Environmental regulation of bioluminescence in Vibrio fischeri ES114
Lyell, Noreen Loretta
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The pheromone-mediated circuitry that governs bioluminescence in Vibrio fischeri is well understood; however, less is known about the environmental conditions that influence pheromone and light production. The environment has been shown to have a profound effect on luminescence in V. fischeri, as cells in symbiotic association with the Hawaiian bobtail squid, Euprymna scolopes, are ~1000-fold brighter than non-symbiotic cells despite reaching similar cell densities. In this dissertation, I show that luminescence is governed by a complex regulatory web and that certain environmental conditions mediate the regulation of pheromone circuitry. My first goal was to identify and characterize previously unidentified regulators that control luminescence in V. fischeri strain ES114. I helped develop and employed a transposon mutagenesis system to discover novel negative regulators of luminescence. In this study, I characterized twenty-eight independent luminescence-up mutants with insertions in 14 loci. This work revealed that environmental conditions such as inorganic phosphate and Mg2+ concentrations are integrated into the regulation of the pheromone-dependent lux system. Furthermore, I showed that competition between the LuxI- and AinS-generated pheromones is an important and density-dependent factor in the level of light produced by V. fischeri ES114 cells, such that C8-HSL inhibits luminescence in dense populations. The second goal of this work was to clarify the role of cAMP receptor protein (CRP) in luminescence. Attempts to study the effects of glucose on V. fischeri luminescence have been contradictory and inconclusive, possibly due to strain-specific effects. I confirmed that both cAMP and glucose modulate light production in ES114, which is consistent with CRP regulation of luminescence. Previous researchers proposed a model wherein CRP regulates transcription of luxR, which encodes a pheromone-dependent activator known to induce luminescence. The data reported in this dissertation indicates that CRP is involved in the expression of not only luxR but also ainS. Using qRT-PCR and purified CRP, I show that CRP significantly increases transcript levels of both luxR and ainS, and that the CRP protein binds the upstream promoter regions of both genes. Taken together, this dissertation illustrates and clarifies how environmentally responsive regulators are integrated with pheromone-mediated circuitry in the regulation of V. fischeri bioluminescence.