Les légumineuses sont capables de développer des organes symbiotiques, les nodules, qui hébergent des bactéries du sol appelées rhizobia. Au sein des nodules les rhizobia intracellulaires se différencient en bactéroïdes capables de réduire l'azote atmosphérique en ammonium au bénéfice de la plante. En contrepartie, la plante alimente la bactérie en sources de carbone. Des études récentes sur le modèle symbiotique Medicago/Sinorhizobium ont montré dans les nodules la forte présence d'une grande diversité de peptides appelés NCR qui sont similaires aux peptides antimicrobiens (AMP) impliqués dans l'immunité innée. Ces NCR sont responsables du maintien de l'homéostasie entre les cellules hôtes et la forte population bactérienne qu'elles contiennent. Bien que certains NCR sont de vrais AMP, capable de tuer des bactéries in vitro, dans les nodules ils induisent plutôt une différenciation terminale caractérisée par une élongation cellulaire, une amplification du génome, une perméabilité membranaire et une perte des capacités de division de la bactérie. Néanmoins le mode d'action des NCR reste à élucider. Au cours de ma thèse j'ai participé à la caractérisation des processus de différenciation dans le modèle Aeschynomene, une légumineuse tropicale, Bradyrhizobium.Dans un premier temps, une nouvelle classe de NCR a été identifiée chez différentes espèces d'Aeschynomene. Ces NCR sont responsables de la différenciation des Bradyrhizobium via un processus similaire à celui décrit chez Medicago. Ces résultats suggèrent une évolution convergente des processus de différenciation chez les Dalbergioïdes (Aeschynomene) et le clade des IRLC (Medicago).Ensuite, pour identifier les fonctions bactériennes requises lors de la différenciation, j'ai criblé 53 mutants Tn5 d'Aeschynomene indica fix- . Huit gènes bactériens dont la mutation inhibe ou affecte le processus de différenciation ont été identifiés. Parmi eux, je me suis focalisé sur la DD-CPase une enzyme de modification du peptidoglycane et sur 2 gènes impliqués dans l'homéostasie du phosphate.La caractérisation du gène DD-CPase1 a permis de démontrer que le remodelage du peptidoglycane est requis pour une différenciation correcte des bactéroïdes chez les plantes hôtes qui produisent des NCR, en général, et chez Aeschynomene en particulier. Ces résultats suggèrent une interaction possible entre DD-CPase1 et des NCR conduisant à l'endoréplication des bactéroïdes.Enfin, j'ai étudié les propriétés physiologiques et symbiotiques des mutants pstC et pstB. Les mutants Tn5 des gènes pstC et pstB de la souche ORS285 de Bradyrhizobium sont sévèrement affectés par la carence en phosphate en culture pure et leurs propriétés symbiotiques (différenciation, réduction de l'azote) sont fortement réduites. Des analyses fonctionnelles plus approfondies de l'opéron Pst devraient permettre une meilleure compréhension du lien entre l'homéostasie du phosphate et l'efficience symbiotique dans l'interaction Aeschynomene-Bradyrhizobium.Mes travaux ont permis d'élargir nos connaissances sur l'évolution de la symbiose en montrant que le modus operandi impliquant des peptides dérivés de l'immunité innée utilisée par certaines légumineuses pour maintenir leur population bactérienne intracellulaire sous contrôle est plus répandue et ancienne qu'on ne le pensait et a été utilisée par l'évolution à plusieurs reprises. De plus différentes cibles bactériennes pouvant participer au processus de différenciation ont également été identifiées. / The legume species are able to form symbiotic organs, the nodules, that house soil bacteria called rhizobia. Within these nodules intracellular rhizobia differentiate into bacteroids, which are able to reduce atmospheric dinitrogen to ammonium for the benefit of the plants. In counterpart, the plants provide carbon sources to the bacteria. Recent studies on symbiotic model Medicago-Sinorhizobium showed that the nodules of M. truncatula produce a massive diversity of peptides called NCRs, which are similar to antimicrobial peptides (AMPs) of innate immune systems. These NCRs are responsible in maintaining the homeostasis between the host cells in the nodules and the large bacterial population they contain. Although many NCRs are genuine AMPs, which kill microbes in vitro, in nodule cells they do not kill the bacteria but induce them into the terminally differentiated bacteroids characterized by cell elongation, genome amplification, membrane permeability and loss of cell division capacity. However, the action mode of NCRs is still an open question. During my PhD thesis I focused on the identification of plant and bacterial functions required for bacteroid differentiation in the Aeschynomene-Bradyrhizobium model.Firstly, a new class of cysteine rich peptides (NCR-like) was identified in tropical aquatic legumes of the Aeschynomene genus, which belong to the Dalbergioid clade. These peptides govern terminal bacteroid differentiation of photosynthetic Bradyrhizobium spp. This mechanism is similar to the one previously described in Medicago suggesting that the endosymbiont differentiation in Dalbergioid and ILRC legumes is convergently evolved.Secondly, in order to identify the bacterial functions involved in bacteroid differentiation, I screened 53 fix- Tn5 mutants of the ORS278 strain on Aeschynomene indica. This screening allowed identify 8 bacterial genes, which inhibit or disorder the bacteroid differentiation. Among these identified genes, I focused on DD-CPase encoding a peptidoglycan-modifying enzyme and two genes pstC and pstB belonging to Pst-system.The characterization of DD-CPase gene demonstrated that the remodeling peptidoglycan enzyme, DD-CPase1, of Bradyrhizobium is required for normal bacteroid differentiation in host legumes that produce NCRs, in general, and in Aeschynomene spp., in particular. This prompts a possibility of direct interaction of DD-CPase1 with NCRs leading to endoreduplication of the bacteroids.Finally, I have investigated the physiological and symbiotic properties of different mutants of pstC and pstB genes. The Tn5 mutants of pstC and pstB genes of Bradyrhizobium sp. strain ORS278 severely affected symbiosis on A. indica and A. evenia. Further functional studies on pst-operon will provide deeper understanding the correlation between phosphate homeostasis and nitrogen fixation efficiency in Aeschynomene-Bradyrhizobium symbiosis.This study broadens our knowledge on the evolution of symbiosis by showing that the modus operandi involving peptides derived from innate immunity used by some legumes to keep their intracellular bacterial population under control is more widespread and ancient than previously thought and has been invented by evolution several times.

A habilidade de sensoriar o ambiente extracelular e responder às suas mudanças é inerente para a maioria das bactérias. As concentrações de nutrientes direcionam os processos metabólicos relacionados à sobrevivência e proliferação. O fosfato inorgânico (Pi) é um dos nutrientes cuja regulação, sensoriamento e sinalização são bastante conservados em bactéria. Um dos mecanismos de captação do íon fosfato com alta afinidade é o sistema Pst, um transportador do tipo ABC (ATP-Binding Cassette) , localizado na membrana interna das células. Este transportador, juntamente com as proteínas PhoR/PhoB que formam um sistema de dois componentes (Two-Component Regulatory System) , são capazes de sensoriar e monitorar os níveis deste íon nas células. Ambos os sistemas pertencem ao chamado regulon Pho, conjunto de genes envolvidos no transporte, captação e metabolização do fosfato. Estudos tem mostrado que a interação entre os sistema Pst e o sistema doiscomponentes PhoR/PhoB é mediada pela proteína PhoU, um regulador negativo cujo gene encontra-se no mesmo operon do transportador. Apesar de muito estudados em Escherichia coli , poucas informações existem sobre as características destes sistemas em Xanthomonas citri subsp. citri , bactéria responsável pelo cancro cítrico e de grande importância econômica para o país. Estudos realizados pelo nosso grupo mostraram que X. citri conserva a maioria dos genes descritos como pertencentes ao regulon Pho, incluindo o sistema Pst, as proteínas PhoR/PhoB e PhoU. Este trabalho, portanto, tem como objetivos, a caracterização funcional e estrutural da proteína PhoU de X. citri e a análise da possível interação de PhoU com a proteína PhoR, a histidina quinase do sistema dois componentes. Para tal, as proteínas foram expressas em linhagens de E. coli Tuner e purificadas por cromatografia de afinidade a metal, seguida de exclusão molecular. Visando a caracterização biofísica e estrutural da proteína PhoU, foram realizados ensaios de dicroísmo circular, cristalização, análises de bioinformática e modelagem molecular. Os resultados de bioinformática mostraram que PhoU conserva características estruturais e funcionais quando comparada com ortólogos. Após sua purificação, a proteína foi produzida na sua forma enovelada e mostrou interação com ligantes, conforme descrito na literatura para ortólogos. A expressão da proteína PhoR também foi obtida e ensaios de pull-down foram realizados para a caracterização da interação entre PhoU-PhoR. Adicionalmente, foram realizados estudos de expressão das proteínas em diferentes condições de cultivo, utilizando-se anticorpos policlonais anti-PhoU e anti-PhoR. Os resultados apresentados neste projeto são de grande importância uma vez que se obteve a padronização dos processos de produção de ambas as proteínas e ensaios biofísicos e estruturais para a futura caracterização do complexo, o que será de grande relevância para a compreensão do papel destes sistemas na fisiologia da bactéria. / The ability to sensor the extracellular environment and respond to its changes is inherent to most bacteria. Nutrient concentrations direct metabolic processes related to survival and proliferation. Inorganic phosphate (Pi) is one of the nutrients whose regulation, sensing and signalling are quite preserved in bacteria. One of the mechanisms for phosphate ion uptake with high affinity is the Pst system, composed by an ABC transporter (ATP-Binding Cassette), located on the inner membrane of the cells. This transporter, along with the PhoR/PhoB proteins, which form a Two Component Regulatory System, are capable of sensing and monitoring the levels of phosphate in cells. Both systems belong to the called regulon Pho, set of genes involved in phosphate transport, uptake and metabolism. Studies have shown that the interaction between the Pst system and the two component PhoR/PhoB system is mediated by the PhoU protein, a negative regulator, whose gene is located in the same operon of Pst system. Although much studied in Escherichia coli , there are few information about of these systems in Xanthomonas citri subsp. citri , the major causative of citric canker. Studies conducted by our group showed that X. citri conserves most of the genes described as belonging to regulon Pho, including the Pst system, the proteins PhoR/PhoB and PhoU. This work, therefore, aimed at performing functional and structural characterization of the X. citri PhoU protein and analyzing the possible interaction of PhoU with the PhoR protein, the histidine kinase of the Two Component System. For this, the proteins were expressed in E. coli Tuner strains and purified by metal affinity chromatography, followed by size exclusion chromatography. Aiming at the biophysical and structural characterization of the PhoU protein, we performed circular dichroism, crystallization, bioinformatics and molecular modeling. The results of bioinformatics showed that PhoU retains structural and functional characteristics when compared with orthologs. After purification, the protein was produced in its folded form and showed interaction with ligands, as described in the literature for orthologs. Expression of the PhoR protein was also obtained and Pull Down assays were performed for the characterization of the interaction between PhoUPhoR. In addition, protein expression studies were carried out under different culture conditions using polyclonal anti-PhoU and anti-PhoR antibodies. The results presented in this project are of great importance, once the standardization of the production processes of both proteins has been obtained, as well as biophysical and structural information. These information will be important for future characterization of the complex, which will be of great relevance for the understanding of the role of these systems in the physiology of bacteria.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal