Comparative analysis reveals regulatory motifs at the ainS/ainR pheromone-signaling locus of Vibrio fischeri

Vibrio fischeri uses the AinS/AinR pheromone-signaling system to control bioluminescence and other symbiotic colonization factors. The Ain system is thought to initiate cell-cell signaling at moderate cell densities and to prime the LuxI/LuxR signaling system. Here we compared and analyzed the ain locus from two V . fischeri strains and a Vibrio salmonicida strain to explore ain regulation. The ainS and ainR genes were predicted to constitute an operon, which we corroborated using RT-PCR. Comparisons between strains revealed a stark area of conservation across the ainS - ainR junction, including a large inverted repeat in ainR . We found that this inverted repeat in cis can affect accumulation of the AinS-generated pheromone N- octanoyl homoserine lactone, which may account for the previously unexplained low-signal phenotype of a ∆ ainR mutant, although the mechanism behind this regulation remains elusive. We also extended the previous observation of a possible “ lux box” LuxR binding site upstream of ainS by showing the conservation of this site as well as a second putative lux box. Using a plasmid-based reporter we found that LuxR can mediate repression of ainS , providing a negative feedback mechanism in the Ain/Lux signaling cascade. Our results provide new insights into the regulation, expression, and evolution of ainSR .