Antibiotic Susceptibility Testing of AHLs

Pure C6HSL, 3OC6HSL, and 3OC12HSL were obtained from Shanghai UDChem Technology Co., Ltd. 3OC8HSL was purchased from Sigma Aldrich Chemicals Co., Ltd (Shanghai, China). The chemical structures of various N-acyl homoserine lactones (AHLs) used in this study are shown in Supplementary Figure S1. Different antibiotics—ampicillin (Amp, 50 mg⋅mL^–1), chloramphenicol (Cm, 30 mg⋅mL^–1), tetracycline (Tc, 5 mg⋅mL^–1), streptomycin (Str, 50 mg⋅mL^–1), gentamicin (Gen, 50 mg⋅mL^–1), and kanamycin (Kan, 50 mg⋅mL^–1)—were used in antimicrobial susceptibility tests. Pesticide streptomycin (98% active ingredient) was used as a positive control at a concentration of 3.3 g⋅mL^–1. Bacterial strains and plasmids used in this study are presented in Table 1.
The bacterial strain was cultured in minimal salt medium (MSM) [composition (g⋅L^–1): (NH₄)₂SO₄ 2.0, Na₂HPO₄⋅12H₂O 1.5, KH₂PO₄ 1.5, MgSO₄⋅7H₂O 0.2, CaCl₂⋅2H₂O 0.01, FeSO₄⋅7H₂O 0.001, pH 6.5] and Luria-Bertani medium (LB) [composition (g⋅L^–1): Yeast extract 5.0, Tryptone 10.0, NaCl 10.0, pH 7.0] (Chen et al., 2011a (link)). Minimal medium (MM) [composition (g⋅L^–1): (NH₄)₂SO₄ 2.0, MgSO₄⋅7H₂O 0.2, CaCl₂ 0.01, FeSO₄ 0.005, MnCl₂ 0.002, K₂HPO₄ 10.5, KH₂PO₄ 4.5, mannitol 2, glycerol 2, pH 6.5] was used for detecting AHL (Liu et al., 2017 (link)). Agar (15 g⋅L^–1) was used to solidify the medium. Media were sterilized at 121 °C for 20 min.