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The Genus Bacillus, Vol UNIVERSIDAD DE LOS ANDES FACULTAD DE CIENCIAS DEPARTAMENTO DE CIENCIAS BIOLÓGICAS DIVERSIDAD DE Bacillus sphaericus EN DIFERENTES HABITAT y REGIONES GEOGRÁFICAS JENNY DUSSÁN GARZÓN TESIS DOCTORAL BOGOTÁ, JULIO 2006 DIVERSIDAD DE Bacillus sphaericus EN DIFERENTES HABITAT y REGIONES GEOGRAFICAS DISERTACIÓN SOMETIDA A LA ESCUELA DE POSTGRADO DEPARTAMENTO DE CIENCIAS BIOLÓGICAS , FACULTAD DE CIENCIAS COMO REQUISITO PARA OBTENER EL GRADO DE DOCTORA EN CIENCIAS BIOLÓGICAS Biología Molecular JENNY DUSSÁN GARZÓN FACULTAD DE CIENCIAS UNIVERSIDAD DE LOS ANDES BOGOTÁ, JULIO 2006 PALABRAS CLAVES: Bacillus sphaericus, Quercus humboldtii, Quercus robur Colombia, España, Proteinas, Proteina-S, Peptidoglicano, RAPDs, RFLPs, 16S rDNA secuencia. A la memoria de mis padres quienes me enseñaron la constancia por el saber y hacer A la memoria de la doctora Grose quien me dio la libertad de dar vuelo a mi imaginación Amis hijitas Camilita y Juanita fuentes de alegria y amor eterno TABLA DE CONTENIDO RESUMEN ................................................................................................................iv ABSTRACT…………………………………………………………………………viii AGRADECIMIENTOS ……………………………………………………………xii LISTA DE TABLA ………………………………………………………………..xiii LISTA DE FIGURAS ………………………………………………………………xiv. INTRODUCCIÓN ………………………………………………………………......1 I . REVISION LITERATURA Bacillus generalidades contexto organismo modelo ……………........................3 Clasificación y Filogénia ………………………………………...........................4 Pared celular y Proteínas capa-S …………………………………………………9 Ecología del Género Bacillus ………………………………………………………...11 II. MATERIALES Y METODOS Localización de sitios de muestreo y cepas de estudio……………………….14 Aislamiento de bacterias y análisis agrológico de suelo ……………………...18 Sincronización de cultivos : Célula vegetativa y Espora………………………..20 Preparación de peptidoglicano de célula vegetativa y asociado a esporas………………...………………………………………………….……..21 Extracción de proteinas y fracciones celulares …………………………………23 Electroblot y secuencia N-terminal de proteina de la capa - S y toxina 41KD ..................... ……………………………………………………25 Preparación de DNA total………………………………. ....................................25 Polimorfismo intraespecifico RAPD´s………….. ……………………...............26 Polimorfismo intraespecifico gen r16S rRNA…..........……….....……………....27 Análisis de datos....................................................................................................27 Número Acceso Genbank …………………………………………………..........28 III RESULTADOS Y DISCUSION PROTEINAS TOTALES Y FRACCIONES CELULARES EN CELULA VEGETATIVA Y ESPORAS ………………………………………………….29 PROTEINA ENTOMOTOXIGÉNICA DE 41KD Y DE LA CAPA-S…………38 PEPTIDOGLICANO DE CELULA VEGETATIVA Y ESPORA ……………..41 POLIMORFISMO INTRAESPECIFICO : RAPD`s ............................................48 POLIMORFISMO INTRAESPECIFICO GEN 16S rRNA RFLPs.....................57 SECUENCIACION de GEN 16S rRNA ……………………………………….59 Bacillus sphaericus ASOCIADO A Quercus humboldtii y Quercus robur Linneo…………………………………………………………………………….62 DISCUSIÓN 65 CONCLUSIONES 70 BIBLIOGRAFIA 73 RESUMEN DIVERSIDAD DE Bacillus sphaericus EN DIFERENTES HABITAT Y REGIONES GEOGRAFICAS JENNY DUSSAN GARZON1 JUAN AYALA, DIRECTOR2 Departamento de Ciencias Biológicas, Universidad de los Andes. Bogotá, Colombia1. Consejo Superior de Investigaciones Científicas. Universidad Autónoma de Madrid, España2 RESUMEN Bacillus sphaericus ha sido reconocido en cinco grupos de homología con base a la hibridización DNA-DNA. Fundamentalmente, las cepas patógenas se han asociado al grupo IIA y las no patógenas a los grupos I, IIB, III, IV y V. Treinta y dos aislamientos de este taxón de diferentes habitat y regiones geográficas en Colombia y España, fueron evaluados a nivel fenotípico por patrones de perfil de peptidoglicano, perfiles proteicos de fracciones celulares y a nivel molecular por polimorfismos por RAPDs, RFLPs, y secuenciación del gen 16S rRNA Dos clados fueron determinados por secuenciación parcial de 16S DNA en relación a su carácter patógeno y ubicación geográfica con una divergencia superior al 4% Los aislamientos patógenos (6/9) se ubicaron en el clado I relacionados con los grupos de referencia I, IIA y IV. Los aislamientos de España asociados a Quercus robur Linneo (6/9) mostraron una agrupación en el clado II sin relación con grupos de homología. iv Los aislamientos no patógenos de Colombia asociados a Quercus humboldtii se relacionaron tanto en el clado I como el II igualmente sin relación con grupos de homología El perfil de muropeptidos de peptidoglicano tanto en célula vegetativa como en espora revelo un patrón similar entre todos los aislamientos. Una tendencia diferenciar se determino en el peptidoglicano de la célula vegetativa por la ausencia de mas de 10 picos de muropeptidos y un patrón más homogéneo en esporas (ausencia de 3 picos). El fenograma comparativo revelo dos grupos claramente definidos en aislamientos patógenos relacionados con las cepas I, IIA y IV y no patógenos tanto de Colombia como de España relacionados con los grupos IIB y V de homología a un nivel de similitud entre el 20% y 50%. El perfil de proteínas en etapas logarítmica, estacionaria y en los extractos de esporas mostraron tres grupos: Patógenas, no patógenos asociadas a Quercus humboldtii (Colombia) y no patógenos asociados a Quercus robur ( España). Se determino que los aislamientos de Colombia se encuentran asociados al grupo IV de homología y los aislamientos de España al grupo V. Los aislamientos patógenos se asociaron al grupo IIA de homología. De igual forma un perfil homogéneo en todos los aislamientos en relación con las fracciones de membrana y citoplasma fue el patrón establecido en las fracciones individuales. En 36 (de 39 total) cepas, el N-terminal de la proteína de 125 KD dio una identidad del 91% y 100% con la proteína de la capa – S de B. sphaericus. Diferencias en la posición 10 del aminoácido serina por cisteina se determino en las cepas de España con respecto a las de Colombia. v Tres oligonucleotidos de 37 probados fueron seleccionados para el análisis de los patrones de RAPDs. Los productos de amplificación revelaron un patrón de bandas heterogéneo (bandas entre 250 pb y las 1500 pb) en los aislamientos no patógenos tanto de Colombia como de España. En contraste para las cepas patógenas, se evidencio un bandeo homogéneo (bandas entre l00 pb y 900 pb) El Dendograma mostró dos grupo, uno relacionado con el grupo V de homología correspondiente a cepas no patógenas de España y Colombia y un segundo agrupamiento de los patógenos relacionado con el grupo IIA y IV de homología con una disimilitud mayor al 70% Se evidencio por análisis comparativo de los parámetros fenotipicos y genotípicos dos grupos claramente definidos: En el grupo I los aislamientos patógenos relacionados con los grupos de homología I, IIA y IV y en el grupo II, los aislamientos no patógenos de Colombia (subgrupo IIa) y España (subgrupo IIb), estos relacionados con el grupo V de homología El polimorfismo por RFLPs revelo para los 32 aislamientos y los grupos de homología pertenecientes a B.sphaericus patrones homogéneos de restricción correspondientes a lo establecido en el genero Bacillus. Los fragmentos generados por la enzima EcoR1 revelaron bandas fuertes de 850, 450 y 300 pb. Para la enzima Ddel1 se obtuvieron dos productos de digestión de 900 pb y 600 pb. Para la enzima Pst1 no se encontraron puntos de digestión en ninguna de las cepas analizadas En B. globisporus (outgroup) con la enzima Ddel1 no hubo restricción en la secuencia 16S rDNA Una relación ecofisiológica fue establecida en asociación con Quercus humboldtii y Quercus robur Linneo. Se determino un numero alto de bacterias del genero Bacillus y específicamente del taxón B. sphaericus en la rizosfera de los cuatro bosques analizados del genero Quercus. Las concentraciones de iones divalentes de Ca, Mn y Fe mostraron una relación directa con el alto porcentaje de esporas (> 70%). vi La inducción de la esporulación en medio suelo sitio especifico con concentraciones de 0.06% de Ca, 0.01% de Fe y 0.015% de Mn para el pool de Bacillus formulado de cada bosque fue superior al 70% en relación a los grupos de homología seleccionados (I, IIA, IV), no obstante la cepa IV dio valores cercanos a los determinados en los aislamientos nativos de cada bosque. Bacillus globisporus outgroup mostró el menor porcentaje de esporulación en las condiciones establecidas para los tres iones divalentes probados. Los resultados obtenidos soportan la gran heterogeneidad fenotípica y genotípica del complejo taxón de Bacillus sphaericus, sin embargo tanto los grupos de homología como los 32 de estudio se agruparon en relación con su carácter no patógenos separadamente de los patógenos independiente del hábitat y región geográfica. Este es el primer reporte de asociación de Bacillus sphaericus con el genero Quercus robur Linneo. vii ABSTRACT Bacillus sphaericus Diversity in Different Habitats and Geographic Regions. JENNY DUSSAN GARZON1 JUAN AYALA, DIRECTOR2 Departamento de Ciencias Biológicas, Universidad de los Andes. Bogotá, Colombia1. Consejo Superior de Investigaciones Científicas. Universidad Autónoma de Madrid, España2 Bacillus sphaericus has been recognized in five groups of homology based on DNA- DNA hybridization patterns. Mainly, the pathogen strains have been associated with the IIA- group and the non-pathogen strains with the groups I, IIB, III, IV and V. Thirty two isolations of this taxon from different habitats and geographical regions
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