Briefly, 100 mg of extracted and purified rhamnolipids were suspended in 5 ml of 50 mM sodium acetate buffer, pH 4.1. To this solution was added 100 mg of naringinase from Penicillum decumbens (Sigma). The mixture was then kept at 50°C for 2 h with gyratory shaking (240 rpm), at which point 20 ml of buffer were added. After 24 h, HSP990 solubility dmso another 150 mg of naringinase were added as well as 25 ml of buffer. The reaction was kept under these conditions for 8 days. A final 50 mg of naringinase

in 20 ml of buffer were added to the mixture and was left for another 24 h. Thereafter, the solution was acidified to pH 3-4 using concentrated HCl and extracted three times with ethyl acetate. The fatty acid moieties generated by naringinase cleavage were then analyzed by LC/MS after the extract had been dried and evaporated. CMC – Surface tension assay Critical micelle concentration and surface tension were measured by the du Noüy ring method [50] using a surface tensiometer (Fisher). The instrument was calibrated against water and assays were performed in triplicate at room temperature. Swarming motility For swarming JQ-EZ-05 cell line assays, cultures were grown overnight, diluted

in fresh medium and subcultured until OD600~6.0 was reached. Swarm plates were prepared as follows: freshly autoclaved medium consisting of NB supplemented with 0.5% dextrose (Fisher) and 0.5% Bacto-agar (Difco) was poured into standard Petri dishes and dried under laminar flow for 30 min, as before [42]. Immediately following the drying period, plates were inoculated at their center with 5 μl of bacterial culture and placed at 30°C. For swarming phenotype restoration, 1, 5, 10 and 25 mg/L of purified B. thailandensis E264 rhamnolipids were deposited (10 μl) at the

center of respective plates and left to dry for 15 minutes before spot inoculation with swarming-deficient ΔrhlA mutant strains. For cross-feeding experiments, either equal parts of the cultures were mixed before being plated at the center on the swarm plate, or cultures were simply spotted side-by-side. Acknowledgements Special thanks to Marie-Christine Groleau and Ludovic Vial for insightful oxyclozanide comments and technical assistance as well as all members of ED laboratory for helpful discussions. This work was funded by NSERC discovery grants to FL and ED. DD was recipient of a Master’s Degree scholarship from The Fondation Armand-Frappier. References 1. Jarvis FG, Johnson MJ: A glyco-lipid produced by Pseudomonas aeruginosa. J Am Oil Chem Soc 1949,71(12):4124–4126. 2. Edwards JR, Hayashi JA: Structure of a rhamnolipid from Pseudomonas aeruginosa. Arch Biochem Biophys 1965,111(2):415–421.CrossRefPubMed 3. Kitamoto D, Isoda H, Nakahara T: Functions and potential applications of glycolipid biosurfactants–from energy-saving materials to gene delivery carriers. J Biosci Bioeng 2002,94(3):187–201.PubMed 4. Rahman PKSM, Gakpe E: Production, characterisation and applications of biosurfactants – Review.