Antisocial luxO Mutants Provide a Stationary-Phase Survival Advantage in Vibrio fischeri ES114

The squid light organ symbiont Vibrio fischeri controls bioluminescence using two acyl-homoserine lactone pheromone-signaling (PS) systems. The first of these systems to be activated during host colonization, AinS/AinR, produces and responds to N-octanoyl homoserine lactone (C(8)-AHL). We screened activity of a P(ainS)-lacZ transcriptional reporter in a transposon mutant library and found three mutants with decreased reporter activity, low C(8)-AHL output, and other traits consistent with low ainS expression. However, the transposon insertions were unrelated to these phenotypes, and genome resequencing revealed that each mutant had a distinct point mutation in luxO. In the wild type, LuxO is phosphorylated by LuxU and then activates transcription of the small RNA (sRNA) Qrr, which represses ainS indirectly by repressing its activator LitR. The luxO mutants identified here encode LuxU-independent, constitutively active LuxO* proteins. The repeated appearance of these luxO mutants suggested that they had some fitness advantage during construction and/or storage of the transposon mutant library, and we found that luxO* mutants survived better and outcompeted the wild type in prolonged stationary-phase cultures. From such cultures we isolated additional luxO* mutants. In all, we isolated LuxO* allelic variants with the mutations P41L, A91D, F94C, P98L, P98Q, V106A, V106G, T107R, V108G, R114P, L205F, H319R, H324R, and T335I. Based on the current model of the V. fischeri PS circuit, litR knockout mutants should resemble luxO* mutants; however, luxO* mutants outcompeted litR mutants in prolonged culture and had much poorer host colonization competitiveness than is reported for litR mutants, illustrating additional complexities in this regulatory circuit.

Importance: Our results provide novel insight into the function of LuxO, which is a key component of pheromone signaling (PS) cascades in several members of the Vibrionaceae. Our results also contribute to an increasingly appreciated aspect of bacterial behavior and evolution whereby mutants that do not respond to a signal from like cells have a selective advantage. In this case, although "antisocial" mutants locked in the PS signal-off mode can outcompete parents, their survival advantage does not require wild-type cells to exploit. Finally, this work strikes a note of caution for those conducting or interpreting experiments in V. fischeri, as it illustrates how pleiotropic mutants could easily and inadvertently be enriched in this bacterium during prolonged culturing.