on, ligation, transformation, plasmid isolation, agarose gel electrophoresis, and preparation of chemically competent E. coli cells as described by Sambrook and Russell. Electrocompetent P. aeruginosa cells were prepared as described by Choi and Shwiezer. Chromosomal DNA was get SU11274 extracted from P. aeruginosa using the DNeasy Tissue Kit. Plasmid DNAs were also prepared from E. coli or P. aeruginosa using a GeneJET Plasmid Miniprep kit according to a protocol provided by the manufacturer. The WizardH SV Gel and PCR Clean-Up System kit was used to purify PCR products and to gel-purify DNA fragments generated by restriction endonuclease digestion. Oligonucleotides were synthesized by Integrated DNA Technologies and nucleotide sequencing was performed by AGCT Corp.. Materials and Methods Bacterial strains and plasmids Susceptibility testing The antimicrobial susceptibilities of the various P. aeruginosa strains were assessed in 96-well microtiter plates using twofold serial dilutions as described. In assessing the impact of PCP exposure on antimicrobial susceptibility, 0.75 mM PCP was included in the growth medium used to prepare the bacterial inoculum and to generate the serial dilutions. Quantitative RT-PCR Overnight cultures of P. aeruginosa strains were subcultured in fresh L-broth and incubated at 37uC with shaking for 2.5 hours, at which time cells were harvested by centrifugation. In some experiments, PCP was added 1 to 1.5 hr before harvesting. Total bacterial RNA was isolated from 1 ml of late-log phase culture using the RiboPureTM Bacteria kit or the High Pure RNA Isolation Kit using the manufacturers’ protocols, and resuspended in 50 ml elution buffer. Samples were treated with Turbo DNA-Free. DNA-free RNA was used to synthesize cDNA using an iScript cDNA Synthesis kit in a reaction mixture formulated as described by the kit manufacturer and incubated for 5 min at 25uC, 30 min at 42uC and 5 min at 85uC. cDNA was then stored until needed at 220uC. Quantification of the cDNA Pentachlorophenol Induction of mexAB-oprM Strain Relevant characteristic Reference or source E. coli DH5a K113 K114 S17-1 Sm10 NovaBlue w80D lacZDM15 endA1 recA1 hsdR17 supE44 thi-1 gyrA96 relA1 F2 DU169 BL21 BL21 thi pro hsdR recA Tra+ thi-1 thr lec tonA lacY supE recA::RP4-2-Tc::Mu; Kmr lpir recA2 endA2 lacIq Novagen P. aeruginosa K767 K1454 K2276 K2568 K3145 K3146 K3130 K3151 Plasmids pET23a pKLE1 pLMS3 pEX18Tc pLC8 pLMS2 pMF1 His-tag expression vector: Apr pET23a::mexR pET23a::nalC Broad-host-range gene replacement vector; sacB; Tcr pEX18Tc::DarmR pEX18Tc::DPA3720 pEX18Tc::DPA3720-DarmR Novagen This study This study This study PAO1 prototroph Spontaneous nalC mutant of K767 K1454 DarmR 
K767 DmexR K767 DarmR K767 DPA3720 K767 DPA3720-DarmR K1454 DPA3720 This study This study This study This study Apr, ampicillin resistant; Kmr, kanamycin resistant; Tcr, tetracycline resistant. doi:10.1371/journal.pone.0032684.t001 was carried out using a Bio-Rad, CFX96TM Real-Time PCR Detection System. PCR amplification reactions were performed in 20 ml reaction volumes each containing 10 ml of SsoFast EvaGreen Supermix, 0.6 mM each of 2 primers per gene being amplified and PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22189973 5 ml of 1:49 diluted cDNA. Following an initial 3-min denaturation at 95uC, the mixture was subjected to 40 cycles of 10 sec at 95uC and 30 sec at 60uC. A melt curve, obtained following an initial 10-sec treatment at 95uC and involving 5-sec incubations of 0.5uC increments beginning at 65uC, was run at the end
