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Triclosan is an antimicrobial found in deodorants, toothpaste, hand lotions, and acne treatments, along with many other household products and is an effective inhibitor of both Gram-positive and Gram-negative species (8). Recently, there has been increased research into the mechanisms whereby bacteria become resistant to the antimicrobial effects of triclosan. McMurry et al. (10,11) identified the enoyl-acyl carrier protein (ACP) reductase, FabI, as the main intracellular enzyme target of triclosan in E. coli. It has also been shown, for example, that some bacteria contain orthologous enoyl-ACP reductases, namely FabL and FabK, which are not inhibited by triclosan (6,7). Bacteria that contain these alternate enzymes are able to tolerate higher concentrations of triclosan than those bacteria that have only the FabI-type enoyl-ACP reductase (6,7). The construction of a vector with a triclosan resistance determinant would make triclosan a useful addition to the selection agents that are currently available. Triclosan is effective against a range of bacteria at less than 10 ppm (8). In E. coli, even mutants with increased resistance are still inhibited at approximately 10 ppm triclosan (10, 11). Triclosan is also nontoxic to humans (3,8). These characteristics make triclosan an attractive selective agent. However, no cloning vectors are currently available with triclosan resistance determinants. Here we report the construction of a broad host range plasmid that is amenable to blue-white screening and that contains a triclosan resistance marker. The enoyl-ACP reductase gene fabL (6), including the promoter, was amplified by PCR from Bacillus subtilis chromosomal DNA using the primers YgaABam (5′-GGATCCTTAAACGAGCAGTGAGCGTCCGCCGTC-3′) (6) and YgaABam3 (5′-GGATTTTAAAGTTCTTGCCA-3′). Both primers were designed with BamHI sites (in bold) on the 5′ ends. PCR product was ligated into pGEM-T® Easy (Promega, Madison, WI, USA) and transformed into One-Shot (Invitrogen, Carlsbad, CA, USA) chemically competent cells following the manufacturers’ instructions. Clones containing the fabL insert were identified through gel electrophoresis of BamHI-digested plasmid. The fabL fragment was purified from the agarose gel and ligated into BglII-digested pBBR1-MCS2 (9). The ligation reaction was electroporated into E. coli S17 cells, and transformants were selected on LB agar containing 50 ppm kanamycin and 10 ppm triclosan. Several transformants were obtained, and three were analyzed by restriction digest with HindIII. All contained fabL in the same orientation. This new vector was called pBBRT (Figure 1). E. coli S17 containing pBBRT was streaked on LB containing up to 10 000 ppm triclosan to determine the level of resistance. Also, growth curves of E. coli S17 with or without pBBRT were determined in liquid LB medium at varied concentrations of triclosan. In a 96well microplate, overnight culture of either S17 or S17 pBBRT was inoculated into LB containing 0–100 ppm triclosan. Absorbance at 600 nm was measured over time in a MicroQuant (Bio-Tek® Instruments, Winooski, VT, USA) spectrophotometer with KC Junior software (Bio-Tek Instruments). E. coli S17 pBBRT was able to grow in liquid media containing up to 25 ppm triclosan. At 50 ppm and higher, growth of S17 pBBRT was slow, although not completely inhibited (data not shown). The parental E. coli S17 strain has a minimum inhibitory concentration of less than 1 ppm (data not shown). On LB agar plates, however, S17 pBBRT was able to grow in the presence of up to 10 000 ppm triclosan, whereas the parent strain could not grow on plates containing 2 ppm triclosan. The tolerance of higher triclosan levels by S17 pBBRT on the plate may be due to diffusional constraints of the excess triclosan, as triclosan is only soluble to approximately 10 ppm in water (4). To demonstrate the utility of this construct in other organisms, pBBRT was also transferred Benchmarks

Construction of a Broad Host Range Cloning Vector Conferring Triclosan Resistance