Coordination of the Arc Regulatory System and Pheromone-Mediated Positive Feedback in Controlling the Vibrio fischeri lux Operon Alecia N. Septer, Eric V. Stabb* Department of Microbiology, University of Georgia, Athens, Georgia, United States of America Abstract Bacterial pheromone signaling is often governed both by environmentally responsive regulators and by positive feedback. This regulatory combination has the potential to coordinate a group response among distinct subpopulations that perceive key environmental stimuli differently. We have explored the interplay between an environmentally responsive regulator and pheromone-mediated positive feedback in intercellular signaling by Vibrio fischeri ES114, a bioluminescent bacterium that colonizes the squid Euprymna scolopes. Bioluminescence in ES114 is controlled in part by N-(3-oxohexanoyl)-L-homoserine lactone (3OC6), a pheromone produced by LuxI that together with LuxR activates transcription of the luxICDABEG operon, initiating a positive feedback loop and inducing luminescence. The lux operon is also regulated by environmentally responsive regulators, including the redox-responsive ArcA/ArcB system, which directly represses lux in culture. Here we show that inactivating arcA leads to increased 3OC6 accumulation to initiate positive feedback. In the absence of positive feedback, arcA-mediated control of luminescence was only ,2-fold, but luxI-dependent positive feedback contributed more than 100 fold to the net induction of luminescence in the arcA mutant. Consistent with this overriding importance of positive feedback, 3OC6 produced by the arcA mutant induced luminescence in nearby wild-type cells, overcoming their ArcA repression of lux. Similarly, we found that artificially inducing ArcA could effectively repress luminescence before, but not after, positive feedback was initiated. Finally, we show that 3OC6 produced by a subpopulation of symbiotic cells can induce luminescence in other cells co-colonizing the host. Our results suggest that even transient loss of ArcA-mediated regulation in a sub-population of cells can induce luminescence in a wider community. Moreover, they indicate that 3OC6 can communicate information about both cell density and the state of ArcA/ArcB. Citation: Septer AN, Stabb EV (2012) Coordination of the Arc Regulatory System and Pheromone-Mediated Positive Feedback in Controlling the Vibrio fischeri lux Operon. PLoS ONE 7(11): e49590. doi:10.1371/journal.pone.0049590 Editor: Mark R. Liles, Auburn University, United States of America Received August 28, 2012; Accepted October 11, 2012; Published November 13, 2012 Copyright: 2012 Septer, Stabb. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by the National Science Foundation (NSF) under grants CAREER MCB-0347317, IOS-0841480, and IOS-1121106. ANS was supported by funds awarded by the University of Georgia Graduate School and the DoD, Air Force Office of Scientific Research, National Defense Science and Engineering Graduate (NDSEG) Fellowship, 32 CFR 168a. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: estabb@uga.edu Introduction Many bacteria regulate gene expression by producing and sensing pheromones. Because these signals can accumulate as culture density increases, pheromone-mediated responses often depend on high cell densities, giving rise to the term ‘‘quorum sensing’’ [1]. However, in many systems pheromone signaling is not simply a function of cell density. Instead, both synthesis of pheromones and responsiveness to them are often context dependent. Environmentally responsive regulators control expres- sion of many pheromone synthases and/or their cognate receptors, rendering such signaling dependent on other parame- ters in addition to cell density [2–27]. Moreover, pheromone signals often stimulate an increased rate of their own synthesis [28– 39]. This positive feedback can mean that even at the same cell density, the concentration and synthesis of a pheromone are partly a function of whether the system has recently been in a stimulated state. Combining context-dependent regulatory control over phero- mone synthesis with pheromone-mediated positive feedback has profound functional implications. Positive feedback can amplify the effects of other regulatory inputs that modulate pheromone synthesis, and the relative strengths of a regulatory input and positive feedback will affect communication, particularly in a population that spans a heterogeneous environment. If positive feedback is strong, a subpopulation of bacteria experiencing an environment that favors pheromone production might elicit a population-wide response, even in cells that would otherwise remain uninduced given their distinct environmental context. If positive feedback is weak, local environmental context becomes a more defining determinant of whether a pheromone system is induced. Understanding how bacteria integrate pheromone sensing, environmentally responsive regulation, and positive feedback to coordinate group responses requires model systems with pheromone-mediated behaviors that are easily observable and induced in natural environments. Vibrio fischeri is an attractive model for studying pheromone- mediated gene regulation and host-microbe symbiosis [40]. Bioluminescence in V. fischeri is regulated in part by the LuxR- LuxI pheromone system [28], and it is induced upon infecting the PLOS ONE | www.plosone.org 1 November 2012 | Volume 7 | Issue 11 | e49590