Novel Biochemical Properties and Physiological Role of the Flavin Mononucleotide Oxidoreductase YhdA from Bacillus subtilis.

Cr(VI) is mutagenic and teratogenic and considered an environmental pollutant of increasing concern. The use of microbial enzymes that convert this ion into its less toxic reduced insoluble form, Cr(III), represents a valuable bioremediation strategy. In this study, we examined the Bacillus subtilis YhdA enzyme, which belongs to the family of NADPH-dependent flavin mononucleotide oxide reductases and possesses azo-reductase activity as a factor that upon overexpression confers protection on B. subtilis from the cytotoxic effects promoted by Cr(VI) and counteracts the mutagenic effects of the reactive oxygen species (ROS)-promoted lesion 8-OxoG. Further, our in vitro assays unveiled catalytic and biochemical properties of biotechnological relevance in YhdA; a pure recombinant His10-YhdA protein efficiently catalyzed the reduction of Cr(VI) employing NADPH as a cofactor. The activity of the pure oxidoreductase YhdA was optimal at 30°C and at pH 7.5 and displayed Km and Vmax values of 7.26 mM and 26.8 µmol·min-1·mg-1 for Cr(VI), respectively. Therefore, YhdA can be used for efficient bioremediation of Cr(VI) and counteracts the cytotoxic and genotoxic effects of oxygen radicals induced by intracellular factors and those generated during reduction of hexavalent chromium.IMPORTANCE Here, we report that the bacterial flavin mononucleotide/NADPH-dependent oxidoreductase YhdA, widely distributed among Gram-positive bacilli, conferred protection to cells from the cytotoxic effects of Cr(VI) and prevented the hypermutagenesis exhibited by a MutT/MutM/MutY-deficient strain. Additionally, a purified recombinant His10-YhdA protein displayed a strong NADPH-dependent chromate reductase activity. Therefore, we postulate that in bacterial cells, YhdA counteracts the cytotoxic and genotoxic effects of intracellular and extracellular inducers of oxygen radicals, including those caused by hexavalent chromium.

Role of Azospirillum brasilense in triggering different Fe chelate reductase enzymes in cucumber plants subjected to both nutrient deficiency and toxicity.

Azospirillum brasilense was reported to up-regulate iron (Fe) uptake mechanisms, such as Fe reduction and rhizosphere acidification, in both Fe sufficient and deficient cucumber plants (Cucumis sativus L.). Strategy I plants take up both Fe and copper (Cu) after their reduction mediated by the ferric-chelate reductase oxidase (FRO) enzyme. Interestingly, in cucumber genome only one FRO gene is reported. Thus, in the present study we applied a bioinformatics approach to identify the member of cucumber FRO gene family and allowed the identification of at least three CsFRO genes, one of which was the already identified, i.e. CsFRO1. The expression patterns of the newly identified transcripts were investigated in hydroponically grown cucumber plants treated with different Fe and Cu nutritional regimes. Gene expression was then correlated with morphological (i.e. root architecture) and physiological (Fe(III) reducing activity) parameters to shed light on: i) the CsFRO homologue responsible of the increased reduction activity in Fe-sufficient plants inoculated with A. brasilense cucumber plants, and ii) the possible effect of A. brasilense in ameliorating the symptoms of Cu toxicity in cucumber plants. The data obtained showed that all the CsFRO genes were expressed in the root tissues of cucumber plants and responded to Cu starvation, combined Cu/Fe deficiency and Cu toxicity. Only CsFRO3 was modulated by the A. brasilense in Fe-sufficient plants suggesting for the first time a different specificity of action of the three isoenzymes depending not only on the nutritional regime (either deficiency or toxicity) but also on the presence of the PGPR. Furthermore, results suggest that the PGPR could even ameliorate the stress symptoms caused by both the double (i.e. Cu and Fe) and Cu deficiency as well as Cu toxicity modulating, on one hand, the growth of the root system and, on the other hand, the root nutrient uptake.

Fatty acid composition from the marine red algae Pterocladiella capillacea (S. G. Gmelin) Santelices & Hommersand 1997 and Osmundaria obtusiloba (C. Agardh) R. E. Norris 1991 and its antioxidant activity

ABSTRACT This study evaluated the chemical composition and antioxidant activity of fatty acids from the marine red algae Pterocladiella capillacea (S. G. Gmelin) Santelices & Hommersand 1997 and Osmundaria obtusiloba (C. Agardh) R. E. Norris 1991. The gas chromatography mass spectrometry (GC-MS) identified nine fatty acids in the two species. The major fatty acids of P. capillacea and O. obtusiloba were palmitic acid, oleic acid, arachidonic acid and eicosapentaenoic acid. The DPPH radical scavenging capacity of fatty acids was moderate ranging from 25.90% to 29.97%. Fatty acids from P. capillacea (31.18%) had a moderate ferrous ions chelating activity (FIC), while in O. obtusiloba (17.17%), was weak. The ferric reducing antioxidant power (FRAP) of fatty acids from P. capillacea and O. obtusiloba was low. As for β-carotene bleaching (BCB), P. capillacea and O. obtusiloba showed a good activity. This is the first report of the antioxidant activities of fatty acids from the marine red algae P. capillacea and O. obtusiloba.

Fatty acid composition from the marine red algae Pterocladiella capillacea (S. G. Gmelin) Santelices & Hommersand 1997 and Osmundaria obtusiloba (C. Agardh) R. E. Norris 1991 and its antioxidant activity.

This study evaluated the chemical composition and antioxidant activity of fatty acids from the marine red algae Pterocladiella capillacea (S. G. Gmelin) Santelices & Hommersand 1997 and Osmundaria obtusiloba (C. Agardh) R. E. Norris 1991. The gas chromatography mass spectrometry (GC-MS) identified nine fatty acids in the two species. The major fatty acids of P. capillacea and O. obtusiloba were palmitic acid, oleic acid, arachidonic acid and eicosapentaenoic acid. The DPPH radical scavenging capacity of fatty acids was moderate ranging from 25.90% to 29.97%. Fatty acids from P. capillacea (31.18%) had a moderate ferrous ions chelating activity (FIC), while in O. obtusiloba (17.17%), was weak. The ferric reducing antioxidant power (FRAP) of fatty acids from P. capillacea and O. obtusiloba was low. As for ß-carotene bleaching (BCB), P. capillacea and O. obtusiloba showed a good activity. This is the first report of the antioxidant activities of fatty acids from the marine red algae P. capillacea and O. obtusiloba.

Analysis of allelic variants of rdxA associated with metronidazole resistance in Helicobacter pylori: detection of common genotypes in rdxA by multiplex allele-specific polymerase chain reaction.

Resistance to metronidazole (Mtz) in Helicobacter pylori is a major problem worldwide, especially in developing countries. Alterations in Mtz nitroreductase enzymes, such as oxygen-insensitive NADPH nitroreductase (RdxA) and NADPH flavin oxidoreductase (FrxA), are the major contributing factors for this resistance. In this study, rdxA and frxA were amplified, sequenced, and analyzed in 34 Mtz-resistant H. pylori isolates (MIC ≥ 8 µg/mL) using multiple allele-specific polymerase chain reaction (MAS-PCR); this method was developed to target the most common genotypes of rdxA in H. pylori. In this study, the rdxA and frxA genes in Mtz-resistant H. pylori strains displayed a large number of point mutations. The rdxA and frxA genes of Mtz-resistant clinical isolates showed a higher percentage of missense mutations (97.1 and 78.6%, respectively) compared to 26695 reference strains; additionally, missense mutations were more common than frameshift (20.6 and 32.1%) and nonsense mutations (8.8 and 10.7%, respectively) in these genes. The most common missense mutations in rdxA were D 59 N (94.1%), T 31 E (88.2%), and R 131 K (85.3%). The most common missense mutations in frxA were F 72 S (57.1%), G 73 S (57.1%), and C 193 S (53.6%). The developed MAS primers, specific to position 175 and 392 of rdxA, successfully amplified the common alleles and distinguished the variants. MAS-PCR could be a useful tool for epidemiological studies of H. pylori, associated with Mtz resistance. rdxA variants must be screened in order to ensure the effectiveness of Mtz-based H. pylori therapies in developing countries.

Discovery of Unusual Biaryl Polyketides by Activation of a Silent Streptomyces venezuelae Biosynthetic Gene Cluster.

Comparative transcriptional profiling of a ΔbldM mutant of Streptomyces venezuelae with its unmodified progenitor revealed that the expression of a cryptic biosynthetic gene cluster containing both type I and type III polyketide synthase genes is activated in the mutant. The 29.5 kb gene cluster, which was predicted to encode an unusual biaryl metabolite, which we named venemycin, and potentially halogenated derivatives, contains 16 genes including one-vemR-that encodes a transcriptional activator of the large ATP-binding LuxR-like (LAL) family. Constitutive expression of vemR in the ΔbldM mutant led to the production of sufficient venemycin for structural characterisation, confirming its unusual biaryl structure. Co-expression of the venemycin biosynthetic gene cluster and vemR in the heterologous host Streptomyces coelicolor also resulted in venemycin production. Although the gene cluster encodes two halogenases and a flavin reductase, constitutive expression of all three genes led to the accumulation only of a monohalogenated venemycin derivative, both in the native producer and the heterologous host. A competition experiment in which equimolar quantities of sodium chloride and sodium bromide were fed to the venemycin-producing strains resulted in the preferential incorporation of bromine, thus suggesting that bromide is the preferred substrate for one or both halogenases.

Nucleotide diversity based on phaseolin and iron reductase genes in common bean accessions of different geographical origins.

Discriminating genotypes within plant collections is imperative, and DNA sequence approaches for detecting single nucleotide polymorphisms (SNPs) have proved essential in any modern analysis of germplasm. By sequencing the α-Phs and PvFRO1 genes that, respectively, encode phaseolin and an iron reductase, we prospected for SNPs in exonic and intronic regions of both genes in a sample of 31 accessions of Phaseolus vulgaris from Mesoamerican and Andean gene pools, and one accession of Phaseolus lunatus, chosen as an outgroup. Sequence alignment showed 95 SNPs in α-Phs and 83 in PvFRO1, but diversity along the nucleotide sequences was not evenly distributed in both genes. Accessions from the same gene pool showed greater similarity than those from different gene pools, and the cluster patterns obtained in this study were consistent with the hierarchical organization into two P. vulgaris gene pools. The polymorphisms detected in the α-Phs gene allowed better discrimination among the accessions within each cluster than the PvFRO1 polymorphisms. Furthermore, some variations within exons changes amino acids in both predicted protein sequences. In an unprecedented result, the phaseolin-predicted amino acid variation allowed most of the accessions to be typified.

Identification and expression analysis of multiple FRO gene copies in Medicago truncatula.

Iron (Fe) is an essential element for plant growth. Commonly, this element is found in an oxidized form in soil, which is poorly available for plants. Therefore, plants have evolved ferric-chelate reductase enzymes (FRO) to reduce iron into a more soluble ferrous form. Fe scarcity in plants induce the FRO enzyme activity. Although the legume Medicago truncatula has been employed as a model for FRO activity studies, only one copy of the M. truncatula MtFRO1 gene has been characterized so far. In this study, we identified multiple gene copies of the MtFRO gene in the genome of M. truncatula by an in silico search, using BLAST analysis in the database of the M. truncatula Genome Sequencing Project and the National Center for Biotechnology Information, and also determined whether they are functional. We identified five genes apart from MtFRO1, which had been already characterized. All of the MtFRO genes exhibited high identity with homologous FRO genes from Lycopersicon esculentum, Citrus junos and Arabidopsis thaliana. The gene copies also presented characteristic conserved FAD and NADPH motifs, transmembrane regions and oxidoreductase signature motifs. We also detected expression in five of the putative MtFRO sequences by semiquantitative RT-PCR analysis, performed with mRNA from root and shoot tissues. Iron scarcity might be a condition for an elevated expression of the MtFRO genes observed in different M. truncatula tissues.

Variation and inheritance of iron reductase activity in the roots of common bean (Phaseolus vulgaris L.) and association with seed iron accumulation QTL.

BACKGROUND: Iron deficiency anemia is a global problem which often affects women and children of developing countries. Strategy I plants, such as common bean (Phaseolus vulgaris L.) take up iron through a process that involves an iron reduction mechanism in their roots; this reduction is required to convert ferric iron to ferrous iron. Root absorbed iron is critical for the iron nutrition of the plant, and for the delivery of iron to the shoot and ultimately the seeds. The objectives of this study were to determine the variability and inheritance for iron reductase activity in a range of genotypes and in a low × high seed iron cross (DOR364 x G19833), to identify quantitative trait loci (QTL) for this trait, and to assess possible associations with seed iron levels. RESULTS: The experiments were carried out with hydroponically grown plants provided different amounts of iron varying between 0 and 20 µM Fe(III)-EDDHA. The parents, DOR364 and G19833, plus 13 other cultivated or wild beans, were found to differ in iron reductase activity. Based on these initial experiments, two growth conditions (iron limited and iron sufficient) were selected as treatments for evaluating the DOR364 × G19833 recombinant inbred lines. A single major QTL was found for iron reductase activity under iron-limited conditions (1 µM Fe) on linkage group b02 and another major QTL was found under iron sufficient conditions (15 µM Fe) on linkage group b11. Associations between the b11 QTL were found with several QTL for seed iron. CONCLUSIONS: Genes conditioning iron reductase activity in iron sufficient bean plants appear to be associated with genes contributing to seed iron accumulation. Markers for bean iron reductase (FRO) homologues were found with in silico mapping based on common bean synteny with soybean and Medicago truncatula on b06 and b07; however, neither locus aligned with the QTL for iron reductase activity. In summary, the QTL for iron reductase activity under iron limited conditions may be useful in environments where beans are grown in alkaline soils, while the QTL for iron reductase under sufficiency conditions may be useful for selecting for enhanced seed nutritional quality.

Molecular and neurochemical mechanisms in PD pathogenesis.

Oxidation of dopamine to aminochrome seems to be a normal process leading to aminochrome polymerization to form neuromelanin, since normal individuals have this pigment in their dopaminergic neurons in the substantia nigra. The neurons lost in individuals with Parkinson's disease are dopaminergic neurons containing neuromelanin. This raises two questions. First, why are those cells containing neuromelanin lost in this disease? Second, what is the identity of the neurotoxin that induces this cell death? We propose that aminochrome is the agent responsible for the death of dopaminergic neurons containing neuromelanin in individuals with Parkinson's disease. The normal oxidative pathway of dopamine, in which aminochrome polymerizes to form neuromelanin, can be neurotoxic if DT-diaphorase is inhibited under certain conditions. Inhibition of DT-diaphorase allows two neurotoxic reactions to proceed: (i) the formation of aminochrome adducts with alpha-synuclein, which induce and stabilize the formation of neurotoxic protofibrils; and (ii) the one electron reduction of aminochrome to the neurotoxic leukoaminochrome o-semiquinone radical. Therefore, we propose that DT-diaphorase is an important neuroprotective enzyme in dopaminergic neurons containing neuromelanin.

Response of Wolfiporia cocos to iron availability: alterations in growth, expression of cellular proteins, Fe3+-reducing activity and Fe3+-chelators production.

AIMS: The main objective of this study was to evaluate the behaviour of the brown-rot fungus Wolfiporia cocos under differential iron availability. METHODS AND RESULTS: W. cocos was grown under three differential iron conditions. Growth, catecholate and hydroxamate production, and mycelial and extracellular Fe3+-reducing activities were determined. Iron starvation slowed fungal growth and accelerated pH decline. Some mycelial proteins of low molecular weight were repressed under iron restriction, whereas others of high molecular weight showed positive iron regulation. Mycelial ferrireductase activity decreased as culture aged, while Fe3+-reducing activity of low molecular reductants constantly increased. Hydroxamates production suffered only limited iron repression, whereas catecholates production showed to be more iron repressible. CONCLUSIONS: W. cocos seems to possess more than one type of iron acquisition mechanism; one involving secretion of organic acids and ferrireductases and/or extracellular reductants, and another relying on secretion of catecholates and hydroxamates chelators. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper is the first to report the kinetic study of brown-rot fungus grown under differential iron availability, and the information provided here contributes to address more traditional problems in protecting wood from brown decay, and also makes a contribution in the general area of the physiology of brown-rot fungi.

Identification of iron-regulated cellular proteins, Fe3+-reducing and -chelating compounds, in the white-rot fungus Perenniporia medulla-panis.

In this paper, we present the responses of the white-rot fungus Perenniporia medulla-panis to iron availability with regard to alterations in growth, expression of cellular proteins, Fe3+-reducing activity, and Fe3+ chelators production. Iron supplementation stimulated fungal growth but did not result in a significant increase in biomass production. Catechol and hydroxamate derivatives were produced mainly under iron deficiency, and their productions were repressed under iron supplementation conditions. Perenniporia medulla-panis showed several cellular proteins in the range of 10-90 kDa. Some of them showed negative iron-regulation. Iron-supplemented medium also repressed both cell surface and extracellular Fe3+-reducing activities; however, the highest cell surface activity was detected at the initial growth phase, whereas extracellular activity increased throughout the incubation period. No significant production of chelators and extracellular Fe3+-reducing activity were observed within the initial growth phase, suggesting that the reduction of Fe3+ to Fe2+ is performed by ferrireductases.

A morphological and cytochemical study of the interaction between Paracoccidiodes brasiliensis and neutrophils.

Paracoccidioidomycosis is a systemic granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis. It is the most prevalent systemic mycosis of Latin America and 80% of the reported cases are from Brazil. Because of the great number of neutrophils found in the P. brasiliensis granuloma, studies have been done to evaluate the role of these cells during the development of the infection. Scanning and transmission electron microscopy of thin sections showed that the neutrophils ingest yeast cells through a typical phagocytic process with the formation of pseudopodes. The pseudopodes even disrupt the connection established between the mother and the bud cells. Neutrophils also associate to each other, forming a kind of extracellular vacuole where large yeast cells are encapsulated. Cytochemical studies showed that once P. brasiliensis attaches to the neutrophil surface, it triggers a respiratory burst with release of oxygen-derived products. Attachment also triggers neutrophils degranulation, with release of endogenous peroxidase localized in cytoplasmic granules. Together, these processes lead to killing of both ingested and extracellular P. brasiliensis.

Physiological modulation of iron metabolism in rainbow trout (Oncorhynchus mykiss) fed low and high iron diets.

Iron (Fe) is an essential element, but Fe metabolism is poorly described in fish and the role of ferrireductase and transferrin in iron regulation by teleosts is unknown. The aim of the present study was to provide an overview of the strategy for Fe handling in rainbow trout, Oncorhynchus mykiss. Fish were fed Fe-deficient, normal and high-Fe diets (33, 175, 1975 mg Fe kg(-1) food, respectively) for 8 weeks. Diets were chosen so that no changes in growth, food conversion ratio, haematology, or significant oxidative stress (TBARS) were observed. Elevation of dietary Fe caused Fe accumulation particularly in the stomach, intestine, liver and blood. The increase in total serum Fe from 10 to 49 micro mol l(-1) over 8 weeks was associated with elevated total Fe binding capacity and decreased unsaturated Fe binding capacity, so that in fish fed a high-Fe diet transferrin saturation increased from 15% at the start of the experiment to 37%. Fish on the high-Fe diet increased Fe accumulation in the liver, which was correlated with elevation of hepatic ferrireductase activity and serum transferrin saturation. Conversely, fish on the low-Fe diet did not show tissue Fe depletion compared with normal diet controls and did not change Fe binding to serum transferrin. Instead, these fish doubled intestinal ferrireductase activity which may have contributed to the maintenance of tissue Fe status. The absence of clear treatment-dependent changes in branchial Fe accumulation and ferrireductase activity indicated that the gills do not have a major role in Fe metabolism. Some transient changes in Cu, Zn and Mn status of tissues occurred.

Characterization of an NADH-linked cupric reductase activity from the Escherichia coli respiratory chain.

Previous results from our laboratory have shown that NADH-supported electron flow through the Escherichia coli respiratory chain promotes the reduction of cupric ions to Cu(I), which mediates damage of the respiratory system by hydroperoxides. The aim of this work was to characterize the NADH-linked cupric reductase activity from the E. coli respiratory chain. We have used E. coli strains that either overexpress or are deficient in the NADH dehydrogenase-2 (NDH-2) to demonstrate that this membrane-bound protein catalyzes the electron transfer from NADH to Cu(II), but not to Fe(III). We also show that purified NDH-2 exhibits NADH-supported Cu(II) reductase activity in the presence of either FAD or quinone, but is unable to reduce Fe(III). The K(m) values for free Cu(II) were 32 +/- 5 pM in the presence of saturating duroquinone and 22 +/- 2 pM in the presence of saturating FAD. The K(m) values for NADH were 6.9 +/- 1.5 microM and 6.1 +/- 0.7 microM in the presence of duroquinone and FAD, respectively. The quinone-dependent Cu(II) reduction occurred through both O(*-)(2)-mediated and O(*-)(2)-independent pathways, as evidenced by the partial inhibitory effect (30-50%) of superoxide dismutase, by the reaction stoichiometry, and by the enzyme turnover numbers for NADH and Cu(II). The cupric reductase activity of NDH-2 was dependent on thiol groups which were accessible to p-chloromercuribenzoate at low, but not at high, ionic strength of the medium, a fact apparently connected to a conformational change of the protein. To our knowledge, this is the first protein with cupric reductase activity to be isolated and characterized in its biochemical properties.