The trimethoprim resistance marker was amplified from the pFTP1 plasmid (a gift from H. P. Schweizer, Colorado State University) using primers rhlATp1F and rhlATp1R [47, 48]. Cells of the single ΔrhlA mutant were rendered competent using DM medium and then exposed to various concentrations of the mutagenic PCR fragment. Double ΔrhlA

mutants were selected on TSB agar containing 150 μg/ml tetracycline and 100 μg/ml trimethoprim. The B. thailandensis ΔrhlA double mutant was confirmed by diagnostic PCR to verify proper recombination and insertion of the resistance marker. Absence of rhamnolipid production by LC/MS analysis also served as a confirmation. Preparation PF-01367338 molecular weight of culture samples for LC/MS analysis To prepare samples for LC/MS analysis, the culture samples were firstly centrifuged to remove cells (16,000 × g, 15 min). To the cell-free supernatant was then added either 16-hydroxyhexadecanoic

acid or deuterium-labeled 4-hydroxy-2-heptylquinoline (HHQ-D4) [49] as internal standards used for quantitative measurements, both at a final concentration of 10 mg/L. For the highly pathogenic B. pseudomallei, cell-free supernatants were Alvocidib solubility dmso obtained by centrifugation (16,000 × g, 15 min) followed by filtration on a 0.22 μm filter. Twenty μl of samples were injected for LC/MS analysis. Quantification was performed by integration of the pseudomolecular and the proper fragment ions and the use of dose-response calibration curves using purified Selleckchem PCI-32765 rhamnolipids. Rhamnolipid analysis (LC/MS) All rhamnolipid quantifications and analyses were performed using a Quattro II (Waters, Mississauga, Ontario, Canada) triple-quadrupole mass spectrometer in negative electrospray ionization mode coupled

to an HP 1100 (Agilent Technologies, Saint Laurent, Quebec, Canada) high-performance liquid chromatograph (HPLC) equipped with a 4.6 × 50 mm 300SB-C3 Zorbax 5 μm (Agilent) reverse-phase column. The HPLC flow rate was set at 400 μl/min and was split to 10% by the means of a Valco Tee prior to being introduced into the mass spectrometer. An acetonitrile-water gradient containing 2 mM of ammonium acetate was used starting with 25% acetonitrile during the first 5 min, raised to 50% by 18 min and 100% by 19 min. This concentration was held until Selleck Erlotinib 22 min, where the initial concentration was resumed and kept until 26 min. Voltage of the capillary was set to 3.5 kV and cone voltage to 30 V. The temperature of the source block was kept at 120°C. Scan mass range was set from 130 to 940 Da. A calibration curve was performed to determine the long chain rhamnolipid response factor. During LC/MS/MS experimentation, fragmentation of the molecules were induced with argon serving as the collision gas at 2 × 10-3 mTorr. Enzymatic hydrolysis of rhamnolipids – Naringinase To study the rhamnolipid congeners produced by B.