The effects of carbon sources on cAMP-CRP-mediated regulation in Vibrio fischeri
Colton, Deanna Marie
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The Proteobacterium Vibrio fischeri induces bioluminescence upon establishing its light-organ symbiosis with the Hawaiian bobtail squid, Euprymna scolopes. This bioluminescence is controlled by pheromone-mediated signaling, which in turn is influenced by many environmental factors, one of which is a carbon source. In particular, glucose affects pheromone regulation, apparently through the activity of the cAMP-receptor protein CRP and the second messenger cAMP, which is generated by CyaA. In the absence of glucose, cAMP-CRP stimulates many catabolic operons in Proteobacteria, and crp is important in host colonization for other vibrios. Therefore, I became interested in cAMP-CRP’s role in the light-organ environment, and how a carbon source translates into changes in cAMP-CRP activity. My first goal was to investigate cAMP-CRP’s role in the symbiosis and during growth in culture. Both ∆cyaA and ∆crp mutants were attenuated in host colonization, and cAMP-CRP activity measured from a CRP-dependent transcriptional reporter was elevated in the symbiosis. In culture, glucose improved the growth of ∆cyaA and ∆crp mutants and decreased cAMP-CRP activity. N-acetylglucosamine (NAG) similarly improved the growth of the mutants, although it did not decrease cAMP-CRP activity. My results suggest that CRP is active in V. fischeri during colonization and that glucose is not a major carbon source in the light organ. My second goal was to determine how cAMP and CRP levels are controlled in V. fischeri. In Escherichia coli, glucose lowers intracellular cAMP and CRP levels. However, despite lowered cAMP-CRP activity, I found higher intracellular cAMP levels in the presence of glucose. I showed that the proteins CyaA, CpdA, CpdP, and EIIAGlc, along with an unknown transporter, control cAMP levels; however, neither cAMP concentration nor CRP levels seemed to explain the decrease in cAMP-CRP activity in glucose-grown cells. I hypothesize that CRP is being regulated by a post-translational mechanism, such as protein acetylation. This dissertation illustrates the importance of cAMP-CRP-mediated regulation in the V. fischeri-E. scolopes symbiosis and suggests there are yet unknown means by which glucose regulates cAMP-CRP activity in Proteobacteria.