Positive Feedback in V. fischeri lux Regulation Figure 5. Inter-strain bioluminescence induction in the host light organ. (A) Relative luminescence induction over time for apo-symbiotic squid (black triangles) and squid infected with either the wild type (gray squares) or the VCW2G7 luxI mutant (empty circles). (B) GFP-labeled DluxCDABEG (EVS102) cells were mixed with RFP-labeled luxI mutant (VCW2G7) cells at a ratio of ,1:1 to inoculate apo-symbiotic juvenile squid. At 24 hr post infection, squid were dissected and imaged by epifluorescence microscopy to determine the spatial location of both cell types within the squid light organ using filters that allowed visualization of green fluorescence (top), red fluorescence (middle), or green and red fluorescence (bottom). Scale bar indicates approximately 100 mm. (C) Squid were then homogenized and plated to determine the CFU per squid for each cell type and to calculate the luminescence per 106 wild-type or luxI mutant cells. Data include nine squid colonized with wild type and six co-colonized squid. Co-colonized squid contained 12–92% of the luxI mutant in the colonization. For reference, squid clonally colonized with the luxI mutant have an average background relative luminescence per 106 CFU of ,104 on this scale. doi:10.1371/journal.pone.0049590.g005 Previous studies showed wild-type V. fischeri cells produce high levels of pheromone in the light organ [48]. Moreover, ArcA does not repress luminescence in symbiotic cells [52], suggesting ArcA most likely does not repress pheromone synthesis in these cells as well. For these reasons, we used arcA+ V. fischeri strains to test for intercellular signaling in the squid light organ. To determine if a subpopulation of cells can induce lumines- cence in the wider light organ community, aposymbiotic juvenile squid were co-infected with a 1:1 mixture of a gfp-labeled dark mutant (DluxCDABEG), that can still synthesize 3OC6, and a rfp- labeled luxI mutant that cannot make 3OC6. Neither strain alone can induce symbiotic luminescence [63] (Fig. 5A). However, when these strains co-colonize the light organ, if sufficient pheromone from the gfp-labeled dark mutant can diffuse into the pheromone deficient rfp-labeled strain, luminescence should be observed. 24 hr after infection, luminescence values were recorded for each squid before it was dissected and imaged with an epifluorescence microscope to visualize the spatial distribution of the two strains within the light organ (Fig. 5B). We observed wild-type levels of luminescence in the rfp-labeled luxI mutant cells in the squid when co-infecting the light organ with a dark 3OC6-producing strain (Fig. 5C). Interestingly, even when the dark pheromone-donor strain (DluxCDABEG) comprised as little as 8% of the light organ population in a mixed infection with the 3OC6-deficient (luxI) strain, the latter achieved luminescence comparable to that of wild-type in a clonal infection, and this robust induction of the luxI mutant by the dark strain occurred despite significant segregation of the two strains in the light organ. These results indicate that 3OC6-mediated intercellular signaling can occur between distinct subpopulations of symbiotic cells and that high levels of 3OC6 synthesis in only a subset of the population are sufficient to induce luminescence fully in the remaining cells occupying the light organ. Discussion Bacterial pheromone signaling often is governed by environ- mental regulators and by pheromone-dependent positive feedback loops, both of which modulate the LuxR/LuxI pheromone signaling system that controls bioluminescence in V. fischeri. The relative strengths of environmentally responsive regulators and positive feedback have important implications for the biological functions of pheromone signaling in nature, and in this study we explored their contributions to lux regulation in the squid symbiotic strain ES114. We previously found that ArcA is a direct repressor of the luxICDABEG operon, and that arcA or arcB mutants are 100- to 1000-fold brighter than ES114 in culture [50,52]. In this study we were surprised to find that in the absence of LuxI-mediated positive feedback, ArcA is actually a very weak repressor of the lux operon, repressing luminescence only about 2 fold. However, its influence over lux expression is amplified by LuxI-dependent positive feedback resulting in more than a hundred-fold additional increase in luminescence (Fig. 2). Given the interest in mathematically modeling the lux circuit, we should note that our data do not necessarily indicate that a simple positive feedback circuit has such a large effect on lux operon transcription. Importantly for quantitative assessment of this circuit, luminescence output does not appear to correspond linearly with transcription from the luxI promoter. In studies either using gfp embedded in the lux operon or measuring transcripts by microarray analysis, conditions that induce luminescence 100- to 1000-fold only had a 10- to 20-fold effect on the GFP reporter or lux mRNA [52,70,71]. Similarly, in Figure 2 we show a ,10-fold effect on expression of a plasmid-borne PluxI-lacZ reporter under conditions where luminescence was affected by at least an additional order of magnitude. Using the strain with gfp added to the lux operon, Perez et al. noted the relationship between fluorescence and luminescence followed a power law, and speculated that this may be due to the association equilibrium of PLOS ONE | www.plosone.org 7 November 2012 | Volume 7 | Issue 11 | e49590