The Burkholderia pseudomallei KHW quorum-sensing systems produced N-octanoyl-homoserine lactone, N-decanoyl-homoserine lactone, N-(3-hydroxy)-octanoyl-homoserine lactone, N-(3-hydroxy)-decanoyl-homoserine lactone, N-(3-oxo)-decanoyl-homoserine lactone, and N-(3-oxo)-tetradecanoyl-homoserine lactone. The extracellular secretion of these acyl-homoserine lactones is dependent absolutely on the function of the B. pseudomallei BpeAB-OprB efflux pump.Burkholderia pseudomallei, a gram-negative soil bacillus, is the causative agent of melioidosis, a severe and potentially fatal emerging tropical infectious disease. Its intrinsic resistance to many antibiotics is attributed mostly to multiple multidrug efflux pumps, such as AmrAB-OprA, BpeAB-OprB, and BpeEF-OprC, which efflux aminoglycosides, macrolides, chloramphenicol, and trimethoprim (4,12,14). We hypothesized that these pumps also efflux physiological compounds that could compromise the fitness of the bacterium when accumulated to high intracellular concentrations and propose that this exerts a positive selection pressure for persistence even in the absence of antimicrobials (11).B. pseudomallei is reported to produce up to six different types of acyl-homoserine lactones (acyl-HSLs), the composition of which may differ slightly from strain to strain. For instance, strain PP844 secreted N-octanoyl-homoserine lac-, and N-(3-hydroxy)-dodecanoyl-homoserine lactone (3-hydroxy-C12HSL), while strain DD503, an amrAB-oprA efflux pump operon deletion mutant derived from 1026b, secreted only C8HSL, 3-hydroxy-C8HSL, and C10HSL (13,(17)(18)(19). It is unclear how acyl-HSLs move across the B. pseudomallei cell membranes and into the extracellular compartment, although in Pseudomonas aeruginosa, the shorter-chain acylHSLs appear to do so by diffusion while the longer-chain acyl-HSLs are secreted by multidrug efflux pumps (1, 15). In B. pseudomallei KHW, the expression of bpeAB-lacZ was induced by acyl-HSLs, and the bpeAB mutant failed to secrete any extracellular acyl-HSLs when it was cross-streaked against the JB525 reporter strain (3).Extracellular secretion of acyl-HSLs is dependent on BpeAB-OprB. In order to ascertain whether a blockage in the efflux mechanism, an inhibition of acyl-HSL synthesis, or both had contributed to the absence of extracellular acyl-HSLs in the bpeAB mutant, we compared the acyl-HSLs produced in cell supernatants of B. pseudomallei before and after the bacterial cells were permeabilized by a freeze-thaw procedure. Wild-type B. pseudomallei KHW, KHW carrying a plasmid overexpressing the BpeR repressor, the bpeAB null mutant, and a complemented bpeAB mutant were used in the comparison (Table 1), and a modified cross-streak bioassay was used for detection of acyl-HSLs (2). The culture supernatants of unpermeabilized wild-type KHW and the complemented bpeAB mutant contained acyl-HSLs but not those of the bpeAB mutant and the bpeR-overexpressing strain, both of which had impaired BpeAB-OprB function (Fig. 1, lanes U). Acyl-HSLs were detected in the cell sup...