Still pictures were taken with a gel doc imager (AlphaInnotech ChemiImager). Time lapse videos were acquired using a Marshall electronics v-1070 surveillance camera, set up in a room acclimatized to 37C. Swarming plates with fluorescently labeled strains were imaged using the Amersham Typhoon 9400 (GE Healthcare). Colony forming units (CFU) were estimated by plating serial dilutions with different strains distinguished by fluorescent color. Data points for number of cells in colony and rhamnose secreted measurements shown in plots represent the median value among all experimental replicates, with error bars representing the 95- and 5-percentile. Cell number ratios were determined by dividing the CFU number of one color by the CFU number of the other color. Error bars for such ratio measurements were estimated from binomial distribution fitting (Johnson et al., 1993 ). rhlA expression in swarming assays was assessed by quantifying the total GFP expression in swarming colonies of a strain containing the PrhlABGFP construct. The total colony fluorescence was measured in Photoshop and normalized by the fluorescence of a colony expressing GFP constitutively (promoter PA1/04/03GFP). Secreted lipids were extracted from growth supernatants using a chloroform/methanol extraction protocol adapted from (Caiazza et al., 2007 (link)). The rhamnolipids in the extract were measured using the anthrone colorimetric assay (Zhu & Rock, 2008 ). The amount of rhamnose in culture supernatant (47.4 mg/L for the WT grown for 24 h in the standard minimal medium) was calibrated using a rhamnose calibration curve. This was converted into rhamnolipid concentration applying a conversion factor of 3.0 to 3.2 (Camilios Neto et al., 2008 (link)), leading to the concentration of biosurfactants of 0.14–0.16 g/L. The concentration of dry mass of cells (0.717 g/L) was measured by gravimetry. Nalgene sterile analytical filter units (Thermo Fisher Scientific, Rochester, NY) with 0.2 μm pore size where pre-dried for 24 h at 65 C and used to filter 120 mL of culture. The filters where then dry for 48 h until mass became stable over time.