FIG. 3.

Comparison of intracellular and extracellular acyl-HSLs produced by wild-type B. pseudomallei KHW and the bpeAB null mutant by reversed-phase HPLC. Radiolabeled [¹⁴C]acyl-HSLs was extracted from 5-ml cultures of KHW, KHW ΔbpeAB mutant, and KHW(pUCP28TbpeR), as described in the legend to Fig. 2. The extract was dried and dissolved in 200 μl of 20% (vol/vol) methanol before the application of 100 μl into a C₁₈ reversed-phase HPLC column. Two-milliliter fractions were collected during elution at a flow rate of 1 ml/min, with an isocratic profile of methanol-water (50:50, vol/vol) for 10 min, followed by a linear gradient of 50 to 90% methanol in water for 25 min and an isocratic profile of 90% methanol over 10 min. (A) [¹⁴C]acyl-HSLs secreted into the culture supernatants of untreated stationary-phase bacterial cultures. (B) [¹⁴C]acyl-HSLs extracted from freeze-thaw-permeabilized bacterial cells. Peaks 1, 2, 3, 4, 5, 6, and 7 were detected with a Radiomatic 500TR Radioisotope HPLC detector (Perkin-Elmer) and correspond to the elution profiles of l-methionine, C8HSL, C10HSL, 3-hydroxy-C8HSL, 3-oxo-C10HSL, 3-hydroxy-C10HSL, and 3-oxo-C14HSL, respectively. The HPLC profiles of acyl-HSLs extracted from culture supernatants of wild-type KHW and its bpeAB deletion mutant are represented by closed and open squares, respectively. C8HSL, C10HSL, and methionine were purchased from Sigma, while 3-oxo-C10HSL and 3-oxo-C14HSL were synthesized by a microwave-assisted solid-phase synthetic route to acyl-homoserine lactones (8) and 3-hydroxy-C8HSL and 3-hydroxy-C10HSL were synthesized by the Reformatsky reaction (9).