Pseudomonas syringae is a pathogen of numerous plant species, including several economically important crops. P. syringae pv. syringae B728a is a resident on leaves of common bean, where it utilizes several well-studied virulence factors, including secreted effectors and toxins, to develop a pathogenic interaction with its host. The B728a genome was recently sequenced, revealing the presence of 1,297 genes with unknown function. This dissertation demonstrates that a 29.9-kb cluster of genes in the B728a genome encodes a novel secretion pathway, the type VI secretion system (T6SS), that functions to deliver at least one protein outside of the bacterial cell. Western blot analyses show that this secretion is dependent on clpV, a gene that likely encodes an AAA+ ATPase, and is repressed by retS, which apparently encodes a hybrid sensor kinase. RetS and a similar protein called LadS are shown to collectively modulate several virulence-related activities in addition to the T6SS. Plate assays demonstrate that RetS negatively controls mucoidy, while LadS negatively regulates swarming motility. A mutation in retS affects B728a population levels on the surface of bean leaves. A model for the LadS and RetS control of B728a virulence activities is proposed, and possible roles for the B728a T6SS are addressed.

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.