Estudio funcional del sistema tiorredoxina de la bacteria extremófila Leptospirillum Grupo II CF-1
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2017
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es
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Universidad Andrés Bello
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Licencia CC
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La biolixiviación se define como el proceso por el cual existe una solubilización de metales a partir de minerales insolubles, mediante oxidación biológica. La aplicación biotecnológica de esta técnica reside en que los microorganismos son capaces de extraer los metales de los minerales de baja ley, mediante un proceso sustentable y que logra controlar la salida de contaminantes al medio ambiente. Actualmente se considera que dentro de los grupos bacterianos responsables más importantes de la biolixiviación del mineral se incluye el género Leptospirillum, el cual contiene microorganismos que se caracterizan por vivir en condiciones extremas tales como pH muy ácido (<pH1) y elevadas concentraciones de metales. Estas condiciones contribuyen a la generación de especies reactivas de oxígeno y consecuentemente a inducir estrés oxidativo. Uno de los blancos de ROS son los tioles de las proteínas, los cuales al oxidarse son regenerados a través de sistemas tioprotectores, como lo son las glutarredoxinas y tiorredoxinas. Estudios bioinformáticos muestran que en el genoma de Leptospirillum Grupo II CF-1 existen múltiples genes que codificarían para el sistema tiorredoxina. Estudios recientes mostraron que bajo condiciones de estrés oxidativo aumenta la actividad tiorredoxina y los niveles de mRNA de algunos genes (trx). No obstante se desconoce cuál es la contribución fisiológica de cada uno estos genes frente a la defensa antioxidante. Los resultados presentados en esta tesis demuestran que bajo distintas condiciones de estrés oxidativo (como lo son estrés por diamida y sulfato férrico), se activan set de genes de trx distintos, dependiendo de las condiciones de estrés a la que se ve sometido el organismo. Así mismo los ensayos de complementación en cepas de E.coli deficientes de este sistema, con genes trx de Leptospirillum CF-1, demuestran que algunos de estos genes son funcionales, ya que restituye el fenotipo de la cepa mutante y que inclusive algunos de ellos, restituyen el fenotipo mejor que la cepa silvestre.
Finalmente, y en análisis bioinformáticos, se logró determinar teóricamente cuales serían las posibles funciones que estarían cumpliendo estas tiorredoxinas en la respuesta ante estrés oxidativo. En conclusión todas estas proteínas estarían participando de manera diferencial y a distintos niveles celulares para así asegurar la sobrevivencia de este microorganismo en su ambiente natural.
Bioleaching is defined as the process by which there is a solubilization of metals from insoluble minerals by biological oxidation. The biotechnological application of this technique resides in that the microorganisms are able to extract the metals of low-grade ores, through a sustainable process and that manages to control the exit of pollutants to the environment. Currently, the most important responsible bacterial groups of the mineral bioleaching are considered to be the genus Leptospirillum, which contains microorganisms that are characterized by living in extreme conditions such as very acidic pH (<pH1) and high concentrations of metals. These conditions contribute the generation of reactive oxygen species (ROS) and consequently to induce oxidative stress. One of the targets of ROS is the thiol of proteins, which oxidized are regenerated through thioprotective systems, such as glutarredoxins and thioredoxin. Bioinformatic studies show that in the genome of Leptospirillum Group II CF-1 there are multiple genes that would code for the thioredoxin system. Recent studies showed that under conditions of oxidative stress increases the activity of thioredoxin and mRNA levels of some genes (trx). However, it is unknown what the physiological contribution of each of these genes is to antioxidant defense. The results presented in this thesis demonstrate that under different conditions of oxidative stress (such as stress by diamide and ferric sulphate), sets of different trx genes are activated, depending on the stress conditions to which the organism is subjected. Complementary assays in E.coli strains deficient in this system, with Leptospirillum CF-1 trx genes, demonstrate that some of these genes are functional, since it restores the phenotype of the mutant strain and that even some of them , restore the phenotype better than the wild-type strain. Finally, and in bioinformatic analyzes, it was possible to determine theoretically what would be the possible functions that would be fulfilling these thiorredoxinas in the response to oxidative stress. In conclusion all these proteins would be participating differentially and at different cellular levels to ensure the survival of this microorganism in its natural environment.
Bioleaching is defined as the process by which there is a solubilization of metals from insoluble minerals by biological oxidation. The biotechnological application of this technique resides in that the microorganisms are able to extract the metals of low-grade ores, through a sustainable process and that manages to control the exit of pollutants to the environment. Currently, the most important responsible bacterial groups of the mineral bioleaching are considered to be the genus Leptospirillum, which contains microorganisms that are characterized by living in extreme conditions such as very acidic pH (<pH1) and high concentrations of metals. These conditions contribute the generation of reactive oxygen species (ROS) and consequently to induce oxidative stress. One of the targets of ROS is the thiol of proteins, which oxidized are regenerated through thioprotective systems, such as glutarredoxins and thioredoxin. Bioinformatic studies show that in the genome of Leptospirillum Group II CF-1 there are multiple genes that would code for the thioredoxin system. Recent studies showed that under conditions of oxidative stress increases the activity of thioredoxin and mRNA levels of some genes (trx). However, it is unknown what the physiological contribution of each of these genes is to antioxidant defense. The results presented in this thesis demonstrate that under different conditions of oxidative stress (such as stress by diamide and ferric sulphate), sets of different trx genes are activated, depending on the stress conditions to which the organism is subjected. Complementary assays in E.coli strains deficient in this system, with Leptospirillum CF-1 trx genes, demonstrate that some of these genes are functional, since it restores the phenotype of the mutant strain and that even some of them , restore the phenotype better than the wild-type strain. Finally, and in bioinformatic analyzes, it was possible to determine theoretically what would be the possible functions that would be fulfilling these thiorredoxinas in the response to oxidative stress. In conclusion all these proteins would be participating differentially and at different cellular levels to ensure the survival of this microorganism in its natural environment.
Notas
Tesis (Magíster en Biotecnología)
Palabras clave
Lixiviación Bacteriana, Sistema Tiorredoxina, Leptospirillum