Gene regulation by Arca and Phob in the light-organ symbiont Vibrio fischeri
Stoudenmire, Julie Lynn
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Bacterial pheromone signaling was discovered in Vibrio fischeri, where it controls activation of the lux operon responsible for bioluminescence and pheromone production. Although a threshold population density is required for pheromone signaling, environmental factors are also critical. For example, when V. fischeri enters its light-organ symbiosis with the Hawaiian Bobtail squid, the symbiotic V. fischeri cells are 1000-fold brighter than cells in laboratory cultures, even at the same population density. This observation prompted interest in understanding the regulators and environmental cues underlying lux regulation. Two such controls are the PhoBR and ArcAB two-component regulatory systems. The response regulators PhoB and ArcA control the lux operon in response to low phosphate and redox conditions, respectively; however it is unknown whether they are relevant to symbiotic bioluminescence induction, nor is the cue underlying Arc activation well understood. In this dissertation, I describe development of sensitive and specific fluorescent reporters to interrogate the regulatory status of PhoB and ArcA in V. fischeri cells both in and out of symbiosis. By adding a modified tag to a green fluorescent protein (GFP) and thereby targeting it for protease degradation, I decreased the half-life of the protein from over 24 hours to 81 minutes, making it a better proxy for recent transcriptional activity. Using a novel, iterative and semi-randomized approach I generated a synthetic transcriptional promoter-gfp fusion optimized to be induced when PhoB is active. This construct was induced in low-phosphate conditions and showed heterogeneous activation in light-organ infections, suggesting non-uniform phosphate availability and the potential for low-phosphate conditions to contribute to symbiotic luminescence. I used the same synthetic promoter-gfp construct in conjunction with a chimeric ArcA-PhoB protein, using the receiver domain from ArcA fused to the DNA binding domain of PhoB, to explore Arc activation. I show that ArcB activates the ArcA-PhoB chimera in culture, but there is also ArcB-independent activation, which surprisingly appears to be dominant during colonization. Finally, I present data suggesting a role for acetyl-phosphate in activating the ArcA-PhoB chimera. Together, these studies pioneer the optimization of transcriptional regulators for assessing V. fischeri regulation in situ.