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Diversidad Filogenómica Y Funcional De Procariotas De Suelos Hipersalinos Tesis Doctoral

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Diversidad Filogenómica Y Funcional De Procariotas De Suelos Hipersalinos Tesis Doctoral Departamento de Microbiologí a y Parasitologí a Facultad de Farmacia Universidad de Sevilla Diversidad filogenómica y funcional de procariotas de suelos hipersalinos Tesis Doctoral Blanca Vera Gargallo Sevilla, 2018 FACULTAD DE FARMACIA Departamento de Microbiologí a y Parasitologí a Memoria que presenta la Lcda. en Farmacia Blanca Vera Gargallo para optar al grado de Doctor por la Universidad de Sevilla La doctoranda: Fdo.: Blanca Vera Gargallo FACULTAD DE FARMACIA Departamento de Microbiologí a y Parasitologí a ANTONIO VENTOSA UCERO, CATEDRÁTICO DE UNIVERSIDAD EN EL DEPARTAMENTO DE MICROBIOLOGÍA Y PARASITOLOGÍA DE LA FACULTAD DE FARMACIA DE LA UNIVERSIDAD DE SEVILLA, CERTIFICA: Que la Tesis Doctoral titulada “Diversidad filogenómica y funcional de procariotas de suelos hipersalinos” presentada por la Licenciada en Farmacia Dª BLANCA VERA GARGALLO para optar al grado de Doctor ha sido realizada en el Departamento de Microbiología y Parasitología de la Facultad de Farmacia de la Universidad de Sevilla, reuniendo los requisitos exigidos, bajo mi dirección. Y para que así conste, se expide el presente certificado en Sevilla, a 15 de junio de 2018. Fdo.: Antonio Ventosa Ucero FACULTAD DE FARMACIA Departamento de Microbiologí a y Parasitologí a MIGUEL ÁNGEL CAVIEDES FORMENTO, PROFESOR TITULAR Y DIRECTOR DEL DEPARTAMENTO DE MICROBIOLOGÍA Y PARASITOLOGÍA DE LA FACULTAD DE FARMACIA DE LA UNIVERSIDAD DE SEVILLA, CERTIFICA: Que la Tesis Doctoral titulada “Diversidad filogenómica y funcional de procariotas de suelos hipersalinos” presentada por la Licenciada en Farmacia Dª BLANCA VERA GARGALLO para optar al grado de Doctor ha sido realizada en el Departamento de Microbiología y Parasitología de la Facultad de Farmacia de la Universidad de Sevilla, reuniendo los requisitos exigidos, bajo la dirección del Doctor D. Antonio Ventosa Ucero. Y para que así conste, se expide el presente certificado en Sevilla, a 15 de junio de 2018. Fdo.: Miguel Ángel Caviedes Formento La realización de esta Tesis Doctoral ha sido financiada mediante los siguientes proyectos de investigación: “Biodiversidad microbiana de suelos salinos: una aproximación molecular y metagenómica” (P10-CVI-6226) subvencionado por la Junta de Andalucía durante el período 2012-2016. “De la metagenómica al cultivo puro: Spiribacter salinus” (CGL2013-46941-P), subvencionado por el Ministerio de Ciencia e Innovación durante el período 2014-2016. “Microbiomas de ambientes hipersalinos: suelos versus salinas” (CGL2017-83385-P), financiado por el Ministerio de Economía y Competitividad durante el período 2018-2020. Parte de los resultados de esta Tesis Doctoral han sido publicados en los siguientes artículos científicos: Vera-Gargallo, B., Navarro-Sampedro, L., Carballo, M., Ventosa, A. (2018). Metagenome sequencing of prokaryotic microbiota from two hypersaline soils of the Odiel Salt Marshes in Huelva, Southwestern Spain. Genome Announcemenents. 6: e00140-18. Vera-Gargallo, B., Ventosa, A. (2018). Metagenomic insights into the phylogenetic and metabolic diversity of the prokaryotic community dwelling in hypersaline soils from the Odiel Saltmarshes (SW Spain). Genes. 9:E15 Vera-Gargallo, B., RoyChowdhury, T., Brown, J., Fansler, SJ, Durán-Viseras, A., Sánchez-Porro C., Bailey, V.L., Jansson J.K., Ventosa, A. Factors governing the spatial distribution of prokaryotic communities in hypersaline soils. Enviado a Scientific Reports. Vera-Gargallo, B., Dumont, M.G., Hernández. M.T., Ventosa, A. Light shapes prokaryotic community structure in hipersaline soils. En preparación. Y en las comunicaciones a congresos: Vera-Gargallo, B., Ventosa, A. Insights into the phylogenetic and metabolic diversity of the prokaryotic community dwelling in hypersaline soils. ASM, 2018. Comunicación en panel. Vera-Gargallo, B., Ventosa, A. Diversidad procariota de suelos hipersalinos de las Marismas del Odiel. REDEX, 2018. Comunicación oral. Vera-Gargallo, B., Sánchez-Porro, C. y Ventosa, A. Characterization of the microbial community form hypersaline soils and its metabolic potential. FEMS-SEM, 2017. Comunicación en panel. Vera-Gargallo, B., RoyChowdhury, T., Brown, J., Sánchez-Porro, C., Bailey, V., Jansson, J.K. y Ventosa, A. Factors influencing the microbial community structure of saline soils from the Odiel Saltmarshes. FEMS-SEM, 2017. Comunicación en panel. Vera-Gargallo, B., Sánchez-Porro, C. y Ventosa, A. Prokaryotic community of hypersaline soils from Odiel saltmarshes unveiled through metagenomics. 3rd Thünen Symposium on Soil Metagenomics, 2016. Comunicación en panel. Vera-Gargallo, B., Sánchez-Porro, C. y Ventosa, A. Insights into functional diversity of hypersaline soils. 3rd Thünen Symposium on Soil Metagenomics, 2016. Comunicación en pan Vera-Gargallo, B., Sánchez-Porro, C. y Ventosa, A. Metabolic diversity of hypersaline environments. XI Reunión del Grupo Especializado de Microbiología Molecular, 2016. Comunicación en panel. Vera-Gargallo B, de la Haba RR, Sánchez-Porro C y Ventosa A. New microbial taxon from hypersaline soils unveiled through metagenomics. 11th International Meeting Halophiles, 2016. Comunicación en panel. Vera-Gargallo B, Sánchez-Porro C y Ventosa A. Análisis metagenómico de un suelo salino del Parque Natural Marismas del Odiel. XXV Congreso Nacional de Microbiología, 2015. Comunicación oral. ÍNDICE I. INTRODUCCIÓN ........................................................................................................... 1 I.1 AMBIENTES EXTREMOS Y LA VIDA EN ELLOS......................................................................... 1 I.2 LA SALINIDAD COMO FACTOR LIMITANTE PARA LA VIDA ........................................................ 5 I.3 AMBIENTES HIPERSALINOS Y MICROORGANISMOS HALÓFILOS .............................................. 6 I.3.1 Tipos de ambientes hipersalinos ............................................................................... 6 I.3.2 Microorganismos halófilos ........................................................................................ 13 I.4 ESTUDIO DEL SUELO COMO SISTEMA BIOLÓGICO ................................................................ 39 I.4.1 Particularidades del suelo como hábitat ................................................................ 39 I.4.2 Aproximaciones ómicas al estudio de la ecología microbiana de suelos ........... 40 II. OBJETIVOS .................................................................................................................. 45 III. MATERIALES Y MÉTODOS ........................................................................................ 47 III.1 MUESTREOS Y ANÁLISIS FISICOQUÍMICO DE LAS MUESTRAS ............................................... 47 III.1.1 Toma de muestras ................................................................................................. 47 III.1.2 Análisis fisicoquímicos de las muestras ............................................................. 48 18 III.2 MARCAJE CON ISÓTOPOS ESTABLES (H2O ) ..................................................................... 49 III.2.1 Tratamiento previo de las muestras .................................................................... 49 18 III.2.2 Diseño del experimento, marcaje con H2O e incubación ............................... 49 III.2.3 Centrifugación isopícnica y separación/fraccionamiento de gradientes ....... 49 III.2.4 qPCR en tiempo real .............................................................................................. 50 III.3 EXTRACCIÓN Y PURIFICACIÓN DE ADN METAGENÓMICO .................................................... 52 III.4 DETERMINACIÓN DE LA INTEGRIDAD, CONCENTRACIÓN Y PUREZA DEL ADN ...................... 52 III.4.1 Electroforesis en gel de agarosa .......................................................................... 52 III.4.2 Espectrofotometría ................................................................................................ 53 III.4.3 Fluorimetría ............................................................................................................. 54 III.5 SECUENCIACIÓN DEL ADN METAGENÓMICO Y LIBRERÍAS DEL GEN ARNR 16S .................. 54 i III.6 BASES DE DATOS ............................................................................................................... 54 III.6.1 Metagenomas ......................................................................................................... 54 III.6.2 Bases de datos de secuencias ............................................................................. 55 III.7 ANÁLISIS BIOINFORMÁTICO DE LAS SECUENCIAS METAGENÓMICAS .................................... 57 III.7.1 Control de calidad de las secuencias .................................................................. 57 III.7.2 Contenido en guanina más citosina (G + C) del ADN ...................................... 57 III.7.3 Frecuencia de aminoácidos .................................................................................. 57 III.7.4 Punto isoeléctrico .................................................................................................. 58 III.7.5 Anotación de secuencias metagenómicas ......................................................... 58 III.7.6 Ensamblado de secuencias metagenómicas ..................................................... 59 III.7.7 Extracción de genomas a partir de metagenomas ............................................ 61 III.7.8 Estimación de la posición filogenética de los genomas extraídos .................. 62 III.7.9 Anotación de los genomas extraídos ..................................................................
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