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PubMed1997

Purification and Characterization of Cystathionine (gamma)-Lyase from Lactococcus lactis subsp. cremoris SK11: Possible Role in Flavor Compound Formation during Cheese Maturation.

A cystathionine (gamma)-lyase (EC 4.4.1.1) ((gamma)-CTL) was purified to homogeneity from a crude cell extract of Lactococcus lactis subsp. cremoris SK11 by a procedure including anion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration chromatography. The activity of SK11 (gamma)-CTL is pyridoxal-5(prm1)-phosphate dependent, and the enzyme catalyzes the (alpha),(gamma)-elimination reaction of L-cystathionine to produce L-cysteine, (alpha)-ketobutyrate, and ammonia. The native enzyme has a molecular mass of approximately 120 to 200 kDa and apparently consists of at least six identical subunits of 20 kDa. In this respect, the SK11 enzyme clearly differs from other bacterial cystathionine lyases, which are all tetrameric proteins with identical subunits of approximately 40 kDa. In addition, the specific activity of purified SK11 (gamma)-CTL toward L-cystathionine is relatively low compared with those reported for other bacterial cystathionine lyases. The SK11 enzyme shows a broad substrate specificity. In the case of L-methionine, the action of SK11 (gamma)-CTL results in the formation of methanethiol, a volatile sulfur compound known to be required in flavor development in cheddar cheese. The (alpha),(beta)-elimination reaction of L-cysteine is also efficiently catalyzed by the enzyme, resulting in the formation of hydrogen sulfide. Although the conditions are far from optimal, cystathionine (gamma)-lyase is still active under cheddar cheese-ripening conditions, namely, pH 5.0 to 5.4 and 5% (wt/vol) NaCl. The possible role of the enzyme in cheese flavor development is discussed.

Journal Article↗
PubMed2004

Development of an automated water toxicity biosensor using Thiobacillus ferrooxidans for monitoring cyanides in natural water for a water filtering plant.

An on-line biosensor consisting of immobilized Thiobacillus ferrooxidans and an oxygen electrode was developed for automated monitoring of acute toxicity in water samples. T. ferrooxidans is an obligatory acidophilic, autotrophic bacterium and derives its energy by the oxidation of ferrous ion, elemental sulfur, and reduced sulfur compounds including metal sulfides. The assay is based on the monitoring of a current increase by addition of toxicoids, which is caused by the inhibition of bacterial respiration and decrease in oxygen consumption. Optimum cell number on the membrane was 5.0 x 10(8) cells. The steady-state current was obtained when concentration of FeSO4 was above 3.6 mM at pH 3. The sensor response of T. ferrooxidans immobilized membrane for 5.0 microM KCN was within an error of 10% for 30 membranes. A linear relationship was obtained at KCN concentration in the range of 0.5-3.0 microM in a flow-type monitoring system. Minimum detectable concentrations of KCN, Na2S, and NaN3 were 0.5, 1.2, and 0.07 microM, respectively. The monitoring system contained two biosensors and these sensors were cleaned with sulfuric acid (pH 1.5) twice a day. This treatment could remove fouling on microbial immobilized membrane by natural water and ferrous precipitation in the flow cell. This flow-type monitoring sensor was operated continuously for 5 months. Also, T. ferrooxidans immobilized membrane can be stored for one month at 4 degrees C when preserved with wet absorbent cotton under argon gas.

Biosensing Techniques↗
PubMed2003

Thialkalivibrio nitratireducens sp. nov., a nitrate-reducing member of an autotrophic denitrifying consortium from a soda lake.

Strain ALEN 2(T) was isolated from a mixed culture capable of complete autotrophic denitrification with thiosulfate as electron donor at pH 10; the mixed culture was enriched from sediment from Lake Fazda (Wadi Natrun, Egypt), a hypersaline alkaline lake. The isolate had large, non-motile, coccoid or barrel-shaped cells with intracellular sulfur globules. The bacterium was obligately chemolithoautotrophic. It grew with reduced sulfur compounds aerobically and anaerobically with nitrate as electron acceptor, nitrate being reduced to nitrite. It was moderately halophilic and obligately alkaliphilic. On the basis of genetic analysis and its unique phenotype, strain ALEN 2(T) (=DSM 14787(T)=UNIQEM 213(T)) is proposed as the type strain of a novel species of the genus Thialkalivibrio, Thialkalivibrio nitratireducens.

Chromatiaceae↗
PubMed2003

Biodegradation of linear alkylbenzene sulfonates in sulfate-leached soil mesocosms.

Aromatic sulfonates (R-SO(3)(-)) can be used as sulfur sources by sulfate-starved bacteria in laboratory cultures and the corresponding phenols are excreted from the cells. The present study was conducted to demonstrate whether such desulfonation reactions also occur in sulfate-leached agricultural soil, where desulfonation of organic sulfur compounds may have agronomic importance as a S source for plants. Xenobiotic linear alkylbenzene sulfonates (LAS) were added to nominal concentrations of 0, 10 and 100 mgkg(-1) dry weight in a sandy soil that was depleted in sulfate by leaching the soil with water (sulfate depletion, approximately 75%). The soil was incubated at 20 degrees C in duplicate 3-dm(3) mesocosms for 8 weeks. Primary degradation of LAS was rapid with half-lives of 1-4 days. Sulfophenylcarboxylates were identified and quantified as intermediates, whereas linear alkylphenols (the expected primary desulfonation products) were not detected by high-pressure liquid chromatography coupled with both fluorescence and electrospray ionization-mass spectrometry. Thus, LAS was used by the bacteria as a source of energy and carbon, rather than as a source of sulfur. Measurements of soil pH, fluorescein diacetate (FDA) hydrolysis and arylsulfatase activity showed that stable microbial conditions prevailed in the soil mesocosms. FDA hydrolysis (a measure of total microbial activity) was transiently inhibited at the highest LAS concentrations. Arylsulfatase activity (i.e., hydrolysis of aromatic sulfate esters) was not significantly affected by the soil incubation, although arylsulfatases may be upregulated in sulfate-starved bacteria. However, an increased production of arylsulfatase may be difficult to detect due to the background of extracellular arylsulfatases stabilised in the soil. Therefore, the present data does not exclude a regulatory response to sulfate depletion by the soil microorganisms. However, the importance of desulfonation reactions in natural environments still needs to be demonstrated.

Agriculture↗
PubMed2004

Facile reduction of arsenate in methanogenic sludge.

Due to the recent enactment of a stricter drinking water standard for arsenate, large quantities of arsenate-laden drinking water residuals will be disposed in municipal landfills. The objective of this study was to determine the role of methanogenic consortia on the conversion of arsenate. Methanogenic conditions commonly occur in mature municipal solid waste landfills. The results indicate the rapid and facile reduction of arsenate to arsenite in methanogenic sludge. Endogenous substrates in the sludge were sufficient to support the reductive biotransformation. However the rates of arsenate reduction were stimulated by the addition of exogenous electron donating substrates, such as H2, lactate or a mixture of volatile fatty acids. A selective methanogenic inhibitor stimulated arsenate reduction in microcosms supplied with H2, suggesting that methanogens competed with arsenate reducers for the electron donor. Rates of arsenate reduction increased with arsenate concentration up to 2 mM, higher concentrations were inhibitory. The electron shuttle, anthraquinone-2,6-disulfonate, used as a model of humic quinone moieties, was shown to significantly increase rates of arsenate reduction at substoichiometric concentrations. The presence of sulfur compounds, sulfate and sulfide, did not affect the rate of arsenate transformation but lowered the yield of soluble arsenite, due to the precipitation of arsenite with sulfides. The results taken as a whole suggest that arsenate disposed into anaerobic environments may readily be converted to arsenite increasing the mobility of arsenic. The extent of the increased mobility will depend on the concentration of sulfides generated from sulfate reduction.

Anthraquinones↗
PubMed2006

[Air stripping-UASB process for the treatment of evaporator condensate from a Kraft pulp mill].

Evaporator condensate from a kraft pulp mill is characterized by high temperature, high strength, poor nutrition, and some odor and inhibitive materials. In this study, air stripping-UASB process was developed to treat the wastewater from a kraft pulp mill. The lab scale study demonstrated that air stripping process removed 70%-80% of the volatile organic sulfur compounds. After that, the UASB reactor showed high efficiency, at the organic loading rate (COD) of 30 kg x (m3 x d)(-1), COD removal was retained about 95%. On the other hand, the inoculated granules were broken in the new surroundings and were replaced with the newly formed granules The scanning electronic microscope (SEM) observation showed wide difference of the predominant anaerobic microorganisms in the seed and newly formed granules.

Bacteria, Anaerobic↗
PubMed1994

Isolation of a dimethylsulfide-utilizing Hyphomicrobium species and its application in biofiltration of polluted air.

The methylotrophic bacterium Hyphomicrobium VS was enriched and isolated, using activated sewage sludge as inoculum in mineral medium containing dimethylsulfide (DMS) at a low concentration to prevent toxicity. DMS concentrations above 1 mM proved to be growth inhibiting. Hyphomicrobium VS could use DMS, dimethylsulfoxide (DMSO), methanol, formaldehyde, formate, and methylated amines as carbon and energy source. Carbon was assimilated via the serine pathway. DMS-grown cells respired sulfide, thiosulfate, methanethiol, dimethyldisulfide and dimethyltrisulfide. To test Hyphomicrobium VS for application in biofiltration of air polluted with volatile sulfur compounds two laboratory scale trickling biofilters with polyurethane and lava stone as carrier material were started up by inoculation with this bacterium. Both methanol- and DMS-grown cells could be used. Only a short adaptation period was needed. Short term experiments showed that high concentrations of DMS (1-2 mumol l-1) were removed very efficiently by the biofilters at space velocities up to 100 h-1.

Air Pollutants↗
PubMed1990

Influence of dithiocarbamates on the metabolism and toxicity of N-nitrosodimethylamine in rats.

Five secondary amines and secondary amino acids were reacted with carbon disulfide to yield dithiocarbamates. These compounds were tested for their influence on biochemical parameters and on some biological effects of N-nitrosodimethylamine (NDMA). The following results were found. (i) All dithiocarbamates tested reduced the activity of N-nitrosodiethylamine-deethylase and completely inhibited the N-nitrosodimethylamine-demethylase in the rat liver. A striking influence on the glutathione content and on the activity of glutathione-S-transferase and glutathione-reductase was not observed. (ii) The excretion of unmetabolized N-nitrosodimethylamine in rat urine during 24 h increased from 0.1% without pretreatment to 3.6% of the given dose on combination with a single dose of dithiocarbamate. (iii) The acute toxicity of NDMA could be reduced with diethanol-dithiocarbamate. After a single simultaneous application of the inhibitor with NDMA, the LD50 was increased from 40 to 66 mg/kg. When the sulfur compound was administered twice, both simultaneously and 24 h after the nitrosamine, lethality was almost completely inhibited. We conclude from these results that dithiocarbamates may be suitable compounds for chemoprevention of nitrosamine-induced tumors.

Animals↗
PubMed2001

Novel genes of the sox gene cluster, mutagenesis of the flavoprotein SoxF, and evidence for a general sulfur-oxidizing system in Paracoccus pantotrophus GB17.

The novel genes soxFGH were identified, completing the sox gene cluster of Paracoccus pantotrophus coding for enzymes involved in lithotrophic sulfur oxidation. The periplasmic SoxF, SoxG, and SoxH proteins were induced by thiosulfate and purified to homogeneity from the soluble fraction. soxF coded for a protein of 420 amino acids with a signal peptide containing a twin-arginine motif. SoxF was 37% identical to the flavoprotein FccB of flavocytochrome c sulfide dehydrogenase of Allochromatium vinosum. The mature SoxF (42,832 Da) contained 0.74 mol of flavin adenine dinucleotide per mol. soxG coded for a novel protein of 303 amino acids with a signal peptide containing a twin-arginine motif. The mature SoxG (29,657 Da) contained two zinc binding motifs and 0.90 atom of zinc per subunit of the homodimer. soxH coded for a periplasmic protein of 317 amino acids with a double-arginine signal peptide. The mature SoxH (32,317 Da) contained two metal binding motifs and 0.29 atom of zinc and 0.20 atom of copper per subunit of the homodimer. SoxXA, SoxYZ, SoxB, and SoxCD (C. G. Friedrich, A. Quentmeier, F. Bardischewsky, D. Rother, R. Kraft, S. Kostka, and H. Prinz, J. Bacteriol. 182:4476-4487, 2000) reconstitute a system able to perform thiosulfate-, sulfite-, sulfur-, and hydrogen sulfide-dependent cytochrome c reduction, and this system is the first described for oxidizing different inorganic sulfur compounds. SoxF slightly inhibited the rate of hydrogen sulfide oxidation but not the rate of sulfite or thiosulfate oxidation. From use of a homogenote mutant with an in-frame deletion in soxF and complementation analysis, it was evident that the soxFGH gene products were not required for lithotrophic growth with thiosulfate.

Amino Acid Sequence↗
PubMed2000

Diversity of L-methionine catabolism pathways in cheese-ripening bacteria.

Enzymatic activities that could be involved in methanethiol generation in five cheese-ripening bacteria were assayed, and the major sulfur compounds produced were identified. L-Methionine and alpha-keto-gamma-methyl-thio-butyric acid demethiolating activities were detected in whole cells and cell extracts (CFEs) of all the bacteria tested. No L-methionine deaminase activity could be detected in any of the ripening bacteria and L-methionine aminotransferase was detected in CFEs of Brevibacterium linens, Micrococcus luteus, and Corynebacterium glutamicum. The results suggest that several pathways for L-methionine catabolism probably coexist in these ripening bacteria.

Arthrobacter↗
PubMed1979

Simultaneous occurrence of xanthine oxidase and sulfite oxidase deficiency. A molybdenum dependent inborn error of metabolism?

In a 3-week old female child with clinical features including neurologic abnormalities and lens dislocation, xanthinuria co-existed with increased excretion of sulfur compounds (sulfite, S-sulfocysteine, taurine and thio-sulfate). Low xanthine oxidase and absent sulfite oxidase activities were found on liver biopsy. No abnormality was detected in either parent. Both the above enzymes are molybdenum-flavoproteins. Normal serum molybdenum concentration seemed to rule out dietary deficiency or impaired absorption. A defect in the incorporation of the metal into flavoproteins is postulated in this case.

Animals↗
PubMed1995

Reaction between metabolically activated acetaminophen and phosphorothioate oligonucleotides.

Assessment of toxic or mutagenic risks associated with phosphorothioate oligonucleotides (PTO) is important. In vitro and in vivo data have shown that PTOs are nontoxic and nonmutagenic. However, these studies do not address interactions between PTOs and other compounds. The sulfur on PTOs may provide a novel reactive center on a DNA molecule for drug interactions. This study chose acetaminophen (ACAP) as a model drug because ACAP is oxidized to the reactive intermediate N-acetyl-p-benzoquinone imine (NAPQI), which reacts with sulfur-containing compounds. Reaction of dCTP(S) with NAPQI or activated ACAP formed a product distinct from the reactants. Analysis of the product by fast atom bombardment mass spectroscopy gave a molecular weight consistent with NAPQI bound to a sulfur. Higher-molecular-weight products were seen on a polyacrylamide gel electrophoresis after incubation of fluorescein-labeled PTO with NAPQI. These products were not seen after incubation of a phosphodiester oligonucleotide with NAPQI. 31P NMR analysis confirmed the existence of a heterogenous mixture of adducts between a PTO and NAPQI. Nonsequence-specific PTOs of various lengths were tested for their ability to reduce ACAP toxicity. Cell viability showed that larger PTOs provided greater protection. We evaluated the ability of NAPQI to cause mutations in the LacZ gene of pBluescript plasmid which contained phosphorothioate linkages at designed locations within the gene. In addition, the ability of ACAP to cause mutations in the HGPRT locus in cells grown in dATP(S)-containing medium was measured. No mutations were seen in either assay. Based upon these results, activated ACAP is reactive with PTOs in vitro, although this interaction is nontoxic and nonmutagenic.

Acetaminophen↗
PubMed2001

Thioalkalimicrobium aerophilum gen. nov., sp. nov. and Thioalkalimicrobium sibericum sp. nov., and Thioalkalivibrio versutus gen. nov., sp. nov., Thioalkalivibrio nitratis sp.nov., novel and Thioalkalivibrio denitrificancs sp. nov., novel obligately alkaliphilic and obligately chemolithoautotrophic sulfur-oxidizing bacteria from soda lakes.

Forty-three strains of obligately chemolithoautotrophic sulfur-oxidizing bacteria were isolated from highly alkaline soda lakes in south-east Siberia (Russia) and in Kenya using a specific enrichment procedure at pH 10. The main difference between the novel isolates and known sulfur bacteria was their potential to grow and oxidize sulfur compounds at pH 10 and higher. The isolates fell into two groups that were substantially different from each other physiologically and genetically. Most of the Siberian isolates belonged to the group with a low DNA G+C content (48.0-51.2 mol%). They were characterized by a high growth rate, a low growth yield, a high cytochrome content, and high rates of oxidation of sulfide and thiosulfate. This group included 18 isolates with a DNA homology of more than 40%, and it is described here as a new genus, Thioalkalimicrobium, with two species Thioalkalimicrobium aerophilum (type species) and Thioalkalimicrobium sibericum. The other isolates, mainly from Kenyan soda lakes, fell into a group with a high DNA G+C content (61.0-65.6 mol%). In general, this group was characterized by a low growth rate, a high molar growth yield and low, but relatively equal, rates of oxidation of thiosulfate, sulfide, elemental sulfur and polythionates. The group included 25 isolates with a DNA homology of more than 30%. It was less compact than Thioalkalimicrobium, containing haloalkalophilic, carotenoid-producing, nitrate-reducing and facultatively anaerobic denitrifying strains. These bacteria are proposed to be assigned to a new genus, Thioalkalivibrio, with three species Thioalkalivibrio versutus (type species), Thioalkalivibrio denitrificans and Thioalkalivibrio nitratis. Phylogenetic analysis revealed that both groups belong to the gamma-Proteobacteria. The Thioalkalimicrobium species were closely affiliated with the neutrophilic chemolithoautotrophic sulfur bacteria of the genus Thiomicrospira, forming a new alkaliphilic lineage in this cluster. In contrast, Thioalkalivibrio was not related to any known chemolithoautotrophic taxa, but was distantly associated with anaerobic purple sulfur bacteria of the genus Ectothiorhodospira.

Alkalies↗
PubMed1994

Enzymatic conversion of dethiobiotin to biotin in cell-free extracts of a Bacillus sphaericus bioB transformant.

The activity of biotin synthase, responsible for biotin synthesis from dethiobiotin, was demonstrated in a completely defined reaction mixture with cell-free extracts of a Bacillus sphaericus bioB transformant. Among the sulfur compounds tested, only S-adenosyl-L-methionine was active, while L-methionine and L-cysteine had no significant effect. Protein concentrations higher than 15 mg/ml in the reaction mixture were needed to detect biotin synthase activity. When dialyzed cell-free extracts were used for the reaction, NADH, NADPH, or FAD among the well-known cofactors tested enhanced the activity, and Fe2+, Mn2+, and Ca2+ among the metal ions tested also had some effects.

Bacillus↗
PubMed2002

[Determination of trace sulfur and mercury in the Chinese traditional medicine].

This paper reports that the sulfur compounds of traditional Chinese medicine were reduced to S2- by Auger reagent, and determined by cold atomic fluorescence. At the same time the trace mercury was determined. The conditions the for quantitative determination of Hg2+ and S2- were obtained. The linear range of the determination is 11.20-55.60 mg.mL-1, the correlation coefficient is 0.999 78. In this method, little reagent is needed, the treatment of the sample is simple, the process of determination is convenient, and the sensitivity is high.

Astragalus Plant↗
PubMed1996

Halitosis. A common oral problem.

Halitosis is caused primarily by bacterial putrefaction and the generation of volatile sulfur compounds. Ninety percent of patients suffering from halitosis have oral causes, such as poor oral hygiene, periodontal disease, tongue coat, food impaction, unclean dentures, faulty restorations, oral carcinomas, and throat infections. The remaining 10 percent of halitosis sufferers have systemic causes that include renal or hepatic failure, carcinomas, diabetes or trimethylaminuria. Modern analytical and microbiological techniques permit diagnosis of bad breath. Management of halitosis involves maintaining proper oral hygiene, and periodontal treatment, including tongue brushing.

Bacteria↗
PubMed2004

Inorganic polysulfides' quantitation by methyl iodide derivatization: dimethylpolysulfide formation potential.

Inorganic polysulfides are important intermediates in the formation of dimethylpolysulfides and possibly other volatile sulfur compounds of environmental significance. Currently, direct determination of these ions in the concentration range of natural systems is practically impossible, particularly under oxic conditions. Polysulfide quantification by derivatization with methyl iodide or d6-methyl iodide is emerging as a valuable alternative method for studies of polysulfide formation in natural systems. This manuscript presents detailed studies aimed at the evaluation of this method. We determined the conversion of the inorganic polysulfides to dimethylpolysulfides by methylation with methyl iodide. Close to 100 per cent of the molar concentration of polysulfide salts were converted to organic polysulfides for very low concentrations of dissolved polysulfide solutions, but only a small recovery was obtained for high concentrations of polysulfide precursors or when the solubility limit was exceeded. The recovery of polysulfides based on the calculated dissolved polysulfide concentration exceeds 1,000 per cent for very low dissolved concentrations of polysulfides. This unexpected dependence is attributed to continuous inorganic polysulfide formation from hydrogen sulfide and sulfur precipitate concurrent with, and in fact driven by, the methylation process.

Environmental Monitoring↗
PubMed2004

Overexpression of Lactobacillus casei D-hydroxyisocaproic acid dehydrogenase in cheddar cheese.

Metabolism of aromatic amino acids by lactic acid bacteria is an important source of off-flavor compounds in Cheddar cheese. Previous work has shown that alpha-keto acids produced from Trp, Tyr, and Phe by aminotransferase enzymes are chemically labile and may degrade spontaneously into a variety of off-flavor compounds. However, dairy lactobacilli can convert unstable alpha-keto acids to more-stable alpha-hydroxy acids via the action of alpha-keto acid dehydrogenases such as d-hydroxyisocaproic acid dehydrogenase. To further characterize the role of this enzyme in cheese flavor, the Lactobacillus casei d-hydroxyisocaproic acid dehydrogenase gene was cloned into the high-copy-number vector pTRKH2 and transformed into L. casei ATCC 334. Enzyme assays confirmed that alpha-keto acid dehydrogenase activity was significantly higher in pTRKH2:dhic transformants than in wild-type cells. Reduced-fat Cheddar cheeses were made with Lactococcus lactis starter only, starter plus L. casei ATCC 334, and starter plus L. casei ATCC 334 transformed with pTRKH2:dhic. After 3 months of aging, the cheese chemistry and flavor attributes were evaluated instrumentally by gas chromatography-mass spectrometry and by descriptive sensory analysis. The culture system used significantly affected the concentrations of various ketones, aldehydes, alcohols, and esters and one sulfur compound in cheese. Results further indicated that enhanced expression of d-hydroxyisocaproic acid dehydrogenase suppressed spontaneous degradation of alpha-keto acids, but sensory work indicated that this effect retarded cheese flavor development.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗