Analysis of the Quorum Sensing Regulons of Vibrio parahaemolyticus BB22 and Pantoea stewartii subspecies stewartii

Abstract

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.