squid Euprymna scolopes in a natural and experimentally tractable symbiosis [40–42]. The luxICDABEG operon (Fig. 1) underlies bioluminescence and encodes the LuxI pheromone synthase, which produces N-(3-oxohexanoyl)-L-homoserine lactone (3OC6) [43]. When 3OC6 accumulates to a threshold concentration, it combines with LuxR to activate transcription of luxICDABEG [44– 46]. Because the LuxI product 3OC6 induces luxI transcription, this ‘‘autoinducer’’ pheromone initiates a positive-feedback loop resulting in both increased 3OC6 production and biolumines- cence. The V. fischeri lux system also is regulated tightly in response to the environment, and such control is especially pronounced in isolates from E. scolopes such as strain ES114. ES114 is dim and produces little 3OC6 in culture, but in the host light organ it is ,1000-fold more luminescent and produces more 3OC6 than in culture, even at similar high cell densities [47–49]. Several regulators modulate Lux expression [27,38,50,51], perhaps none more impressively than the redox-responsive ArcA/ArcB two- component regulatory system [52]. ArcA is a direct repressor of lux and a V. fischeri arcA mutant is 100- to 1000-fold brighter than ES114 in culture, achieving nearly symbiotic luminescence levels [52]. In this study, we used V. fischeri and ArcA-mediated control of lux to examine the interplay between direct regulation by an environmentally-responsive regulator and the positive feedback inherent in pheromone production. We also explored the potential for intercellular signaling by distinct symbiotic V. fischeri popula- tions. Our data illustrate important potential roles for 3OC6- mediated communication beyond sensing a quorum. Figure 1. ArcA/ArcB and LuxR-LuxI-mediated regulation of bioluminescence in V. fischeri. LuxI synthesizes 3OC6, a diffusible pheromone that upon reaching a sufficient concentration combines with LuxR. 3OC6-LuxR binds to the ‘‘lux box’’ and stimulates transcription of the luxICDABEG operon, which produces more 3OC6 and bioluminescence. The ArcA/ArcB two-component regulatory system responds to reducing conditions, and ArcA-P binds near the lux box, effectively inhibiting bioluminescence. Two other autoinducer pheromones AI-2 and C8-HSL are not shown, although the latter can also function with LuxR. doi:10.1371/journal.pone.0049590.g001 Positive Feedback in V. fischeri lux Regulation Materials and Methods Media and Growth Conditions V. fischeri was grown at 28uC or 24uC in one of three rich media depending on the application, as indicated below. The media used were LBS medium [53], ASWT medium [54], or SWTO medium [52]. E. coli strains were grown in either LB medium [55] or Brain Heart Infusion (Difco) at 37uC. Antibiotic selection for V. fischeri and E. coli strains was performed as described previously [56]. Plasmid and Strain Construction Bacterial strains, plasmids, and oligonucleotides used in this study are presented in Table 1. Plasmids were maintained in E. coli strain DH5a [57] except for plasmids that contained only the R6Kc origin of replication (oriVR6K), which were maintained in strain DH5alpir [56], or in strain CC118lpir [58] in the case of plasmid pEVS104. Plasmids that were stably maintained in V. fischeri were derived from shuttle vectors that contain both oriVR6K and the replication origin from V. fischeri plasmid pES213 (oriVpES213) [56,59]. These shuttle vectors were maintained in E. coli DH5alpir prior to introducing them into V. fischeri. Plasmids were mobilized from E. coli into V. fischeri by triparental mating using CC118lpir pEVS104 as a conjugative helper and exploiting the RP4 origin of transfer (oriTRP4) as previously described [60]. To generate mutations in V. fischeri, mutant alleles were mobilized on unstable plasmids into recipients, and allelic exchange was screened using appropriate antibiotic resistance markers and PCR. To construct a DluxI mutant, sequence upstream of luxI was PCR amplified using primers ASLUX1 and ASLUX2 and cloned into pEVS122 at the SmaI site resulting in plasmid pAS2. pAS2 was fused at the NheI site to NheI-digested pEVS148k, which contains sequence downstream of luxI, resulting in the DluxI deletion construct pAS3. The DluxI allele on pAS3 was exchanged into ES114 to generate strain ANS3. To make the DarcA luxI double mutant strain ANS7, plasmid pAJ4 containing the DarcA allele was fused to pBluescript at their respective SpeI sites, resulting in plasmid pAS6, and the DarcA allele on pAS6 was exchanged into VCW2G7 (luxI point mutant). To construct the DarcA::erm DluxCDABEG strain JB33, the DluxCDABEG allele on plasmid pEVS153 was exchanged into the DarcA::erm strain AMJ1. To construct the V. fischeri DluxICDABEG mutant, pAS2 was fused at the NheI site to NheI-digested pEVS149k, which contains sequence downstream of luxG, resulting in the luxICDABEG deletion allele on plasmid pAS4. This DluxICDABEG allele was exchanged into ES114, generating strain ANS5. To make the DarcA::erm DluxICDABEG strain ANS6, the DarcA::erm allele on plasmid pJLB169 was exchanged into strain ANS5. To construct plasmid pAS104 with an inducible arcA, the arcA gene including 24 bp upstream of the ATG and 21 bp downstream of the TAA stop codon, was PCR amplified using primers ASind_arcAF and ASind_arcAR and directionally cloned into the SacI and XbaI sites of plasmid pAKD601B. Luminescence Assays To assay luminescence, V. fischeri cultures were grown overnight in LBS medium and diluted 1:1000 into either 25 ml SWTO medium in 125 ml flasks or 50 ml SWTO medium in 250 ml flasks. Within each experiment, the same flask and medium volume combinations were used for all strains and treatments. Media was supplemented with 2 mM isopropyl beta D-thioga- lactopyranoside (IPTG) (Sigma-Aldrich, St. Louis, MO) or 50 nM 3OC6 (Sigma-Aldrich) where indicated. Cultures were incubated at 24uC with shaking at 200 rpm. At indicated time points, 0.5-ml samples were removed and the cell density was estimated by PLOS ONE | www.plosone.org 2 November 2012 | Volume 7 | Issue 11 | e49590