Made available in DSpace on 2014-12-17T14:05:21Z (GMT). No. of bitstreams: 1 FabioTD_TESE.pdf: 3340647 bytes, checksum: f53a49b5ef7773a351040d0690797be2 (MD5) Previous issue date: 2012-03-30 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / The ?-proteobacterium Chromobacterium violaceum is a Gram-negative, free-living, saprophytic and opportunistic pathogen that inhabits tropical and subtropical ecosystems among them, in soil and water of the Amazon. It has great biotechnological potential, and because of this potential, its genome was completely sequenced in 2003. Genome analysis showed that this bacterium has several genes with functions related to the ability to survive under different kinds of environmental stresses. In order to understand the physiological response of C. violaceum under oxidative stress, we applied the tool of shotgun proteomics. Thus, colonies of C. violaceum ATCC 12472 were grown in the presence and absence of 8 mM H2O2 for two hours, total proteins were extracted from bacteria, subjected to SDS-PAGE, stained and hydrolysed. The tryptic peptides generated were subjected to a linear-liquid chromatography (LC) followed by mass spectrometer (LTQ-XL-Orbitrap) to obtain quantitative and qualitative data. A shotgun proteomics allows to compare directly in complex samples, differential expression of proteins and found that in C. Violaceum, 131 proteins are expressed exclusively in the control condition, 177 proteins began to be expressed under oxidative stress and 1175 proteins have expression in both conditions. The results showed that, under the condition of oxidative stress, this bacterium changes its metabolism by increasing the expression of proteins capable of combating oxidative stress and decreasing the expression of proteins related processes bacterial growth and catabolism (transcription, translation, carbon metabolism and fatty acids). A tool with of proteomics as an approach of integrative biology provided an overview of the metabolic pathways involved in the response of C. violaceum to oxidative stress, as well as significantly amplified understanding physiological response to environmental stress. Biochemical and "in silico" assays with the hypothetical ORF CV_0868 found that this is part of an operon. Phylogenetic analysis of superoxide dismutase, protein belonging to the operon also showed that the gene is duplicated in genome of C. violaceum and the second copy was acquired through a horizontal transfer event. Possibly, not only the SOD gene but also all genes comprising this operon were obtained in the same manner. It was concluded that C. violaceum has complex, efficient and versatile mechanisms in oxidative stress response / A ?-proteobact?ria Chromobacterium violaceum ? um bacilo Gram-negativo, de vida livre,sapr?fito e pat?geno oportunista que habita em ecossistemas tropicais e subtropicais dentre eles no solo e na ?gua da Amaz?nia. Possui grande potencial biotecnol?gico e, devido a esse potencial, seu genoma foi totalmente sequenciado em 2003. A an?lise do genoma mostrou que essa bact?ria possui v?rios genes com fun??es relacionadas ? capacidade de sobreviver sob diferentes tipos de estresse ambiental. Objetivando entender a resposta fisiol?gica de C. violaceum sob condi??o de estresse oxidativo, foi aplicada a ferramenta de prote?mica de shotgun. Sendo assim, col?nias de C. violaceum ATCC 12472 foram cultivadas na presen?a e na aus?ncia de 8 mM de H2O2 por duas horas, as prote?nas totais da bact?ria foram extra?das, submetidas ? SDS-PAGE, coradas e hidrolisadas. Os pept?deos tr?pticos gerados foram submetidos a uma cromatografia l?quida (LC)-linear seguido de espectr?metro de massa (LTQ-XL-Orbitrap) para a obten??o de dados quantitativos e qualitativos. A prote?mica de shotgun permite comparar diretamente, em amostras complexas, express?o diferencial de prote?nas e revelou que, em C. violaceum, 131 prote?nas s?o expressas exclusivamente na condi??o controle, 177 prote?nas passaram a ser expressas sob estresse oxidativo e 1175 prote?nas possuem express?o em ambas condi??es. A an?lise dos resultados mostrou que, na condi??o de estresse oxidativo, essa bact?ria muda seu metabolismo, aumentando a express?o de prote?nas capazes de combater o estresse oxidativo e diminuindo a express?o de prote?nas de processos relacionados com o crescimento bacteriano e catabolismo (transcri??o, tradu??o, metabolismo de carbono e de ?cidos graxos). A ferramenta de prote?mica com uma abordagem de biologia integrativa forneceu uma vis?o geral das vias metab?licas envolvidas na resposta de C. violaceum ao estresse oxidativo, bem como amplificou significativamente a compreens?o resposta fisiol?gica ao estresse ambiental. Ensaios bioqu?micos e in silico com a ORF hipot?tica CV_0868 constataram que esta faz parte de um operon. A an?lise filogen?tica de super?xidos dismutase, prote?na pertencente tamb?mb ao operon, mostrou que esse gene est? duplicado no genona de C. violaceum e que a segunda c?pia foi aquirida atrav?s de um evento de transfer?ncia horizontal. Possivelmente, n?o s? o gene sod, mas tamb?m todos os genes que comp?em este operon foram adquiridos da mesma maneira. Concluiu-se que C. violaceum possui mecanismos complexos, eficientes e vers?teis em resposta estresse oxidativo