Vibrio parahaemolyticus is considered to be an emerging, yet understudied, human pathogen. The V. parahaemolyticus BB22OP genome was sequenced to allow for a comparative analysis between the genome of BB22OP and another previously sequenced, pathogenic strain of V. parahaemolyticus, RIMD2210633. V. parahaemolyticus BB22OP is interesting because it exhibits a spontaneous phenotypic switch in colony morphology due to the loss of a functional OpaR; this also influences virulence. OpaR is the major quorum-sensing regulator in V. parahaemolyticus homologous to LuxR from V. harveyi. When opaR is removed from the RIMD2210633 genome, the same phenotypic switch is not seen indicating a difference between the quorum-sensing systems in these two strains. Understanding the regulatory variation in these two strains has the potential to provide key insights into the control of pathogenesis in this organism. Initially, the BB22OP genome sequencing results aligned into 125 contigs. The genome has now been assembled into two distinct chromosomes with only two gaps remaining to be filled. These gaps are located in the integron region, which is difficult to assemble due to its structure. The integron is a series of gene cassettes separated by inverted repeats that facilitate recombination events that build the integron. The integron region is further evidence of genetic differences between the two strains. The integron in the RIMD2210633 strain is comprised of 69 gene cassettes, while the BB22OP integron contains at least 86 gene cassettes. There are 313 genes novel to the BB22OP genome, which could result in the phenotypic differences seen in these two strains. Additionally five of the 313 genes are predicted to be transcriptional regulators indicating the potential for differential gene regulation. Further comparative analysis will likely reveal more phenotypic divergence between the physiology of RIMD2210633 and BB22OP. Additionally, the CsrA regulatory network was explored in RIMD2210633. CsrA was first characterized in E. coli as a global regulator of carbon storage and metabolism. RIMD2210633 contains a CsrA homolog and was predicted to contain four CsrA-regulating sRNAs (CsrB1-3 and CsrC), and this work confirmed that these sRNAs regulate CsrA in the same manner as in E. coli. CsrA and the same CsrA-regulating sRNAs were found in the BB22OP genome as well. Since CsrA is known to regulate glycogen production, a qualitative iodine-staining plate assay and a quantitative glycogen assay were used to indirectly measure CsrA activity in the presence and absence of individual regulatory sRNAs. The RIMD2210633 CsrA, CsrB1, CsrB2, CsrB3 and CsrC were shown to have the predicted physiological role in recombinant E. coli, with higher glycogen levels observed when CsrA was active and lower levels when each of the sRNAs was overexpressed. CsrA is also known to regulate biofilm production and virulence factors. In an attempt to develop a screening method for potential CsrA targets, a transcriptional/translational fusion system was developed. Transcriptional and translational fusions to β-galactosidase were created to PdksA, PglgC1 and PtoxR from RIMD2210633. CsrA or CsrB2 was overexpressed in recombinant E. coli containing each of the fusion constructs in order to see what happens to the gene expression from these promoters at low and high CsrA activity levels. Surprisingly, changing the activity levels of CsrA impacted both transcriptional and translational levels making the results of the assay difficult to interpret. Collectively these efforts have enhanced our understanding of V. parahaemolyticus. In particular, the sequencing of BB22OP has allowed for a comparative analysis between the BB22OP and RIMD2210633 strains. These strains have remarkably conserved genomes despite the phenotypic differences they exhibit. It appears there is variation in the quorum-sensing systems of these two strains. Further analysis will reveal how the quorum-sensing regulons differ and how this impacts the virulence of these two pathogenic V. parahaemolyticus strains. / 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.