We examined luxI point mutant VCW2G7 and found that, as predicted, it achieved the same luminescence as the wild type under anaerobic conditions with added 3-oxo-C6-HSL (data not shown). It was suggested that a putative FNR box upstream of luxR might underpin

the FNR-mediated regulation of luminescence in MJ1 (Muller-Breikreutz & Winkler, 1993); however, attempts to define a footprint using FNR*, an E. coli FNR derivative that is active aerobically (Kiley & Reznikoff, 1991), failed to show binding to this site (A.M. Stevens, pers. commun.). SB203580 To further explore how FNR might affect luminescence, we conducted a ‘Virtual Footprint’ analysis with the PRODORIC database (Munch et al., 2005), searching the V. fischeri genome for FNR boxes using a weighted consensus matrix based on data from E. coli. As expected, high Position Weight Matrix (PWM) scores (≥7.0) were skewed toward intergenic regions. Such putative

FNR boxes numbered in the hundreds, consistent with FNR’s global role in E. coli, and these included intergenic regions upstream of genes involved in anaerobic metabolism (e.g. upstream of nitrate and nitrite reductase genes). However, the best FNR box matches in the lux intergenic region of MJ1 and ES114 returned scores of 6.73 and only 5.88, respectively. To put this in perspective, >25 000 Selumetinib sites with no skew toward intergenic regions returned scores ≥5.9. Although we cannot rule out the possibility that FNR directly binds to the lux intergenic region, we believe this model is unlikely, especially in strain ES114. Virtual Footprinting did suggest a possible indirect effect of FNR on luminescence. Mirabegron The highest PWM score returned in this analysis (7.67) was found in six intergenic regions, one of which was upstream

of arcA. In E. coli, FNR activates arcA (Compan & Touati, 1994), and in ES114, ArcA strongly represses the lux operon (Bose et al., 2007). If FNR activates arcA in V. fischeri, this might explain FNR’s repressive effect on luminescence. Using ParcA-lacZ transcriptional reporters, we found that fnr was responsible for an ∼2–4-fold activation of the arcA promoter(s) anaerobically in ES114 and MJ1 backgrounds (Fig. 3). We tested whether FNR was important for symbiotic colonization by ES114 using established measures of symbiotic competence (Adin et al., 2009). The onset of symbiotic luminescence (Fig. 4a), colonization levels (Fig. 4b), and colonization competitiveness (Fig. 4c) were similar for ES114 and fnr mutant JB1 during the first 2 days of infection. The fnr mutant was also equally competitive up to 90 h after inoculation (data not shown). Furthermore, the fnr mutation did not appear to affect the symbiosis in a ΔarcA mutant background (data not shown).