FIG. 2.

Intracellular accumulation of exogenous [¹⁴C]acyl-HSLs. Radiolabeled [¹⁴C]acyl-HSLs were prepared by adding 5 μCi of l-[1-¹⁴C]methionine (Sigma) to a 16-h culture of B. pseudomallei in AB medium containing 20 mM glycerol for 30 min. [¹⁴C]acyl-HSLs were then extracted from the culture supernatants by acidified ethyl acetate, according to a procedure described previously (16). Two hundred microliters of the [¹⁴C]acyl-HSLs was added to early-log-phase cells (optical density at 600 nm, ∼0.2), and 0.2-ml aliquots were removed after 30, 60, and 90 min for scintillation counting. The cells were washed three times with saline containing 100 nM C8HSL to remove extracellular radiolabeled acyl-HSLs, air dried, and solubilized in 2 ml of scintillation cocktail (Amersham Biosciences) for liquid scintillation counting with an LS6500 multipurpose scintillation counter (Beckman Instruments Inc., Fullerton, CA). White bars, wild-type B. pseudomallei KHW; black bars, KHW ΔbpeAB mutant; stippled bars, KHW bpeR::Km null mutant; gray bars, KHW(pUCP28TbpeR).