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Biodiversidad Bacteriana Marina: Nuevos Taxones Cultivables

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Biodiversidad Bacteriana Marina: Nuevos Taxones Cultivables Departamento de Microbiología y Ecología Colección Española de Cultivos Tipo Doctorado en Biotecnología Biodiversidad bacteriana marina: nuevos taxones cultivables Directores de Tesis David Ruiz Arahal Mª Jesús Pujalte Domarco Mª Carmen Macián Rovira Teresa Lucena Reyes Tesis Doctoral Valencia, 2012 Dr. David Ruiz Arahal , Profesor Titular del Departamento de Microbiología y Ecología de la Universidad de Valencia, Dra. María Jesús Pujalte Domarco , Catedrática del Departamento de Microbiología y Ecología de la Universidad de Valencia, y Dra. Mª Carmen Macián Rovira , Técnico Superior de Investigación de la Colección Española de Cultivos Tipo de la Universidad de Valencia, CERTIFICAN: Que Teresa Lucena Reyes, Licenciada en Ciencias Biológicas por la Universidad de Valencia, ha realizado bajo su dirección el trabajo titulado “Biodiversidad bacteriana marina: nuevos taxones cultivables”, que presenta para optar al grado de Doctor en Ciencias Biológicas por la Universidad de Valencia. Y para que conste, en el cumplimiento de la legislación vigente, firman el presente certificado en Valencia, a. David Ruiz Arahal Mª Jesús Pujalte Domarco Mª Carmen Macián Rovira Relación de publicaciones derivadas de la presente Tesis Doctoral Lucena T , Pascual J, Garay E, Arahal DR, Macián MC, Pujalte MJ (2010) . Haliea mediterranea sp. nov., a marine gammaproteobacterium. Int J Syst Evol Microbiol 60 , 1844-8. Lucena T, Pascual J, Giordano A, Gambacorta A, Garay E, Arahal DR, Macián MC, Pujalte MJ (2010) . Euzebyella saccharophila gen. nov., sp. nov., a marine bacterium of the family Flavobacteriaceae . Int J Syst Evol Microbiol 60 , 2871-6. Lucena T, Ruvira MA, Pascual J, Garay E, Macián MC, Arahal DR, Pujalte MJ (2011) . Photobacterium aphoticum sp. nov., isolated from coastal water. Int J Syst Evol Microbiol 61 , 1579-84. Lucena T, Pujalte MJ, Ruvira MA, Garay E, Macián MC, Arahal DR (2012). Tropicibacter multivorans sp. nov., an aerobic Alphaproteobacterium isolated from surface seawater. Int J Syst Evol Microbiol 62 , 844-848. Lucena T, Ruvira MA, Garay E, Macián MC, Arahal DR, Pujalte MJ (2012). Actibacterium mucosum gen. nov., sp. nov., a new marine Alphaproteobacterium from Mediterranean seawater. Int J Syst Evol Microbiol doi:10.1099/ijs.0.038026-0. Lucena T, Ruvira MA, Arahal DR, Macián MC, Pujalte MJ. Vibrio aestivus sp. nov. and Vibrio quintilis sp. nov., related to Marisflavi and Gazogenes clades, respectively. System Appl Microbiol (En revisión) Ruvira MA, Lucena T, Garay E, Pujalte MJ, Arahal DR, Macián MC. Marinifilum flexuosus sp. nov., a new Bacteroidetes isolated from coastal Mediterranean Sea water and emended description of the genus Marinifilum Na et al., 2009. System Appl Microbiol (En revisión) Agradecimientos Este trabajo de Tesis no se podría haber realizado sin el esfuerzo de muchas personas. Me gustaría agradecerles a todas ellas el apoyo recibido. En primer lugar, a mis directores por transmitirme su amor por la ciencia y en concreto por la microbiología. Por el apoyo personal recibido en todo momento, por ser tan cercanos. Al DR. David R. Arahal, por haber confiado en mí desde el principio. Por transmitirme su entusiasmo y buena praxis. A la Dra. Mª Jesús Pujalte por su valioso conocimiento en bacteriología y por concederme la oportunidad de formar parte de su grupo de investigación. A la Dra. Mª Carmen Macián, por su infinita paciencia y enseñarme a desenvolverme en el laboratorio. Por compartir su mesa conmigo, por entenderme. A todos mis compañeros de la colección Adrián, Ana, Aurora, Bea, Begoña, Chemi, Inma, Jordi, Laura, Mª José, Mapy, María Rosa y Rosa, sin vuestra ayuda el trabajo habría sido más largo y pesado. Gracias por esos almuerzos al sol. Por supuesto, agradecer a nuestra directora Esperanza el haberme acogido en la CECT y haber hecho de la colección lo que es ahora. Sobre todo, agradecer a Amparo ser más que una compañera, por aguantarme y ser mi apoyo diario. Espero ser de la misma utilidad. Por último, a mis compañeros de departamento Alberto, Amparo, David, Eva, Javi y Paco, por sus consejos y estar ahí en las horas muertas. Gracias a todos aquellos que de una manera u otra han hecho posible esta Tesis. ÍNDICE INTRODUCCIÓN .............................................................................................1 1. Taxonomía de procariotas ........................................................................1 1_1. Fuentes de información en taxonomía de procariotas ......................... 5 1_2. Publicación efectiva de propuestas taxonómicas................................. 7 1_3. Papel de las colecciones de cultivo en taxonomía de procariotas ....... 8 1_4. Aproximaciones metodológicas utilizadas en taxonomía de procariotas ........................................................................................... 9 Caracterización fenotípica....................................................................9 Caracterización genotípica.................................................................12 Análisis filogenético basado en secuencias génicas..........................15 Filogenómica......................................................................................20 2. Diversidad bacteriana marina .................................................................23 2_1. Principales grupos bacterianos quimioheterótrofos marinos.............. 24 Filo Proteobacteria .............................................................................24 Filo “ Bacteroidetes ”............................................................................50 Otros filos que albergan taxones marinos..........................................55 OBJETIVOS ..................................................................................................57 MATERIAL Y MÉTODOS..............................................................................59 1. Material Biológico: cepas bacterianas ...................................................59 Cepas de referencia .................................................................................... 61 2. Caracterización fenotípica ......................................................................63 2_1. Características morfológicas celulares y coloniales........................... 63 2_2. Caracterización bioquímica y fisiológica ............................................ 65 2_3. Pruebas nutricionales......................................................................... 70 2_4. Caracterización quimiotaxonómica .................................................... 72 2_5. MALDI-TOF MS ................................................................................. 73 3. Caracterización genotípica .....................................................................74 3_1. Extracción de DNA............................................................................. 74 3_2. Determinación del contenido en Guanina y Citosina (G+C) del DNA 74 3_3. Secuenciación del gen 16S rRNA...................................................... 74 3_4. Secuenciación de otros genes esenciales ......................................... 76 4. Análisis de secuencias ...........................................................................78 5. Secuenciación genómica ........................................................................78 6. Comparación de los genomas parciales ...............................................79 RESULTADOS Y DISCUSIÓN......................................................................81 1. Identificación presuntiva de los aislados ..............................................81 1.1. Cepas de agua de mar de la playa de la Malvarrosa......................... 81 1.2. Cepas de agua de mar y de ostra de la costa de Vinaroz.................. 88 2. Selección de las cepas de interés ..........................................................90 3. Caracterización taxonómica de las cepas seleccionadas ...................92 Estudio de la cepa 7SM29: Haliea mediterranea sp. nov............................ 93 Estudio de la cepa 7SM30 : Euzebyella saccharophyla gen. nov ., sp. nov.100 Estudio de la cepa M46: Photobacterium aphoticum sp. nov. ................... 108 Estudio de la cepa MD5: Tropicibacter multivorans sp. nov. ..................... 120 Estudio de la cepa R46: Actibacterium mucosum gen. nov. sp. nov. ........ 127 Estudio de la cepa M30: Marinifilum flexuosus sp. nov ............................. 137 Estudio de las cepas M22, M61 y M62: Vibrio aestivus sp. nov. y Vibrio quintilis sp. nov.......................................................................................... 147 Estudio de las cepas M17, M49, M76, M15Ø-3, 2OM6 y R37: Phaeomarinomonas mediterranea gen. nov., sp. nov. y Phaeomarinomonas costicola sp. nov....................................................... 160 Estudio de la cepa 4SM10: Roseovarius litoralis sp. nov. ......................... 170 CONCLUSIONES ........................................................................................177 BIBLIOGRAFÍA...........................................................................................179 ANEXO I ......................................................................................................203 ANEXO II .....................................................................................................213 Índice de Tablas Tabla 1. Clasificación jerárquica de los filos Proteobacteria y Bacteroidetes (http://www.bacterio.cict.fr)............................ 36 Tabla 2. Cepas procedentes de la campaña llevada a cabo en la Playa de la Malvarrosa,
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