C8 hsl

AHL standards (C6-HSL, 3-oxo-C6-HSL, C8-HSL, 3-oxo-C8-HSL and 3-oxo-C10-HSL) were purchased from the Cayman Chemical Company (Ann Arbor, Michigan, USA). Chromatographic-grade methanol and 99.9% ethyl acetate were purchased from Sigma-Aldrich (Buchs, Switzerland). The standards were dissolved in methanol at a concentration of 1 mM and stored at −20 °C.

XY-85 was cultured in marine broth 2,216 (MB; BD Difo^™) at 28°C. E. coli strains DH5α and BL21 (DE3) were cultured on Luria–Bertani (LB) agar at 37°C and used as hosts for expressing the protein whose encoding gene was cloned into pET-28a (Ke Lei Biotechnology Co., Ltd). The AHL biosensors Chromobacterium violaceum CV026 and VIR24 (McClean et al., 1997 (link); Someya et al., 2009 (link)), used to detect short-chain (C₄ to C₈) and long-chain (C₈ to C₁₄) AHLs, were maintained on LB agar at 28°C. Pectobacterium caratovorum subsp. caratovorum (Pcc, provided by Dr. Junna He at China Agricultural University) was cultured on Luria–Bertani (LB) agar at 28°C. Kanamycin was added at 50 μg mL⁻¹. C₄-HSL, C₆-HSL, 3-oxo-C₆-HSL, and C₈-HSL were purchased from Cayman Chemical Company (Ann Arbor, MI, United States); 3-oxo-C₈-HSL, C₁₀-HSL, 3-oxo-C₁₀-HSL, C₁₂-HSL, 3-oxo-C₁₂-HSL, C₁₄-HSL, and 3-oxo-C₁₄-HSL were purchased from Sigma-Aldrich (St. Louis, MO, United States). All of the AHL stock solutions (10 to 500 mM) were prepared in dimethyl sulfoxide (DMSO) and stored under −20°C.

To evaluate the expression levels of hchA-smoR promoter, ß-galactosidase assays were performed for the strains E77 wild type and ΔsmoR mutant harboring either the vectors pBBR1MCS-5-PsmoR:lacZ or pBBR1MCS-5-lacZ—the latter used as a control– during growth in LB medium at 30°C, following the protocol described by Miller (1972 ). All bacterial cultures were started with an initial inoculum corresponding to an optical density at 550 nm (OD₅₅₀) of 0.05. To determine the activity of the hchA-smoR promoter during growth curve, 0.1 ml-samples were taken at different times from 4 to 48 h. To investigate the effect of the presence of AHL molecules in the activity of the hchA-smoR promoter, initial cultures were supplemented with various synthetic AHLs (Cayman Chemical) –C6-HSL, oxo-C8-HSL, C8-HSL, and C10-HSL– with different concentrations (1 up to 10 μM), and 0.1 ml samples were taken and measured after 24 h and 48 h of incubation at 30°C. After analyzing the data we determined β-galactosidase specific activities in Miller Units (Miller, 1972 ). All AHL stocks were solubilized in 70% acetonitrile/water acidified with 0.1 M HCl final concentration. All experiments were performed by triplicate and comparison of ß-galactosidae activity was performed by One-Way analysis of variance (ANOVA) with a Bonferroni's multiple comparison post-test.