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

Abstract

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. IMPORTANCEOur 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. Many bacteria use pheromones to regulate group behaviors (1). These pheromone-signaling (PS) systems generally require sufficiently high cell density for pheromone accumulation and are also regulated in response to the environment (2-6). Accordingly, PS outputs depend on both population density and an appropriate context. The importance of such control is easily rationalized, given the energetic cost of many PS-activated processes, such as bioluminescence (7,8). This cost of inducing PS-controlled...

Environmental Viscosity Modulates Interbacterial Killing during Habitat Transition

“…To ensure that these regulatory proteins function in ES401 in high viscosity as expected, we grew wild-type and vasA_2, luxR, and luxU mutant strains in high-viscosity medium and monitored cell density and luminescence over time. Consistent with the known role for these genes (50,53), luminescence values were lower for the luxR mutant and higher for the luxU mutant than for the wild type ( Fig. S2B).…”

Quorum Sensing Gene Regulation by LuxR/HapR Master Regulators in Vibrios

“…These mutants can outcompete wild-type cells in vitro, suggesting that such mutations could easily spread in the environment. A similar effect is observed for V. fischeri litR and luxO mutants (34,109). However, bacterial populations can police themselves against cheaters via a number of mechanisms, including toxin production and metabolite exclusion (110,111).…”

Host-selected mutations converging on a global regulator drive an adaptive leap by bacteria to symbiosis

“…Nor would these mutants be expected to rise in frequency to the point of detection considering alleles that confer enhanced fitness in squid often carried a fitness tradeoff during growth in broth culture ( Figure 2E). However, the improved survival of spontaneous qrr1-enhancing luxO mutants during stationary growth suggests a plausible mechanism for ancestral population enrichment of binK alleles that enhance qrr1 expression ( Figure 2E) (Miyashiro et al 2010;Kimbrough and Stabb 2015).…”