Phenazine detection and quantification.

(i) For phenazine extraction from liquid cultures, 1 ml of liquid subcultures was washed three times in 1× PBS and resuspended in 1 ml 1× PBS. Washed cells were diluted 1:100 in 200 μl of the respective growth medium in a clear, flat-bottom polystyrene 96-well plate (VWR 82050-716) and incubated at 37°C with continuous shaking on the medium setting in a Synergy 4 plate reader (BioTek). Growth was measured by taking OD readings at 500 nm every 30 min, and cultures were monitored to determine onset of stationary phase. Five hours after each culture reached stationary phase, samples were collected into a microcentrifuge tube and centrifuged for 2 min at 16,873 relative centrifugal force (rcf) to pellet cells. The supernatant of each sample was applied to a 0.22-μm Spin-X column (VWR 29442-754) and centrifuged for 2 min at 16,873 rcf, and 200 μl of the resulting cell-free flowthrough was loaded into an HPLC vial for analysis. For each growth condition, n = 10.

(ii) For phenazine extraction from biofilms, 1 ml of liquid subcultures was washed twice in 1× PBS and resuspended in 1 ml of 1× PBS. A 25-mm filter disk with a pore size of 0.2 μm (GE Healthcare 110606) was placed into the center of one 35- by 10-mm round petri dish (VWR 25373-041) filled with 4 ml of 1% tryptone agar, MOPS-glucose agar, MOPS-succinate agar, or MOPS–α-KG agar, and 10 μl of the washed cells was spotted onto the filter disk. Colony biofilms were grown for 3 days in the dark at 25°C with > 90% humidity, after which point each colony and filter were lifted off their plate. The agar upon which the biofilm had developed was placed into a 5-ml aliquot of 100% methanol in a polypropylene conical tube, and phenazines were extracted from the agar overnight at room temperature in the dark. Three hundred microliters of the phenazine extraction was filtered through a 0.22-μm Spin-X column as described above, and 200 μl of the cell-free flowthrough were loaded into an HPLC vial for analysis. For each growth condition, n ≥ 6.

(iii) Phenazines were identified using high-performance liquid chromatography (Agilent 1100 HPLC system) as described previously (43, 60) and by comparing sample peaks to peaks of pure phenazine standards run as controls. The height of each peak (arbitrary units [AU]) was used to determine the concentration of each phenazine. For phenazine quantification from biofilms, the calculated phenazine concentration was multiplied by 1.25 to account for dilution during the phenazine extraction step. Dilution standards of purified PYO, PCA, and PCN were prepared at known concentrations, and extinction coefficients (ε) were generated for each: ε_PYO = 1.34 μM/AU, ε_PCA = 7.686 μM/AU, and ε_PCN = 7.518 μM/AU.