N acyl homoserine lactone

E. coli strains used in this study were always grown in Luria-Bertani (LB) medium at 37 °C. The antibiotics kanamycin (50 μg mL⁻¹), ampicillin (150 μg mL⁻¹) and gentamicin (20 μg mL⁻¹) were added when necessary. In a typical experiment, for each of the biological replicates, a single colony from the strain of interest was taken from a fresh plate and cultured overnight in 1 mL of liquid medium in 24-well plates (500 rpm, PMS-1000i Microplate Shaker, Grant, Shepreth, UK). These cultures were diluted 500-fold in the same medium and further grown in the same conditions. Once they reached the exponential phase (OD₆₀₀ = 0.2–0.3), 2 μL of the culture were transferred into 1 mL of fresh medium containing IPTG (Sigma-Aldrich, St. Louis, MO, USA, final concentrations of 0.1, 0.2 and 0.5 mM) and/or N-acyl homoserine lactone (AHL, Sigma-Aldrich, St. Louis, MO, USA, final concentrations of 1.25, 2.5, 5, 10, 20 nM) and cultured in the same way as in the previous two steps.

QS signaling molecules, such as N-acyl-homoserine-lactone, N-butanoyl- (C₄-HSL), N-3-hydroxybutanoyl- (HC₄-HSL), N-hexanoyl- (C₆-HSL), N-heptanoyl- (C₇-HSL), N-octanoyl- (C₈-HSL), N-decanoyl- (C₁₀-HSL), N-dodecanoyl- (C₁₂-HSL), N-3-oxo-hexanoyl- (3-oxo-C₆-HSL), N-3-oxo-octanoyl-(3-oxo-C₈-HSL), and N-3-oxo-dodecanoyl-(3-oxo-C₁₂-HSL) homoserine lactone, were procured from Sigma Aldrich (Buchs, Switzerland). Analytical grade acetonitrile and formaldehyde were purchased from Sisco Research Pvt. Lit. (India). Working concentrations of the AHLs were prepared by dissolving them in acetonitrile (CH₃CN) at a concentration of 1 mg/ml and then storing them at −20°C.