AHL Extraction and Measurement

Acyl-homoserine lactone extraction was performed using a modified method of ethyl acetate liquid-liquid extraction (Elasri et al., 2001). From 1 mL of MOPS bacterial culture, cells were removed by centrifugation (10,000 × g, 5 min) and the supernatant was extracted once with ethyl acetate (1:1 v/v). The organic upper phase was evaporated to dryness, and the residues were resuspended into 15 μL of DMSO. As blank, sterile MOPS medium was extracted the same way.

Acyl-homoserine lactones were quantified in the extract using two reporter strains: P. putida KS35 and C. violaceum CV026. P. putida KS35 harbored an integrated transposon carrying a lasR gene controlled by a lac promoter and a gfp gene fused to lasB promoter which can be activated by LasR binding 3-oxo-C₁₂ HSL (Steidle et al., 2001). C. violaceum CV026 do not produce AHLs (cvI inactivated) but able to detect short-chain AHLs (McClean et al., 1997).

For 3-oxo-C₁₂ HSL detection, P. putida KS35 was precultivated overnight in LB (supplemented with 50 μg/mL kanamycin) at 30°C and then diluted to 1/10 in 1 mL of fresh LB with 5 μL of extract. After 8 h of culture at 30°C, the fluorescence was measured using a plate reader (SynergyHT, BioTek) with an excitation wavelength of 485 nm and emission detection at 528 nm. For C₄ HSL, C. violaceum CV026 was precultivated overnight in LB at 30°C and then diluted to 1/1,000 in 1 mL of fresh LB with 10 μL of extract. After 24 h of culture at 30°C, the violacein was quantified using a method based on ethyl acetate extraction (Collins et al., 1980). Briefly, 0.5 mL of cell culture was vigorously mixed with ethyl acetate (1:1 v/v) and then 200 μL of the organic upper phase were transferred into a quartz 96 well plate and the OD at 565 nm was measured. Results for each AHL measurement were plotted after background noise removal.