coli strains. The strains E. coli W4680AE (ΔacrA, ΔacrB, ΔacrE, and ΔacrF) and E. coli strain 5X RND (ΔacrB, ΔacrD, ΔacrF, ΔmdtF, and ΔmdtBC) were used for further analysis in addition to E. coli W4680AD. Escherichia coli W4680AD expressing

the gold-efflux system from pGesAB exhibited strong resistance toward 12 chemicals (Table 2). The classes of compounds included β-lactams, the bacteriostatics chloramphenicol and thiamphenicol, several other antimicrobials, a surfactant, and a protein kinase C inhibitor. Although the cloning vector contained coding sequences for β-lactamase and chloramphenicol acetyltransferase (Soncini et al., 1995), the resistance observed for β-lactams, chloramphenicol, and thiamphenicol was much greater than the empty vector control. Moderate resistance was detected in approximately the same BYL719 cost number of chemicals and consisted mostly of antibiotics, fungicides, a cationic surfactant, and a DNA mutagen. Of the chemicals initially identified from the Biolog screen, chloramphenicol, chlorquinaldol, and dichlofluanid were chosen for further analysis. Methylene blue and crystal violet were previously reported to be substrates of GesABC in Salmonella typhimurium (Nishino et al., 2006; Pontel et al., 2007), and were also tested here. All three E. coli strains expressing GesAB showed chloramphenicol resistance in the liquid

media tests (Fig. S3). MIC analysis Buparlisib molecular weight showed that the level of resistance increased fourfold in E. coli strain 5X RND and eightfold in strains W4680AD and W4680AE (Table 4). Chloramphenicol had not been identified as a substrate of the Ges system previously. Moreover,

chlorquinaldol resistance was detected in all the tested strains expressing GesAB in liquid media tests (data cAMP not shown). Escherichia coli strains W4680AD and W4680AE carrying pGesAB were resistant to chlorquinaldol with a twofold increase MIC value, via the agar results. The discrepancy between growth in the Biolog assay and MIC assays in LB medium could be attributed to differing growth conditions (media, incubation time, detection method). Crystal violet and methylene blue, which was not present in the Biolog panels, were tested because GesABC conferred resistance to both compounds in Salmonella (Nishino et al., 2006; Pontel et al., 2007). Previous studies have shown that the MIC values for crystal violet and methylene blue are 8- and 16-fold greater when the gold efflux system is overexpressed in a ΔgesABC, ΔacrB knockout (Nishino et al., 2006). Here, only the MIC value of E. coli W4680AD containing pGesAB exposed to crystal violet was greater than the control, but gesAB expressed in E. coli strains W4680AE and 5X RND did not show any difference from the vector control. It is possible that the level of expression of gesAB in these backgrounds is not sufficient to detect a difference in the MIC values when compared with the vector controls.