measuring the optical density at 595 nm (OD595) using a BioPhotometer (Brinkman Instruments, Westbury, NY). The cuvette was then shaken to aerate the sample and luminescence was measured using a GLOMAX 20/20 luminometer (Promega, Madison, WI) with a 10 sec integration setting. Luminescence values were normalized to cell density (OD595) unless indicated otherwise. ß-galactosidase Assays for Promoter Reporter Activity V. fischeri strains harboring the PluxI-lacZ transcriptional reporter plasmid pJLB171 or the promoterless-lacZ vector parent pAKD702 were grown as described above for luminescence assays. At peak luminescence (OD595 ,2.0–2.5) cells were collected by centrifugation and the supernatant was discarded. Cell pellets were frozen at 220uC overnight and ß–galactosidase assays were performed to determine Miller units as described previously [61]. 3OC6 Bioassays Previous studies assayed 3OC6 by adding samples to V. fischeri wild-type ES114, and endogenous 3OC6 production did not impede its utility in assaying exogenous 3OC6 [48,62]. However, it has more recently been found that ES114 produces N-octanoyl homoserine lactone (C8) at much higher levels than 3OC6, and C8 can affect LuxR expression and activity [63–65]. For our bioassay we therefore used strain NL11 (Dains; litR::kan), which lacks both the C8 synthase (AinS) and the LitR regulator that modulates LuxR in response to C8 (or to the LuxS-generated AI- 2). To determine the level of 3OC6, V. fischeri cultures were grown in SWTO medium in aerobic shake flasks to an OD595 of 2.0 when cells were near peak luminescence. Cells were removed by centrifugation and the supernatant was extracted using equal parts supernatant and acidified ethyl acetate (1:1000 acetic acid in ethyl acetate). The ethyl acetate layer was removed and allowed to evaporate in sterile glass flasks. After evaporation the extracted HSL was resuspended in a volume of SWTO equal to the original culture volume, and this extract-amended SWTO was inoculated 1:1000 with V. fischeri bioassay strain NL11. Cultures were incubated at 24uC with shaking at 200 rpm, and cell density and luminescence were measured over time. Luminescence values for each sample extract were compared to those for 3OC6 standards to quantify the pheromone levels. For standards, 3OC6 (Sigma-Aldrich) was added into culture supernatant from a DluxI deletion strain (ANS3) to known concentrations (0, 1, 12.5, 25, 50, and 100 nM) and these 3OC6-amended supernatants were extracted and processed as described above. C8 Bioassays C8 bioassays employed a Ralstonia solanacearum bioassay strain (AW1-AI8,395) that is responsive to unsubstituted HSLs with acyl chains of eight or more carbons [66,67] and has been used previously to assay C8 levels in V. fischeri [27,38,66]. V. fischeri cultures were grown and HSLs extracted as described for the 3OC6 bioassays above. After evaporation the extracted HSL was resuspended in an equal volume of bioassay medium (per liter: 10 g tryptone, 5 g yeast extract, 0.5% glucose) and then diluted 1:100 into 2 ml of bioassay medium. Strain AW1-AI8,395 was inoculated to an OD595 of ,0.1 and tubes were incubated at 28uC with shaking at 200 rpm for ,5 hr until cultures reached an OD595 of ,0.4–0.6. Cells were collected to assay for ß– galactosidase activity as described previously [61] and Miller units for each sample extract were compared to those for C8 standards to quantify pheromone levels. For C8 standards, C8 (Sigma- Positive Feedback in V. fischeri lux Regulation Aldrich) was added to culture supernatant from a V. fischeri ainS mutant (CL21) to known concentrations (0, 1, 5, 10 nM) and these C8-amended supernatants were extracted and processed as described above. We used a Student’s t-test to determine whether strains had significantly different C8 accumulation. Squid Colonization Assays V. fischeri cultures were grown in ASWT to an OD595 of 0.3–0.7 and diluted in Instant Ocean (United Pet Group Inc., Cincinnati, OH) to 660–3000 CFU ml21. Aposymbiotic squid were added to the inoculum water overnight. The next morning squid were transferred to V. fischeri-free Instant Ocean. To measure the onset of luminescence in symbiotic animals, the luminescence per squid was measured using a LS6500 scintillation counter (Beckman Coulter, Fullerton, CA). At designated time points, squid were anesthetized with MgCl2, dissected and imaged using a Nikon (Melville, NY) Eclipse E600 epifluorescence microscope with a Nikon 96157 red filter cube, a Nikon 41017 green filter cube, and a Nikon Coolpix 5000 camera. After imaging, squid were homogenized and plated to determine CFU per squid. Results The arcA Mutant Produces High Levels of 3OC6 Pheromone Previous work in V. fischeri showed arcA and arcB mutants have bright luminescence in culture relative to the parent strain [50,52]. Since luxI, the 3OC6 synthase gene, is encoded in the same operon as the genes directly involved in generating bioluminescence, we predicted that an arcA mutant would also produce more 3OC6 pheromone than wild type. We found that at an OD595 of 2.0 when cultures are near peak luminescence, DarcA mutant cultures contained on average 55 nM 3OC6 pheromone while the wild- type, luxI, and DarcA luxI mutant cultures were below the level of detection for the assay (,1 nM). Thus, as predicted, ArcA mediates repression of not only bioluminescence but also 3OC6 pheromone synthesis. Bright Luminescence of the arcA Mutant is Mostly due to 3OC6-mediated Positive Feedback The ,500-fold increase in luminescence in arcA mutants (documented previously [52]) should be a combined effect of the loss of direct, ArcA-dependent repression of the lux operon together with the 3OC6-mediated positive feedback inherent in the LuxR-LuxI regulatory circuit. To test the relative importance of these two effects we first assayed luminescence in a DarcA mutant strain with or without a functional luxI 3OC6 synthase gene. It is important to note that under these broth culture conditions, LuxR-mediated activation of the lux operon in ES114 is stimulated primarily by C8 [63], which is the product of AinS [68,69]. C8 measurements ranged from 150 to 500 nM in different experiments but were never significantly different between ES114 and the DarcA mutant (p.0.2). The arcA mutant was .350-times more luminescent than wild type; however, in the luxI mutant background, the arcA mutation had only a 2-fold effect on luminescence (Fig. 2A). Similarly, we saw a much greater effect of the DarcA allele on a Plux-lacZ reporter in the wild-type background (10-fold) than in a luxI mutant (2-fold) (Fig. 2B). These results indicate that in the absence of ArcA-dependent repression of lux, bright bioluminescence is mediated primarily through the 3OC6-dependent positive feedback regulation of the lux genes. To further explore the relative regulatory strengths of 3OC6- mediated positive feedback and direct repression of lux by ArcA, we tested whether addition of 3OC6 at concentrations found in PLOS ONE | www.plosone.org 4 November 2012 | Volume 7 | Issue 11 | e49590