UNIVERSIDADE FEDERAL DE MINAS GERAIS INSTITUTO DE CIcNCIAS BIOLGICAS DEPARTAMENTO DE BIOLOGIA GERAL PROGRAMA DE PS-GRADUAÇIO EM GENTICA Biogeografia e caracteriza o molecular e fisiolgica de bact rias heterotrficas, com nfase em Chromobacterium , dos biomas Mata Atlntica e Cerrado CLÁUDIA IRACEMA LIMA BITTENCOURT ORIENTADORA: Andr a Maria Amaral Nascimento CO-ORIENTADOR: Edmar Chartone de Souza BELO HORIZONTE Maio - 2011 CLÁUDIA IRACEMA LIMA BITTENCOURT Biogeografia e caracteriza o molecular e fisiolgica de bact rias heterotrficas, com nfase em Chromobacterium , dos biomas Mata Atlntica e Cerrado Tese de Doutorado apresentado ao Programa de Ps- Gradua o em Gen tica do Departamento de Biologia Geral do Instituto de Cincias Biolgicas da Universidade Federal de Minas Gerais, como requisito parcial obten o do ttulo de Doutor em Gen tica. ORIENTADORA: Andr a Maria Amaral Nascimento CO-ORIENTADOR: Edmar Chartone de Souza BELO HORIZONTE UFMG M 2011 ii OResilience describes the speed with which a community returns to its former state after it has been perturbed and displaced from that state.P Ecology: from individuals to ecosystems / Michael Begon, Colin R. Townsend, John L. Harper.N4th Ed. iii Dedico esta Tese ao meu pai, minha m e, minha irm zinha e ao meu marido, sem vocs nada disso teria sido possvel. iv AGRADESCIMENTOS H Andr a Amaral, ao Edmar Chartone, meus orientadores. Aos colaboradores Francisco Barbosa, Cludia Teixeira Guimar es (EMBRAPA) e Ubiraci Gomes P. Lana (EMBRAPA) pela grande contribui o neste trabalho. Hs professoras Mnica e Adlane e ao departamento de biologia geral pelo constante compartilhamento do conhecimento. Aos colegas de laboratrio por tornarem as horas mais divertidas, principalmente, ao Alexandre , Patrcia , Mariana, Juliana, Isabela, Flaviane e Magna. Hs Andr a Reis, Maria Rosa e Paix o pela manuten o do laboratrio. Aos colegas, amigos e parentes, que estiveram presentes ao longo desta trajetria, principalmente Carla e Sofia. H minha famlia e ao meu marido, Sueide , por me fazerem acreditar que os obstculos devem ser sempre superados. Ao CNPq pelo apoio financeiro. Hqueles que n o atrapalharam, eu agrade o, pois muito ajudaram. LISTA DE FIGURAS ......................................................................................................... 1 LISTA DE TABELAS ........................................................................................................ 4 LISTA DE ABREVIATURAS ........................................................................................... 5 RESUMO ............................................................................................................................ 8 ABSTRACT ...................................................................................................................... 10 ESTRUTURA DA TESE .................................................................................................. 12 I) INTRODUÇIO GERAL .............................................................................................. 13 Diversidade de Procariotos ........................................................................................... 13 M todos moleculares para acessar diversidade bacteriana ........................................... 15 Conceito de esp cie ....................................................................................................... 16 Biogeografia Microbiana .............................................................................................. 17 Ecossistemas Aquticos de Água Doce ........................................................................ 23 Gnero Chromobacterium ............................................................................................. 28 II) OBJETIVOS ................................................................................................................ 33 Objetivo Geral ............................................................................................................... 33 Objetivos Especficos .................................................................................................... 33 III) CAPTULOS .............................................................................................................. 34 CAPTULO 1 M Comparative biogeography of Chromobacterium from the neotropics . 34 CAPTULO 2 M A survey on cultivable heterotrophic bacteria inhabiting a thermally unstratified water column in Atlantic Forest Lake ............................................................ 51 CAPTULO 3 M Phylogeny of cultivable heterotrophic bacteria derived from mixed colonies ............................................................................................................................. 92 CAPTULO 4 M Characterization of a Chromobacterium haemolyticum population from a natural tropical lake ......................................................................................................... 126 IV) CONCLUSIO GERAL ........................................................................................... 139 V) REFERcNCIAS ......................................................................................................... 140 1 LISTA DE FIGURAS I) INTRODUÇIO GERAL 1 Mapa do Parque Nacional da Serra do Cip...................................................... 24 2 Imagem do Parque Estadual do Rio Doce, pelo sat lite Landsat (Miranda & Coutinho, 2004).................................................................................................. 25 3 Inferncia filogen tica baseada em sequncias do gene de rRNA 16S, mostrando os principais txons bacterianos presentes em ecossistemas de gua..................................................................................................................... 28 4 Anlise filogen tica baseada em sequncias de rRNA 16S depositadas no Genbank com o m todo de minimum evolution (1000 repeti es de Bootstrap)........................................................................................................... 29 CAPTULO 1 1 Sampling site locations along the Indai Stream in the National Park of Serra do CipGGGGGGGGGGGGGGGGGGGGGGGGGGG... 36 2 Cluster analysis of Chromobacterium sp. isolates and of C. violaceum ATCC 12472 according to BIOLOG GN2 microplates profilesGGGGGGGG... 40 3 Cluster analysis of Chromobacterium sp. isolates and of C. violaceum ATCC 12472 according to antimicrobial susceptibility profilesGGGGGGGG... 42 4 Phylogenetic tree based on 16S rRNA gene sequences of all Chromobacterium sp. isolates from water and soilGGGGGGGGGGG 45 5 Principal component analysis ordination plot for the 16S rRNA geneGGG.. 46 6 Cluster analysis of Chromobacterium sp. isolates and of C. violaceum ATCC 12472 according to BOX-PCR fingerprintingGGGGGGGGGGGGG 47 CAPTULO 2 1 Rio Doce State Park and the sampling site location, Carioca LakeGGGG... 81 2 Correlation of the abiotic parameters biplot with the first two axes of principal component analysis (PCA) and distributions of communities sampled in June and AugustGGGGGGGGGGGGGGGGGGG... 82 3 UPGMA cluster analysis of microbial communities based on metabolic diversity obtained through BIOLOG EcoPlates after incubation at 28:C for 2 72 hGGGGGGGGGGGGGGGGGGGGGGGGGGGGG. 83 4 Correlation of the BIOLOG EcoPlates CLPP biplot with the first two axes of principal component analysis (PCA) and distributions of communities sampled in June and AugustGGGGGGGGGGGGGGGGGGG... 84 5 Triplot of Ecoplate CLPP, environmental variables and distributions of communities sampled in June and August along the first two axes produced by the Canonical correspondence analyses (CCA)GGGGGGGGGGG 85 6 Compressed tree of the evolutionary relationships of phylaGGGGGGG.. 86 7 Rarefaction curves for each bacterial community based on the 16S rRNA gene sequencesGGGGGGGGGGGGGGGGGGGGGGGG... 87 8 Relative abundance of phyla in Carioca Lake based on sequence analysis of the 16S rRNA geneGGGGGGGGGGGGGGGGGGGGGGG. 88 9 Relative abundance of genera in Carioca Lake based on sequence analyses of the 16S rRNA geneGGGGGGGGGGGGGGGGGGGGGGG. 89 10 dendrogram, generated by UniFrac, of isolates from 18 bacterial communities from Carioca LakeGGGGGGGGGGGGGGGGGGGGGGG.. 90 11 Principal components analysis ordination plot for the 16S rRNA geneGGG. 91 CAPTULO 3 1 UPGMA cluster environmental analysis, generated by UniFrac, of isolates from the 1%, 10% and 100% euphotic gradientsGGGGGGGGGGG... 113 2 Principal component analysis ordination plot for 16S rRNA gene sequences... 114 3 Media-joining network showing the haplotype distribution into groups according to phylaGGGGGGGGGGGGGGGGGGGGGGG.. 115 S1 Distribution of the number of associated isolates died, identified and unidentified, throughout the euphotic gradientGGGGGGGGGGGG.. 119 S2 Distribution of the number of original colonies that harbored associated isolatesGGGGGGGGGGGGGGGGGGGGGGGGGGGG 120 S3a Phylogenetic tree of isolates from the 1% euphotic gradient based on 16S rRNA gene sequencesGGGGGGGGGGGGGGGGGGGGGG. 121 S3b Phylogenetic tree of isolates from the 1% euphotic gradient based on 16S rRNA gene sequencesGGGGGGGGGGGGGGGGGGGGGG. 122 3 S4 Phylogenetic tree of isolates from the 10% euphotic gradient based on 16S rRNA gene sequencesGGGGGGGGGGGGGGGGGGGGGG. 123 S5 Phylogenetic tree of isolates from the 100% euphotic gradient based on 16S rRNA gene sequencesGGGGGGGGGGGGGGGGGGGGGG. 124 S6 Dendrogram showing the genetic relatedness among associated isolates as determined by (GTG)5-PCR fingerprint analysisGGGGGGGGGGG.. 125 CAPTULO