RESUMEN
Tellurium oxyanions are chemical species of great toxicity and their presence in the environment has increased because of mining industries and photovoltaic and electronic waste. Recovery strategies for this metalloid that are based on micro-organisms are of interest, but further studies of the transport systems and enzymes responsible for implementing tellurium transformations are required because many mechanisms remain unknown. Here, we investigated the involvement in tellurite uptake of the putative phosphate transporter PitB (PP1373) in soil bacterium Pseudomonas putida KT2440. For this purpose, through a method based on the CRISPR/Cas9 system, we generated a strain deficient in the pitB gene and characterized its phenotype on exposing it to varied concentrations of tellurite. Growth curves and transmission electronic microscopy experiments for the wild-type and ΔpitB strains showed that both were able to internalize tellurite into the cytoplasm and reduce the oxyanion to black nano-sized and rod-shaped tellurium particles, although the ΔpitB strain showed an increased resistance to the tellurite toxic effects. At a concentration of 100 µM tellurite, where the biomass formation of the wild-type strain decreased by half, we observed a greater ability of ΔpitB to reduce this oxyanion with respect to the wild-type strain (~38 vs ~16â%), which is related to the greater biomass production of ΔpitB and not to a greater consumption of tellurite per cell. The phenotype of the mutant was restored on over-expressing pitB in trans. In summary, our results indicate that PitB is one of several transporters responsible for tellurite uptake in P. putida KT2440.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Transporte de Fosfato/metabolismo , Pseudomonas putida/metabolismo , Telurio/metabolismo , Proteínas Bacterianas/genética , Transporte Biológico , Biomasa , Biotransformación , Mutación , Nanoestructuras/química , Nanoestructuras/toxicidad , Proteínas de Transporte de Fosfato/genética , Pseudomonas putida/efectos de los fármacos , Pseudomonas putida/crecimiento & desarrollo , Telurio/química , Telurio/toxicidadRESUMEN
Alongside the Brazil nut's role as an excellent source of vitamins, oil, fatty acids, lipids and nutrients, it is also recognized as a rich source of selenium. The pathway along which selenium and sulfur are metabolized in plants is theorized to be the same as that used for tellurium. Total tellurium content and its bioaccessibility are then evaluated by ICP-MS. Interferences and sample preparation are evaluated for the accurate determination of tellurium, and the accuracy determined through analysis of the certified reference material 1643e. A concentration of 4.02⯱â¯0.391â¯ngâ¯g-1 is obtained as an average concentration through external and internal calibrations. Through this reliable result, tellurium bioaccessibility in Brazil nuts is obtained via an in vitro validated unified bioaccessibility method. Values of 32% and 30% of total tellurium are available in the gastric and gastrointestinal fractions, respectively.
Asunto(s)
Nueces/química , Nueces/metabolismo , Telurio/análisis , Telurio/metabolismo , Brasil , Espectrometría de MasasRESUMEN
The increasing industrial utilization of tellurium has resulted in an important environmental pollution with the soluble, extremely toxic oxyanion tellurite. In this context, the use of microorganisms for detoxifying tellurite or tellurium biorecovery has gained great interest. The ability of different Shewanella strains to reduce tellurite to elemental tellurium was assessed; the results showed that the reduction process is dependent on electron transport and the ∆pH gradient. While S. baltica OS155 showed the highest tellurite resistance, S. putrefaciens was the most efficient in reducing tellurite. Moreover, pH-dependent tellurite transformation was associated with tellurium precipitation as tellurium dioxide. In summary, this work highlights the high tellurite reduction/detoxification ability exhibited by a number of Shewanella species, which could represent the starting point to develop friendly methods for the recovery of elemental tellurium (or tellurium dioxide).
Asunto(s)
Biodegradación Ambiental , Inactivación Metabólica/fisiología , Shewanella/metabolismo , Telurio/metabolismo , Transporte de Electrón , Oxidación-ReducciónRESUMEN
The tellurium oxyanion tellurite is harmful for most microorganisms. Since its toxicity occurs chiefly once the toxicant reaches the intracellular compartment, unveiling the toxicant uptake process is crucial for understanding the whole phenomenon of tellurium toxicity. While the PitA phosphate transporter is thought to be one of the main paths responsible for toxicant entry into Escherichia coli, genetic and physiological evidence have identified the ActP acetate carrier as the main tellurite importer in Rhodobacter capsulatus. In this work, new background on the role of these transporters in tellurite uptake by E. coli is presented. It was found that, similar to what occurs in R. capsulatus, ActP is able to mediate toxicant entry to this bacterium. Lower reactive oxygen species levels were observed in E. coli lacking the actP gene. Antioxidant enzyme catalase and fumarase C activity was almost unchanged after short exposure of E. coli ΔactP to sublethal tellurite concentrations, suggesting a low antioxidant response. In this strain, tellurite uptake decreased significantly during the first 5 min of exposure and inductively coupled plasma optical emission spectroscopy assays using an actP-overexpressing strain confirmed that this carrier mediates toxicant uptake. Relative gene expression experiments by qPCR showed that actP expression is enhanced at short times of tellurite exposure, while pitA and pitB genes are induced later. Summarizing, the results show that ActP is involved in tellurite entry to E. coli and that its participation occurs mainly at early stages of toxicant exposure.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas de Transporte de Fosfato/metabolismo , Telurio/metabolismo , Transporte Biológico , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Eliminación de Gen , Perfilación de la Expresión Génica , Transportadores de Ácidos Monocarboxílicos/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Activación Transcripcional/efectos de los fármacosRESUMEN
Exposure to the tellurium oxyanion tellurite (TeO3(2-)) results in the establishment of an oxidative stress status in most microorganisms. Usually, bacteria growing in the presence of the toxicant turn black because of the reduction of tellurite (Te(4+)) to the less-toxic elemental tellurium (Te(0)). In vitro, at least part of tellurite reduction occurs enzymatically in a nicotinamide dinucleotide-dependent reaction. In this work, we show that TeO3(2-) reduction by crude extracts of Escherichia coli overexpressing the zwf gene (encoding glucose-6-phosphate dehydrogenase) takes place preferentially in the presence of NADPH instead of NADH. The enzyme responsible for toxicant reduction was identified as 6-phosphogluconate dehydrogenase (Gnd). The gnd gene showed a subtle induction at short times after toxicant exposure while strains lacking gnd were more susceptible to the toxicant. These results suggest that both NADPH-generating enzymes from the pentose phosphate shunt may be involved in tellurite detoxification and resistance in E. coli.
Asunto(s)
Farmacorresistencia Bacteriana , Escherichia coli/enzimología , Escherichia coli/metabolismo , NADP/metabolismo , Fosfogluconato Deshidrogenasa/metabolismo , Telurio/metabolismo , Escherichia coli/efectos de los fármacos , Inactivación Metabólica , Oxidación-Reducción , Telurio/toxicidadRESUMEN
Tellurium, a metalloid belonging to group 16 of the periodic table, displays very interesting physical and chemical properties and lately has attracted significant attention for its use in nanotechnology. In this context, the use of microorganisms for synthesizing nanostructures emerges as an eco-friendly and exciting approach compared to their chemical synthesis. To generate Te-containing nanostructures, bacteria enzymatically reduce tellurite to elemental tellurium. In this work, using a classic biochemical approach, we looked for a novel tellurite reductase from the Antarctic bacterium Pseudomonas sp. strain BNF22 and used it to generate tellurium-containing nanostructures. A new tellurite reductase was identified as glutathione reductase, which was subsequently overproduced in Escherichia coli. The characterization of this enzyme showed that it is an NADPH-dependent tellurite reductase, with optimum reducing activity at 30°C and pH 9.0. Finally, the enzyme was able to generate Te-containing nanostructures, about 68 nm in size, which exhibit interesting antibacterial properties against E. coli, with no apparent cytotoxicity against eukaryotic cells.
Asunto(s)
Antibacterianos/biosíntesis , Proteínas Bacterianas/metabolismo , Glutatión Reductasa/metabolismo , Nanoestructuras/análisis , Pseudomonas/enzimología , Telurio/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Biotransformación , Estabilidad de Enzimas , Glutatión Reductasa/química , Glutatión Reductasa/genética , Oxidación-Reducción , Pseudomonas/química , Pseudomonas/genética , Pseudomonas/metabolismoRESUMEN
Tellurite, the most soluble tellurium oxyanion, is extremely harmful for most microorganisms. Part of this toxicity is due to the generation of reactive oxygen species that in turn cause oxidative stress. However, the way in which tellurite interferes with cellular processes is not well understood to date. Looking for new cellular tellurite targets, we decided to evaluate the functioning of the electron transport chain in tellurite-exposed cells. In this communication we show that the E. coli ndh gene, encoding NDH-II dehydrogenase, is significantly induced in toxicant-exposed cells and that the enzyme displays tellurite-reducing activity that results in increased superoxide levels in vitro.
Asunto(s)
Membrana Celular/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Oxidorreductasas/metabolismo , Superóxidos/metabolismo , Telurio/metabolismo , Telurio/farmacología , Membrana Celular/metabolismo , Escherichia coli/citología , Escherichia coli/metabolismo , Oxidación-Reducción , Oxidorreductasas/genéticaRESUMEN
The vast application of fluorescent semiconductor nanoparticles (NPs) or quantum dots (QDs) has prompted the development of new, cheap and safer methods that allow generating QDs with improved biocompatibility. In this context, green or biological QDs production represents a still unexplored area. This work reports the intracellular CdTe QDs biosynthesis in bacteria. Escherichia coli overexpressing the gshA gene, involved in glutathione (GSH) biosynthesis, was used to produce CdTe QDs. Cells exhibited higher reduced thiols, GSH and Cd/Te contents that allow generating fluorescent intracellular NP-like structures when exposed to CdCl(2) and K(2)TeO(3). Fluorescence microscopy revealed that QDs-producing cells accumulate defined structures of various colors, suggesting the production of differently-sized NPs. Purified fluorescent NPs exhibited structural and spectroscopic properties characteristic of CdTe QDs, as size and absorption/emission spectra. Elemental analysis confirmed that biosynthesized QDs were formed by Cd and Te with Cd/Te ratios expected for CdTe QDs. Finally, fluorescent properties of QDs-producing cells, such as color and intensity, were improved by temperature control and the use of reducing buffers.
Asunto(s)
Compuestos de Cadmio/metabolismo , Escherichia coli/metabolismo , Glutatión/metabolismo , Nanopartículas/química , Telurio/metabolismo , Citratos/farmacología , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Genes Bacterianos/genética , Microscopía Fluorescente , Nanopartículas/ultraestructura , Tamaño de la Partícula , Puntos Cuánticos , Espectrometría de Fluorescencia , Espectroscopía Infrarroja por Transformada de Fourier , Temperatura , Difracción de Rayos XRESUMEN
(S)-dimethyl 2-(3-(phenyltellanyl) propanamido) succinate, a new telluroamino acid derivative, showed remarkable glutathione peroxidase (GPx)-like activity, attesting to its antioxidant potential. However, the stability and toxicity of this compound has not yet been investigated. The present study was designed to investigate the pharmacological/toxicological properties of this compound in vitro and in vivo. In vitro, this telluroamino acid derivative significantly blocked spontaneous and Fe(II)-induced TBARS formation in rat brain homogenates, demonstrating high antioxidant activity. In addition, it exhibited GPx-like and thiol oxidase activities. However, when subcutaneously administered to mice, (S)-dimethyl 2-(3-(phenyltellanyl) propanamido) succinate indicated genotoxic and mutagenic effect in adult male mice. Considering the differential effects of (S)-dimethyl 2-(3-(phenyltellanyl) propanamido) succinate in vitro and in vivo, additional experiments are needed to elucidate the mechanism(s) by which this compound displays its antioxidant/toxicological effects.
Asunto(s)
Antioxidantes/farmacología , Ácido Aspártico/análogos & derivados , Succinatos/farmacología , Administración Oral , Análisis de Varianza , Animales , Ácido Aspártico/toxicidad , Ensayo Cometa , Daño del ADN , Compuestos Ferrosos/metabolismo , Glutatión Peroxidasa/metabolismo , Dosificación Letal Mediana , Masculino , Ratones , Compuestos Organometálicos/metabolismo , Compuestos Organometálicos/farmacología , Compuestos Organometálicos/toxicidad , Ratas , Ratas Wistar , Succinatos/toxicidad , Telurio/metabolismo , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
The soluble tellurium oxyanion, tellurite, is toxic for most organisms. At least in part, tellurite toxicity involves the generation of oxygen-reactive species which induce an oxidative stress status that damages different macromolecules with DNA, lipids and proteins as oxidation targets. The objective of this work was to determine the effects of tellurite exposure upon the Escherichia coli pyruvate dehydrogenase (PDH) complex. The complex displays two distinct enzymatic activities: pyruvate dehydrogenase that oxidatively decarboxylates pyruvate to acetylCoA and tellurite reductase, which reduces tellurite (Te(4+)) to elemental tellurium (Te(o)). PDH complex components (AceE, AceF and Lpd) become oxidized upon tellurite exposure as a consequence of increased carbonyl group formation. When the individual enzymatic activities from each component were analyzed, AceE and Lpd did not show significant changes after tellurite treatment. AceF activity (dihydrolipoil acetyltransferase) decreased ~30% when cells were exposed to the toxicant. Finally, pyruvate dehydrogenase activity decreased >80%, while no evident changes were observed in complex's tellurite reductase activity.
Asunto(s)
Escherichia coli/enzimología , Estrés Oxidativo/efectos de los fármacos , Complejo Piruvato Deshidrogenasa/metabolismo , Telurio/toxicidad , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Oxidación-Reducción , Oxidorreductasas/metabolismo , Carbonilación Proteica , Telurio/metabolismoRESUMEN
The Geobacillus stearothermophilus V cobA gene encoding uroporphyrinogen-III C-methyltransferase (also referred to as SUMT) was cloned into Escherichia coli and the recombinant enzyme was overexpressed and purified to homogeneity. The enzyme binds S-adenosyl-L-methionine and catalyzes the production of III methyl uroporphyrinogen in vitro. E. coli cells expressing the G. stearothermophilus V cobA gene exhibited increased resistance to potassium tellurite and potassium tellurate. Site-directed mutagenesis of cobA abolished tellurite resistance of the mesophilic, heterologous host and SUMT activity in vitro. No methylated, volatile derivatives of tellurium were found in the headspace of tellurite-exposed cobA-expressing E. coli, suggesting that the role of SUMT methyltransferase in tellurite(ate) detoxification is not related to tellurium volatilization.
Asunto(s)
Escherichia coli/metabolismo , Geobacillus stearothermophilus/enzimología , Metiltransferasas , Telurio/metabolismo , Secuencia de Aminoácidos , Clonación Molecular , Metiltransferasas/análisis , Metiltransferasas/biosíntesis , Metiltransferasas/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/biosíntesis , S-Adenosilmetionina/metabolismo , Uroporfirinógenos/biosíntesisRESUMEN
Potassium tellurite (K(2)TeO(3)) is extremely toxic for most forms of life and only a limited number of organisms are naturally resistant to the toxic effects of this compound. Crude extracts prepared from the environmental isolate Aeromonas caviae ST catalize the in vitro reduction of TeO32- in a NADH-dependent reaction. Upon fractionation by ionic exchange column chromatography three major polypeptides identified as the E1, E2, and E3 components of the pyruvate dehydrogenase (PDH) complex were identified in fractions exhibiting tellurite-reducing activity. Tellurite reductase and pyruvate dehydrogenase activities co-eluted from a Sephadex gel filtration column. To determine which component(s) of the PDH complex has tellurite reductase activity, the A. caviae ST structural genes encoding for E1 (aceE), E2 (aceF), and E3 (lpdA) were independently cloned and expressed in Escherichia coli and their gene products purified. Results indicated that tellurite reductase activity lies almost exclusively in the E3 component, dihydrolipoamide dehydrogenase. The E3 component of the PDH complex from E. coli, Zymomonas mobilis, Streptococcus pneumoniae, and Geobacillus stearothermophilus also showed NADH-dependent tellurite reductase in vitro suggesting that this enzymatic activity is widely distributed among microorganisms.
Asunto(s)
Aeromonas/enzimología , Proteínas Bacterianas/metabolismo , Dihidrolipoamida Deshidrogenasa/metabolismo , Oxidorreductasas/metabolismo , Telurio/metabolismo , Aeromonas/efectos de los fármacos , Aeromonas/genética , Clonación Molecular , Dihidrolipoamida Deshidrogenasa/genética , Escherichia coli/enzimología , Escherichia coli/genética , Oxidación-Reducción , Oxidorreductasas/química , Telurio/toxicidadRESUMEN
The glutamatergic system is an important target in many neurodegenerative diseases and for several neurotoxic drugs. Organotellurium compounds are often very good free radical scavengers' agents. Recently, we reported that diethyl-2-phenyl-2-tellurophenyl vinylphosphonate is a compound with low toxicity in vitro and in vivo, as well as also possesses antioxidant activity against iron-induced lipid peroxidation. The aim of this study was to evaluate in vitro the antioxidant and mitochondrial protective effect of this organotellurium compound against quinolinic acid (QA) and sodium nitroprusside (SNP), and to evaluate the in vitro actions of this organotellurium compound in the glutamatergic system in brain of rats. We observed that the telluro vinylphosphonate possess an antioxidant activity against QA and SNP at micromolar concentrations. When tested at antioxidant concentrations (from 2 to 10muM), the compound does not affect the mitochondrial viability and [3H]glutamate uptake in slices from cerebral cortex, hippocampus and striatum, [3H]glutamate release from synaptosomal preparations and [3H]glutamate binding in membrane preparation. Our data suggest that the telluro vinylphosphonate act as an antioxidant in the central nervous system in vitro with no effects on the glutamatergic system; nevertheless more studies in different models of brain injury must be performed in order to corroborate our findings.
Asunto(s)
Antioxidantes , Encéfalo/metabolismo , Neurotoxinas/metabolismo , Organofosfonatos , Telurio , Compuestos de Vinilo , Animales , Antioxidantes/química , Antioxidantes/metabolismo , Encéfalo/anatomía & histología , Ácido Glutámico/metabolismo , Peroxidación de Lípido , Mitocondrias/metabolismo , Donantes de Óxido Nítrico/metabolismo , Nitroprusiato/metabolismo , Organofosfonatos/química , Organofosfonatos/metabolismo , Ácido Quinolínico/metabolismo , Ratas , Ratas Wistar , Sinaptosomas/metabolismo , Telurio/química , Telurio/metabolismo , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo , Compuestos de Vinilo/química , Compuestos de Vinilo/metabolismoRESUMEN
Reactive oxygen species damage intracellular targets and are implicated in cancer, genetic disease, mutagenesis, and aging. Catalases are among the key enzymatic defenses against one of the most physiologically abundant reactive oxygen species, hydrogen peroxide. The well-studied, heme-dependent catalases accelerate the rate of the dismutation of peroxide to molecular oxygen and water with near kinetic perfection. Many catalases also bind the cofactors NADPH and NADH tenaciously, but, surprisingly, NAD(P)H is not required for their dismutase activity. Although NAD(P)H protects bovine catalase against oxidative damage by its peroxide substrate, the catalytic role of the nicotinamide cofactor in the function of this enzyme has remained a biochemical mystery to date. Anions formed by heavy metal oxides are among the most highly reactive, natural oxidizing agents. Here, we show that a natural isolate of Staphylococcus epidermidis resistant to tellurite detoxifies this anion thanks to a novel activity of its catalase, and that a subset of both bacterial and mammalian catalases carry out the NAD(P)H-dependent reduction of soluble tellurite ion (TeO(3)(2-)) to the less toxic, insoluble metal, tellurium (Te(o)), in vitro. An Escherichia coli mutant defective in the KatG catalase/peroxidase is sensitive to tellurite, and expression of the S. epidermidis catalase gene in a heterologous E. coli host confers increased resistance to tellurite as well as to hydrogen peroxide in vivo, arguing that S. epidermidis catalase provides a physiological line of defense against both of these strong oxidizing agents. Kinetic studies reveal that bovine catalase reduces tellurite with a low Michaelis-Menten constant, a result suggesting that tellurite is among the natural substrates of this enzyme. The reduction of tellurite by bovine catalase occurs at the expense of producing the highly reactive superoxide radical.
Asunto(s)
Catalasa/metabolismo , Oxidorreductasas/metabolismo , Secuencia de Aminoácidos , Animales , Catalasa/genética , Bovinos , Farmacorresistencia Bacteriana/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Escherichia coli/genética , Genes Bacterianos , Técnicas In Vitro , Cinética , Hígado/enzimología , Mutación , NAD/metabolismo , NADP/metabolismo , Oxidorreductasas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Staphylococcus epidermidis/enzimología , Staphylococcus epidermidis/genética , Especificidad por Sustrato , Superóxidos/metabolismo , Telurio/metabolismo , Telurio/farmacologíaRESUMEN
O-Acetylserine (thiol)-lyase (cysteine synthase) was purified from Azospirillum brasilense Sp7. After hydrolysis of the purified protein, amino acid sequences of five peptides were obtained, which permitted the cloning and sequencing of the cysK gene. The deduced amino acid sequence of cysteine synthase exhibited homology with several putative proteins from Alpha- and Gammaproteobacteria. Azospirillum brasilense Sp7 cysK exhibited 58% identity (72% similarity) with Escherichia coli K12 and Salmonella enterica serovar Typhimurium cysteine synthase proteins. An E. coli auxotroph lacking cysteine synthase loci could be complemented with A. brasilense Sp7 cysK. The 3.0-kb HindIII-EcoRI fragment bearing cysK contained two additional ORFs encoding a putative transcriptional regulator and dUTPase. Insertional disruption of the cysK gene did not produce a cysteine auxotroph, indicating that gene redundancy in the cysteine biosynthetic or other biosynthetic pathways exists in Azospirillum, as already described in other bacteria. Nitrogen fixation was not altered in the mutant strain as determined by acetylene reduction. However, this strain showed an eight-fold reduction in tellurite resistance as compared to the wild-type strain, which was only observed during growth in minimal medium. These data confirm earlier observations regarding the importance of cysteine metabolism in tellurite resistance.
Asunto(s)
Azospirillum brasilense/genética , Liasas de Carbono-Oxígeno/genética , Liasas de Carbono-Oxígeno/aislamiento & purificación , Telurio/metabolismo , Telurio/farmacología , Azospirillum brasilense/efectos de los fármacos , Azospirillum brasilense/enzimología , Secuencia de Bases , Liasas de Carbono-Oxígeno/química , Liasas de Carbono-Oxígeno/metabolismo , Cromatografía , Clonación Molecular , Farmacorresistencia Fúngica , Prueba de Complementación Genética , Mutación , Oxidación-Reducción , FenotipoRESUMEN
A 3.8-kb fragment of chromosomal DNA of Geobacillus stearothermophilus V cloned in pSP72 (p1VH) confers resistance to potassium tellurite (K(2)TeO(3)) and to potassium tellurate (K(2)TeO(4)) when the encoded genes are expressed in Escherichia coli K-12. The nt sequence of the cloned fragment predicts three ORFs of 780, 399, and 600 bp, whose encoded protein products exhibit about 80% similarity with the SUMT methyltransferase and the BtuR protein of Bacillus megaterium, and with the UbiE methyltransferase of Bacillus anthracis A2012, respectively. In addition, E. coli/p1VH cells evolved dimethyl telluride, which was released into the headspace gas above liquid cultures when amended with K(2)TeO(3) or with K(2)TeO(4). After 48 h of growth in the presence of these compounds, a protein of about 25 kDa was found at a significantly higher level when crude extracts were analyzed by SDS-PAGE. The N-terminal amino acid (aa) sequence of this protein, obtained by Edman degradation, matched the deduced aa sequence predicted by the G. stearothermophilus V ubiE gene. This gene was amplified by PCR, subcloned in pET21b, and transformed into E. coli JM109(DE3). Interestingly, DMTe evolution occurred when these modified cells were grown in K(2)TeO(4) - but not in K(2)TeO(3) - amended media. These results may be indicative that the two Te oxyanions could be detoxified in the cell by different metabolic pathways.
Asunto(s)
Bacillaceae/química , Proteínas Bacterianas/metabolismo , Escherichia coli K12/efectos de los fármacos , Telurio/metabolismo , Telurio/farmacología , Aniones/química , Proteínas Bacterianas/genética , Secuencia de Bases , Cromosomas/genética , Medios de Cultivo , ADN/química , Electroforesis en Gel de Poliacrilamida , Escherichia coli K12/enzimología , Escherichia coli K12/genética , Datos de Secuencia Molecular , Peso Molecular , Telurio/química , Factores de TiempoRESUMEN
Plasmids of the H incompatibility complex confer protection against all known channel-forming colicins (PacB character) and resistance to potassium tellurite (Te(r)) to Escherichia coli strains. A DNA clone (2.2 kbp) from plasmid Mip233 (IncHI3) expressing PacB-Te(r) phenotypes was studied. DNA sequence analysis revealed a high degree of homology with the enzyme O-acetylserine sulfhydrylase. Size of the PacB-Te(r) transcript was estimated as 1200 bases. A single polypeptide was found on SDS-polyacrylamide gel with a molecular mass estimated of 34 kDa. The effect of channel-forming colicins and tellurite was analyzed at physiological and transcriptional levels. Results suggest that the pacB gene product could be a reductase-like enzyme. It is also suggested that presence of the PacB character among H plasmid confers selective advantage on cells sharing an ecological niche.