AntisocialluxOMutants Provide a Stationary-Phase Survival Advantage in Vibrio fischeri ES114
ABSTRACTThe squid light organ symbiontVibrio fischericontrols 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 toN-octanoyl homoserine lactone (C8-AHL). We screened activity of a PainS-lacZtranscriptional reporter in a transposon mutant library and found three mutants with decreased reporter activity, low C8-AHL output, and other traits consistent with lowainSexpression. However, the transposon insertions were unrelated to these phenotypes, and genome resequencing revealed that each mutant had a distinct point mutation inluxO. In the wild type, LuxO is phosphorylated by LuxU and then activates transcription of the small RNA (sRNA) Qrr, which repressesainSindirectly by repressing its activator LitR. TheluxOmutants identified here encode LuxU-independent, constitutively active LuxO* proteins. The repeated appearance of theseluxOmutants suggested that they had some fitness advantage during construction and/or storage of the transposon mutant library, and we found thatluxO* mutants survived better and outcompeted the wild type in prolonged stationary-phase cultures. From such cultures we isolated additionalluxO* 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 theV. fischeriPS circuit,litRknockout mutants should resembleluxO* mutants; however,luxO* mutants outcompetedlitRmutants in prolonged culture and had much poorer host colonization competitiveness than is reported forlitRmutants, illustrating additional complexities in this regulatory circuit.IMPORTANCEOur results provide novel insight into the function of LuxO, which is a key component of pheromone signaling (PS) cascades in several members of theVibrionaceae. 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 inV. fischeri, as it illustrates how pleiotropic mutants could easily and inadvertently be enriched in this bacterium during prolonged culturing.