Bacterial cultivation and quorum biosensor assay

All Pseudomonas wild-type and mutant strains in this study were routinely cultured in LB (lysogeny broth) media at 28 °C. Detailed information on all strains used in this study can be found in Table S1. The quorum-sensing biosensor assay was carried out by restreaking single colonies of wild-type and mutant strains on LB with 1.5% agar next to a streak of Chromobacterium violaceaum CV026, then incubating overnight at 28 °C [19, 20]. For selected strains in the Pseudomonas brassicacearum clade (as well as the ∆luxI_LPQ N2C3 mutant), we also performed a quantitative assessment of violacein production as previously described [21]. Briefly, each strain of interest was streaked three times next to a streak of C. violaceum CV026 on LB agar and incubated overnight. The next day, each CV026 streak was scraped off the plate using an inoculation loop and resuspended in 1 mL 10 mM MgSO₄. One hundred and fifty microliters of the suspension was diluted into 10 mM MgSO₄ prior to an absorbance reading at 764 nm to measure cell density. To normalize cell density, 764 nm was used because violacein absorbs near the 600 nm wavelength normally used for optical density (OD). The remaining suspension was centrifuged for 1 min at 6000 rcf, the supernatant was removed, and the pellet was resuspended in 1 mL dimethyl sulfoxide and incubated for 30 min at room temperature. After incubation, samples were centrifuged at 15,000 rcf for 15 mins. Five hundred microliters of the supernatant was mixed with 500 µL 10 mM MgSO₄ and the absorbance was measured at violacein’s absorbance peak of 585 nm. Relative violacein production was reported as the ratio of the OD₅₈₅/OD₇₆₄ measurements to account for differences in cell density.