FIG. 1.

Detection of extracellular and intracellular acyl-HSLs produced by B. pseudomallei with the E. coli JB525 bioassay. Overnight (16-h) cultures of B. pseudomallei in 5 ml of AB medium (6) containing 0.2% glucose were centrifuged at 3,000 rpm for 15 min. Secreted acyl-HSLs were detected by adding 50 μl of the cell supernatant to the wells labeled U on LB agar. The cell pellets, each resuspended in 1 ml of fresh AB medium, were freeze-thawed (five cycles at −80°C for 3 min and 37°C for 1 min) to permeabilize the cells, and 50 μl of the cell extract was then added to the adjacent wells, labeled P. A gradient of acyl-HSLs diffusing from the wells after incubation at 30°C for 20 h was detected as green fluorescence by a row of 2-μl spots of E. coli JB525 when illuminated under UV. The direction of diffusion from the well is indicated by the arrow. E. coli DH5α was included as a negative control.