Characterization of NaCl tolerance mechanism and its relation with the antioxidant mechanisms in the acidophilic bacterium Leptospirillum ferriphilum DSM 14647

Chloride bioleaching is considered a promising alternative method to recover copper from chalcopyrite and other primary copper sulfides, because it favors the leaching kinetics and avoids passivation of minerals. Nevertheless, chloride ions are highly toxic for iron-oxidizing microorganisms that par...

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Detalles Bibliográficos
Autor: Rivera-Araya, Javier Ignacio
Tipo de recurso: tesis doctoral
Estado:Versión publicada
Fecha de publicación:2019
País:Chile
OAI Identifier:oai:repositorio.anid.cl:10533/246430
Acceso en línea:https://hdl.handle.net/10533/246430
Access Level:acceso abierto
Palabra clave:Ciencias Naturales
Biotecnología Industrial
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dc.title.es_CL.fl_str_mv Characterization of NaCl tolerance mechanism and its relation with the antioxidant mechanisms in the acidophilic bacterium Leptospirillum ferriphilum DSM 14647
Caracterización del mecanismo de tolerancia a NaCl y su relación con los mecanismos antioxidantes en la bacteria acidófila Leptospirillum ferriphilum DSM 14647
title Characterization of NaCl tolerance mechanism and its relation with the antioxidant mechanisms in the acidophilic bacterium Leptospirillum ferriphilum DSM 14647
spellingShingle Characterization of NaCl tolerance mechanism and its relation with the antioxidant mechanisms in the acidophilic bacterium Leptospirillum ferriphilum DSM 14647
Rivera-Araya, Javier Ignacio
Ciencias Naturales
Biotecnología Industrial
title_short Characterization of NaCl tolerance mechanism and its relation with the antioxidant mechanisms in the acidophilic bacterium Leptospirillum ferriphilum DSM 14647
title_full Characterization of NaCl tolerance mechanism and its relation with the antioxidant mechanisms in the acidophilic bacterium Leptospirillum ferriphilum DSM 14647
title_fullStr Characterization of NaCl tolerance mechanism and its relation with the antioxidant mechanisms in the acidophilic bacterium Leptospirillum ferriphilum DSM 14647
title_full_unstemmed Characterization of NaCl tolerance mechanism and its relation with the antioxidant mechanisms in the acidophilic bacterium Leptospirillum ferriphilum DSM 14647
title_sort Characterization of NaCl tolerance mechanism and its relation with the antioxidant mechanisms in the acidophilic bacterium Leptospirillum ferriphilum DSM 14647
dc.creator.none.fl_str_mv Rivera-Araya, Javier Ignacio
author Rivera-Araya, Javier Ignacio
author_facet Rivera-Araya, Javier Ignacio
author_role author
dc.contributor.advisor.none.fl_str_mv Levicán, Gloria Paz
dc.contributor.institution.es_CL.fl_str_mv UNIVERSIDAD DE CHILE
dc.subject.oecd1n.es_CL.fl_str_mv Ciencias Naturales
topic Ciencias Naturales
Biotecnología Industrial
dc.subject.oecd2n.es_CL.fl_str_mv Biotecnología Industrial
description Chloride bioleaching is considered a promising alternative method to recover copper from chalcopyrite and other primary copper sulfides, because it favors the leaching kinetics and avoids passivation of minerals. Nevertheless, chloride ions are highly toxic for iron-oxidizing microorganisms that participate in the bioleaching process. In addition to the osmotic imbalance, chloride can also induce acidification of the cytoplasm in these microorganisms. We predicted that intracellular acidification produces an increase in respiratory rate and reactive oxygen species generation, and therefore oxidative stress can also be induced. The general goal of this study was to characterize the NaCl tolerance molecular mechanism and establish its relation with the antioxidant mechanism in Leptospirillum ferriphilum DSM 14647. First, the participation of canonical systems of tolerance to osmotic stress and the antioxidant systems, as an early response mechanism, were studied. By bioinformatic analysis, it was determined that genes for a complete or partial repertoire of K+ transporters, the biosynthesis pathways and transporter for compatible solutes (hidroxi)ectoine and trehalose were found in most of the acidophilic iron-oxidizing microorganisms. Additionally, the exposition of L. ferriphilum to 100 mM NaCl immediately up-regulated kdpC and kdpD genes coding for potassium transporters. A prolonged exposure to NaCl also increased the expression of genes encoding for biosynthesis of compatible solutes (hydroxy)ectoine (ectC and ectD) and trehalose (otsB). As a consequence, the intracellular levels of both hydroxyectoine and trehalose increased significantly, suggesting a strong response to keep osmotic homeostasis. On the other hand, the intracellular pH significantly decreased from 6.7 to pH 5.5 and oxygen consumption increased significantly when the cells were exposed to NaCl stress. Furthermore, this stress condition led to a significant increase of the intracellular content of reactive oxygen species, and to a rise of the antioxidative cytochrome c peroxidase (CcP) and thioredoxin (Trx) activities. In agreement with these results, ccp and trx genes were up-regulated under this condition, suggesting that this bacterium displays a transcriptionally regulated response against oxidative stress induced by chloride. In parallel, L. ferriphilum was adapted to 180 mM NaCl to identify the late response strategy. The analysis by transcriptomic profile revealed that the principal mechanisms involved in the adaptation were related with genes associated to the cell membrane integrity, respiration and antioxidant proteins, probably to conserve the pH and redox homeostasis. Inspection of these parameters in the adapted culture proved an increase in the respiratory rate and the maintain of the intracellular ROS levels. On contrary, genes associated with biosynthesis of hydroxyectoine (ectB, ectC, ectD) were repressed, coincident with the lack of detection of this compound in cell extract. Thus, these data suggest that the cells were not under osmotic stress. Finally, according with these results, we were able to conclude that chloride has a dramatic multifaceted effect on acidophile physiology that involves osmotic, acidic and oxidative stresses. The early response mechanism was composed by the osmotic response, pH homeostasis by cell respiration and antioxidant response. Instead, the late response mechanism involved pH homeostasis and antioxidant response.
publishDate 2019
dc.date.issued.es_CL.fl_str_mv 2019
dc.date.accessioned.none.fl_str_mv 2020-11-13T19:15:57Z
2022-08-16T19:13:46Z
dc.date.available.none.fl_str_mv 2020-11-13T19:15:57Z
2022-08-16T19:13:46Z
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spelling UNIVERSIDAD DE CHILERivera-Araya, Javier Ignacio2019https://hdl.handle.net/10533/246430http://purl.org/coar/access_right/c_abf2Biotecnología IndustrialCiencias NaturalesCharacterization of NaCl tolerance mechanism and its relation with the antioxidant mechanisms in the acidophilic bacterium Leptospirillum ferriphilum DSM 14647Levicán, Gloria PazUNIVERSIDAD DE CHILEChileRivera-Araya, Javier Ignacio2020-11-13T19:15:57Z2022-08-16T19:13:46Z2020-11-13T19:15:57Z2022-08-16T19:13:46Z2019Chloride bioleaching is considered a promising alternative method to recover copper from chalcopyrite and other primary copper sulfides, because it favors the leaching kinetics and avoids passivation of minerals. Nevertheless, chloride ions are highly toxic for iron-oxidizing microorganisms that participate in the bioleaching process. In addition to the osmotic imbalance, chloride can also induce acidification of the cytoplasm in these microorganisms. We predicted that intracellular acidification produces an increase in respiratory rate and reactive oxygen species generation, and therefore oxidative stress can also be induced. The general goal of this study was to characterize the NaCl tolerance molecular mechanism and establish its relation with the antioxidant mechanism in Leptospirillum ferriphilum DSM 14647. First, the participation of canonical systems of tolerance to osmotic stress and the antioxidant systems, as an early response mechanism, were studied. By bioinformatic analysis, it was determined that genes for a complete or partial repertoire of K+ transporters, the biosynthesis pathways and transporter for compatible solutes (hidroxi)ectoine and trehalose were found in most of the acidophilic iron-oxidizing microorganisms. Additionally, the exposition of L. ferriphilum to 100 mM NaCl immediately up-regulated kdpC and kdpD genes coding for potassium transporters. A prolonged exposure to NaCl also increased the expression of genes encoding for biosynthesis of compatible solutes (hydroxy)ectoine (ectC and ectD) and trehalose (otsB). As a consequence, the intracellular levels of both hydroxyectoine and trehalose increased significantly, suggesting a strong response to keep osmotic homeostasis. On the other hand, the intracellular pH significantly decreased from 6.7 to pH 5.5 and oxygen consumption increased significantly when the cells were exposed to NaCl stress. Furthermore, this stress condition led to a significant increase of the intracellular content of reactive oxygen species, and to a rise of the antioxidative cytochrome c peroxidase (CcP) and thioredoxin (Trx) activities. In agreement with these results, ccp and trx genes were up-regulated under this condition, suggesting that this bacterium displays a transcriptionally regulated response against oxidative stress induced by chloride. In parallel, L. ferriphilum was adapted to 180 mM NaCl to identify the late response strategy. The analysis by transcriptomic profile revealed that the principal mechanisms involved in the adaptation were related with genes associated to the cell membrane integrity, respiration and antioxidant proteins, probably to conserve the pH and redox homeostasis. Inspection of these parameters in the adapted culture proved an increase in the respiratory rate and the maintain of the intracellular ROS levels. On contrary, genes associated with biosynthesis of hydroxyectoine (ectB, ectC, ectD) were repressed, coincident with the lack of detection of this compound in cell extract. Thus, these data suggest that the cells were not under osmotic stress. Finally, according with these results, we were able to conclude that chloride has a dramatic multifaceted effect on acidophile physiology that involves osmotic, acidic and oxidative stresses. The early response mechanism was composed by the osmotic response, pH homeostasis by cell respiration and antioxidant response. Instead, the late response mechanism involved pH homeostasis and antioxidant response.La biolixiviación con cloruro se considera un método alternativo prometedor para recuperar el cobre de la calcopirita y otros minerales sulfurados de cobre, porque favorece la cinética de la lixiviación y evita la pasivación de los minerales. Sin embargo, los iones cloruro son altamente tóxicos para los microorganismos biolixiviantes. Además del desequilibrio osmótico, el cloruro también puede inducir la acidificación del citoplasma en estos microorganismos. Por consiguiente, es posible predecir que la acidificación intracelular produce un aumento de la frecuencia respiratoria y la generación de especies reactivas de oxígeno, por lo que se podría inducir estrés oxidativo. El objetivo general de este estudio fue caracterizar el mecanismo molecular de tolerancia al NaCl y establecer su relación con el mecanismo antioxidante en Leptospirillum ferriphilum DSM 14647. Inicialmente, se estudió la participación de los sistemas canónicos de tolerancia al estrés osmótico y los sistemas antioxidantes en la respuesta temprana. Utilizando herramientas bioinformáticas, se determinó que la mayoría de los microorganismos acidófilos oxidantes de hierro poseen un set completo o parcial de genes para los transportadores de K+ y las vías de síntesis y transportadores de los solutos compatibles (hidroxi)ectoína y trehalosa. También la exposición de L. ferriphilum a 100 mM de NaCl reguló de forma positiva los genes kdpC y kdpD que codifican para transportadores de potasio. La exposición prolongada a NaCl llevó también a la expresión de genes para la biosíntesis de los solutos compatibles (hidroxi)ectoína (ectC y ectD) y trehalosa (otsB). Como consecuencia, los niveles intracelulares de estos solutos aumentaron significativamente, lo que sugiere una respuesta para mantener la homeostasis osmótica. Por otro lado, el pH intracelular disminuyó significativamente de 6.7 a pH 5.5 y el consumo de oxígeno aumentó cuando las células se expusieron al estrés con NaCl. Además, esta condición de estrés llevó a un aumento significativo del contenido intracelular de especies reactivas de oxígeno (ROS), y a un aumento de las actividades antioxidantes citocromo c peroxidasa (CcP) y tiorredoxina (Trx). En concordancia con estos resultados, los genes ccp y trx fueron sobre-expresados bajo esta condición, lo que sugiere que esta bacteria muestra una respuesta regulada transcripcionalmente contra el estrés oxidativo inducido por el cloruro. En paralelo, se adaptó L. ferriphilum a NaCl 180 mM para identificar la estrategia de respuesta tardía. El análisis del perfil transcriptómico reveló que los principales mecanismos involucrados en la adaptación estaban relacionados con los genes asociados a la integridad de la membrana celular, la respiración y las proteínas antioxidantes, probablemente para mantener el pH y la homeostasis redox. El estudio de las células adaptadas demostró un aumento en la tasa respiratoria, sin embargo, el nivel de ROS intracelular se mantuvo en niveles basales. Por el contrario, los genes asociados con la biosíntesis de hidroxiectoína (ectB, ectC, ectD) fueron reprimidos, lo cual coincide con la falta de detección de este compuesto en extractos celulares. Por consiguiente, estos datos indican que las células no se encontrarían expuestas a un estrés osmótico. Finalmente, de acuerdo con estos resultados, se puede concluir que el cloruro tiene un efecto multifacético dramático en la fisiología de acidófilos que involucra estrés osmótico, ácido y oxidativo. El mecanismo de respuesta temprana estuvo compuesto por la respuesta osmótica, la homeostasis de pH a través de la respiración celular y la respuesta antioxidante. En su lugar, el mecanismo de respuesta tardía involucró la homeostasis de pH y la respuesta antioxidante.21151185https://hdl.handle.net/10533/246430instname: Conicytreponame: Repositorio Digital RI2.0info:eu-repo/grantAgreement//21151185info:eu-repo/semantics/dataset/hdl.handle.net/10533/93488info:eu-repo/semantics/openAccessCiencias NaturalesBiotecnología IndustrialCharacterization of NaCl tolerance mechanism and its relation with the antioxidant mechanisms in the acidophilic bacterium Leptospirillum ferriphilum DSM 14647Caracterización del mecanismo de tolerancia a NaCl y su relación con los mecanismos antioxidantes en la bacteria acidófila Leptospirillum ferriphilum DSM 14647info:eu-repo/semantics/doctoralThesisinfo:eu-repo/semantics/publishedVersionTesisTesishttps://hdl.handle.net/10533/2464307990160c-3052-411c-b094-77727a51a614virtual::57918-17990160c-3052-411c-b094-77727a51a614virtual::57918-1ORIGINALTesis Javier Rivera.pdfArticulo principalapplication/pdf4135370https://repositorio.anid.cl/bitstreams/a175f1ca-fd85-4e97-84c5-2d0b5e728374/download9e3a3cd0882b8c98196f2b860524b5f5MD51Tabla S1 .xlsxMaterial suplementarioapplication/vnd.openxmlformats-officedocument.spreadsheetml.sheet19822https://repositorio.anid.cl/bitstreams/0cc5bf27-c907-413d-a460-ab136db102aa/download74a6013a5f598db4fde133444de437b6MD52LICENSElicense.txttext/plain1779https://repositorio.anid.cl/bitstreams/48afbc73-b257-49e4-91f2-b0fbbca4b90f/download593a6e7305c66c56041a9f9e15a649c1MD53TEXTTesis Javier Rivera.pdf.txtExtracted texttext/plain204184https://repositorio.anid.cl/bitstreams/538c9234-b431-4aa7-b75b-dc4939e473e1/download0fa9db78a94dfef8d845c227e2162df0MD54THUMBNAILTesis Javier Rivera.pdf.jpgIM Thumbnailimage/jpeg4441https://repositorio.anid.cl/bitstreams/18cbccd2-d01d-4684-b800-8d59364abdb9/download12071a18d13890e97c7d38f6fbd7a943MD5510533/246430oai:repositorio.anid.cl:10533/2464302023-07-24 08:40:34.265https://repositorio.anid.clRepositorio ANIDaletelier@anid.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