Pantoea stewartii subsp. stewarti is the causative agent of Stewart's wilt disease in maize. Disease symptoms develop after the bacteria grow to high cell densities in the plant xylem and secrete an abundance of exopolysaccharide (EPS). EPS production is regulated by quorum sensing. Two regulatory proteins are key to the process of quorum sensing, the LuxI and LuxR homologues EsaI and EsaR. Most LuxR homologues function as activators of transcription in the presence of their cognate acylated homoserine lactone signal (AHL). EsaR utilizes an AHL-response opposite of the majority of the LuxR homologues. EsaR represses EPS production at low cell densities. However, at high cell densities when high concentrations of AHL are present, EsaR is inactivated and derepression of EPS production occurs. The mechanism that enables EsaR to respond to AHL in a manner opposite to that of most LuxR homologues remains elusive. A comparative study of EsaR and the well characterized quorum-sensing regulators LuxR from Vibrio fischeri and TraR from Agrobacterium tumefaciens was initiated. Previous studies demonstrated that in the absence of AHL, EsaR retains the ability to function as a weak activator of the lux operon in recombinant Escherichia coli. This thesis research further characterized the role of EsaR as an activator. Variant forms of EsaR with deletions or single residue substitutions were generated and their ability to regulate transcription was examined in vivo. Furthermore, a native EsaR-activated promoter has been identified, which controls expression of a putative regulatory sRNA in P. stewartii. It is apparent that EsaR functions as a transcription factor at low concentrations of AHL as demonstrated by its ability to inhibit EPS production. At high concentrations, the AHL appears to bind and cause a conformational shift in the protein leading to its inactivation. The second goal of this study was to further elucidate the mechanism by which AHL regulates EsaR. Pulse-chase experiments demonstrated that EsaR is resistant to proteases with or without AHL in vivo. Limited proteolytic digestions in vitro suggest that the protein does undergo conformational changes in response to AHL. Gel filtration chromatography, sucrose gradient ultracentrifugation, and cross-linking experiments proved that this conformational change does not impact the multimeric state of EsaR. To better understand the mechanism of regulation by AHL, the final goal of this project was to examine the interactions which result in EsaR-responsiveness to AHL. Several individual amino acid substitutions were identified that cause EsaR to function in an AHL-independent manner, by which variants retain the ability to bind and block gene expression in the presence of AHL. These residues have been mapped onto a homology model of EsaR and their role has been examined in vitro. The ability of these EsaR* variants to bind AHL and an analysis of the effects individual mutations have on the overall conformation of the protein was performed. Overall this study has revealed several unique aspects of the quorum-sensing system in P. stewartii whereby gene expression is regulated at both low and high cell density. Studies were also initiated to examine the mechanism of AHL-responsiveness of EsaR. The mechanism by which AHL modulates most LuxR homologues remains elusive. The ability to purify EsaR +/- its cognate AHL may prove critical in elucidating this mechanism. / Ph. D.

The LuxR protein family is a class of quorum-sensing regulated bacterial transcription factors that alter gene expression as a function of ligand detection. This coincides with a high population density and/or a low rate of signal ligand diffusion. The majority of LuxR proteins are activated only in the presence of the signal ligand, an acyl-homoserine lactone (AHL). EsaR, from the corn pathogen Pantoea stewartii, represents a subset of LuxR homologues that are active in the absence of AHL and deactivated by its presence. The mechanism by which EsaR responds to AHL in a manner opposite to that of the majority of LuxR homologues remains elusive. Unlike the majority of LuxR homologues, which require AHL for purification, EsaR can be purified and biochemically investigated in the absence and presence of AHL. This work sought to answer questions regarding the structure-function relationship of the LuxR homologue, EsaR. Fluorescence anisotropy was used to determine the relative DNA-binding affinity of wild type EsaR and three AHL-independent EsaR variants in the presence and absence of AHL. This enabled for quantitative analysis of the relative binding affinities of these AHL-independent variants for the EsaR binding site, the esa box. The results demonstrate that one AHL-independent EsaR variant has a slightly higher affinity for the esa box in the presence, rather than the absence of AHL. The affinity of the other two for the DNA is not impacted by AHL, potentially due to an inability to transduce the signal of ligand detection to the DNA binding domain. Constructs containing only the EsaR N-terminal domain (NTD) were also developed. These constructs circumvented solubility issues associated with the full-length protein, allowing for additional biochemical analysis. It was determined that the EsaR NTD alone is sufficient for multimerization and ligand binding. Additionally, preliminary X-ray crystallography efforts have established some of the early parameters required to solve the crystal structure of the EsaR ligand binding domain in both the presence and absence of AHL. If pursued, these structures would be the first solved of a LuxR homologue ligand binding domain in both the presence and absence of the native AHL, potentially demonstrating the conformational change that occurs as a result of ligand binding. Collectively, these findings have established some of the groundwork required to resolve the question of what sort of conformational changes occur in EsaR as a result of ligand binding. / Master of Science

The LuxR family of proteins serves as quorum-sensing transcriptional regulators in proteobacteria. At high population densities, a small acyl-homoserine lactone (AHL) molecule, produced by a LuxI homologue, accumulates in the environment. The LuxR proteins bind to their respective AHL when the ligand accumulates to sufficient levels. Once bound to AHL, the holoproteins usually become functional as transcriptional activators. However, there is a subset of LuxR homologues, the EsaR subfamily, which is active without the AHL ligand and becomes inactivated once bound to it. EsaR is the best understood member of this subfamily. It controls virulence in the corn pathogen Pantoea stewartii ssp. stewartii. Solubility issues have previously limited structural studies of LuxR homologues as the proteins could not be purified without the AHL ligand. A soluble recombinant EsaR protein, HMGE, is biologically active and can be purified in the absence and presence of AHL, unlike most other LuxR homologues. Using HMGE, amino acid substitutions and Förster resonance energy transfer (FRET), experimental methods were designed for determining the dimerization interface of EsaR and for testing the hypothesis that EsaR undergoes a conformational shift when presented with the AHL ligand. To identify residues of the dimerization interface, heterodimerization assays were designed, involving either coexpression or coincubation of wild-type EsaR and variant HMGE proteins. In this assay, the inability of the proteins to copurify by nickel affinity chromatography would indicate that the modified residue(s) are important for dimerization of EsaR. To determine the conformational change that EsaR undergoes when bound to the AHL ligand, a FRET assay was developed to estimate the distances between amino acid residues in the absence and presence of AHL. Future work will have to include a few modifications to the methods and/or control experiments. This study provides the basis upon which the present methods can be further developed and later used for structural studies of EsaR. / Master of Science

Pantoea stewartii subsp. stewartii is the causative agent of Stewart's wilt disease in maize plants. The bacteria are injected into the plant by corn flea beetles during feeding. They colonize the xylem and overproduce a capsular exopolysaccharide (EPS) at high cell densities. The production of EPS is regulated by an EsaI/EsaR quorum sensing mechanism, homologous to the LuxI/R system. Although activation of the EPS encoding genes by EsaR occurs after it complexes to the AHL (3-oxo-C6-HSL), unlike the LuxI/R system, this activation occurs by a different mechanism. At low cell densities, dimerized EsaR acts as a repressor. At a high cell population, derepression of the EPS genes occurs via an unknown mechanism once the AHL complexes to EsaR. Hence, a random mutagenesis genetic approach to isolate EsaR* variants that are immune to the effects of AHL has been utilized. Error-prone PCR and site-directed mutagenesis were used to generate desired mutants, which were subsequently screened for their ability to repress transcription in the presence of AHL. Several individual amino acids playing a critical role in the AHL-insensitive phenotype have been identified and mapped onto a homology model of EsaR. A separate study attempted to localize the dimerization region and analyze the stability of the N-terminal domain of EsaR. Truncations of EsaR at amino acids 169 and 178, without and with the extended linker region respectively, were generated using PCR. Dimerization assays similar to those by Choi and Greenberg in 1991 were performed but proved to be unsuccessful. However, the N-terminal domain is stable as determined by western blotting, which may facilitate its future structural analysis. Together, these efforts have contributed to the molecular understanding of AHL-dependent derepression of EsaR. / Master of Science

Quorum sensing is utilized by many different proteobacteria, including the two studied for this dissertation work, Vibrio parahaemolyticus and Pantoea stewartii subsp. stewartii. V. parahaemolyticus causes acute gastroenteritis in people who eat contaminated raw or undercooked shellfish. It is found in warmer marine waters and in rare cases, causes systemic infections when bacteria enter the body through open wounds. P. stewartii, on the other hand, is a phytopathogen that causes Stewart's wilt in maize. It is found in soil or the mid-gut of the corn flea beetle, its insect vector. Both V. parahaemolyticus and P. stewartii utilize quorum sensing to control their pathogenicity. Quorum sensing enables coordinate gene expression across a bacterial population. The V. parahaemolyticus quorum-sensing system utilizes the master regulator OpaR, which is homologous to the V. harveyii LuxRVh and the P. stewartii system contains EsaR which is homologous to the V. fischeri LuxRVf regulator. While the two systems differ in the molecular details of their mechanistic control, they are both forms of cell density dependent regulation that are either directly or indirectly controlled by small signaling molecules. Three different signaling molecules are found in V. parahaemolyticus, and only one signal is used in P. stewartii. The focus of this dissertation has been on understanding the downstream targets of OpaR and EsaR in their respective quorum-sensing systems. Prior to this work, it was known that when OpaR is not present or is nonfunctional V. parahaemolyticus changes from an opaque to a translucent colony morphology phenotype and the cells also become swarm proficient and more pathogenic. The complete genome of the V. parahaemolyticus BB22OP strain was assembled and annotated (Chapter 2). RNA-Seq was then used to analyze the transcriptomes of OpaR-active and OpaR-deficient strains of V. parahaemolyticus and identify genes that were regulated via quorum sensing (Chapter 3). Similarly, P. stewartii was also analyzed using RNA-Seq to identify genes controlled by EsaR in the transcriptome that had not been detected through prior proteomic studies. The initial RNA-Seq work confirmed the control of some previously identified direct targets of EsaR and newly identified ten other genes also directly controlled by EsaR (Chapter 4). Two direct targets of EsaR, rcsA and lrhA, became the focus of additional studies to further define the hierarchy of gene control downstream of the quorum-sensing regulator EsaR. RcsA controls capsule production, while LrhA controls motility and adhesion in P. stewartii. The regulons of rcsA and lrhA were defined by RNA-Seq, which also revealed multi-level control of rcsA gene expression (Chapter 5). Tight coordinated and temporal control of virulence factors is important for successful disease progression by pathogens. This dissertation work aims to enable a better understanding of the quorum-sensing hierarchy of genetic control in V. parahaemolyticus and P. stewartii. / Ph. D.

Pantoea stewartii subsp. stewartii is a bacterium that causes Stewart’s wilt disease in corn plants. The bacteria are transmitted to the plants via an insect vector, the corn flea beetle Chaetocnema pulicaria. Once in the plant, the bacteria migrate to the xylem and grow to high cell densities, forming a biofilm by secreting excess capsular exopolysaccharide, which blocks water transport and causes wilting. The timing of virulence factor synthesis is regulated by the cell-density dependent quorum sensing (QS) system. Such temporal regulation is crucial in establishing infection and is orchestrated by the QS-dependent transcriptional regulator EsaR. EsaR represses expression of capsular exopolysaccharide at low cell densities. At high cell densities, an acylated homoserine lactone (AHL) molecule produced during growth by the cognate AHL-synthase EsaI accumulates. The AHL binds to and inactivates EsaR, causing derepression of capsule production. EsaR is a member of the LuxR family of QS-dependent transcriptional factors. Most LuxR homologs are unstable and/or insoluble in the absence of AHL which has hindered structural studies. Chapter Two describes the changes in the structure of EsaR due to binding of AHL ligand as determined through biochemical methods. EsaR was found to be stable and retain its multimeric state in the absence or presence of AHL, but intra- and inter-domain changes occurred that affect its DNA-binding capacity. Apart from repressing expression of capsule at low cell-densities, EsaR represses its own expression and activates production of a small RNA, EsaS, with unknown function. In Chapter Three a proteomic approach was used to identify an additional 30 QS-controlled proteins. Genes encoding three of these proteins are directly regulated by EsaR and the EsaR binding sites in the respective promoters were defined. In Chapter Four, a high-throughput RNA-Seq method identified even more genes in the QS regulon that the proteomic approach overlooked. RNA-Seq analysis of rRNA-depleted RNA from two strains of P. stewartii was used as a screen to help identify 11 promoters, subsequently shown to be directly regulated by EsaR in vitro. Most of the genes controlled by QS grouped into three major physiological responses, capsule & cell wall production, surface motility & adhesion and stress response. In Chapter Five, the role of two QS regulated genes, dkgA (encoding 2, 5-diketo-D-gluconate) and lrhA (encoding a repressor of chemotaxis, adhesion and motility), in plant virulence were examined. These studies have better characterized the QS regulator EsaR and its interaction with the AHL ligand, and shown that QS has a more global response in P. stewartii than previously recognized. Further characterization of the genes identified in this study could facilitate identification of factors crucial in plant pathogenesis or insect-vector symbiosis and aid in the development of molecular-based approaches for possible disease intervention. / Ph. D.

La présente étude élaborée en fonction des données de la psychopédagogie actuelle propose un instrument d'analyse des jouets et du matériel de jeu à l'intention des éducateurs; elle répond en outre plus spécialement dans ses modalités d'application, aux besoins d'analyse et de classement des services de prêts de jouets et de matériel de jeu. Cette recherche appliquée présente d'abord le milieu des ludothèques et passe en revue les nombreux modèles de classement des jeux, des jouets et du matériel de jeu tirés de la littérature ancienne et contemporaine et les regroupe pour en faire une analyse critique. Le modèle proposé s'appuie sur un plan de classification emprunté au langage psychologique et articulé avec le support des techniques documentaires et informatisées. Il élabore un ensemble de catégories générales et spécifiques disposées dans un ordre cumulatif et hiérarchique. Ces catégories se présentent sous forme de répertoire de descripteurs c'est-à -dire de mots-clés spécialisés servant d'unités d'analyse conformes aux réalités décrites. Le modèle de classement ESAR est constitué de facettes complémentaires représentant autant d'aspects du savoir jouer. Le nom même du modèle est tiré du premier volet du système. Il correspond aux grandes catégories de jeux inspirées de l'approche piagétienne et couvre l'ensemble du développement de l'activité ludique, de la petite enfance à l'âge adulte; ce sont les jeux d'exercice (E), les jeux symboliques (S), les jeux d'assemblage (A) et les jeux de règles simples et de règles complexes (R). Les modalités d'application du modèle de classement ESAR prévoient un protocole d'analyse régi par des règles précises, un guide de travail , des bordereaux d'analyse et un fichier de définitions de chacun des 151 mots clés composant le thésaurus de manière à uniformiser le processus d'analyse . Cette étude qui associe de façon inédite à la fois un langage psychologique précis et cohérent et les techniques du traitement documentaire fait également appel au langage informatisé. Le modèle de classement ESAR a été utilisé à l'étape d'indexation dans la mise sur pied d'une banque d'analyse informatisée de jouets et de matériel de jeu et a été expérimenté, par la Centrale des bibliothèques du ministère de l'Éducation du Québec. La méthodologie d'application de cette expérience est décrite dans la dernière partie de l'étude. Les informations concernant le jeu et les jouets contenues dans cette banque de données automatisée sont accessibles pour interrogation directe par les usagers (éducateurs en garderies, en maternelles, orthopédagogues, parents, ludothécaires, etc.) ou sous forme de publications courantes distribuées par la Centrale des bibliothèques qui se propose d'enrichir périodiquement la banque de données et de la tenir à jour. La présente étude apporte donc à plusieurs groupes d'éducateurs différents, un instrument d'analyse du jeu et de ses accessoires, instrument à la fois cohérent sur le plan psychopédagogique et à la fine pointe des techniques de traitement documentaire et automatisé. / Québec Université Laval, Bibliothèque 2014

Québec Université Laval, Bibliothèque 2015