Orientador: Jesus Aparecido Ferro / Coorientador: Marcelo Luiz de Laia / Banca: Agda Paula Facincani / Banca: José Belasque Junior / Banca: Haroldo Alves Pereira Junior / Banca: Eliana Gertrudes de Macedo Lemos / Resumo: O cancro cítrico é uma das principais doenças da cultura do citros, provocando lesões nas folhas, ramos e frutos, tendo como consequência a queda dos frutos e folhas, o que leva à perdas significativas na produção. A partir do sequenciamento completo do genoma da bactéria gram-negativa Xanthomonas citri subsp. citri (Xac), agente causal do cancro cítrico, abriu-se a possibilidade da utilização de estratégias de análise genômica funcional no estudo da função de genes da bactéria relacionados com a infecção na planta e com o desenvolvimento da doença. Uma das estratégias utilizadas foi a obtenção de mutantes de Xac contendo genes relacionados à patogenicidade e virulência interrompidos pelo método de mutagênese insercional aleatória utilizando o transposon Tn5 (LAIA et al., 2009). No presente trabalho a técnica de microarranjos de DNA foi utilizada para avaliar a expressão global de genes de dois mutantes de Xac 72 h após a infecção in planta. Em um dos mutantes (8B7) o gene interrompido foi o xrvA, um regulador de virulência, e no outro mutante (18D6) o gene interrompido codifica uma histidina quinase híbrida sensora que faz parte de um sistema de transdução de sinal de dois componentes. Os resultados das hibridizações revelaram um total de 553 genes diferencialmente expressos para os dois mutantes estudados quando comparado com o genótipo selvagem (Xac 306), sendo 323 no mutante 8B7 e 230 no mutante 18D6. Esses genes foram divididos em diferentes categorias funcionais e uma análise funcional comparativa revelou que eles podem desempenhar um papel importante no processo de patogenicidade / Abstract: Citrus canker is a major disease affecting citrus crops worldwide, causing lesions on leaves, branches and fruits that results in the falling of fruit and leaves, leading to significant losses in orange production. The complete genome sequencing of Xanthomonas citri subsp. citri (Xac), a Gram-negative bacteria and the causal agent of citrus canker, allowed the possibility of using functional genomic strategies to study the function of genes related to plant infection and disease development in this bacteria. One strategy was to produce mutants for phatogenicity and virulence genes by random insertional mutagenesis using Tn5 Transposon (LAIA et al., 2009). In the present work DNA microarray analysis was used to evaluate the global gene expression profile of two Xac mutants after 72 hours of plant infection. One mutant (8B7) carry a mutation in the xrvA gene (XAC1495), a virulence regulator, and the other (18D6) carry a mutation in a hybrid histidine quinase sensor of a two-component signal transduction system. The results revealed a total of 553 differentially expressed genes for the two mutant strains compared with Xac wild type, with 323 in the mutant 8B7, and 230 in the mutant 18D6. These genes were allocated into several functional categories and a comparative functional analysis showed that they can play an important role in the pathogenicity and virulence of Xac / Doutor

No description available.

Bacteria may live in harsh environments where they face changing and new conditions. Therefore, the ability to maintain homeostasis in cells may be vital for survival. Transition metals such as iron, zinc, and copper are essential nutrients for cell survival, but become toxic if in excess amount. In order to survive, bacteria have developed defensive mechanisms to protect themselves. Copper and silver levels need to be carefully maintained within cells to balance cellular needs with potential toxicity. This dissertation focuses on the Cus copper and silver efflux system in E. coli. The E. coli cus system is composed of two divergently transcribed operons, cusCFBA and cusRS. The cusCFBA genes encode for a tripartite metal efflux pump CusCBA and a metallochaperone CusF. The cusRS genes encode a two-component system CusS-CusR that regulates the expression of the cusCFBA genes in response to elevated levels of copper or silver in the periplasm. The histidine kinase CusS senses and binds to metals on its periplasmic sensor domain and transduces signal into the cytoplasm to further communicate with its cognate response regulator CusR through histidyl-aspartyl phosphotransfer event. CusR then outputs cellular response by activating the upregulation of the cusCFBA genes, which then turn on the CusCBA efflux pump to eliminate excess copper or silver in the periplasm. While bacterial two-component systems have been widely studied, the mechanisms of ligand-induced signal transduction by histidine kinases remain unclear. It is now known that cusS is essential for copper and silver resistance, and CusS directly binds metal ions in the periplasmic sensor domain and dimerizes upon metal binding. Thus, the goal of this research is to characterize the metal binding properties in the sensor domain, and to elucidate the signal transduction and autophosphorylation mechanisms of CusS upon metal binding. The data from this work reveal that there are two distinct metal binding sites, interface and internal binding sites, in the sensor domain of CusS, and the interface binding site is functionally more important in metal resistance in E. coli. Furthermore, metal-induced dimerization through the interface metal binding site plays an important role in CusS kinase activity. Together, these findings aid in our understanding of the molecular details in metal binding within the sensor domain of CusS. Based on these data, we propose a model for the signal transduction mechanism and histidine phosphorylation mechanism of the histidine kinase CusS.

Two-component systems (TCS) are the predominant signal transduction pathways in prokaryotes, being present also in eukaryotic organisms, such as algae, fungi and yeast, and higher plants. TCSs play an important role in environmental signal perception and response, essentially implementing adaptation to the surrounding environment. Histidine Kinase 2 (Hik2) in cyanobacteria is a typical sensor histidine kinase, one component of a TCS, and has been identified to be a homologue protein of Arabidopsis Chloroplast Sensor Kinase (CSK). Previous research has elucidated Hik2 to regulate photosynthetic gene transcription with two response regulators, Rre1 and RppA via phosphorylation. A typical histidine kinase contains a variable sensor domain and a conserved kinase domain. It usually functions as a homodimer. This thesis describes the structural characterisation of Hik2, probing particularly its discovered oligomeric states. Results obtained from size exclusion chromatography, native-PAGE, chemical cross-linking analyses and mass spectrometry, amongst others, have shown a variety of Hik2 structural populations exist, further validated by negative stain transmission electron microscopy coupled to single particle analysis. Hik2 protein exists predominantly as a hexamer in low salt conditions, and adding NaCl dissociates hexamers into tetramers, critical for the autophosphorylation activity of Hik2. Thus, a model is proposed for the constitution change of Hik2 oligomers when salt concentration differs. In addition, the sensor domain is typically responsible for detecting environmental input, however, it is not yet clear how Hik2 and CSK sense signals. In this thesis, the structures of Hik2 and CSK sensor domains were analysed and discussed, to aid our understanding of their mechanism of signal perception and transduction.

Made available in DSpace on 2014-06-11T19:32:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-07-08Bitstream added on 2014-06-13T21:03:52Z : No. of bitstreams: 1 souza_ec_dr_jabo.pdf: 821266 bytes, checksum: 46390da0d02b9c37aa41e4af297432aa (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O cancro cítrico é uma das principais doenças da cultura do citros, provocando lesões nas folhas, ramos e frutos, tendo como consequência a queda dos frutos e folhas, o que leva à perdas significativas na produção. A partir do sequenciamento completo do genoma da bactéria gram-negativa Xanthomonas citri subsp. citri (Xac), agente causal do cancro cítrico, abriu-se a possibilidade da utilização de estratégias de análise genômica funcional no estudo da função de genes da bactéria relacionados com a infecção na planta e com o desenvolvimento da doença. Uma das estratégias utilizadas foi a obtenção de mutantes de Xac contendo genes relacionados à patogenicidade e virulência interrompidos pelo método de mutagênese insercional aleatória utilizando o transposon Tn5 (LAIA et al., 2009). No presente trabalho a técnica de microarranjos de DNA foi utilizada para avaliar a expressão global de genes de dois mutantes de Xac 72 h após a infecção in planta. Em um dos mutantes (8B7) o gene interrompido foi o xrvA, um regulador de virulência, e no outro mutante (18D6) o gene interrompido codifica uma histidina quinase híbrida sensora que faz parte de um sistema de transdução de sinal de dois componentes. Os resultados das hibridizações revelaram um total de 553 genes diferencialmente expressos para os dois mutantes estudados quando comparado com o genótipo selvagem (Xac 306), sendo 323 no mutante 8B7 e 230 no mutante 18D6. Esses genes foram divididos em diferentes categorias funcionais e uma análise funcional comparativa revelou que eles podem desempenhar um papel importante no processo de patogenicidade / Citrus canker is a major disease affecting citrus crops worldwide, causing lesions on leaves, branches and fruits that results in the falling of fruit and leaves, leading to significant losses in orange production. The complete genome sequencing of Xanthomonas citri subsp. citri (Xac), a Gram-negative bacteria and the causal agent of citrus canker, allowed the possibility of using functional genomic strategies to study the function of genes related to plant infection and disease development in this bacteria. One strategy was to produce mutants for phatogenicity and virulence genes by random insertional mutagenesis using Tn5 Transposon (LAIA et al., 2009). In the present work DNA microarray analysis was used to evaluate the global gene expression profile of two Xac mutants after 72 hours of plant infection. One mutant (8B7) carry a mutation in the xrvA gene (XAC1495), a virulence regulator, and the other (18D6) carry a mutation in a hybrid histidine quinase sensor of a two-component signal transduction system. The results revealed a total of 553 differentially expressed genes for the two mutant strains compared with Xac wild type, with 323 in the mutant 8B7, and 230 in the mutant 18D6. These genes were allocated into several functional categories and a comparative functional analysis showed that they can play an important role in the pathogenicity and virulence of Xac

No description available.

Several published functions associated with the CHK1 histidine kinase of Candida albicans resemble those of the MAPK Cek1p and its cognate receptor Sho1p (SSU81). To explore this further, we have compared mutants lacking the proteins mentioned above and have constructed a double sho1/chk1Δ null mutant to determine relationships among these proteins. We observed that the sensitivity to Congo red (CR), calcofluor white (CW), as well as clumping of cells, was slightly increased in the double mutant compared to the single chk1Δ or sho1Δ mutants. However, Cek1p phosphorylation via Sho1p, which occurs during log phase growth in the presence or absence of CR in Wt cells, does not require Chk1p. These data suggest that Chk1p and Sho1p are components of parallel but independent signal pathways. In addition, bulk mannan of strains was analyzed by GLC/MS and GPC MALLS and NMR. Compared to Wt and a CHK1 gene-reconstituted strain (CHK23) that contained high, intermediate and low Mw mannan species, we found that the mannan of strains CHK21 (chk1Δ null), the cek1Δ null, and the double mutant consisted only of low Mw mannan. The sho1Δ null mutant only demonstrated a reduced intermediate type of mannan. Alcian blue binding was lower in cek1Δ, chk1Δ, and the double sho1/chk1Δ null mutant lacking high and intermediate Mw mannan than in the sho1Δ null which had a partial loss of intermediate Mw mannan only. We conclude that the Chk1p HK is part of a functionally similar but parallel pathway to the Sho1p-Cek1p pathway that confers resistance to the cell wall inhibitors CR and CW. However, a functional relationship in mannan biosynthesis of Chk1p and Cek1p exists that only partially requires Sho1p.

No description available.

Les systèmes à deux composants jouent un rôle prépondérant dans l'adaptation des bactéries à leur environnement. L'objectif de ce travail de thèse était d'identifier et de caractériser des systèmes à deux composants chez la bactérie phytostimulatrice Azospirillum nécessaires à l'adaptation à la rhizosphère de sa plante-hôte. L'analyse de la distribution génomique des gènes appartenant à la famille des systèmes à deux composants dans les génomes d'Azospirillum disponibles a révélé l'existence d'un grand nombre de gènes codant des hisitidine kinases hybrides, et une analyse plus approfondie a montré une organisation multidomaines complexe de cette famille de protéines. Afin de comprendre leur rôle chez Azospirillum, nous avons, dans un premier temps, sélectionné et inactivé quatre gènes codant des histidine kinases hybrides présentant une architecture multidomaines complexe. A l'aide d'une approche multidisciplinaire combinant génétique, biochimie et phylogénie, nous avons mis en évidence pour la première fois chez Azospirillum, un système atypique à trois-composants nommé PreSKR contrôlant un grand nombre de processus impliqués dans la survie et la colonisation de la rhizosphère, qui agirait en modulant le taux intracellulaire de c-di-GMP. Dans un second temps, nous nous sommes focalisés sur une histidine kinase hybride exprimée au contact de la plante hôte ; cette protéine, appelée RsiK, s'avère être impliquée dans la perception de surfaces et la régulation de la formation de biofilms. L'analyse du régulon par RNA-seq a révèlé que 78 gènes étaient contrôlés par ce système. La prévalence de la famille des histidine kinases hybrides chez Azospirillum couplée à l'approche fonctionnelle réalisée sur deux d'entre elle souligne l'importance des phosphorelais encore largement méconnus chez les bactéries rhizosphériques / Bacterial two-component systems play an important role in the ability of bacteria to adapt to various environments. The aim of this thesis was to identify and characterize two-component systems involved in the adaptation of the phytostimulatory bacteria Azospirillum to its host plant. Analysis of the genomic distribution of genes encoding two-component systems across Azospirillum available genomes revealed the existence of a high number of genes encoding hybrid histidine kinases, and further analyses highlighted a complex multi-domain organization of this family of proteins. In order to understand their role in Azospirillum, as a first step we selected and inactivated four genes encoding complex hybrid histidines kinases. Using a multidisciplinary approach which combines genetics, biochemistry and phylogeny, we brought to light for the first time in Azospirillum, an atypical three-component system named PreSKR which controls a wide variety of processes involved in survival and rhizosphere colonization likely by modulating c-di-GMP levels. As a second step, we focused on a gene encoding a hybrid histidine kinase named RsiK which is induced in contact with its host plant. RsiK is involved in surface sensing and biofilm formation regulation. Transcriptomic analysis of rsiK regulon by RNA-seq showed that 78 genes were under the control of this system. The prevalence of genes encoding hybrid histidine kinase family in Azospirillum, coupled with the functional characterization of two of them, highlight the importance of phosphorelays, still largely unrecognized in rhizospheric bacteria

Les cytokinines sont des hormones régulant de nombreux processus physiologiques. Elles sont en particulier impliquées dans certaines interactions plantes-microorganismes pathogènes. Leur perception est assurée par les récepteurs Histidine Kinases (HK) dont la fonction est prédominante dans la transduction de signaux moléculaires et environnementaux. Ce travail a permis une caractérisation complète des cinq récepteurs aux cytokinines de type CHASE-Histidine Kinase (MdCHK) et d’un osmosenseur (MdHK1) chez le pommier, espèce d’intérêt économique soumise à l’attaque de nombreux pathogènes. La combinaison d’approches moléculaires et bio-informatiques a révélé les particularités fonctionnelles et les rôles spécifiques des MdCHK. Ces connaissances ont permis de sélectionner une souche de levures exprimant le récepteur MdCHK2 en tant que biosenseur de cytokinines. Ce nouvel outil biotechnologique optimisé pour la détection rapide de cytokinines a conduit à la mise en évidence d’une production de ces composés par Erwinia amylovora, bactérie pathogène du pommier. Le criblage de bactéries pathogènes humaines grâce au biosenseur a également révélé leur production par Staphylococcus aureus et Streptococcus agalactiae. Afin d’optimiser ce biosenseur, une approche mécanistique de MdCHK2 a été entreprise et dévoile l’importance de certains domaines dans ses spécificités de perception. / Cytokinins are hormones regulating numerous physiological processes. They are particularly involved in some plant-pathogen interactions. Their perception is ensured by Histidine Kinase (HK) receptors which play a prevailing role in transducing molecular and environmental signals. This work enabled the full characterization of the five cytokinin CHASE-Histidine Kinase receptors (MdCHK) and the osmosensor (MdHK1) in apple tree, a species of economic interest subjected to many pathogen attacks. Combining molecular and bioinformatics approaches led us to reveal the functional features and specific roles of the MdCHK. This knowledge allowed us to select a yeast expressing the MdCHK2 receptor as a cytokinin biosensor. This new biotechnological tool optimized for fast cytokinin detection led us to expose the production of such molecules in Erwinia amylovora, a pathogenic bacterium of apple. Screening human pathogenic bacteria with the biosensor also unveiled their production in Staphylococcus aureus and Streptococcus agalactiae. In order to optimize this biosensor, we initiated a mechanistic approach on MdCHK2 and showed the importance of some domains in its perception specificities.