cue in a host microenvironment is found in a gradient. For example, if oxygen, reactive oxygen species or another good electron acceptor were provided by the host but also consumed by the bacteria (e.g. oxygen consumed by luciferase), there could be a relatively steep gradient of this cue. Perhaps symbionts nearer the host epithelium are exposed to a sufficiently oxidative environment to turn off Arc while others deeper in the crypt lumen experience more reduced conditions wherein Arc is active. The first group, with inactive Arc, might then initiate LuxI-mediated pheromone synthesis, producing a signal that spreads through the population. Quite the opposite of the original environmental cue, the pheromone signal would not be destroyed by other symbiont cells but rather it would be amplified by positive feedback, leading to a much more shallow gradient. Put together, this scenario could allow cell-cell signaling to induce a broader population response based on an environment only experienced by a subpopulation. Our model in Figure 6B in part invokes a scenario wherein 3OC6 signal from a subpopulation in the light organ initiates a population-wide response, and while this seemed plausible to us it had never been tested. In Figure 5, we observed pheromone- mediated intercellular communication in the squid light organ, where dark 3OC6-producing cells induced luminescence in co- colonizing, 3OC6-deficient cells. This inter-strain signaling in the light organ was particularly remarkable in that only a relatively small and segregated portion of the population needed to be producing 3OC6 in order to induce luminescence fully in the remaining cells. Moreover, it is worth noting that only the 3OC6- producing cells retain positive feedback regulation of pheromone synthesis, and therefore such positive feedback was not required for the subpopulation receiving the signal to induce luminescence fully. These data are proof in principle that symbiotic lumines- cence induction could originate from a regulatory response by a subpopulation. We have previously shown that symbiotic lux induction is spatially heterogeneous, indicating that subpopula- tions in different light organ microenvironments experience different regulatory cues [59]. The regulators and environmental conditions underlying this heterogeneity, and whether in fact subpopulations are responsible for the ultimate population-wide induction of luminescence, are fertile areas for future research. Pheromone production is required for luminescence induction in V. fischeri cells colonizing the squid; however, as noted above the environmental cues and regulators responsible for activating symbiotic pheromone synthesis and luminescence remain uncer- tain. Previous work showed that arcA mutant cells are brighter than wild type in culture but not in the squid [52], consistent with a model in which inactivation of ArcA/ArcB in the host may be important for luminescence derepression in symbiotic cells. Future work will focus on directly testing the role of ArcA/ArcB in symbiotic luminescence induction in V. fischeri and on elucidating the physiological conditions that control the activity of this two- component system in V. fischeri. Studies of E. coli have indicated References 1. Fuqua WC, Winans SC, Greenberg EP (1994) Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators. J Bacteriol 176: 269–275. 2. Schuster M, Hawkins AC, Harwood CS, Greenberg EP (2004) The Pseudomonas aeruginosa RpoS regulon and its relationship to quorum sensing. Mol Microbiol 51: 973–985. 3. Van Delden C, Comte R, Bally M (2001) Stringent response activates quorum sensing and modulates cell density-dependent gene expression in Pseudomonas aeruginosa. J Bacteriol 183: 5376–5384. 4. Moris M, Braeken K, Schoeters E, Verreth C, Beullens S, et al. (2005) Effective symbiosis between Rhizobium etli and Phaseolus vulgaris requires the alarmone ppGpp. J Bacteriol 187: 5460–5469. Positive Feedback in V. fischeri lux Regulation that oxygen itself is not perceived by ArcA/ArcB [75], and varied reports have suggested that ArcB may sense and respond to fermentation acids [76,77] and/or the redox state of the quinone pool [78,79]. The predicted ArcA regulon in V. fischeri [80] appears to be very similar to that of E. coli [80], and the V. fischeri arcA gene complements an E. coli arcA mutant [52], suggesting functional conservation of this two-component system across these two bacterial species. However, the mechanism underlying ArcB sensing of redox state in V. fischeri remains uncertain. Future work in this area will elucidate both how Arc functions and how it is integrated with pheromone signaling in a natural infection. Our results are also consistent with a different regulatory input overpowering ArcA-mediated lux repression. Figure 2C directly illustrates that 3OC6 can induce luminescence and render ArcA- mediated lux repression insignificant. This result shows that if another regulator induces 3OC6-mediated positive feedback in symbiotic cells, whether or not Arc is repressing lux may not matter. We are actively investigating the Arc status of symbiotic V. fischeri cells, but we are also elucidating regulators other than Arc that could play key roles in symbiotic pheromone signaling. A recent mutant screen identified transposon-insertion mutants in thirteen loci other than arc that led to increased luminescence of ES114 in culture [50]. These and other regulatory inputs should also be considered with respect to lux induction in the symbiotic environment. The lux pheromone system in V. fischeri is controlled by environmental regulators, and we have shown that such inputs can be powerfully amplified by the positive feedback inherent in the lux circuitry. Because bacterial pheromones are often regulated in response to environmental conditions and also subject to positive feedback, we expect that these results may reflect signaling systems in many other host-associated bacteria. Although phero- mone signaling may require a sufficiently high cell density for effective communication, the pheromones cannot be considered simply as census-taking molecules. Scenarios similar to the model presented in Figure 6B may explain the combination of environmental regulation and positive feedback in the pheromone signaling systems of host-associated bacteria, and this possibility should be considered as we seek to understand the biological roles of these systems. Acknowledgments We would like to thank Jeffery L. Bose for use of pJLB169 and JB33, Lawrence Shimkets for helpful discussions, and anonymous reviewers for insightful comments. Author Contributions Conceived and designed the experiments: ANS EVS. Performed the experiments: ANS. Analyzed the data: ANS EVS. Contributed reagents/ materials/analysis tools: EVS. Wrote the paper: ANS EVS. 5. Chatterjee J, Miyamoto CM, Zouzoulas A, Lang BF, Skouris N, et al. (2002) MetR and CRP bind to the Vibrio harveyi lux promoters and regulate luminescence. Mol Microbiol 46: 101–111. 6. Liang W, Pascual-Montano A, Silva AJ, Benitez JA (2007) The cyclic AMP receptor protein modulates quorum sensing, motility and multiple genes that affect intestinal colonization in Vibrio cholerae. Microbiology 153: 2964–2975. 7. Liang W, Sultan SZ, Silva AJ, Benitez JA (2008) Cyclic AMP post- transcriptionally regulates the biosynthesis of a major bacterial autoinducer to modulate the cell density required to activate quorum sensing. FEBS Lett 582: 3744–3750. 8. Wang L, Hashimoto Y, Tsao CY, Valdes JJ, Bentley WE (2005) Cyclic AMP (cAMP) and cAMP receptor protein influence both synthesis and uptake of extracellular autoinducer 2 in Escherichia coli. J Bacteriol 187: 2066–2076. PLOS ONE | www.plosone.org 9 November 2012 | Volume 7 | Issue 11 | e49590