Positive Feedback in V. fischeri lux Regulation Figure 2. Effect of luxI and 3OC6 on derepression of the lux operon in an arcA mutant. Luminescence or lux reporter expression was measured in strains ES114 (WT), AMJ2 (DarcA), VCW2G7 (luxI mutant), or ANS7 (DarcA luxI). (A) Peak specific luminescence (luminescence per OD595) of strains grown in SWTO in aerobic shake flasks. Error bars indicate standard deviation (n = 3). (B) Plasmid based PluxI-lacZ transcriptional reporter assays. Strains containing pJLB171 or the promoterless vector pAKD702 were grown in duplicate aerobic shake flasks in SWTO medium. Cells were harvested at peak luminescence for ß–galactosidase assays. Error bars indicate standard deviation (n = 2). Asterisk indicates p-value of ,0.05 with a Student’s t-test comparing the arcA mutant to its respective parent strain. (C) Peak specific luminescence values for aerobic cultures grown in SWTO medium with 50 nM 3OC6 added where indicated. Error bars (some too small to see) indicate standard deviation (n = 2). Each panel is representative of at least three independent experiments. doi:10.1371/journal.pone.0049590.g002 cultures of the DarcA mutant could overcome ArcA-mediated repression of lux in wild-type cells. When 50 nM 3OC6 (the amount accumulated in arcA mutant cultures) was added to wild- type cultures, luminescence increased to levels comparable to that found in the DarcA mutant (Fig. 2C). Taken together, the results above suggested that ArcA-mediated repression of lux might be rendered inconsequential if cells have previously induced LuxI- mediated positive feedback or if a distinct population of neighboring cells lacks active ArcA-mediated repression of lux. Below we describe tests of both of these ideas. 3OC6-mediated Positive Feedback Results in Irreversible Luminescence Induction Because an arcA mutant produces high levels of 3OC6 and exogenous 3OC6 can overcome ArcA-mediated lux repression, we asked whether ArcA can repress luminescence once the LuxI- mediated positive feedback circuit is initiated. To test this, we constructed an IPTG-inducible arcA expression vector (pAS104) and moved it into an arcA mutant to control when arcA is expressed. When the arcA mutant carrying pAS104 was grown in aerobic shake flasks without IPTG, luminescence reached the same high level observed when the empty vector was present (data not shown), suggesting ArcA expression in the absence of IPTG was low enough to have little regulatory impact. When IPTG was present from the start of the experiment (T0), luminescence was repressed to a level similar to that of the wild-type control (Fig. 3). However, when IPTG was added to cultures after luminescence was induced (T1), arcA expression was no longer able to repress luminescence, resulting in cultures with bright luminescence similar to that in the DarcA mutant without IPTG (Fig. 3). These results suggest that once 3OC6-mediated positive feedback is Figure 3. Effect of controlled ArcA expression on biolumines- cence. Cultures of either the DarcA mutant with the inducible-arcA vector pAS104 (circles and squares) or wild type (diamonds) were grown in SWTO medium in duplicate aerobic shake flasks. Specific luminescence (luminescence per OD595) was observed for cultures grown with no addition (black) or with 2 mM IPTG added at the time of inoculation (T0, gray squares) or when cultures reached an OD595 of ,1.0 (T1, gray circles). doi:10.1371/journal.pone.0049590.g003 initiated fully by the loss of Arc-dependent repression, expression of ArcA cannot reverse this effect. PLOS ONE | www.plosone.org 5 November 2012 | Volume 7 | Issue 11 | e49590