El gen acr3 de Exiguobacterium sp. SH31 es capaz de generar un fenotipo resistente a As en conjunto con los genes del operón ars en Escherichia coli
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2018
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es
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Universidad Andrés Bello
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El salar de Huasco corresponde a un humedal salino del altiplano chileno, considerado una zona desafiante para el desarrollo de vida debido a sus características climáticas, geológicas y geográficas. El norte de Chile presenta una elevada riqueza mineral, fomentando las actividades relacionadas a la extracción de los mismos. La particularidad del conjunto de condiciones que pueden co-ocurrir en los nichos del altiplano chileno está evidenciada por la gran diversidad de microorganismos presentes y la capacidad de estos mismos de resistir o resistir estas extremas condiciones, lo que ha generado gran interés en la comunidad científica respecto al estudio los mecanismos moleculares de adaptación a ambientes extremos. Debido a la alta toxicidad de este metaloide y a la diversidad de microorganismos capaces de desarrollarse en su presencia, se ha considerado que estos, presentan potencial biotecnológico, particularmente en el tratamiento de aguas residuales y suelos contaminados. Entre las bacterias que han sido reportadas como resistentes a altas concentraciones de arsénico se encuentra el género Exiguobacterium, este ha sido aislado de una diversidad de ambientes extremos, incluyendo el altiplano andino, demostrando su plasticidad y adaptación. Una de las cepas más resistentes es la S17, aislada del altiplano argentino, en la cual fue reportada por primera vez la presencia del gen acr3 que codifica para una bomba accesoria de expulsión de As(III), sugiriendo que esta sería el responsable de la superior resistencia de esta cepa, con respecto a otras del género. Nuestro grupo de trabajo aisló la cepa Exiguobacterium sp. SH31 del Salar del Huasco que presenta un nivel equivalente de resistencia a As (III) y As(V) que la cepa S17. Estudios realizados con la cepa SH31 demostraron que también porta el gen acr3 y que este responde a la presencia de arsénico. El mecanismo más estudiado y frecuente a través del cual los microorganismos resisten la presencia de arsénico en su ambiente es la reducción del As(V) dada por la presencia y expresión del operón ars, basado en la arseniato reductasa (arsC) y la bomba de eflujo de As(III) (arsB). El requerimiento de este operón en la resistencia a arsénico, ha sido demostrado utilizando la cepa de Escherichia coli AW3110 que tiene delecionado el operón ars de Escherichia coli K-12. Entonces, considerando estos antecedentes, se propone demostrar la participación del gen acr3 de Exiguobacterium sp. SH31 en la resistencia a As y su posible efecto en conjunto con los genes del operón ars en la cepa Escherichia coli AW3110 hipersensible, mediante la expresión heteróloga de los genes de interés, mediante experimentos bioquímicos y moleculares.
The Salar of Huasco corresponds to a saline wetland of the Chilean highlands, considered a challenging area for the development of life due to its climatic, geological and geographical characteristics. The north of Chile has a high mineral wealth, promoting activities related to the extraction of them. The economic benefits brought by the mining exploitation, caused the increase of public health problems related to poisoning, mainly by drinking water contaminated by Arsenic (As). It was reported that drinking water in this area exceeded 86 times the limit established by the WHO standards. The particularity of the conditions that can co-occur in the niches of the Chilean highlands is evidenced by the great diversity of microorganisms present and their ability to resist or tolerate these extreme conditions, which has generated great interest in the scientific community regarding the study of the molecular mechanisms of adaptation to extreme environments. Due to the high toxicity of this metalloid, and the diversity of microorganisms capable of developing in its presence, it has been considered that these has a biotechnology potential, particularly in the treatment of wastewater and contaminated soils. Among the bacteria that have been reported as resistant to high concentrations of arsenic, is the genus Exiguobacterium, which has been isolated from a variety of extreme environments, including the Andean high plateau, demonstrating its plasticity and adaptation to extreme enviroments. One of the most resistant strains is S17, isolated from the Argentine highlands, which the presence of the acr3 gene, encoding for an accessory pump for expulsion of As (III) was reported for the first time, suggesting that this would be responsible for the high resistance of this strain, with respect to others of the genus. Our working group isolated the strain Exiguobacterium sp. SH31 from Salar del Huasco that presents an equivalent level of resistance to As (III) and As (V) as the strain S17. Studies carried out with the SH31 strain showed that it also presents the acr3 gene and that it responds to the presence of arsenic. The most studied and frequent mechanism through which microorganisms tolerate or resist the presence of arsenic in their environment is the reduction of As (V), given by the presence and expression of the ars operon, based on arsenate reductase (arsC) and the As (III) efflux pump (arsB). The participation and importance of this operon in the resistance has been demonstrated by the use of the strain of Escherichia coli AW3110 which has deleted the ars operon of Escherichia coli K-12 strain. Considering this background, it is proposed to demonstrate the participation of the acr3 gene of Exiguobacterium sp. SH31 in the resistance to As and the possible synergistic effect that it may have in conjunction with the genes of the ars operon in the Escherichia coli AW3110 strain, through the heterologous expression of the genes of interest, biochemical and molecular experiments.
The Salar of Huasco corresponds to a saline wetland of the Chilean highlands, considered a challenging area for the development of life due to its climatic, geological and geographical characteristics. The north of Chile has a high mineral wealth, promoting activities related to the extraction of them. The economic benefits brought by the mining exploitation, caused the increase of public health problems related to poisoning, mainly by drinking water contaminated by Arsenic (As). It was reported that drinking water in this area exceeded 86 times the limit established by the WHO standards. The particularity of the conditions that can co-occur in the niches of the Chilean highlands is evidenced by the great diversity of microorganisms present and their ability to resist or tolerate these extreme conditions, which has generated great interest in the scientific community regarding the study of the molecular mechanisms of adaptation to extreme environments. Due to the high toxicity of this metalloid, and the diversity of microorganisms capable of developing in its presence, it has been considered that these has a biotechnology potential, particularly in the treatment of wastewater and contaminated soils. Among the bacteria that have been reported as resistant to high concentrations of arsenic, is the genus Exiguobacterium, which has been isolated from a variety of extreme environments, including the Andean high plateau, demonstrating its plasticity and adaptation to extreme enviroments. One of the most resistant strains is S17, isolated from the Argentine highlands, which the presence of the acr3 gene, encoding for an accessory pump for expulsion of As (III) was reported for the first time, suggesting that this would be responsible for the high resistance of this strain, with respect to others of the genus. Our working group isolated the strain Exiguobacterium sp. SH31 from Salar del Huasco that presents an equivalent level of resistance to As (III) and As (V) as the strain S17. Studies carried out with the SH31 strain showed that it also presents the acr3 gene and that it responds to the presence of arsenic. The most studied and frequent mechanism through which microorganisms tolerate or resist the presence of arsenic in their environment is the reduction of As (V), given by the presence and expression of the ars operon, based on arsenate reductase (arsC) and the As (III) efflux pump (arsB). The participation and importance of this operon in the resistance has been demonstrated by the use of the strain of Escherichia coli AW3110 which has deleted the ars operon of Escherichia coli K-12 strain. Considering this background, it is proposed to demonstrate the participation of the acr3 gene of Exiguobacterium sp. SH31 in the resistance to As and the possible synergistic effect that it may have in conjunction with the genes of the ars operon in the Escherichia coli AW3110 strain, through the heterologous expression of the genes of interest, biochemical and molecular experiments.
Notas
Tesis (Ingeniero en Biotecnología)
Esta tesis se realizó en el Laboratorio de Microbiología Molecular de la Facultad de Ciencias Biológicas de la Universidad Andrés Bello y fue financiada por el proyectos de Investigación con Financiamiento Interno (Núcleo UNAB) bajo el título del proyecto “Evaluación de mecanismos de resistencia presentes en microorganismos que prosperan en ambientes naturales con arsénico: caso del género Exiguobacterium en el Salar de Huasco” y el v proyecto de cooperación internacional Chile- Francia (ECOS) titulado “Comparision of molecular regulation strategies for two bacterial lifestyle models in response to niche oxidative stress challenges”, en donde las principales cepas de estudios correspoden a Salmonella Typhimurium 14028s y ExiguobacteriumSH31.
Esta tesis se realizó en el Laboratorio de Microbiología Molecular de la Facultad de Ciencias Biológicas de la Universidad Andrés Bello y fue financiada por el proyectos de Investigación con Financiamiento Interno (Núcleo UNAB) bajo el título del proyecto “Evaluación de mecanismos de resistencia presentes en microorganismos que prosperan en ambientes naturales con arsénico: caso del género Exiguobacterium en el Salar de Huasco” y el v proyecto de cooperación internacional Chile- Francia (ECOS) titulado “Comparision of molecular regulation strategies for two bacterial lifestyle models in response to niche oxidative stress challenges”, en donde las principales cepas de estudios correspoden a Salmonella Typhimurium 14028s y ExiguobacteriumSH31.
Palabras clave
Escherichia Coli, Arsénico, Genes, Microorganismos