Features of iron accumulation at high concentration in pulcherrimin-producing Metschnikowia yeast biomass.

In previous studies it was found that the antimicrobial properties of pulcherrimin-producing Metschnikowia species are related to the formation of a red pigment-pulcherrimin and sequestration of free iron from their growth medium. For strains of Metschnikowia pulcherrima, M. sinensis, M. shaxiensis, and M. fructicola, at a high, ≈80 mg/kg, elemental Fe concentration in agar growth media we observed the essentially different (metal luster, non-glossy rust like, and colored) yeast biomass coatings. For the studied strains the optical and scanning electron microscopies showed the increased formation of chlamydospores that accumulate a red pigment-insoluble pulcherrimin rich in iron. The chlamydospore formation and decay depended on the iron concentration. In this study pulcherrimin in biomass of the selected Metschnikowia strains was detected by Mössbauer spectroscopy. At ≈80 mg/kg elemental Fe concentration the Mössbauer spectra of biomass of the studied strains were almost identical to these of purified pulcherrimin. Iron in pulcherrimin reached ≈1% of biomass by weight which is very high in comparison with elemental Fe percentage in growth medium and is not necessary for yeast growth. The pulcherrimin in biomass was also observed by Mössbauer spectroscopy at lower, ≈5 mg/kg, elemental Fe concentration. Through chemical binding of iron pulcherrimin sequestrates the soluble Fe in the growth media. However, at high Fe concentrations, the chemical and biochemical processes lead to the pulcherrimin accumulation in biomass chlamydospores. When soluble iron is sequestrated or removed from the growth media in this way, it becomes inaccessible for other microorganisms.

Distribution and possible biosynthetic pathway of non-protein sulfur amino acids in legumes.

Some grain legumes store sulfur in the form of non-protein amino acids in seed. γ-Glutamyl-S-methylcysteine is found in Phaseolus and several Vigna species. γ-Glutamyl-S-ethenylcysteine, an antinutritional compound, is present in Vicia narbonensis. In P. vulgaris, free S-methylcysteine levels are higher at early stages of seed development followed by a decline. γ-Glutamyl-S-methylcysteine accumulates later, in two phases, with a lag during reserve accumulation. The concentration of total S-methylcysteine, quantified after acid hydrolysis, is positively regulated by sulfate nutrition. The levels of both γ-glutamyl-S-methylcysteine and γ-glutamyl-S-ethenylcysteine are modulated in response to changes in seed protein composition. A model is proposed whereby the majority of γ-glutamyl-S-methylcysteine in P. vulgaris is synthesized via the intermediate S-methylhomoglutathione. Knowledge of the biosynthesis of non-protein sulfur amino acids is required for metabolic engineering approaches, in conjunction with manipulation of the protein sink, to increase the concentration of nutritionally essential methionine and cysteine. This would improve protein quality of some important legume crops.

Novel biosynthetic pathway for sulfur amino acids in Cryptococcus neoformans.

We elucidated a unique feature of sulfur metabolism in Cryptococcus neoformans. C. neoformans produces cysteine solely by the O-acetylserine pathway that consists of serine-O-acetyl transferase and cysteine synthase. We designated the gene encoding the former enzyme CYS2 (locus tag CNE02740) and the latter enzyme CYS1 (locus tag CNL05880). The cys1Δmutant strain was found to be avirulent in a murine infection model. Methionine practically does not support growth of the cys1Δ strain, and cysteine does not serve as a methionine source, indicating that the transsulfuration pathway does not contribute to sulfur amino acid synthesis in C. neoformans. Among the genes encoding enzymes catalyzing the reactions from homoserine to methionine, the gene corresponding to the Saccharomyces cerevisiae MET17 encoding O-acetylhomoserine sulfhydrylase (Met17p) had remained to be identified in C. neoformans. By genetic analysis of Met- mutants obtained by Agrobacterium tumefaciens-mediated mutagenesis, we concluded that Cnc01220, most similar to Str2p (36% identity), cystathionine-γ-synthase, in the Saccharomyces genome, is the C. neoformans version of O-acetylhomoserine sulfhydrylase. We designated CNC01220 as MET17. The C. neoformans met3Δ mutant defective in the first step of the sulfate assimilation pathway, sulfate adenylyltransferase, barely uses methionine as a sulfur source, whereas it uses cysteine efficiently. The poor utilization of methionine by the met3Δ mutant is most probably due to the absence of the transsulfuration pathway, causing an incapability of C. neoformans to produce cysteine and hydrogen sulfide from methionine. When cysteine is used as a sulfur source, methionine is likely produced de novo by using hydrogen sulfide derived from cysteine via an unidentified pathway. Altogether, the unique features of sulfur amino acid metabolism in C. neoformans will make this fungus a valuable experimental system to develop anti-fungal agents and to investigate physiology of hydrogen sulfide.

Chickpea-Derived Prebiotic Substances Trigger Biofilm Formation by Bacillus subtilis.

Chickpea-based foods are known for their low allergenicity and rich nutritional package. As an essential dietary legume, chickpea is often processed into milk or hummus or as an industrial source of protein and starch. The current study explores the feasibility of using the chickpea-derived prebiotic substances as a scaffold for growing Bacillus subtilis (a prospective probiotic bacterium) to develop synbiotic chickpea-based functional food. We report that the chickpea-derived fibers enhance the formation of the B. subtilis biofilms and the production of the antimicrobial pigment pulcherrimin. Furthermore, electron micrograph imaging confirms the bacterial embedding onto the chickpea fibers, which may provide a survival tactic to shield and protect the bacterial population from environmental insults. Overall, it is believed that chickpea-derived prebiotic substances provide a staple basis for developing functional probiotics and synbiotic food.

The antagonistic Metschnikowia andauensis produces extracellular enzymes and pulcherrimin, whose production can be promoted by the culture factors.

Biological control against microbial infections has a great potential as an alternative approach instead of fungicidal chemicals, which can cause environmental pollution. The pigment producer Metschnikowia andauensis belongs to the antagonistic yeasts, but details of the mechanism by which it inhibits growth of other microbes are less known. Our results confirmed its antagonistic capacity on other yeast species isolated from fruits or flowers and demonstrated that the antagonistic capacity was well correlated with the size of the red pigmented zone. We have isolated and characterized its red pigment, which proved to be the iron chelating pulcherrimin. Its production was possible even in the presence of 0.05 mg/ml copper sulphate, which is widely used in organic vineyards because of its antimicrobial properties. Production and localisation of the pulcherrimin strongly depended on composition of the media and other culture factors. Glucose, galactose, disaccharides and the presence of pectin or certain amino acids clearly promoted pigment production. Higher temperatures and iron concentration decreased the diameter of red pigmented zones. The effect of pH on pigment production varied depending of whether it was tested in liquid or solid media. In addition, our results suggest that other mechanisms besides the iron depletion of the culture media may contribute to the antagonistic capacity of M. andauensis.

From sulfur to homoglutathione: thiol metabolism in soybean.

Sulfur is an essential plant nutrient and is metabolized into the sulfur-containing amino acids (cysteine and methionine) and into molecules that protect plants against oxidative and environmental stresses. Although studies of thiol metabolism in the model plant Arabidopsis thaliana (thale cress) have expanded our understanding of these dynamic processes, our knowledge of how sulfur is assimilated and metabolized in crop plants, such as soybean (Glycine max), remains limited in comparison. Soybean is a major crop used worldwide for food and animal feed. Although soybeans are protein-rich, they do not contain high levels of the sulfur-containing amino acids, cysteine and methionine. Ultimately, unraveling the fundamental steps and regulation of thiol metabolism in soybean is important for optimizing crop yield and quality. Here we review the pathways from sulfur uptake to glutathione and homoglutathione synthesis in soybean, the potential biotechnology benefits of understanding and modifying these pathways, and how information from the soybean genome may guide the next steps in exploring this biochemical system.

The level of sulfane sulfur in the fungus Aspergillus nidulans wild type and mutant strains.

The interdependence of the sulfane sulfur metabolism and sulfur amino acid metabolism was studied in the fungus Aspergillus nidulans wild type strain and in mutants impaired in genes encoding enzymes involved in the synthesis of cysteine (a precursor of sulfane sulfur) or in regulatory genes of the sulfur metabolite repression system. It was found that a low concentration of cellular cysteine leads to elevation of two sulfane sulfurtransferases, rhodanase and cystathionine gamma-lyase, while the level of 3-mercaptopyruvate sulfurtransferase remains largely unaffected. In spite of drastic differences in the levels of biosynthetic enzymes and of sulfur amino acids due to mutations or sulfur supplementation of cultures, the level of total sulfane sulfur is fairly stable. This stability confirms the crucial role of sulfane sulfur as a fine-tuning regulator of cellular metabolism.

Rck2 is required for reprogramming of ribosomes during oxidative stress.

Rck2 is a mitogen-activated protein kinase-activated protein kinase in yeast implicated in translational regulation. rck2Delta mutants are mildly sensitive to oxidative stress, a condition that causes dissociation of actively translating ribosomes (polysomes). In rck2Delta cells, polysomes are lost to an even higher degree than in the wild-type upon stress. Cells overexpressing the catalytically inactive rck2-kd allele are highly sensitive to oxidative stress. In such cells, dissociation of polysomes upon stress was instead greatly delayed. The protein synthesis rate decreased to a similar degree as in wild-type cells, however, indicating that in rck2-kd cells, the polysome complexes were inactive. Array analyses of total and polysome-associated mRNAs revealed major deregulation of the translational machinery in rck2 mutant cells. This involves transcripts for cytosolic ribosomal proteins and for processing and assembly of ribosomes. In rck2Delta cells, weakly transcribed mRNAs associate more avidly with polysomes than in wild-type cells, whereas the opposite holds true for rck2-kd cells. This is consistent with perturbed regulation of translation elongation, which is predicted to alter the ratio between mRNAs with and without strong entry sites at ribosomes. We infer that imbalances in the translational apparatus are a major reason for the inability of these cells to respond to stress.

The regulation of the sulfur amino acid biosynthetic pathway in Cryptococcus neoformans: the relationship of Cys3, Calcineurin, and Gpp2 phosphatases.

Cryptococcosis is a fungal disease caused by C. neoformans. To adapt and survive in diverse ecological niches, including the animal host, this opportunistic pathogen relies on its ability to uptake nutrients, such as carbon, nitrogen, iron, phosphate, sulfur, and amino acids. Genetic circuits play a role in the response to environmental changes, modulating gene expression and adjusting the microbial metabolism to the nutrients available for the best energy usage and survival. We studied the sulfur amino acid biosynthesis and its implications on C. neoformans biology and virulence. CNAG_04798 encodes a BZip protein and was annotated as CYS3, which has been considered an essential gene. However, we demonstrated that CYS3 is not essential, in fact, its knockout led to sulfur amino acids auxotroph. Western blots and fluorescence microscopy indicated that GFP-Cys3, which is expressed from a constitutive promoter, localizes to the nucleus in rich medium (YEPD); the addition of methionine and cysteine as sole nitrogen source (SD-N + Met/Cys) led to reduced nuclear localization and protein degradation. By proteomics, we identified and confirmed physical interaction among Gpp2, Cna1, Cnb1 and GFP-Cys3. Deletion of the calcineurin and GPP2 genes in a GFP-Cys3 background demonstrated that calcineurin is required to maintain Cys3 high protein levels in YEPD and that deletion of GPP2 causes GFP-Cys3 to persist in the presence of sulfur amino acids. Global transcriptional profile of mutant and wild type by RNAseq revealed that Cys3 controls all branches of the sulfur amino acid biosynthesis, and sulfur starvation leads to induction of several amino acid biosynthetic routes. In addition, we found that Cys3 is required for virulence in Galleria mellonella animal model.

Identification and High-level Production of Pulcherrimin in Bacillus licheniformis DW2.

Pulcherrimin, a potential biocontrol agent produced by microorganisms, has the promising applications in the agricultural, medical, and food areas, and the low yield of pulcherrimin has hindered its applications. In this study, the red pigment produced by Bacillus licheniformis DW2 was identified as pulcherrimin through the spectrometry analysis and genetic manipulation, and the component of the medium used for pulcherrimin production was optimized. Based on our results, the addition of 1.0 g L-1 Tween 80 could improve the yield of pulcherrimin, and glucose and (NH4)2SO4 were served as the optimal carbon and nitrogen sources for pulcherrimin synthesis, respectively. Furthermore, an orthogonal array design was applied for optimization of the medium. Under optimized condition, the maximum yield of pulcherrimin was 331.17 mg L-1, 5.30-fold higher than that of the initial condition, which was the maximum yield reported for pulcherrimin production. Collectively, this study provided a promising strain and a feasible approach to achieve the high-level production of antimicrobial pulcherrimin.

Enzymatic synthesis of S-aminoethyl-L-cysteine from pantetheine.

The recently characterized compound S-aminoethylcysteine ketimine can be synthesized from purified S-aminoethylcysteine by enzymatic systems (transaminases or L-amino acid oxidase) present in mammalian tissues. S-Aminoethylcysteine, which could be considered as the natural precursor of the ketimine, is produced from L-serine and cysteamine by the action of the enzyme cystathionine-beta-synthase. We demonstrate in this paper that pantetheine, a normal cellular component, is an efficient cysteamine donor for the synthesis of S-aminoethylcysteine and of S-aminoethylcysteine ketimine in the place of free cysteamine, and we describe the enzymatic system, composed of partially purified enzymes, for the in vitro synthesis of S-aminoethylcysteine ketimine from pantetheine. This seems to indicate a new biological role for pantetheine.

Sulfur-containing amino acid metabolism in parasitic protozoa.

Sulfur-containing amino acids play indispensable roles in a wide variety of biological activities including protein synthesis, methylation, and biosynthesis of polyamines and glutathione. Biosynthesis and catabolism of these amino acids need to be carefully regulated to achieve the requirement of the above-mentioned activities and also to eliminate toxicity attributable to the amino acids. Genome-wide analyses of enzymes involved in the metabolic pathways of sulfur-containing amino acids, including transsulfuration, sulfur assimilatory de novo cysteine biosynthesis, methionine cycle, and degradation, using genome databases available from a variety of parasitic protozoa, reveal remarkable diversity between protozoan parasites and their mammalian hosts. Thus, the sulfur-containing amino acid metabolic pathways are a rational target for the development of novel chemotherapeutic and prophylactic agents against diseases caused by protozoan parasites. These pathways also demonstrate notable heterogeneity among parasites, suggesting that the metabolism of sulfur-containing amino acids reflects the diversity of parasitism among parasite species, and probably influences their biology and pathophysiology such as virulence competence and stress defense.

O-Acetylhomoserine sulfhydrylase of the fission yeast Schizosaccharomyces pombe: partial purification, characterization, and its probable role in homocysteine biosynthesis.

A crude extract of Schizosaccharomyces pombe cells catalyzed sulfhydrylation of both O-acetyl-L-serine and O-acetyl-L-homoserine with H2S, but did not synthesize cystathionine from O-acetyl-L-homoserine and L-cysteine. The O-acetylhomoserine sulfhydrylase [EC 4.2.99.10] was very unstable; however, it could be stabilized by the addition of 25% (w/w) sucrose or glycerol. The optimal pH for activity was 8.0 and that for stability was 7.0. The enzyme was purified approximately 300-fold from an ammonium sulfate-precipitated fraction. L-Methionine was the most effective inhibitor among the amino acids examined. It inhibited the enzyme competitively with respect to OAH with a Ki value of 2.6 mM. Sulfhydrylase activity was inhibited to various extents by some carbonyl reagents, but sulfhydryl reagents such as p-chloromercuribenzoic acid, 5,5'-dithio-bis(2-nitrobenzoic acid), and monoiodoacetic acid had no inhibitory effect. The enzyme also reacted with O-succinylhomoserine and L-homoserine to synthesize homocysteine directly, but could not utilize cysteine as a co-substrate in place of H2S. In the sulfhydrylation reactions, Km values for the substrates ranged from 10.4-12.5 mM. The enzyme was resolved to the apoenzyme by incubation with phenylhydrazine and reactivated by the addition of pyridoxal 5'-phosphate, whose Km value was 0.083 microM. The molecular weight of the enzyme was estimated to be approximately 186,000 by gel filtration and 170,000 by ultracentrifugation in sucrose density gradients. The isolectric point of the protein was pH 4.1. The characteristics of this enzyme are compared with those of physiologically functional sulfhydrylases reported for other organisms, and the possibility of the enzyme functioning as a homocysteine synthase is discussed.

Selenoamino acids in tissues of rats administered inorganic selenium.

There are conflicting reports in the literature concerning the synthesis of selenoamino acids from inorganic selenium in animals, and this work was undertaken to further investigate this. Pronase digests of acetone powders of liver and kidney tissue from rats administered 75SeO3= were subjected to fractionation by cation exchange chromatography using current methods for separating the various amino acids. Very little, if any, selenocystine was found in the digests. However, good evidence was obtained for the occurrence of 2,7-diamino-4-thia-5-selenaoctanedioic acid. It is suggested that the selenocysteine portion of this compound was formed by the reduction of the selenite to selenide with its subsequent incorporation into the amino acid by the action of serine hydrolase (E C 4.2.1.22). No selenomethionine was found under the conditions of this study.

Tauropine dehydrogenase from the starfish Asterina pectinifera (Echinodermata: Asteroidea): presence of opine production pathway in a deuterostome invertebrate.

Tauropine dehydrogenase (tauropine:NAD oxidoreductase; TaDH) was purified to homogeneity from the body wall of the starfish Asterina pectinifera Müller at Troschel(Echinodermata: Asteroidea) by means of (NH4)2SO4 precipitation followed by column chromatographies in DEAE-cellulose, Sephadex G75, Macro-prep ceramic hydroxyapatite, PBE 94, and Toyopearl HW50S. The enzyme was a monomeric protein of approximately 42000 Da and pI 5.2. The maximum rate of the tauropine biosynthetic reaction was observed at pH 6.0, and that of the tauropine catabolic reaction was at pH 8.7-9.2. Taurine and pyruvate were the preferred substrates. The tauropine catabolic reaction was inhibited by the substrate tauropine: the peak rate was observed at 12.5 mM. Apparent Km values for NADH, taurine, and pyruvate were 0.036 +/- 0.002, 21.3 +/- 1.6, and 0.46 +/- 0.02 mM, respectively, and for tauropine and NAD+ were 2.64 +/- 0.73 and 0.068 +/- 0.005 mM, respectively. The molecular and catalytic properties of the starfish TaDH were basically similar to those of TaDH from other species belonging to the lower invertebrate phyla and the middle phyla of Prostostomia. Tauropine accumulation in vivo during experimental anoxia was also demonstrated. These results gave clear evidence of opine production pathway in deutrostome invertebrate.

Possible roles of sulfur-containing amino acids in a chemoautotrophic bacterium-mollusc symbiosis.

Invertebrate hosts of chemoautotrophic symbionts face the unique challenge of supplying their symbionts with hydrogen sulfide while avoiding its toxic effects. The sulfur-containing free amino acids taurine and thiotaurine may function in sulfide detoxification by serving as sulfur storage compounds or as transport compounds between symbiont and host. After sulfide exposure, both taurine and thiotaurine levels increased in the gill tissues of the symbiotic coastal bivalve Solemya velum. Inhibition of prokaryotic metabolism with chloramphenicol, inhibition of eukaryotic metabolism with cycloheximide, and inhibition of ammonia assimilation with methionine sulfoximine reduced levels of sulfur-containing amino acids. Chloramphenicol treatment inhibited the removal of sulfide from the medium. In the absence of metabolic inhibitors, estimated rates of sulfide incorporation into taurine and thiotaurine accounted for nearly half of the sulfide removed from the medium. In contrast, amino acid levels in the nonsymbiotic, sulfide-tolerant molluscs Geukensia demissa and Yoldia limatula did not change after sulfide exposure. These findings suggest that sulfur-containing amino acids function in sulfide detoxification in symbiotic invertebrates, and that this process depends upon ammonia assimilation and symbiont metabolic capabilities.

Functional sulfur amino acid production and seawater remediation system by sterile Ulva sp. (Chlorophyta).

Sterile Ulva, which is a macroalga, has the potential to grow stably; therefore, this seaweed is expected to be an efficient resource of functional food containing various nutrients such as sulfur amino acids, proteins, carbohydrates, and minerals. Ulva latuca was selected from the "Marine Park" in Tokyo Bay, and its growth rate (g-dry/[m2.d]) was measured using model reactors located on the land or on the surface of the sea at Yokohama. The growth rate of U. lactuca was recorded to be approx 20 g-dry/(m2.d), which is estimated to be 10 times greater than that in a natural field in the Marine Park. In addition, this growth rate was higher than that of conventional crops such as corn and rice on a farm or paddy. These data led us to newly design and propose a floating type of labor-efficient U. lactuca production system. d-Cysteinolic acid, which is included in U. lactuca as a major sulfur amino acid, inhibited the Fenton reaction, resulting in suppression of hydroxyl radical production and singlet oxygen. Addition of the sulfur amino acid (1 microM) to HepG2 cells markedly decreased the intracellular triglyceride level. Hence, this proposed facility also has the potential for industrial production of a valuable resource for the primary prevention of lifestyle-related diseases using enriched or eutrophied seawater.

The complete genome sequence and analysis of the epsilonproteobacterium Arcobacter butzleri.

BACKGROUND: Arcobacter butzleri is a member of the epsilon subdivision of the Proteobacteria and a close taxonomic relative of established pathogens, such as Campylobacter jejuni and Helicobacter pylori. Here we present the complete genome sequence of the human clinical isolate, A. butzleri strain RM4018. METHODOLOGY/PRINCIPAL FINDINGS: Arcobacter butzleri is a member of the Campylobacteraceae, but the majority of its proteome is most similar to those of Sulfuromonas denitrificans and Wolinella succinogenes, both members of the Helicobacteraceae, and those of the deep-sea vent Epsilonproteobacteria Sulfurovum and Nitratiruptor. In addition, many of the genes and pathways described here, e.g. those involved in signal transduction and sulfur metabolism, have been identified previously within the epsilon subdivision only in S. denitrificans, W. succinogenes, Sulfurovum, and/or Nitratiruptor, or are unique to the subdivision. In addition, the analyses indicated also that a substantial proportion of the A. butzleri genome is devoted to growth and survival under diverse environmental conditions, with a large number of respiration-associated proteins, signal transduction and chemotaxis proteins and proteins involved in DNA repair and adaptation. To investigate the genomic diversity of A. butzleri strains, we constructed an A. butzleri DNA microarray comprising 2238 genes from strain RM4018. Comparative genomic indexing analysis of 12 additional A. butzleri strains identified both the core genes of A. butzleri and intraspecies hypervariable regions, where <70% of the genes were present in at least two strains. CONCLUSION/SIGNIFICANCE: The presence of pathways and loci associated often with non-host-associated organisms, as well as genes associated with virulence, suggests that A. butzleri is a free-living, water-borne organism that might be classified rightfully as an emerging pathogen. The genome sequence and analyses presented in this study are an important first step in understanding the physiology and genetics of this organism, which constitutes a bridge between the environment and mammalian hosts.