Caracterización del gen yhjA y su contexto génico en la defensa durante estrés oxidativo frente a Fe3+ o H2O2 en Leptospirillum rubarum
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Fecha
2013
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Idioma
es
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Universidad Andrés Bello
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Licencia CC
Licencia CC
Resumen
La biolixiviación es un proceso que permite la recuperación de metales desde
minerales sulfurados gracias a la acción de microorganismos, los cuales juegan un rol
indirecto regenerando el ion Fe3
+ necesario para la solubilización de los metales. En este
proceso, los miembros del género Leptospirillum son importantes debido a su capacidad
para oxidar ión ferroso (Fe2+) a ión férrico (Fe3+). Los miembros de este género y otras
bacterias biolixiviantes están usualmente expuestas a condiciones altamente oxidantes,
causadas principalmente por altas concentraciones de hierro y otros metales, y pHs
extremadamente bajos ( <1 ). Estas condiciones pueden favorecer la generación de
especies reactivas de oxígeno (ROS), como el peróxido de hidrógeno (H20 2), superóxido
(02") y el radical hidroxilo (Olf), las cuales pueden inducir estrés oxidativo a los
microorganismos. Adicionalmente, las bacterias oxidantes de hierro poseen elevadas
tasas respiratorias que también pueden favorecer la generación de ROS. Todos estos
factores pueden afectar la actividad y sobrevivencia de los microorganismos, no
obstante, los mecanismos que ayudan a mantener la homeostasis redox de estas bacterias
son muy poco conocidos.
Con el objetivo de conocer los elementos moleculares involucrados en la
respuesta antioxidante de Leptospirillum rubarum se realizó un análisis bioinformático,
que reveló la presencia de genes codificantes para peroxidasas, enzimas que constituyen
una barrera de protección frente a H20 2. El gen yhjA codifica para una citocromo e
peroxidasa (CcP) y se localiza contiguamente a los genesfur y prx que codifican para un
regulador de la homeostasis de hierro y una peroxirredoxina, respectivamente. Mediante
RT -PCR se determinó que estos genes se ce-transcriben, incluyendo además al gen adr
que codifica para una acireductona dioxigenasa, conformando así al denominado
"operón ccp". Un análisis de RT-qPCR reveló que estos genes del operón incrementan
levemente su expresión en células expuestas a estrés oxidativo por Fe3+ o H20 2 .
Adicionalmente un análisis de actividad de la enzima CcP en extractos celulares de L.
rubarum reveló que la enzima es funcional in vivo y que su actividad incrementa
significativamente al inducir estrés oxidativo con Fe3+ o H20 2. Los resultados obtenidos
sugieren que CcP constituye un elemento de respuesta frente a estrés oxidativo y que su
actividad podría estar regulada a nivel transcripcional por Fe3+ o H20 2 intracelular.
Bioleaching is a process which allows recovery of metals from sulphide ores by the action of microorganisms, which play an indirect role in regenerating the ion Fe3 + necessary for the solubilization of the metals. In this process, Leptospirillum genus's members are important because oftheir ability to oxidize ferrous ion (Fe2+) to ferric iron (Fe3+). The members ofthis genus and other bioleaching bacteria are usually exposed to highly oxidizing conditions, mainly caused by high concentrations of iron and other metals, and extremely low pH ( <1 ). These conditions may favor the generation of reactive oxygen species (ROS) such as hydrogen peroxide (H202), superoxide (02) and hydroxyl radical (OR), which may induce oxidative stress in microorganisms. Additionally, high respiration rates of iron oxidizing bacteria may also favor the generation of ROS. All these factors may affect the activity and survival of microorganisms, however the mechanisms involved in maintenance of redox homeostasis of these bacteria are very poorly understood. In order to understand the molecular elements involved in the antioxidant response of Leptospirillum rubarum a bioinformatic analysis was performed, which revealed the presence of genes coding for peroxidases, enzymes that constitute the main protection barrier against H20 2 . The yhjA gene which encodes a cytochrome e peroxidase (CcP) and is located adjacently to fur and prx which encode for a regulator of iron homeostasis anda peroxiredoxin, respectively. By RT-PCR it was determined that these genes are co-transcribed, further comprising at gene adr encoding a acireductone dioxygenase, thereby forming between all the so-called "operon ccp". An analysis RT-qPCR revealed that these operon's genes slightly increase its expression in cells exposed to oxidative stress triggered by H20 2 or Fe3 +. Additionally, an analysis of the CcP activity in cell extracts of L. rubarum revealed that the enzyme is functional in vivo and its activity significantly increases after induction of oxidative stress with Fe3 + or H20 2. The results suggest that CcP is an element of the oxidative stress response and that its activity might be regulated at transcriptionallevel by intracellular Fe3+ and H20 2.
Bioleaching is a process which allows recovery of metals from sulphide ores by the action of microorganisms, which play an indirect role in regenerating the ion Fe3 + necessary for the solubilization of the metals. In this process, Leptospirillum genus's members are important because oftheir ability to oxidize ferrous ion (Fe2+) to ferric iron (Fe3+). The members ofthis genus and other bioleaching bacteria are usually exposed to highly oxidizing conditions, mainly caused by high concentrations of iron and other metals, and extremely low pH ( <1 ). These conditions may favor the generation of reactive oxygen species (ROS) such as hydrogen peroxide (H202), superoxide (02) and hydroxyl radical (OR), which may induce oxidative stress in microorganisms. Additionally, high respiration rates of iron oxidizing bacteria may also favor the generation of ROS. All these factors may affect the activity and survival of microorganisms, however the mechanisms involved in maintenance of redox homeostasis of these bacteria are very poorly understood. In order to understand the molecular elements involved in the antioxidant response of Leptospirillum rubarum a bioinformatic analysis was performed, which revealed the presence of genes coding for peroxidases, enzymes that constitute the main protection barrier against H20 2 . The yhjA gene which encodes a cytochrome e peroxidase (CcP) and is located adjacently to fur and prx which encode for a regulator of iron homeostasis anda peroxiredoxin, respectively. By RT-PCR it was determined that these genes are co-transcribed, further comprising at gene adr encoding a acireductone dioxygenase, thereby forming between all the so-called "operon ccp". An analysis RT-qPCR revealed that these operon's genes slightly increase its expression in cells exposed to oxidative stress triggered by H20 2 or Fe3 +. Additionally, an analysis of the CcP activity in cell extracts of L. rubarum revealed that the enzyme is functional in vivo and its activity significantly increases after induction of oxidative stress with Fe3 + or H20 2. The results suggest that CcP is an element of the oxidative stress response and that its activity might be regulated at transcriptionallevel by intracellular Fe3+ and H20 2.
Notas
Tesis (Magister en Biotecnología)
Proyecto FONDECYT 11085045
Proyecto FONDECYT 1120746
Proyecto FONDECYT 11085045
Proyecto FONDECYT 1120746
Palabras clave
Lixiviación Bacteriana, Estrés Oxidativo