Positive Feedback in V. fischeri lux Regulation Figure 4. Inter-strain bioluminescence induction in mixed and spatially separated arcA+ and arcA mutant cells. Wild-type cells were mixed with either the dark arcA DluxCDABEG strain (JB33) able to produce 3OC6 (A) or the dark arcA DluxICDABEG strain (ANS6) lacking the luxI 3OC6 synthase gene (B). Wild-type and arcA mutant cultures and co-cultures were grown in SWTO broth in duplicate aerobic shake flasks to peak luminescence. A sample was removed and dilution plated to determine the CFU ml21 and the percent of wild-type cells in each co-culture (52% for A, 68% for B). Luminescence values are presented as luminescence per 108 CFU. Luminescence values presented for co- cultures are luminescence per 108 wild-type CFU. (C) Cultures of wild type or the luxI mutant were streaked onto SWTO agar plates next to spotted culture of the dark arcA DluxCDABEG strain (JB33) able to produce 3OC6. Inoculated plates were incubated at room temperature overnight and negative images of bioluminescence were captured with a BioRad Fluor-S MultiImager. Scale bar indicates approximately 5 mm. doi:10.1371/journal.pone.0049590.g004 3OC6 from arcA Mutant Cells can Induce Luminescence in Neighboring Cells in Culture We next asked whether 3OC6 from arcA mutant cells could induce luminescence in neighboring wild-type cells, overcoming ArcA-mediated repression of lux in the wild-type population. To examine this possibility we tested whether a dark (DluxCDABEG) arcA mutant could induce luminescence in wild-type cells in a 3OC6-dependent manner. When wild-type cells are co-cultured in the presence of dark (DluxCDABEG) arcA mutant cells in shake flasks, the specific luminescence of the wild-type cells increases nearly 500-fold (Fig. 4A), and this luminescence induction is dependent on luxI and 3OC6 pheromone production in the arcA mutant cells (Fig. 4B). Additionally, we asked whether pheromone produced by the arcA mutant could be communicated across a distance when strains were spatially segregated. To test this we streaked wild-type and luxI mutant cultures onto an agar plate next to a spot of dark arcA mutant culture. After incubation, the wild-type streak showed high luminescence nearest the pheromone-producing arcA mutant, with elevated but diminishing luminescence in cells further from the pheromone source extending ,15 mm (Fig. 4C). In contrast, the luxI mutant streak showed increased luminescence only nearest to the arcA mutant (Fig. 4C). The luminescence induction observed in both wild-type and luxI mutant streaks was dependent on luxI and 3OC6 synthesis in the dark arcA mutant (data not shown). The difference between luminescence of the wild-type and luxI streaks in Figure 4C presumably reflects induction in the luxI streak only by 3OC6 diffusing from the arcA mutant, whereas cells in the wild- type streak amplify the 3OC6 signal by positive feedback to extend the distance over which luminescence is induced. These results indicate that LuxI-mediated positive feedback considerably expands the range of this Arc-controlled signaling response. 3OC6 from One Population of Symbiotic Cells can Induce Luminescence in 3OC6-deficient Cells in the Squid Light Organ Theoretically, inter-strain induction of luminescence such as that shown in Figure 4 could reflect a similar phenomenon in the light organ, with a subpopulation inducing luminescence in a broader population through amplification and diffusion of pheromone originating from a distinct subgroup. Therefore, we were interested in testing whether a subpopulation of symbiotic cells can induce luminescence in the wider community in the squid light organ. While the intercellular signaling experiments de- scribed above used arcA mutants as a subpopulation of cells with altered regulation of pheromone synthesis, we did not use arcA mutants in the squid colonization experiment described below. PLOS ONE | www.plosone.org 6 November 2012 | Volume 7 | Issue 11 | e49590