Date of Degree
PhD (Doctor of Philosophy)
Linda L. McCarter
The gram-negative marine bacterium Vibrio parahaemolyticus is a pathogen and a common worldwide cause of seafood-associated gastroenteritis. When grown on a surface, V. parahaemolyticus undergoes a dramatic differentiation to an elongated, highly flagellated swarmer cell from the short rod typical of swimming cells. Swarming motility is a complex form of adaptation to growth on a surface, and we developed a set of microarray experiments to examine the global gene expression changes that occur upon differentiation to the swarmer cell. We hypothesized that growth on a surface would elicit a specific response involving genes for motility and surface colonization and not the broad changes in physiology suggested by others to be co-regulated with swarming motility.
By taking advantage of the two known signals required for swarmer cell induction (inhibiting polar flagellar rotation and limiting iron), the swarming response was artificially induced in liquid and used to define the set of genes associated with surface sensing by transcriptome analysis. This approach avoided the confounding physiological differences between growth in liquid and growth on a surface. Fifteen microarrays performed with different strains and growth conditions were used to define a concise set of about 70 genes that comprise the core set of surface-induced genes. This set includes genes encoding the surface motility system lateral flagella and virulence factors including a type three secretion system (T3SS1). I showed a biological consequence of the increased expression of T3SS1 genes, as surface-induced cells were more toxic in a tissue culture infection than either liquid-grown or surface-grown non-swarming mutants.
I explored the role of calcium signaling in regulating the surface sensing network, as calcium seemed a pertinent signal to a marine organism and low calcium is a known inducing signal for T3SS in other organisms. Calcium was shown to enhance swarming motility and lateral flagellar gene expression. Microarrays were used to analyze the transcriptome response to growth with EGTA (a cation chelator commonly used to generate low calcium) or calcium. Surprisingly, both low and high calcium induced T3SS1 gene expression. The EGTA effect was determined to be the result of iron limitation, which was thus shown to be a new inducing signal for T3SS1. I overexpressed the master transcriptional regulator of the T3SS system, encoded by exsA, to define the entire set of T3SS1-associated genes. I found that ExsA was also a new regulator of the surface sensing regulon, which was repressed when exsA was overexpressed. Microarray analysis showed that calcium is a global regulator, controlling transcription of about 50 genes under the conditions tested. I characterized a new calcium-regulated transcription factor that we named CalR, and showed that CalR repressed swarming motility and T3SS1 gene expression.
The transcription factor OpaR was previously known to repress swarming genes and control colony opacity. It is homologous to the output regulators of the quorum sensing pathway in other Vibrio species. I used microarray analysis and mutant strains to explore the functionality of the quorum sensing cascade in V. parahaemolyticus and define the OpaR regulon during growth on a surface. I showed that the quorum sensing regulator LuxO when active silences opaR as it does in other Vibrios, using a translational reporter fusion in opaR. I used microarray analysis to show that 323 genes are induced or repressed by OpaR. The surface-sensing regulon is repressed by OpaR. Many genes encoding proteins involved in virulence, signal transduction, and modulation of the signaling molecule cyclic dimeric GMP are regulated by OpaR. The quorum sensing controlled network of gene expression in V. parahaemolyticus is quite distinct from other Vibrios, with respect to both the specific nature as well as the direction of regulation of genes controlled by OpaR.
calcium, iron, quorum sensing, swarming, type three secretion, Vibrio parahaemolyticus
x, 208 pages
Includes bibliographical references (pages 198-208).
Copyright 2011 Cindy Jean Gode