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Pontificia Universidad Javeriana Facultad De Ciencias Doctorado En Ciencias Biológicas Análisis Comparativo De La Expresión D

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Pontificia Universidad Javeriana Facultad De Ciencias Doctorado En Ciencias Biológicas Análisis Comparativo De La Expresión D PONTIFICIA UNIVERSIDAD JAVERIANA FACULTAD DE CIENCIAS DOCTORADO EN CIENCIAS BIOLÓGICAS ANÁLISIS COMPARATIVO DE LA EXPRESIÓN DE PROTEÍNAS DE Tistlia consotensis EN RESPUESTA A CAMBIOS EN LA SALINIDAD EXTERNA CAROLINA RUBIANO LABRADOR TESIS Presentada como requisito parcial para optar por el titulo de DOCTOR EN CIENCIAS BIOLÓGICAS Bogotá D.C., Colombia Junio 09 de 2014 NOTA DE ADVERTENCIA "La Universidad no se hace responsable por los conceptos emitidos por sus alumnos en sus trabajos de tesis. Solo velará por que no se publique nada contrario al dogma y a la moral católica y por que las tesis no contengan ataques personales contra persona alguna, antes bien se vea en ellas el anhelo de buscar la verdad y la justicia". Artículo 23 de la Resolución No13 de Julio de 1946. ANÁLISIS COMPARATIVO DE LA EXPRESIÓN DE PROTEÍNAS DE Tistlia consotensis EN RESPUESTA A CAMBIOS EN LA SALINIDAD EXTERNA CAROLINA RUBIANO LABRADOR APROBADO: ANÁLISIS COMPARATIVO DE LA EXPRESIÓN DE PROTEÍNAS DE Tistlia consotensis EN RESPUESTA A CAMBIOS EN LA SALINIDAD EXTERNA CAROLINA RUBIANO LABRADOR Concepción Judith Puerta Bula, Ph.D Alba Alicia Trespalacios Rangel, Ph.D Decana Director de Posgrado Facultad de Ciencias Facultad de Ciencias El mundo no es perfecto, y la ley está incompleta. El intercambio equivalente no abarca todo lo que ocurre aquí, pero todavía quiero creer en su principio: Que todas las cosas llegan a un precio, que hay un flujo, un ciclo. Que el dolor que hemos pasado tendrá una recompensa y que cualquiera que sea perseverante obtendrá algo hermoso a cambio, aunque no sea lo esperado AGRADECIMIENTOS A la Unidad de Saneamiento y Biotecnología Ambiental (USBA) de la Pontifica Universidad Javeriana que permitió el desarrollo de este proyecto. A mi directora de tesis Sandra Baena por permitirme formar parte de su grupo de investigación, por la confianza que depositó en mí, por su apoyo incondicional, así como por sus sabios y prudentes consejos durante todo este proceso. A la Vicerrectoría Académica de la Pontificia Universidad Javeriana y al Centro de Genómica de Ambientes Extremos - GeBiX por su apoyo para la financiación, planeación, y ejecución de este proyecto. A el Laboratoire de Biochimie des Systèmes Perturbés (LBSP) del CEA (Commissariat à l’énergie atomique et aux énergies alternatives) que permitió el desarrollo de mi pasantía. A Ricardo Acuña y David Rubio de Cenicafe por sus valiosas orientaciones en el aprendizaje de las técnicas de 2-DE. A Colciencias por su apoyo financiero mediante el programa de créditos condonables para el desarrollo de doctorados nacionales. A mi asesor externo Jean Armengaud por su confianza, motivación y valiosos aportes a este trabajo. A Erica Hartmann, Celine Bland, Damien Joulié, Guylaine Miotello, Jean-Charles Gaillard, y François Allain por su apoyo en los análisis proteómicos y su constante interés en el desarrollo del estudio. A Luisa “La Peluquita” Bernal por su valiosa amistad, por su apoyo, por cada palabra de aliento y por ser la hermanita menor mas paciente e incondicional. A Carito Díaz, Karitol Rodríguez, Javier Gómez, Gina Lopez y Fredy Sánchez por su apoyo en todo momento y por hacer que todos los días fueran más amenos. A Daniel Borda, Johan Saez, Diana Garzón, José Salvador Montana, Andrés Vela, Yady Tovar y Habib Yanine, por su ayuda y colaboración incondicional. A mi hermano Juan Sebastián por su amor, gran ejemplo y apoyo invaluable. A mis padres, María Doris y Alfredo, porque sus esfuerzos y sacrificios permitieron que yo logrará mis sueños, por motivarme y darme la mano cuando sentí que el camino se terminaba. A Demián Felipe por ser la razón de mi vida. RESUMEN En este estudio se evaluó el perfil de expresión de proteínas de Tistlia consotensis bajo condiciones variables de salinidad (0 y 40 g·l-1 NaCl) empleando dos estrategias proteómicas. En la primera estrategia, se utilizó la técnica de electroforesis bidimensional para realizar un análisis cualitativo de la expresión de proteínas de T. consotensis bajo las salinidades evaluadas y posteriormente se realizó un estudio detallado utilizando una estrategia proteogenómica, que consistió en una anotación rápida de los genes cuyos productos se detectaron mediante espectrometría de masas. El análisis cualitativo de la expresión de proteínas de T. consotensis en respuesta a cambios en la salinidad del medio permitió determinar que esta bacteria halotolerante responde al estrés osmótico con cambios en los niveles de expresión de sus proteínas. Asimismo, el análisis del exoproteoma y del proteoma celular de T. consotensis, utilizando el enfoque proteogenómico, permitió la detección de proteínas específicas a 0 y 40 g·l-1 NaCl involucradas en los mecanismos de adaptación de T. consotensis bajo estrés hipo e hiper- osmótico a través de la comparación con el perfil de expresión en la condición de crecimiento a la salinidad óptima, 5 g·l-1 NaCl. En ausencia de sal, T. consotensis aumenta la producción de proteínas de estrés general y proteínas implicadas en la modulación de la composición de membrana celular. En respuesta a un incremento de la salinidad, T. consotensis aumenta la abundancia de proteínas involucradas con procesos de señalización, responsables de detectar eficientemente cambios en la concentración de Cl-, y proteínas relacionadas con diferentes tipo de sistemas de transporte encargados de acumular solutos compatibles y mantener el exceso de Na+ fuera de la célula. Este estudio proporcionó una visión detallada de la adaptación celular, en términos de perfiles proteómicos, a cambios en la salinidad externa en T. consotensis, sugiriendo que esta bacteria halotolerante combina diferentes estrategias novedosas para contrarrestar los cambios de salinidad en su hábitat. ABSTRACT In this study the protein expression profile of Tistlia consotensis under different salinities (0 to 40 g·l-1 NaCl) were evaluated using two proteomic strategies. In the first strategy, two- dimensional electrophoresis technique was used to perform a qualitative analysis of protein expression of T. consotensis under salinities evaluated and subsequently a detailed study was performed using proteogenomics strategy, which consisted of a quick annotation of genes whose products were detected by mass spectrometry. Qualitative analysis of protein expression of T. consotensis in response to changes in the salinity allowed determining that this halotolerant bacterium respond to osmotic stress with changes in the expression levels of proteins. Moreover, analysis of exoproteome and proteome cellular of T. consotensis, using the proteogenómico approach, allowed the detection of specific proteins to 0 and 40 g·l -1 NaCl involved in adaptation mechanisms of T. consotensis under hypo and hyperosmotic stress by comparison with the expression profile in the condition of the optimal growth salinity, 5 g·l-1 NaCl. In the absence of salt, T. consotensis increases the production of proteins of general stress and proteins involved in the modulation of cell membrane composition. In response to an increase of salinity, T. consotensis increases the abundance of proteins involved in signaling processes, responsible for efficiently detect changes in the concentration of Cl-, and proteins related to different types of transport systems responsible for accumulating compatible solutes and maintaining excess Na+ out of the cell. This study provided a detailed overview of cell adaptation, in terms of proteomic profiles, to changes in the external salinity in T. consotensis, suggesting that this halotolerant bacterium combines different innovative strategies to counteract changes in salinity in their habitat. Tabla de contenido ÍNDICE DE FIGURAS ...................................................................................................................... 2 ÍNDICE DE ANEXOS ....................................................................................................................... 4 1. INTRODUCCIÓN ......................................................................................................................... 5 2. MARCO TEÓRICO ...................................................................................................................... 8 2.1. AMBIENTES SALINOS COMO HÁBITATS DE COMUNIDADES MICROBIANAS ... 8 2.1.2. Ambientes salinos de origen terrestre ...................................................................... 10 2.2. DIVERSIDAD MICROBIANA DE AMBIENTES SALINOS ........................................... 11 2.2.1. Dominio Archaea ......................................................................................................... 13 2.2.2. Dominio Bacteria ......................................................................................................... 14 2.2.3. Dominio Eucaria .......................................................................................................... 18 2.3. DIVERSIDAD MICROBIANA DE AMBIENTES SALINOS COLOMBIANOS ............ 18 2.4. Tistlia consotensis .............................................................................................................. 20 2.5. CARACTERÍSTICAS MOLECULARES RELACIONADAS CON LA ADAPTACIÓN A AMBIENTES SALINOS ............................................................................................................. 21 2.5.1. Características moleculares a nivel de ADN ........................................................... 21 2.5.2. Características moleculares a nivel de proteínas ................................................... 25 2.5.3. Proteínas
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