FIG. 2.

Maps of plasmids used for allelic exchange. The I-SceI delivery plasmids pEXKm4 and pEXKm5 contain the following common features: gusA, gene encoding E. coli glucuronidase; lacZα, E. coli LacZα peptide-encoding gene for X-Gal-based blue-white screening with the indicated unique cloning sites; nptII, gene coding for neomycin phosphotransferase, which confers kanamycin resistance; ori, E. coli pMB9 origin of replication; oriT, RK2-derived origin for conjugal plasmid transfer; P1, Pseudomonas integron promoter; P_lac, E. coli lac operon promoter; T₀T₁, transcriptional terminators T₀ and T₁ from bacteriophage λ and E. coli rrnB operon, respectively. pEXKm5 additionally contains sacB, a Bacillus subtilis levansucrase-encoding gene optimized for expression and localization in B. pseudomallei by inclusion of the predicted B. pseudomallei groES promoter (P_groES) and the B. mallei sacB leader sequence (9). The I-sceI source vector pBADSce also contains the following features: I-sceI, gene encoding I-SceI endonuclease; ble, bleomycin resistance gene, which confers zeocin resistance; ori₁₆₀₀, pRO1600 origin of replication requiring the rep(Ts)-encoded replication protein, which confers a temperature-sensitive phenotype in Burkholderia spp. at temperatures of 37°C and above; P_BAD, the arabinose-inducible E. coli araBAD operon promoter controlled by the araC-encoded regulatory protein; P_EM7, synthetic prokaryotic promoter.