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| Author(s): |
Letícia Khater
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Campinas, SP. |
| Institution: | Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia |
| Defense date: | 2006-10-02 |
| Examining board members: |
Carlos Henrique Inacio Ramos;
Jose Camillo Novello;
Gonçalo Amarante Guimarães Pereira;
Michel Georges Albert Vincentz;
Ana Paula Ulian de Araujo
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| Advisor: | Carlos Henrique Inacio Ramos |
| Abstract | |
The complete sequencing of a genome is a powerful method to investigate the biology of this organism and an automatic gene annotation is a valid tool to an initial investigation of the sequenced genome. Genes are annotated by homology and the global picture provided gives enormous information about the organism phylogeny, genome organization, metabolic pathways, etc. The genome sequence of the phytopathogen Xanthomonas axonopodis pv. citri made open the possibility to understand its biology and its mechanisms of infection, which will help to provide systems of control and treatment of plagues and illnesses. The careful analysis of the annotation is necessary to identify proteins that do not present homology among the genes already sequenced and therefore were labeled as hypothetical. Amongst these, are the proteins that assist the translocation of other proteins, designed secretion chaperones, which were not identified in the phytopathogen Xanthomonas axonopodis. We took to identify such proteins. First, a two-hybrid library was made from a previous set of 40 genes identified as potential secretion chaperones in the Xanthomonas axonopodis genome, which were selected by bioinformatics based on the common characteristics of chaperones from this family. This library was co-transformed with a Xanthomonas axonopodis genomic library allowing the identification of interactions between potential secretion chaperones and other proteins of this bacterium. Second, the most interesting interactions were selected and the cDNA of six proteins were cloned into pET-based plasmids for recombinant production: the hypothetical flagellar chaperone XAC1990 (15046.1) and its substrate FlgK; XAC1977 (6776.1), the hypothetical proteins XACb0033 (10073.2) and XACb0032 (11005.1) that belong to the type four secretion system, the hypothetical protein XACa0025 (10039.1) and the transposase XAC3248 (40041.17). Proteins XACb0032 (11005.1), XACa0025 (10039.1) and the transposase XAC3248 (40041.17) were produced insoluble, impairing their purification. Protein XACb0033 (10073.2) was purified soluble and crystallization trials were set up. The potential flagellar chaperone XAC1990 (15046.1), FlgN, and its substrate FlgK were also purified and characterized. Crystallization trials were set up only for FlgK but not for FlgN because of self-degradation, which happens in within few days. The interaction observed in vivo (by two hybrid assay) was confirmed in vitro (by analytical gel filtration and pull down assays) and the conformational state of the proteins was characterized by spectroscopy experiments (circular dichroism and emission fluorescence of the tryptophan). These experiments lead to the identification of FlgN in Xanthomonas axonopodis pv. citri, and by homology also in Xanthomonas oryzae pv. oryzae, Xanthomonas campestris pv. campestris e Xanthomonas campestris pv. vesicatoria. Soundly, such identification represents an initial step to clarify the biology of flagellar formation in Xanthomonas (AU) | |