Stagonospora nodorum is a necrotrophic fungal pathogen that is the causal agent of leaf and glume blotch on wheat. Very little is currently known about the molecular mechanisms required for pathogenicity of S. nodorum, despite its major impact on Australian agriculture. S. nodorum is a polycyclic pathogen. Rain-splashed pycnidiospores attach to and colonise wheat tissue and subsequently sporulate within 2-3 weeks. Several cycles of infection are needed to build up inoculum for the damaging infection of flag leaves and heads, sporulation is therefore a critical component of the infection cycle of S. nodorum; our aim is to determine the genetic and biochemical requirements for sporulation for development of control of the pathogen. Disease progression of S. nodorum on wheat cv. Amery was monitored by light microscopy to determine the time point when pycnidia development began. Early pycnidia development was evident 12 days post-infection. This information was used to guide a genomics and a metabolomics based approach to determine the requirements for sporulation in S. nodorum. The genomics approach utilised two cDNA libraries created from sporulating and non-sporulating cultures. EST frequency was used to determine highly expressed genes under the two developmental states. Gene expression from the most highly represented genes during sporulation were confirmed using quantitative PCR. A gene encoding an arabitol 4-dehydrogenase (Abd1), was mutagenised, in its absence sporulation was reduced by approximately 20%. The metabolomics approach isolated metabolites from both in planta infection and in vitro growth. Rapid changes in the abundance of metabolites were detected during the onset of sporulation. Key fungal metabolites identified include mannitol and trehalose. The concentration of both mannitol and trehalose increased dramatically in concert with pycnidia formation. Both mannitol and trehalose have also been linked to pathogenicity in filamentous fungi. Creation of deletion mutants of the gene encoding trehalose 6-phosphate synthase showed the synthesis of trehalose is required for full sporulation of S. nodorum in planta and in vitro.

Diss. (sammanfattning). Uppsala : Sveriges lantbruksuniv., 2003. / Härtill 4 uppsatser.

Resumo: Objetivou-se o ajuste de metodologia e seleção de genótipos de guandu para resistência a Macrophomina phaseolina a partir de material obtido pela Embrapa Pecuária Sudeste, e verificar o desenvolvimento micelial e esporulação do fungo em meios de cultura. O trabalho foi conduzido em casa de vegetação na UNESP/Jaboticabal no período de agosto de 2004 a dezembro de 2005. Para o ajuste de metodologia e seleção de genótipos resistentes ao fungo as sementes foram submetidas a escarificação com lixa d'água e inoculação artificial através do método de exposição das mesmas ao patógeno por diferentes períodos, que variaram de O a 72 horas. Foram avaliadas porcentagem de plantas sobreviventes e massa fresca. Já para o crescimento micelial e esporulação do fungo foi utilizado o método de sobreposição de discos de diferentes hospedeiros no meio de cultura. A escarificação das sementes contribuiu para a penetração do fungo nas mesmas o período de 24h de exposição das sementes ao fungo são suficientes para detectar diferenças no grau de resistência dos genótipos. Os genótipos mais resistentes são g167-97, g124-95, g27-94, g40-95, g154-95, g127-97 e g9m-97, e os mais suscetíveis são g48-95, g123-95, g8-95, g168-99 e g1m-95. A sobreposição de discos foliares de guandu em meio BDA e folha de papel de filtro em meio sojinha proporcionam um incremento na esporulação de M. phaseolina. / Abstract: This work had the objective of determining the best schedule for artificial inoculation and select pigeon pea genotypes resistant to Macrophomina phaseolina in material obtained by Embrapa Pecuária Sudeste, and verify the mycelial growth and sporulation of the fungi in middle of culture. The work were carried in greenhouse at the UNESP/Jaboticabal, from August 2004 to December 2005. For the methodology and selection adjustment of resistant genotypes to the fungi the seeds were submitted scarified with water sandpaper and artificial inoculation the seeds were the contact method to fungi for different periods, which varied from O to 72 hours. They were evaluated percentage of surviving plants and fresh mass. For the mycelial growth and sporulation of the fungi was used the superposition of disks method of different hosts in the middle of culture. The scarified of the seeds contributed for penetration of the fungi at the seeds; the period of 24h of contact of the seeds to the fungi enough to detect differences in the resistance degree ofthe genotypes. The genotypes g167-97, g124-95, 927-94, g40-95, g154-95, g127-97 and g9m-97 were found to be the most resistant and most susceptible were g48-95, g123-95, g8-95, g168-99 and g1m-95. The treatment with superposition of the leaf disks of pigeon pea in BDA and disks of filter paper in middle of soybean extract were the treatments that provided better sporulation levei in the conditions of that experiment were half. / Orientador: Rita de Cássia Panizzi / Coorientador: Rodolfo Godoy / Banca: Antonio de Goes / Banca: Patrícia Menezes Santos / Mestre

Spore formation is an essential step in the fungal life cycle that contributes to the dispersal of the organism and also to survival under harsh environmental conditions. The morphology of spores shows an astonishing diversity in the fungal kingdom and varies from very simple round and small spores to very complex multi-armed or sigmoid structures. With exception of the regulation of ascospore formation in Saccharomyces cerevisiae and Schizosaccharomyces pombe, which are well-characterized model organisms for spore development in fungi, little is currently known about the regulation of more complex spore morphologies. In this study, the filamentous ascomycete Ashbya gossypii is used as a model system for the investigation of a complex and composite spore morphology. A. gossypii produces linear, needle-shaped spores possessing a length of 30 µm, which can be divided into three major segments: a rigid tip segment, a more fragile membrane compartment and a stable tail-cap. Furthermore, the different compartments were shown to correlate with distinct materials. While the tip segment and the tail-cap of the spores consist of stabilizing materials like chitin and chitosan, these materials are absent from the compartment in the middle. The actin cytoskeleton plays an essential role in several steps of spore formation in A. gossypii. Different regions of actin accumulation were identified that directly correlate with the developing spores. Especially the developing tip segment is characterized by heavy-bundled linear actin structures. Furthermore, proteins of the formin family, a class of actin organizing proteins, were identified to be directly involved in spore formation in A. gossypii. The formin AgBnr2 fulfills an actin-related key function during spore development by linking actin to the spindle pole body during sporulation. Downregulation of AgBNR2 leads to severe sporulation defects, indicating a central function in spore development. Moreover, AgBni1, another representative of the formin family, also has a regulatory function in size determination of the typical needle-shaped spores of A. gossypii. Using a modified yeast two-hybrid approach, four potential activators of the formin AgBni1 were identified: the Rho-type GTPases AgRho1a, AgRho1b, AgRho3 and AgRho4. The interaction of AgBni1 with the two Rho1 GTPases plays an important role during spore development. In this study, the Rho binding domain of AgBni1 was further examined to identify amino acids that are essential for the interaction with the Rho-type GTPases. Using random mutagenesis combined with a two-hybrid screen, the point mutation S250P in the Rho binding domain of AgBni1 was identified to reduce the interaction of the formin with the Rho1 GTPases. Integration of AgBni1 S250P causes an increase in spore length, suggesting a direct effect of this signaling pathway in spore length determination. An actin-regulating protein network that includes the formin AgBni1, the Rho-type GTPases AgRho1a and AgRho1b and the paxillin-like protein AgPxl1 was identified to be mainly involved in the regulation of the spore length. Thereby, this network seems to be involved in the arrangement of the different spore compartments via the actin cytoskeleton.

Nuclear divisions during sporulation of 15 predominantly two-spored and 2 predominantly four-spored yeasts were followed by Giemsa staining and light microscopy. The number of nuclei present per ascus was related to the presence or absence of conjugation at the time of germination of the spores. Plating experiments and a modification of the Finder Slide technique were used to determine whether progeny cells that developed from single spores of 2 two-spored and one four-spored strain could sporulate. The ploidy of these yeasts at different stages of their life cycles was estimated by DNA extractions. The spores of the two-spored yeasts were very difficult to separate following removal of their ascus walls, and electron microscopy was employed in an attempt to account for this. / Thesis / Master of Science (MSc)

Clostridium perfringens is a leading cause of food poisoning annually in the United States. Ingested C. perfringens vegetative cells respond to the acidic conditions of the stomach by initiating sporulation. The process of sporulation is essential in the formation of an enterotoxin (CPE) that is responsible for the symptoms of acute food poisoning. During sporulation, the cell must differentiate into the mother cell and the forespore. Studies in Bacillus subtilis have shown that gene expression during sporulation is compartmentalized, with different genes expressed in the mother cell and the forespore. The cell-specific RNA polymerase sigma factors coordinate the development of the differentiating cell. These sigma factors are Ï F, Ï E, Ï G, and Ï K. The C. perfringens cpe gene, encoding the enterotoxin CPE, is transcribed from three promoters, P1, P2, and P3. P2 and P3 were previously proposed to be Ï E-dependent, and P1 was proposed to be Ï K-dependent based on consensus recognition sequences. In this study, mutations were introduced into the sigE and sigK genes of C. perfringens. In the sigE and sigK mutants, promoter fusion assays indicated that there was no transcription of cpe in either mutant. We also determined through transcriptional analyses that Ï E-associated RNA polymerase and Ï K-associated RNA polymerase co-regulate the transcription of each other. RT-PCR analyses indicated that Ï K is a very early acting sigma factor. The evidence provided here shows that the regulation of sporulation in C. perfringens is not the same as it is in B. subtilis, as previously proposed. / Master of Science

L'azote est connu pour influencer la sensibilité de certaines plantes à diverses maladies. Dans le cas des maladies causées par Botrytis cinerea, le rôle de la fertilisation azotée semble être variable, avec des niveaux élevés favorisant ou réduisant la gravité en fonction des études. Pour vérifier si cette variabilité pourrait être due à des différences possibles entre plantes hôtes, à la pression d'inoculum ou à un comportement différent de différentes souches de l'agent pathogène, des études ont été menées pour évaluer l'effet de différents régimes de fertilisation azotée sur la sensibilité de la tomate et de la laitue à six isolats de B. cinerea. Des effets épidémiologiques éventuels de la fertilisation azotée à travers la sporulation du pathogène et la pathogénicité de l'inoculum secondaire ont également été étudiés sur la tomate. Les plantes ont été cultivées dans un système hors-sol fertirrigué au goutte à goutte. Une nutrition azotée différentielle allant de 0,5 à 30 mM de nitrate a été appliquée pendant les quatre dernières semaines avant l'inoculation des plantes sur des feuilles (laitue) ou sur des plaies d'effeuillage (tomates). Après inoculation, les plantes ont été incubées dans des conditions propices au développement de la maladie. Sur la tomate, l'apparition de la maladie a été retardée et la sévérité globale des symptômes était plus faible pour tous les isolats aux doses de fertilisation azotée les plus élevées, indépendamment de la concentration d'inoculum. Toutefois, le taux d'expansion des lésions sur tige a été affecté différemment selon les souches, diminuant avec des niveaux croissants de fertilisation azotée pour les isolats les plus agressifs, mais augmentant pour les isolats moins agressifs. En contraste avec la tomate, la fertilisation azotée a augmenté la sévérité de la maladie sur la laitue pour tous les isolats testés. La sporulation de B. cinerea sur tomate a diminué significativement avec l'augmentation de la fertilisation azotée des plantes jusqu'à 15-30 mM de nitrate et la pathogénicité des spores a été fortement influencée par l'état nutritionnel de leur substrat de production. Elle était la plus élevée pour les spores produites sur des plantes ayant reçu des niveaux de fertilisation azotée très faibles ou très élevés (0,5 ou 30 mM nitrate) et la plus faible pour celles produites sur des plantes ayant reçu une fertilisation azotée modérée. La fertilisation des plantes a aussi fortement affectée l'efficacité de deux agents de lutte biologique (Trichoderma atroviride et Microdochium dimerum) à protéger les plaies d'effeuillage de la tomate contre B. cinerea. Les plus hauts niveaux de protection ont été obtenus avec la fertilisation azotée élevée et ceci a pu être lié à un retard dans le développement des symptômes sur les tiges, parfois associé à un ralentissement de l'expansion des lésions. Des études histologiques ont montré que la diminution de la gravité de la maladie sous fertilisation azotée élevée a été associée à une altération structurelle des cellules du mycélium de Botrytis. En présence d'un agent de lutte biologique, l'effet de l'agent pathogène a été en outre associé à une vacuolisation, dépôt de glycogène et mort des cellules mycéliennes. Les hypothèses pour expliquer ces résultats sont discutées à la lumière des effets physiologiques possibles de la fertilisation azotée sur la disponibilité des nutriments pour l'agent pathogène dans les tissus de l'hôte et de la production possible de métabolites de défense de la plante. Ces résultats ouvrent de nouvelles perspectives pour manipuler la fertilisation azotée comme un outil pour la protection intégrée des cultures maraîchères / Nitrogen (N) fertilization is known to influence the susceptibility of many plants to a variety of diseases. In the case of diseases caused by Botrytis cinerea, the role of N fertilization appears to be variable, with high levels either fostering or reducing severity depending on the studies. To test whether this variability could be due to possible differences in the host plants, inoculum pressure or in the behavior of different strains of the pathogen, studies were carried out to investigate the effect of different N fertilization regimes on the susceptibility of tomato and lettuce to six isolates of B. cinerea. Possible epidemiological effects of N fertilization through the sporulation of the pathogen and on the pathogenicity of resulting secondary inoculum were also investigated on tomato. Plants were grown in a soil-less drip-irrigation system. Differential N nutrition ranging from 0.5 to 30 mM NO3- was applied for the last four weeks prior to inoculation on the leaves (lettuce) or on leaf pruning wounds (tomato) and incubation of the plants in conditions conducive to disease development. On the tomato stems, disease onset was delayed and overall severity was lower for all isolates on plants with higher N inputs, regardless of inoculum concentration. However, the rate of stem lesion expansion was differentially affected depending on the strains, decreasing with increasing N fertilization levels for the more aggressive isolates, while increasing for the less aggressive isolates.In contrast with tomato, high N fertilization increased disease severity on lettuce for all isolates tested. On tomato plant tissue, sporulation of B. cinerea decreased significantly with increasing N fertilization up to 15-30 mM NO3- and the pathogenicity of the spores was significantly influenced by the nutritional status of their production substrate. It was highest for spores produced on plants with very low or very high N fertilization (0.5 or 30 mM NO3-) and lowest for those from plants with moderate levels of N fertilization. Plant fertilization also strongly affected the efficacy of two biocontrol agents (Trichoderma atroviride and Microdochium dimerum) to protect pruning wounds of tomato against B. cinerea. The highest levels of protection were obtained with high N fertilization and related to a delay in symptom development on the stems, sometimes associated with a slowdown in lesion expansion. Histological studies showed that the decrease in disease severity at high N fertilization was associated to structural alteration of Botrytis mycelial cells. In the presence of a biocontrol agent, the effect on the pathogen was further associated to vacuolisation, glycogen deposition and mycelial cell death. Hypotheses to explain these results are discussed in light of the possible physiological effects of nitrogen fertilization on nutrient availability for the pathogen in the host tissue and of possible production of defense metabolites by the plant. These results also open new possibilities for including the manipulation of N fertilization as a tool for the integrated protection of vegetable crops

Le groupe Bacillus cereus est composé de huit espèces de bactéries à Gram positif sporulantes qui peuvent coloniser plusieurs niches écologiques. Les espèces les plus importantes sont B. cereus, une bactérie ubiquitaire du sol et un pathogène opportuniste; B. thuringiensis, un entomopathogène très utilisé comme biopesticide; et B. anthracis l’agent de la maladie du charbon. Bien que ces espèces présentent différents phénotypes, elles sont étroitement liées génétiquement et leurs facteurs de virulences principaux sont portés par des plasmides. Le cycle infectieux de B. thuringiensis dans la larve d’insecte est régulé par l’activation séquentielle de systèmes de quorum sensing de la famille RNPP. Parmi eux, les systèmes Rap-Phr, caractérisés chez B. subtilis, ont très peu été étudiés dans le groupe B. cereus. Ces systèmes régulent divers processus bactériens importants dont la sporulation. L’objectif de cette étude est d’analyser les systèmes Rap-Phr dans le groupe B. cereus, pour connaitre leur distribution, leur localisation et leur diversité afin d’obtenir une vue globale de ces systèmes chez ces bactéries. De plus, leur possible implication dans la régulation du processus de sporulation a été prédite sur la base de données structurales décrites chez RapH de B. subtilis. Les gènes rap, toujours associés à un gène phr, sont présents dans toutes les souches étudiées avec une moyenne de six gènes rap-phr par souche et avec 30% de ces systèmes qui sont portés par des plasmides. Les souches de B. thuringiensis portent six fois plus de systèmes Rap-Phr plasmidiques que les souches de B. cereus. Par ailleurs, les souches phylogénétiquement proches possèdent un profil de gènes rap-phr similaire. Un tiers des protéines Rap sont prédites pour inhiber la sporulation et ces protéines sont préférentiellement localisées sur les plasmides et donc plus fréquemment présentes chez B. thuringiensis que chez B. cereus. Cette prédiction a été partiellement validée par des tests de sporulation suggérant que les résidus impliqués dans cette activité chez B. subtilis sont conservés mais insuffisants pour prédire cette fonction. Le système Rap63-Phr63 porté par le plasmide pAW63 de la souche B. thuringiensis HD73 a ensuite été caractérisé. La protéine Rap63 a un effet modéré sur la sporulation et retarde l’expression des gènes régulés par Spo0A. La Rap63 est inhibée par son peptide Phr63, dont la forme mature correspond à l’extrémité C-terminale du pro-peptide. Les résultats de sporulation dans l’insecte suggèrent une activité synergique des systèmes Rap63-Phr63 et Rap8-Phr8 (porté par le pHT8_1) dans la régulation de la sporulation. Malgré la similarité entre les Phr63 et Phr8 aucun cross-talk n’a pu être mis en évidence, ce qui confirme la spécificité de ces systèmes de communication cellulaire. L’ensemble de ces résultats démontre la grande diversité des systèmes Rap-Phr dans le groupe B. cereus et souligne l’impact des systèmes plasmidiques dans le développement de ces bactéries. Par conséquent, les plasmides sont des éléments importants pour l’adaptation et la survie de ces bactéries et particulièrement pour B. thuringiensis. / The Bacillus cereus group of Gram positive spore forming bacteria is comprised by eight species that are able to colonize several ecological niches. The most important species are B. cereus, a ubiquitous soil bacterium and an opportunistic pathogen; B. thuringiensis, an entomopathogen widely used as biopesticide; and B. anthracis, the causative agent of anthrax. Even if they present different phenotypes, they are genetic closely related and their main virulence factors are encoded on plasmids. The infectious cycle of B. thuringiensis in the insect larvae is regulated by the sequential activation of quorum sensing systems from the RNPP family. Among them, the Rap-Phr was extensively studied in B. subtilis but just punctually in B. cereus group species. The Rap-Phr systems were shown to regulate various bacterial processes, including the sporulation. The objective of this study was to analyze the Rap-Phr systems in the B. cereus group, regarding their distribution, location and diversity to achieve an overview of these systems in these bacteria. Moreover, their possible involvement in the control of the sporulation process was predicted based on structural data described for RapH in B. subtilis. The rap genes, always associated with a phr gene, were present in all 49 studied strains with an average of six rap-phr genes per strain and 30% were located on plasmids. Comparison among B. cereus and B. thuringiensis strains revealed that the last one harbors six-fold more plasmid rap-phr system then the former. Moreover, phylogenetic closer strains possess a similar profile of rap-phr genes. Interestingly, 32% of the Rap proteins were predicted to inhibit sporulation and these proteins were preferentially located on plasmids and therefore in B. thuringiensis strains. This prediction was partially validated by sporulation efficiency assays suggesting that residues identified in B. subtilis as involved in the phosphatase activity are conserved but not sufficient to predict the sporulation function. Then, the plasmid-borne Rap63-Phr63 system from pAW63 plasmid of B. thuringiensis HD73 strain was further studied. The Rap63 protein moderately inhibits the sporulation and delays the expression of Spo0A-regulated genes. Rap63 is counteracted by its cognate Phr63 peptide, which mature form corresponds to the C-terminal end of the pro-peptide. Sporulation assays in insect larvae suggest a synergistic activity of Rap63-Phr63 and Rap8-Phr8 (from pHT8_1 of B. thuringiensis HD73 strain) systems on sporulation efficiency. Despite the similarities of Phr63 and Phr8 no cross-talk was found between these two systems, confirming their specificity. Altogether, these results reveal the high diversity of the Rap-Phr systems in the B. cereus group and highlight the relevance of the plasmid-borne systems to cell development. Therefore, the results demonstrated the importance of the plasmids in the adaptation and the survival of these bacteria, especially for B. thuringiensis.

Le mécanisme de la motilité de type gliding chez Myxococcus xanthus est longtemps resté incompris, du fait que ce type de déplacement ne requière aucune organelle extracellulaire. Nous avons démontré que le gliding est énergisée par un canal à protons, composé par les protéines AglRQS. Ce moteur coopère avec le cytosquelette d’actine bactérien pour transporter de manière directionnelle le complexe de l’enveloppe Glt à la surface de la cellule. Ce transport est traduit en motilité car les complexes Glt transportés interagissent avec un polysaccharide de surface qui agit comme une colle et immobilise les complexes Glt transportés contre le substrat.Nous avons également fait l’étonnante découverte que le moteur AglRQS est également essentiel à la sporulation, processus cellulaire durant lequel les cellules s’arrondissent et sont recouvertes d’un épais polysaccharide (le spore coat), qui leur confère une résistance face à des conditions défavorables. Nous avons démontré une interaction directe entre le moteur AglRQS et le complexe de l’enveloppe Nfs, un proche homologue du complexe Glt. Nous avons démontré que le moteur AglRQS transporte le complexe Nfs de manière directionnelle autour de la spore. Le spore coat étant sécrété en différents foci autour de la surface de la spore, son transport par la machinerie Agl-Nfs assure la formation d’une couche de « spore coat » compacte autour de la future spore.Ces résultats démontrent l’existence d’un moteur bactérien impliqué dans le transport directionnel de complexes protéiques associés à des sucres. Ces moteurs modulaires pourraient être adaptés à des fonctions spécifiques, en fonction du complexe avec lequel ils interagissent. / How gliding motility on solid surfaces is achieved in Myxococcus xanthus has long remained enigmatic, mostly because movement does not involve obvious extracellular organelles. Recently, we demonstrated that motility in M. xanthus is driven by a proton channel composed by the AglRQS proteins. This motor cooperates with the bacterial actin cytoskeleton to transport an envelope-spanning Glt motility complexes at the cell surface directionally. Motility is produced as a motility machinery surface tip-bound polysaccharide acts like a glue to immobilize the transported Glt complexes against the substratum.In the course of this study, we also made the surprising discovery that the AglRQS motor is essential not only for motility but also for sporulation, a cellular process during which the cells become surrounded by a thick polysaccharide (the spore coat) that confers resistance during unfavourable conditions. We demonstrated a direct interaction between the AglRQS motor and the Nfs envelope complex, a close homolog of the Glt complex. Transmission electron microscopy, time-lapse microscopy and localization studies, showed that the AglRQS motor rotates the Nfs complex directionally around the spore surface. Since the main spore coat polymer is secreted at discrete sites around the spore surface, its transport by the Agl-Nfs machinery ensures the formation of a compact spore coat layer around the future spore.These results highlight the existence of new class of bacterial motors involved in intracellular and directional transport of sugar-associated complex. These modular motors can be adapted to specific functions based which output complex they interact with.

Les bactéries utilisent un mode de communication appelé quorum sensing pour régulerl’expression des gènes en fonction de la densité de population et contrôler ainsi de façonmulticellulaire des processus tels que la sporulation, la compétence ou la virulence. Chez les bactériesà Gram-positif, le quorum sensing repose principalement sur la production, la sécrétion et la détectionde petits peptides de signalisation.Le projet porte sur l’étude du système quorum sensing: NprR/NprX chez Bacillus cereus, oùNprR est l’effecteur qui reconnait spécifiquement le peptide de signalisation NprX. NprR est uneprotéine bi-fonctionnelle. Seule, elle agit en tant qu’inhibiteur de la sporulation, en complexe avecNprX, elle perd sa fonction initiale au profit d’une activité facteur de transcription impliquée dans lavirulence. NprR appartient à une famille d’effecteurs de quorum sensing appelée RNPP (Rap, NprR,PlcR et PrgX) encore mal caractérisée au niveau structural. Mon projet de thèse a consisté en l’analysestructure-fonction du système NprR/NprX.Pour comprendre la régulation fonctionnelle de NprR par NprX, des études en solution (SECMALSet DLS) ont permis de mettre en évidence un switch moléculaire qui repose sur un changementd’oligomérisation. Ainsi NprX fait basculer NprR d’une conformation Apo dimérique à uneconformation compléxée tétramérique.L’étude structurale par cristallographie a aboutit à la résolution de la structure du complexeNprR/NprX. L’analyse de ce tétramère suggère la reconnaissance de 2 sites distincts sur l’ADN.L’étude structurale par SAXS, a quant à elle, permis de proposer une conformation dimérique de laforme Apo NprR, modèle conforté grâce à une étude par mutagénèse dirigée des résidus d’interface. Ils’agit d’un mode de dimérisation semblable à celui des protéines Rap (membres de la famille RNPP).La caractérisation par ITC de l’interaction NprR/NprX avec différentes formes du peptide,ainsi que l’analyse de la poche de fixation du complexe, ont permis de mieux comprendre la spécificitéd’interaction et de mettre en évidence deux résidus clés de l’effecteur : l’Asn275 essentielle à lafixation du peptide et l’Arg 126 essentielle à l’activation de la fonction facteur de transcription.Ces travaux ont contribué à une meilleure compréhension du système quorum sensingNprR/NprX grâce à l’élucidation du switch moléculaire contrôlé par NprX mais aussi à une meilleureconnaissance de la famille d’effecteurs RNPP. / Bacteria use a communication mode named quorum sensing to regulate gene expression depending on the population density and thus to control processes such as sporulation, competence or virulence in a multicellular manner. In Gram-positive bacteria, the quorum sensing relies mostly on the production, the secretion and the detection of small signaling peptides. The project focuses on the study of the quorum sensing system NprR/NprX in Bacillus cereus, where NprR is the effector, which recognizes specifically the signaling peptide NprX. NprR is a bi-functional protein. In the absence of peptide, it acts as a sporulation inhibitor while in complex with NprX, it acts as transcription factor implicated in virulence. NprR belongs to a family of quorum sensing effectors named RNPP (for the first identified members: Rap, NprR, PlcR and PrgX) still not well characterized at a structural level. My PhD project consisted to perform the structure/function analysis of the NprR/NprX system. To understand the functional regulation of NprR by NprX, I carried out different studies in solution (SEC-MALS and DLS). These results allowed me to highlight a molecular switch based on a changing of the oligomerisation state of the protein. NprX binding switches NprR from an Apo dimeric conformation to a tetrameric complex. The structural study by crystallography led to the resolution of the tetrameric NprR/NprX complex structure. The analysis of this tetramer suggests the recognition of 2 DNA binding sites. The structural study of the dimeric conformation of Apo NprR by SAXS, allowed me to propose a model similar to that of the Rap dimers (members of RNPP family). This model is supported by a directed mutagenesis study of interface residues. The characterization by ITC of the NprR/NprX interaction with different forms of the peptide, as well as the analysis of the binding pocket in the complex, led to a better understanding of the specificity of the interaction. Two key residues of the effector were highlighted: Asn275, essential to peptide binding and Arg126, essential to the activation of the transcription factor function. These results have contributed to a better understanding of the NprR/NprX quorum sensing system thanks to the elucidation of the molecular switch controlled by NprX but also in a better knowledge of the RNPP effectors family.