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<jats:title>ABSTRACT</jats:title><jats:p>The squid light organ symbiont<jats:named-content content-type="genus-species">Vibrio fischeri</jats:named-content>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<jats:italic>N</jats:italic>-octanoyl homoserine lactone (C<jats:sub>8</jats:sub>-AHL). We screened activity of a P<jats:sub><jats:italic>ainS</jats:italic></jats:sub>-<jats:italic>lacZ</jats:italic>transcriptional reporter in a transposon mutant library and found three mutants with decreased reporter activity, low C<jats:sub>8</jats:sub>-AHL output, and other traits consistent with low<jats:italic>ainS</jats:italic>expression. However, the transposon insertions were unrelated to these phenotypes, and genome resequencing revealed that each mutant had a distinct point mutation in<jats:italic>luxO</jats:italic>. In the wild type, LuxO is phosphorylated by LuxU and then activates transcription of the small RNA (sRNA) Qrr, which represses<jats:italic>ainS</jats:italic>indirectly by repressing its activator LitR. The<jats:italic>luxO</jats:italic>mutants identified here encode LuxU-independent, constitutively active LuxO* proteins. The repeated appearance of these<jats:italic>luxO</jats:italic>mutants suggested that they had some fitness advantage during construction and/or storage of the transposon mutant library, and we found that<jats:italic>luxO</jats:italic>* mutants survived better and outcompeted the wild type in prolonged stationary-phase cultures. From such cultures we isolated additional<jats:italic>luxO</jats:italic>* 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<jats:named-content content-type="genus-species">V. fischeri</jats:named-content>PS circuit,<jats:italic>litR</jats:italic>knockout mutants should resemble<jats:italic>luxO</jats:italic>* mutants; however,<jats:italic>luxO</jats:italic>* mutants outcompeted<jats:italic>litR</jats:italic>mutants in prolonged culture and had much poorer host colonization competitiveness than is reported for<jats:italic>litR</jats:italic>mutants, illustrating additional complexities in this regulatory circuit.</jats:p><jats:p><jats:bold>IMPORTANCE</jats:bold>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<jats:named-content content-type="genus-species">Vibrionaceae</jats:named-content>. 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<jats:named-content content-type="genus-species">V. fischeri</jats:named-content>, as it illustrates how pleiotropic mutants could easily and inadvertently be enriched in this bacterium during prolonged culturing.</jats:p>
- 出版日期2016-2
