Positive Feedback in V. fischeri lux Regulation Figure 6. Models of how subpopulations with heterogeneous Arc status could coordinate population-wide responses. Triangles indicate 3OC6 pheromone, white cells indicate no luminescence, and blue cells indicate luminescent cells. Panel A illustrates an interpretation of the data in Figure 4C, and panel B illustrates how similar signaling might occur in the host environment. In panel B we propose that even if only a subset of cells in the light organ experience physiological changes that silence ArcB kinase activity, this could result in loss of ArcA-dependent repression of lux followed by increased production of 3OC6 pheromone signals. This 3OC6 could then diffuse into neighboring ArcA-repressed cells to initiate positive feedback regulation of lux, thereby inducing luminescence in a population-wide response to conditions sensed by ArcB in a sub-population of symbionts. doi:10.1371/journal.pone.0049590.g006 LuxA and LuxB, which must dimerize to form active luciferase [71]. Unpublished data also suggest the lux operon may be post- transcriptionally controlled by a regulator that can be titrated with sufficient mRNA, which would also lead to a non-linear response between lux transcription and luminescence. Given the overriding strength of positive feedback, we speculated that ArcA-mediated repression of lux would be rendered insignificant after a culture has engaged LuxI- (3OC6-) mediated positive feedback, or if lux in neighboring cells was highly expressed, for example following deactivation of the Arc system. Figures 3 and 4 illustrate support for these two ideas. Taken together these results have significant implications for the symbiotic role of pheromone signaling in at least two ways. The first functional implication of our results is that the data in Figure 3 indicate an environmental condition in the host necessary for induction of 3OC6 signaling need not be maintained over time, because once the lux system is induced it essentially becomes more difficult to turn off. In the case of the ArcA/ArcB two component system, ArcB renders ArcA more active under reducing conditions and less active under oxidizing conditions. Bose et al. previously proposed that initial V. fischeri colonists might experience an oxidizing environment, leading to inactivation of ArcA/ArcB and induction of the lux operon but that later in infection, cells would become more crowded in the light-organ crypts, consume O2, and generate more reducing conditions that activate ArcA/ArcB [52]. They suggested that while ArcA might regulate other genes at this time later in infection, it would no longer effectively repress lux due to the increased synthesis of LuxI, accumulation of 3OC6, and positive feedback. More recently, studies by Williams et al. using transgenic E. coli strains also noted autoregulatory feedback by LuxR leading to more of this pheromone receptor in the cell and hysteresis [72], which may occur in strain ES114. The model proposed by Bose et al. reconciled the observations that ArcA did not repress lux in symbiotic cells but did appear to have a role in symbiont fitness later in infection [52]. The results of our study lend support for this model; however, our data also underscore the need to understand regulatory inputs during colonization of the host light organ. Given the discovery that direct regulation of the lux promoter by ArcA is quite weak, it is possible that the effect of another regulator is amplified by positive feedback and overpowers the repression by ArcA. The Arc activation state in symbiotic cells remains unknown, but as discussed below it is a priority for future research. A second key implication of our results is that not all symbiotic cells would have to experience a stimulatory environment in the host for there to be a population-wide induction of luminescence. As noted above, high cell density alone will not induce luminescence in ES114, but the combination of a quorum and some aspect of the host environment foster induction of lux and bioluminescence. In Figure 4 we show that in the absence of ArcA- dependent repression of lux, 3OC6 can diffuse into nearby cells and induce luminescence despite ArcA actively repressing the lux operon in the cells receiving 3OC6. Superficially, the experiment shown in Figure 4C resembles many others wherein added pheromone or a pheromone-producing strain stimulates a response in cells lacking pheromone production, with the signal diffusing over millimeter distances [73,74]. However, the key difference is that in Figure 4C an environmentally responsive regulator is what distinguishes pheromone levels in the adjacent strains. In short, Figure 4 shows that in a heterogeneous population, either mixed or spatially segregated, derepression of pheromone synthesis and the ensuing positive feedback override direct repression. These results imply that a group response can be coordinated based on physiological conditions that are not necessarily being experienced by the entire population. Figure 6 further illustrates how we interpret the data shown in Figure 4C, and how this result could reflect luminescence induction in the symbiosis. In our model, some environmental PLOS ONE | www.plosone.org 8 November 2012 | Volume 7 | Issue 11 | e49590