Bioassays of AHLs.

AHL bioactivity was determined by measuring the β-galactosidase bioactivity of the ultrasensitive AHL biosensor strain A. tumefaciens KYC55 (38). Cultures of recombinant E. coli BL21(DE3)(pET-pdeI), wild-type PD1222, and ΔpdeI and ΔpdeR mutants were centrifuged at 5,000 × g for 5 min to pellet the cells. A 0.22-μm syringe filter was used to filter assay supernatants, and the cell-free culture supernatants were stored at −20°C. A. tumefaciens KYC55 (5 × 10⁷) was inoculated into 2 ml of AT culture medium containing 200 μl of supernatant from the growth of recombinant E. coli. The supernatant of the E. coli strain carrying pET-30(a) without the pdeI gene was used as a negative control. The OD₆₀₀ of each sample was recorded after approximately 10 h at 28°C, and 200 μl of the supernatant of the AT culture medium was then combined with 0.8 ml of Z buffer (60 mM Na₂HPO₄, 40 mM NaH₂PO₄, 10 mM KCl, 1 mM MgSO₄, and 50 mM 2-mercaptoethanol [pH 7.0]). Two drops of 0.05% SDS solution and 3 drops of chloroform were added to this solution in 2-ml microcentrifuge tubes. The samples were vortexed vigorously for 10 s, 0.1 ml of 4 mg/ml ortho-nitrophenyl-β-d-galactopyranoside (ONPG) was added, and the samples were then placed into a 30°C water bath for 10 min. Reactions were stopped by the addition of 0.6 ml of 1 M Na₂CO₃. Cell debris were removed by centrifugation for 3 min at 16,000 × g and room temperature, and the OD₄₂₀ of the supernatant was then measured. β-Galactosidase units were calculated according to the following equation: Miller units = (1,000 × OD₄₂₀)/(OD₆₀₀ × 10 × 0.2), where the OD₄₂₀ was read from the supernatant of the reaction mixture.