ABSTRACT Sclerotinia sclerotiorum is a ubiquitous ascomycetous fungal pathogen that causes disease in over 400 crop species, specifically in soybean and canola plants, where stem rot is the most common disease symptom. Pseudomonas chlororaphis PA23 was previously isolated from the rhizosphere of soybean and has demonstrated excellent antifungal activity against S. sclerotiorum in vitro, greenhouse and field experiments. To elucidate the molecular mechanisms involved in PA23 biocontrol, random mutagenesis experiments were initiated. Several mutants were isolated that could be divided into three general classes. Biocontrol activity of various Pseudomonas spp. is highly regulated by a GacS/GacA two-component global regulatory system. Class I PA23 mutants harboured Tn5 insertions in the gacS-coding region, resulting in pleiotropic defects including deficiency in secondary metabolite production and biocontrol activity. Complementation with the wild type gacS allele in trans restored wild type phenotypes. These findings suggest that the ability of P. chlororaphis PA23 to suppress S. sclerotiorum causing stem rot in canola is dependent on a functional GacS/GacA global regulatory system. This is the first study assessing disease symptoms on canola (Brassica napus L.) plants inoculated with a gacS minus strain of P. chlororaphis. Phenazine compounds are considered to be a key secondary metabolite contributing to the antagonistic and antifungal activity of P. chlororaphis. In P. chlororaphis PA23, mutations in phenazine biosynthetic genes exhibited equal or more antifungal activity in vitro, compared to the wild type. To assess the effect of the deficiency in phenazine production, a Class II mutant , harbouring a Tn5 insertion in phzE was tested for a number of biocontrol traits including secondary metabolite production, motility, and suppression of Sclerotinia pathogenic traits. Since no other traits were markedly affected beyond phenazine production, it was concluded that phenazine is not the major product contributing to S. sclerotiorum biocontrol. A single Class III mutant was isolated harbouring a Tn5 insertion in a gene encoding a transcriptional regulator of the LysR family. This mutant exhibited no antifungal activity on plate assays and was unable to protect against S. sclerotiorum in green house assays. A number of secondary metabolites were no longer produced by this mutant, suggesting that this LysR-type transcriptional regulator is either directly or indirectly involved in controlling several genes in P. chlororaphis PA23. / February 2006

ABSTRACT Sclerotinia sclerotiorum is a ubiquitous ascomycetous fungal pathogen that causes disease in over 400 crop species, specifically in soybean and canola plants, where stem rot is the most common disease symptom. Pseudomonas chlororaphis PA23 was previously isolated from the rhizosphere of soybean and has demonstrated excellent antifungal activity against S. sclerotiorum in vitro, greenhouse and field experiments. To elucidate the molecular mechanisms involved in PA23 biocontrol, random mutagenesis experiments were initiated. Several mutants were isolated that could be divided into three general classes. Biocontrol activity of various Pseudomonas spp. is highly regulated by a GacS/GacA two-component global regulatory system. Class I PA23 mutants harboured Tn5 insertions in the gacS-coding region, resulting in pleiotropic defects including deficiency in secondary metabolite production and biocontrol activity. Complementation with the wild type gacS allele in trans restored wild type phenotypes. These findings suggest that the ability of P. chlororaphis PA23 to suppress S. sclerotiorum causing stem rot in canola is dependent on a functional GacS/GacA global regulatory system. This is the first study assessing disease symptoms on canola (Brassica napus L.) plants inoculated with a gacS minus strain of P. chlororaphis. Phenazine compounds are considered to be a key secondary metabolite contributing to the antagonistic and antifungal activity of P. chlororaphis. In P. chlororaphis PA23, mutations in phenazine biosynthetic genes exhibited equal or more antifungal activity in vitro, compared to the wild type. To assess the effect of the deficiency in phenazine production, a Class II mutant , harbouring a Tn5 insertion in phzE was tested for a number of biocontrol traits including secondary metabolite production, motility, and suppression of Sclerotinia pathogenic traits. Since no other traits were markedly affected beyond phenazine production, it was concluded that phenazine is not the major product contributing to S. sclerotiorum biocontrol. A single Class III mutant was isolated harbouring a Tn5 insertion in a gene encoding a transcriptional regulator of the LysR family. This mutant exhibited no antifungal activity on plate assays and was unable to protect against S. sclerotiorum in green house assays. A number of secondary metabolites were no longer produced by this mutant, suggesting that this LysR-type transcriptional regulator is either directly or indirectly involved in controlling several genes in P. chlororaphis PA23.

ABSTRACT Sclerotinia sclerotiorum is a ubiquitous ascomycetous fungal pathogen that causes disease in over 400 crop species, specifically in soybean and canola plants, where stem rot is the most common disease symptom. Pseudomonas chlororaphis PA23 was previously isolated from the rhizosphere of soybean and has demonstrated excellent antifungal activity against S. sclerotiorum in vitro, greenhouse and field experiments. To elucidate the molecular mechanisms involved in PA23 biocontrol, random mutagenesis experiments were initiated. Several mutants were isolated that could be divided into three general classes. Biocontrol activity of various Pseudomonas spp. is highly regulated by a GacS/GacA two-component global regulatory system. Class I PA23 mutants harboured Tn5 insertions in the gacS-coding region, resulting in pleiotropic defects including deficiency in secondary metabolite production and biocontrol activity. Complementation with the wild type gacS allele in trans restored wild type phenotypes. These findings suggest that the ability of P. chlororaphis PA23 to suppress S. sclerotiorum causing stem rot in canola is dependent on a functional GacS/GacA global regulatory system. This is the first study assessing disease symptoms on canola (Brassica napus L.) plants inoculated with a gacS minus strain of P. chlororaphis. Phenazine compounds are considered to be a key secondary metabolite contributing to the antagonistic and antifungal activity of P. chlororaphis. In P. chlororaphis PA23, mutations in phenazine biosynthetic genes exhibited equal or more antifungal activity in vitro, compared to the wild type. To assess the effect of the deficiency in phenazine production, a Class II mutant , harbouring a Tn5 insertion in phzE was tested for a number of biocontrol traits including secondary metabolite production, motility, and suppression of Sclerotinia pathogenic traits. Since no other traits were markedly affected beyond phenazine production, it was concluded that phenazine is not the major product contributing to S. sclerotiorum biocontrol. A single Class III mutant was isolated harbouring a Tn5 insertion in a gene encoding a transcriptional regulator of the LysR family. This mutant exhibited no antifungal activity on plate assays and was unable to protect against S. sclerotiorum in green house assays. A number of secondary metabolites were no longer produced by this mutant, suggesting that this LysR-type transcriptional regulator is either directly or indirectly involved in controlling several genes in P. chlororaphis PA23.

Pseudomonas aeruginosa (Pa) est une bactérie multi-résistante responsable de plus de 10% des infections nosocomiales en Europe. Pa réagit aux signaux environnementaux en activant un système de régulation complexe qui permet à la bactérie d’adopter une mode de vie planctonique (infection aigue) ou sessile (infection chronique caractérisée par la formation des biofilms). Le système à deux composants GacS/GacA joue un rôle central dans la permutation entre ces deux modes. Pendant une infection chronique, GacS HK forme des homodimeres permettant l’activation de la formation du biofilm. Nos travaux ont confirmé le rôle du domaine périplasmique de GacS HK (GacSp) dans la détection du signal et l’activation de la voie GacS/GacA. Malgré l’instabilité de GacSp, J’ai résolu la structure 3D de ce domaine par la spectroscopie RMN. GacSp adopte un repliement PDC qui caractérise un grand nombre de domaines senseurs connus pour fixer une large gamme de ligands. L’étude de la structure m’a permis de proposer un site de fixation du ligand. Les résultats présentés dans cette thèse montrent l’importance de GacSp et propose une nouvelle piste pour les études thérapeutiques visant à éradiquer le biofilm.La deuxième partie de ma thèse présente la structure cristalline du glycoside hydrolase 39 du B. cellulosilyticus WH2 présente dans le côlon (GH39wh2). GH39wh2 partage la même architecture avec les autres membres de la famille 39, cependant, elle possède un site actif plus large et plus exposé. Les analyses structurales et bioinformatiques ont permis d’attribuer GH39wh2 à un nouveau sous-groupe caractérisé par une fonction endoglycosidase inconnue. / Pseudomonas aeruginosa (Pa) is a Multidrug-Resistant bacterium responsible for more than 10% of nosocomial infections in Europe. Interestingly, Pa can switch its lifestyle between planktonic (acute infection) and sessile lifestyle (biofilm-type chronic infection) through different regulatory pathways, in which GacS/GacA Two Component System (TCS) plays a central decisive role. An active homodimer form of GacS Histidine Kinase (HK) leads to biofilm formation, while the inhibition of GacS by the hybrid HK RetS via cytoplasmic hetero-dimerization promotes acute infection. During my thesis, we unveiled for the first time the sensing role of the periplasmic detection domain of GacS HK (GacSp). I further solved the 3D structure of GacSp using NMR spectroscopy revealing its PDC-like fold, which characterizes a large number of detection domains known to bind a wide range of ligands. The structural analyses of the new GacSp structure suggest a conserved ligand-binding pocket. All together, my results present structural and functional understanding of GacSp role, which form the basis to consider this domain as a new target for anti-biofilm therapy.In the second part of my thesis, I report a 2.5Å resolution crystal structure of a family 39 glycoside hydrolase member (GH39wh2) from the human gut bacteria B. cellulosilyticus WH2. GH39wh2 shares a similar architecture as found in other related GH39 members but unveils an atypical shallow solvent-exposed groove. Complementary biochemical and bioinformatics analyses assign GH39wh2 to a new GH39 subgroup harboring a yet unknown endoglycosidase activity.

Two component systems (TCSs) are regulatory pathways in bacteria and lower eukaryotes that integrate multiple stimuli and bring about appropriate responses to promote adaptation of the bacteria to their niches. They are commonly insulated from cross-talk and form discrete regulatory systems where the sensor histidine kinase (SK) and the response regulator (RR) share high fidelity for one another. The GacS network controls the switch between acute and chronic virulence of P. aeruginosa. The network is unusual in having a 'core' SK, GacS, which is modulated directly by one other SK, RetS. Here the complex relationship between GacS and RetS is dissected to reveal three distinct mechanisms by which they interact. Two of these mechanisms involve the dephosphorylation of GacS-P by RetS and it is these mechanisms that are important in vivo for the regulation of biofilm formation, rsmY and rsmZ expression, swarming, and virulence in both Galleria mellonella and an acute model of infection in mice. This study reveals an unprecedented level of complexity in the ability of RetS to interact with GacS and suggests that RetS has a number of mechanisms by which it can downregulate the GacS network output. Furthermore, the interactions of additional SKs that have previously been linked to the GacS network were investigated. Here I demonstrate that many of these kinases can interact with one another but that RetS remained the only kinase tested that could directly interact with GacS. The interactions observed between kinases could be either stimulatory, having a synergistic impact on phosphorylation levels, or inhibitory. I also show that kinase-kinase interactions allow for the regulation of phosphorylation of downstream proteins. Finally, we searched for additional SKs that may be able to interact with the GacS network. Here I identify three new kinases, which show differing interactions with the kinases of the GacS network. The discovery of additional SKs in the GacS network indicates that the network is likely to respond to a far greater number of different signals than previously realised as it decides between acute and chronic virulence.

Part I: Signaling Role of the Sensor Kinase RetS in Biofilm formation Regulation of Pseudomonas aeruginosa-<br />The opportunistic human pathogen Pseudomonas aeruginosa causes both acute and chronic infections in predisposed individuals. Acute infections require a functional Type Three Secretion System (TTSS), which mediates the translocation of select cytotoxins into host cells. Chronic infections, the leading cause of death among cystic fibrosis patients, are characterized by drug-resistant biofilms formation. To regulate gene expression, Pseudomonas aeruginosa utilizes two-component regulatory systems (TCS). Specifically, we focus on the TCS signaling kinase RetS, which is a critical repressor of biofilm formation. The signaling mechanism of RetS is unusual. According to recent findings and one hypothesis, RetS employs a novel signaling mechanism involving direct binding to the signaling kinase GacS, thereby repressing the GacS-induced biofilm formation. RetS is believed to be regulated by the interaction of its periplasmic sensory domain (RetSperi) with an unknown ligand. As such, RetSperi is a potential drug target. We hypothesized that ligand-binding shifts the equilibrium between the formation of a RetS homo-dimer and the RetS-GacS complex by tuning the homo-dimerization of the RetSperi. While the molecular signal that regulates RetS is unknown, our structural studies of the sensory domain suggest that this ligand is a carbohydrate-based moiety. Unchanged biofilm-EPS production phenotype of RetSperi ligand binding site mutants indicates that the natural ligand is not from Pseudomonas aeruginosa.<br />Additional experiments unambiguously determined that the sensory domain forms a stable homodimer. Adding to the complexity of the system, we have identified<br />two possible dimer interfaces in our in vitro assays. However, inconsistent with the current model, elimination of RetSperi results in a slightly increased biofilm EPS production phenotype. Therefore, with the previous demonstration that RetS is able to dephosphorylate GacS, we propose an alternative hypothesis: the RetS kinase domain serves as a phosphatase for phosphorylated GacS; this phosphatase activity is tuned by signaling sensing on RetSperi. Finally, to provide an important piece of information for understanding the molecular basis of RetS-GacS signaling, we have developed a crystallization-based structure determination strategy in order to reveal the precise RetS-GacS interaction pattern.<br /><br />PartII: The catalytic domain of the germination-specific lytic transglycosylase SleB from Bacillus anthracis displays a unique active site topology-<br />germination-specific lytic enzymes (GSLEs) that degrade the unique cortex peptidoglycan to permit resumption of metabolic activity and outgrowth. We report the first crystal structure of the catalytic domain of a GSLE, SleB. The structure revealed a transglycosylase fold with unique active site topology and permitted identification of the catalytic glutamate residue. Moreover, the structure provided insights into the molecular basis for the specificity of the enzyme for muramic-"?lactam-containing cortex peptidoglycan. The protein also contains a metal-binding site that is positioned directly at the entrance of the substrate-binding cleft. / Ph. D.