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Abstract
The regulation of bioluminescence in Vibrio fischeri relies on the transcriptional regulator LuxR to activate lux genes in response to two main pheromone signals: N-3-oxo-hexanoyl-L-homoserine lactone (3OC6-HSL) and N-octanoyl-L-homoserine lactone (C8-HSL). Related pheromone-signaling (PS) systems are commonly found in Pseudomonadota (formerly Proteobacteria) and are responsible for regulating processes like biofilm formation, virulence, and other community-based behaviors relevant to agriculture, industry, and healthcare. V. fischeri bioluminescence serves as an excellent model system for studying LuxR-family transcriptional regulators due to its genetic tractability and the easily measurable nature of luminescence as an output. In this dissertation I explore the notable genetic and functional divergence between luxR from twenty-one strains of V. fischeri and close relatives. I first tested the HSL responsiveness of eight V. fischeri luxR variants using an otherwise isogenic V. fischeri background engineered to be disconnected from other PS regulation, to have constitutive luxR expression, and to lack endogenous HSL synthesis. Strains expressing each LuxR variant produced a unique response to HSL signals added singly and in combination. I then explored whether the differences in luxR coding sequences impact its mRNA or LuxR protein levels and determined that these levels varied by luxR sequence and by the presence of HSL. These differences correlated with the predicted stability of luxR mRNA 5’ secondary structures, which in other genes is known to affect translation initiation efficiency. LuxR variants also differed in the extent to which HSL increased LuxR protein stability. Finally, I tested the HSL responsiveness of an additional four luxR sequences, specifically luxR1 and luxR2 from the V. fischeri relatives Vibrio logei and Vibrio salmonicida. These luxR sequences also exhibited substantial evolutionary divergence, and they could not effectively induce bioluminescence in our engineered V. fischeri background. However, when the LuxR-binding “lux box” sequence was swapped for one from V. salmonicida, that bacterium’s LuxR2 was able to activate a small amount of bioluminescence. These results highlight species-specific regulatory adaptations and coevolution occurring between luxR and the lux box. Taken together, our findings expand our understanding of luxR evolution and regulation of the PS systems in V. fischeri.