the genus Thioalkalivibrio. The rod-shaped isolate differed from the other isolates by its ability to accumulate large amounts of elemental sulfur inside its cells and by its ability to oxidize carbon disulfide. Despite its low DNA homology with and substantial phenotypic differences from the vibrio-shaped strains, this isolate also belonged to the genus Thioalkalivibrio according to a phylogenetic analysis. The heterotrophic and autotrophic alkaliphiles that grew with thiocyanate as an N source possessed a relatively high level of cyanase activity which converted cyanate (CNO⁻) to ammonia and CO₂. On the other hand, cyanase activity either was absent or was present at very low levels in the autotrophic strains grown on thiocyanate as the sole energy and N source. As a result, large amounts of cyanate were found to accumulate in the media during utilization of thiocyanate at pH 10 in batch and thiocyanate-limited continuous cultures. This is a first direct proof of a “cyanate pathway” in pure cultures of thiocyanate-degrading bacteria. Since it is relatively stable under alkaline conditions, cyanate is likely to play a role as an N buffer that keeps the alkaliphilic bacteria safe from inhibition by free ammonia, which otherwise would reach toxic levels during dissimilatory degradation of thiocyanate.     

Identification of the microbial community responsible for thiocyanate and thiosulfate degradation in an activated sludge process

An activated sludge reactor fed with thiocyanate and/or thiosulfate was operated to examine the characteristics of its microbial community. Terminal-restriction fragment length polymorphism analyses were conducted to detect shifts in the microbial community structure corresponding to influent conditions. Then, clone library analyses and RNA-based stable-isotope probing were conducted to identify sulfur-oxidizing bacteria (SOB) responsible for the degradation of each substrate. The results suggested that there were two types of SOB: thiocyanate-degrading bacteria (that can utilize both thocyanate and thiosulfate) and thiosulfate-specific bacteria (that cannot utilize thiocyanate). Thiocyanate-degrading SOB, however, were outcompeted by thiosulfate-specific SOB when the influent contained only thiosulfate. Of the sequenced clones, Marinicella-related (with 98.7% identity) and Methylobacter-related (with 91.3% identity) bacteria were identified as thiocyanate-degrading SOB, whereas Thiomicrospira thermophila-related (with 100% identity over 903 bp) bacteria were identified as thiosulfate-specific SOB.     

Antibacterial Activity of the Lactoperoxidase System in Milk Against Pseudomonads and Other Gram-Negative Bacteria

Products of thiocyanate oxidation by lactoperoxidase inhibit gram-positive bacteria that produce peroxide. We found these products to be bactericidal for such gram-negative bacteria as Pseudomonas species and Escherichia coli, provided peroxide is supplied exogenously by glucose oxidase and glucose. By the use of immobilized glucose oxidase the bactericidal agent was shown to be dialyzable, destroyed by heat and counteracted, or destroyed by reducing agents. Because the system is active against a number of gram-negative bacteria isolated from milk, it may possibly be exploited to increase the keeping quality of raw milk.     

Degradation of thiocyanate by a bacterial coculture

Summary A bacterial coculture capable of growing on thiocyanate has been isolated from thiocyanate adapted bacterial suspension of urban sewage treatment plant. The coculture is composed of two bacteria identified as species Acinetobacter johnsonii and Pseudomonas diminuta. The two end products of thiocyanate conversion are ammonia and sulfate. The thiosulfate has been identified as the sulfur intermediate product of the conversion of thiocyanate to sulfate.     

The reaction of clostridial ferredoxin with cyanide

Treatment of clostridial ferredoxin with cyanide caused bleaching of the protein and formation of thiocyanate. The rate of bleaching was increased by urea, heat, or alkali. In experiments with C. acidi-urici [³⁵S]sulfide-ferredoxin, it was shown that cyanolysis converts 70–80% of the sulfide to [³⁵S]thiocyanate. Apoferredoxin_ox, other disulfides, or Na₂S alone did not yield thiocyanate under these conditions. However, the apoprotein, as well as 2-mercaptoethanol disulfide, forms thiocyanate when Na₂S is added. The addition of Na₂S also increases the amount of thiocyanate formed from ferredoxin. The specific activity of the thiocyanate formed from [³⁵S]sulfide-ferredoxin in the presence of added unlabeled Na₂S is greatly decreased. The specific activity of the thiocyanate formed from the cyanolysis of [³⁵S]sulfide-ferredoxin in the presence of urea and excess sulfide increased with time. Bleaching of ferredoxin during cyanolysis in the presence of urea led to the release of inorganic sulfide prior to the formation of thiocyanate. These observations suggest that it is likely that thiocyanate formation from ferredoxin and cyanide results from the production of a persulfide bond between the apoprotein and the released sulfide. Therefore, thiocyanate production from ferredoxin treated with cyanide does not constitute evidence for the occurrence of the persulfide group in the native protein.     

Response of nitrifying bacterial communities to the increased thiocyanate concentration in pre-denitrification process

Changes in process performance and the nitrifying bacterial community associated with an increase of thiocyanate (SCN⁻) loading were investigated in a pre-denitrification process treating industrial wastewater. The increased SCN⁻ loading led to the concentration of total nitrogen (TN) in the final effluent, but increasing the internal recycling ratio as an operation parameter from 2 to 5 resulted in a 21% increase in TN removal efficiency. In the aerobic reactor, we found that the Nitrosomonas europaea lineage was the predominant ammonia oxidizing bacteria (AOB) and the percentages of the AOB population within the total bacteria increased from about 4.0% to 17% with increased SCN⁻ concentration. The increase of nitrite loading seemed to change the balance between Nitrospira and Nitrobacter, resulting in the high dominance of Nitrospira over Nitrobacter. Meanwhile, a Thiobacillus thioparus was suggested to be the main microorganism responsible for the SCN⁻ biodegradation observed in the system.     

Effect of Chemicals on the Early Stages of Tobacco Mosaic Virus Protein Polymerization

Osmotic pressure and sedimentation velocity techniques have been used to investigate the effect of KSCN, thiourea, EDTA, acetamide, and sucrose on the 53,000 molecular weight A protein at pH 6.5-7.0. In the presence of all the compounds except thiocyanate, the number average molecular weight lies between 50,000 and 56,000 which corresponds to a trimer of three chemical subunits. In the presence of thiocyanate, the molecular weight decreases initially sharply with increasing concentration of thiocyanate to 0.1 M, then dissociation proceeds with less efficiency with increasing concentration of KSCN until a molecular weight close to a monomer (21,700) is obtained at 0.59 M KSCN. Sedimentation data agree with osmotic pressure data since, in the absence of thiocyanate, the measured values of S_20,w indicate that the favored state is that of a trimer. In the presence of thiocyanate, however, S_20,w decreases with thiocyanate concentration to S_20,w = 1.9, which is the value reported for the monomer.     

ATP-dependent chloride transport in plasma membrane vesicles from Aplysia intestine

A Cl⁻-stimulated ATPase activity, which is sensitive to both thiocyanate and vanadate, has been localized to the plasma membrane of Aplysia enterocytes. Utilizing plasma membrane vesicles from Aplysia enterocytes, ATP stimulated Cl⁻ uptake to approximately 2.5-times that of control in a Na⁺, K⁺ and HCO₃⁻-free medium. This ATP-dependent Cl⁻ uptake was sensitive to both thiocyanate and vanadate. These results are consistent with the hypothesis that the active Cl⁻ absorptive process in Aplysia intestine could be a Cl⁻-stimulated ATPase found in the enterocyte plasma membrane.     

Degradation of glucosinolates of Nasturtium officinale seeds

Nasturtium officinale contains four glucosinolates, the major representative being 2-phenethylglucosinolate. On autolysis of seeds or leaves, isothiocyanates were the main products of glucosinolate degradation but no thiocyanate was detected. The application of heat during extraction caused an increase in nitrile formation to dominance over isothiocyanates. A (benzyl) thiocyanate-forming extract of Lepidium sativum seeds did not provoke generation of any thiocyanate from glucosinolates of N. officinale (or Barbarea praecox), but it did impose accentuated nitrile-forming properties on the systems. The conclusion is reached that some glucosinolate-containing Cruciferae are predominantly nitrile-producing and some predominantly isothiocyanate-producing, all other factors being constant.     

The inhibition of streptococci by lactoperoxidase,thiocyanate and hydrogen peroxide. The effect of the inhibitory system on susceptible and resistant strains of group N streptococci

1. The growth of the lactoperoxidase-sensitive Streptococcus cremoris 972 in a synthetic medium was inhibited by lactoperoxidase and thiocyanate. The glycolysis and oxygen uptake of suspensions of Strep. cremoris 972 in glucose or lactose were also inhibited. The lactoperoxidase-resistant Strep. cremoris 803 was not inhibited under these conditions but was inhibited in the absence of a source of energy. 2. Lactoperoxidase (EC 1.11.1.7), thiocyanate and hydrogen peroxide completely inhibited the hexokinases of non-metabolizing suspensions of both strains. The inhibition was reversible, hexokinase and glycolytic activities of Strep. cremoris 972 being restored by washing the cells free from inhibitor. The aldolase and 6-phosphogluconate-dehydrogenase activities of Strep. cremoris 972 were partially inhibited but several other enzymes were unaffected. 3. The resistance of Strep. cremoris 803 to inhibition was not due to the lack of hydrogen peroxide formation, to the destruction of peroxide, to the inactivation of lactoperoxidase or to the operation of alternative pathways of carbohydrate metabolism. 4. A `reversal factor'', which was partially purified from extracts of Strep. cremoris 803, reversed the inhibition of glycolysis of Strep. cremoris 972. The `reversal factor'' also catalysed the oxidation of NADH₂ in the presence of an intermediate oxidation product of thiocyanate and was therefore termed the NADH₂-oxidizing enzyme. 5. The NADH₂-oxidizing enzyme was present in lactoperoxidase-resistant streptococci but was absent from lactoperoxidase-sensitive streptococci.     

Volatile components of cocoa with particular reference to glucosinolate products

The aroma volatiles of raw, fermented and roasted cocoa beans were extracted and concentrated to valid essences using well-established techniques. Analysis by GC and GC/MS showed at least 84 components of which 13 were identified for the first time as cocoa volatiles. In total, ca 5,66 and 65 μg of aroma components were obtained per g of raw, fermented and roasted cocoa beans, respectively. The most abundant groups of volatiles from fermented beans were alcohols (ca40%w/w of the total volatiles) and esters (ca 32%), whilst those from roasted beans were pyrazines (ca 40%) and aldehydes (ca 23%). Trimethyl- and tetramethylpyrazine were also detected in fermented beans, and it is suggested that they contribute to the noticeable cocoa/chocolate aroma of fermented unroasted beans. Phenylacetonitrile, benzyl isothiocyanate and benzyl thiocyanate were all identified amongst cocoa volatiles, together showing the presence of precursor benzylglucosinolate in cocoa. Glucosinolate products were detected in roasted beans, and it seems likely that the enzyme thioglucoside glucohydrolase survived the conditions of roasting. Benzyl thiocyanate was detected only in raw beans, showing that the glucosinolate ‘thiocyanate–forming factor’ did not withstand conditions of fermentation     

Effect of temperature on proton efflux from isolated chloroplast thylakoids

Temperature-induced changes in the decay of the light-induced proton gradient of chloroplast thylakoids isolated from chilling-resistant and chilling-sensitive plants have been examined. In the presence of N-methylphenazonium methosulfate, the thylakoids isolated from chilling-resistant barley (cv. Kanby) and pea (cv. Alaska) and chilling-sensitive mung bean (cv. Berken) plants showed temperature-induced changes at approximately 8.6, 13.3, and 14.0 C, respectively. Barley thylakoids assayed in the presence of sodium thiocyanate also showed a change at 8.6 C, whereas with no addition or upon the inclusion of both N-methylphenazonium methosulfate and sodium thiocyanate the change occurred at approximately 11.5 C.     

Isothiocyanates,nitriles and thiocyanates as products of autolysis of glucosinolates in Cruciferae

Glucosinolates from seventy-nine 8-week-old plant species were hydrolysed and the volatile products identified by GC-MS and related to previous published findings. Known compounds, identified in new plant sources, were 4-methylthiobutyl thiocyanate in Alyssum, 4-methylthiobutyl isothiocyanate in Diplotaxis and Eruca and isopropyl isothiocyanate and 5-vinyl-2-oxazolidinethione in Plantago.     

Cloning of Genes Coding for the Three Subunits of Thiocyanate Hydrolase of Thiobacillus thioparus THI 115 and Their Evolutionary Relationships to Nitrile Hydratase

Thiocyanate hydrolase is a newly found enzyme from Thiobacillus thioparus THI 115 that converts thiocyanate to carbonyl sulfide and ammonia (Y. Katayama, Y. Narahara, Y. Inoue, F. Amano, T. Kanagawa, and H. Kuraishi, J. Biol. Chem. 267:9170–9175, 1992). We have cloned and sequenced the scn genes that encode the three subunits of the enzyme. The scnB, scnA, and scnC genes, arrayed in this order, contained open reading frames encoding sequences of 157, 126, and 243 amino acid residues, respectively, for the β, α, and γ subunits, respectively. Each open reading frame was preceded by a typical Shine-Dalgarno sequence. The deduced amino-terminal peptide sequences for the three subunits were in fair agreement with the chemically determined sequences. The protein molecular mass calculated for each subunit was compatible with that determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. From a computer analysis, thiocyanate hydrolase showed significant homologies to bacterial nitrile hydratases known to convert nitrile to the corresponding amide, which is further hydrolyzed by amidase to form acid and ammonia. The two enzymes were homologous over regions corresponding to almost the entire coding regions of the genes: the β and α subunits of thiocyanate hydrolase were homologous to the amino- and carboxyl-terminal halves of the β subunit of nitrile hydratase, and the γ subunit of thiocyanate hydrolase was homologous to the α subunit of nitrile hydratase. Comparisons of the catalytic properties of the two homologous enzymes support the model for the reaction steps of thiocyanate hydrolase that was previously presented on the basis of biochemical analyses.     

Light-scattering and infrared-spectrophotometric studies of chitin and chitin derivatives

A sample of chitin isolated from the shell of the crab Scylla serrata had, when in lithium thiocyanate solution, an average, weight-average molecular weight (1) of 1.036 x 10⁶ daltons, an intrinsic dissymmetry (2) of 1.93, and a Z-average radius of gyration (3) of 64.14 nm. Carboxymethylchitin and glycol chitin, in 0.5M sodium chloride, had, respectively, (1) 1.896 and 1.819 x 10⁶ daltons, (2) 3.25 and 4.31. and (3) 143.49 and 251.57 nm. They had similar degrees of polymerization, they underwent dissociation as the concentration of sodium chloride was increased to 2.5M, and the molecular packing of the chains was essentially side-by-side. Chitin in 5.55M lithium thiocyanate and carboxymethylchitin in 2.5M sodium chloride had similar degrees of polymerization. It is concluded that a small but significient number of the amino groups in the chitin molecule are not acetylated.     

Isolation of an aboriginal bacterial community capable of utilizing cyanide,thiocyanate, and ammonia from metallurgical plant wastewater

An aboriginal bacterial community capable of degrading cyanide (10 mg/l) and thiocyanate (2 g/l) and eliminating ammonia (120 mg/l) had been isolated from recycled water samples after blast-furnace gas purification of a metallurgical plant wastewater. It was shown that the optimal conditions for this bacterial community were as follows: temperature, 34°C; pH, 8.8–9.0; available organic matter concentration (glucose equivalent), 5 g/l; and dissolved O₂ concentration, 8–10 mg/l. This aboriginal community was formed by the bacteria belonging to the genus Pseudomonas.     

A highly catalytically active γ-carbonic anhydrase from the pathogenic anaerobe Porphyromonas gingivalis and its inhibition profile with anions and small molecules

Carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the γ-class are present in archaea, bacteria and plants but, except the Methanosarcina thermophila enzymes CAM and CAMH, they were poorly characterized so far. Here we report a new such enzyme (PgiCA), the γ-CA from the oral cavity pathogenic bacterium Porphyromonas gingivalis, the main causative agent of periodontitis. PgiCA showed a good catalytic activity for the CO₂ hydration reaction, comparable to that of the human (h) isoform hCA I. Inorganic anions such as thiocyanate, cyanide, azide, hydrogen sulfide, sulfamate and trithiocarbonate were effective PgiCA inhibitors with inhibition constants in the range of 41–97 μM. Other effective inhibitors were diethyldithiocarbamate, sulfamide, and phenylboronic acid, with K_Is of 4.0–9.8 μM. The role of this enzyme as a possible virulence factor of P. gingivalis is poorly understood at the moment but its good catalytic activity and the possibility to be inhibited by a large number of compounds may lead to interesting developments in the field.     

Changes in the populations of microorganisms associated with the application of soil amendments to control Sclerotium rolfsii Sacc.

Populations of microorganisms from soil treated with guanidine thiocyanate, guanylurea sulfate, thiourea, or furfural were compared with those of untreated soil. The materials effected quantitative and/or qualitative changes in composition of the soil microflora depending on the compound used. Guanidine thiocyanate (Gt) significantly (p0.05) increased total fungal populations relative to populations of other treatments. Populations of Penicillium purpurogenum were markedly higher in Gt-treated soil. Gt also increased total bacterial populations, and was the only compound that increased actinomycete populations. The relative percentage of Trichoderma harzianum was significantly higher in soil treated with thiourea than in the other treatments. Furfural increased the percentage of P. purpurogenum with respect to total fungi, and was as effective as guanylurea sulfate in increasing chitinolytic bacteria and those in the Pseudomonas cepacia-group. Thiourea most effectively promoted proliferation of coryneform bacteria. Chitinolytic fungi increased synergistically when Gt and guanylurea sulfate were applied in combination.     

Lactoferrin,myeloperoxidase, and ceruloplasmin: complementary gearwheels cranking physiological and pathological processes

Copper-containing plasma protein ceruloplasmin (Cp) forms a complex with lactoferrin (Lf), an iron-binding protein, and with the heme-containing myeloperoxidase (Mpo). In case of inflammation, Lf and Mpo are secreted from neutrophil granules. Among the plasma proteins, Cp seems to be the preferential partner of Lf and Mpo. After an intraperitoneal injection of Lf to rodents, the “Cp–Lf” complex has been shown to appear in their bloodstream. Cp prevents the interaction of Lf with protoplasts of Micrococcus luteus. Upon immunoprecipitation of Cp, the blood plasma becomes depleted of Lf and in a dose-dependent manner loses the capacity to inhibit the peroxidase activity of Mpo, but not the Mpo-catalyzed oxidation of thiocyanate in the (pseudo)halogenating cycle. Antimicrobial effect against E. coli displayed by a synergistic system that includes Lf and Mpo–H₂O₂–chloride, but not thiocyanate, as the substrate for Mpo is abrogated when Cp is added. Hence, Cp can be regarded as an anti-inflammatory factor that restrains the halogenating cycle and redirects the synergistic system Mpo–H₂O₂–chloride/thiocyanate to production of hypothiocyanate, which is relatively harmless for the human organism. Structure and functions of the “2Cp–2Lf–Mpo” complex and binary complexes Cp–Lf and 2Cp–Mpo in inflammation are discussed.     

Synthesis and characterisation of ammonium mediated assembly of two neutral nickel(II) Schiff base fragments

The 1:2 condensation of o-phenelenediamine and o-vanilline yields a compartmental N₂O₄ ligand N,N′-(1,2-Phenylene)-bis(3-methoxysalicylideneimine) [H₂L]. When nickel(II) thiocyanate is added to the methanol solution of H₂L, followed by addition of ammonium thiocyanate, an unusual nickel(II) compound, [NH₄(NiL)₂SCN]·H₂O (1), is separated out in which an ammonium ion is sandwiched between two neutral square planner NiL moieties. Hydrogen bonding interactions are observed among the ammonium ion, NiL moieties, the thiocyanate anion and the water of crystallization. The compound is characterized by C, H, N analysis, UV-VIS and IR spectroscopy, room temperature magnetic susceptibility measurement and X-ray crystal diffraction study. The compound crystallizes in monoclinic space group P2_1/n with a = 13.8636(7) Å, b = 14.0267(7) Å, c = 22.2715(10) Å and β = 94.301(3)°.