of 40 cycles to test for the presence of a unique PCR product. The expression levels of mexA and PA3720 were normalized to that of the reference gene rpsL. Biological duplicates and technical triplicates were performed for all samples. To confirm the absence of genomic DNA contamination, `no-template’ controls were performed in technical triplicates for all primer sets employed. Expression and purification of polyhistidine -tagged NalC protein The nalC gene was amplified by PCR using primers: nalC-FwdNdeI and nalC-Rev-SalI in a 50 ml PCR reaction that contained 1 mg P. aeruginosa strain K767 chromosomal DNA, 0.2 mM each dNTP, 3% DMSO, 16 PhusionH High Fidelity buffer and 1 U PhusionH polymerase. The reaction mixture was heated for 30 sec at 94uC followed by 30 cycles of 30 sec at 94uC, 30 sec at 54uC and 30 sec at 72uC, before finishing with 10 min at 72uC. The nalCcontaining PCR product was purified, digested with SalI and NdeI and cloned into NdeI-XhoI-restricted pET23a to yield pLMS3 encoding His-tagged NalC. Following nucleotide sequencing of the cloned gene to confirm the absence of PCR-generated mutations, plasmid pLMS3 was introduced into E. coli BL21 carrying the pLysS plasmid and NalC-His production induced with IPTG using a modified version of a previously-described protocol. Briefly, an overnight culture of E. coli BL21 carrying pLysS and pLMS3 was diluted 1:49 in LB supplemented with the appropriate antibiotics and incubated at 37uC until the optical density at 
600 nm reached 0.50.6, at which time IPTG was added and incubation continued a further 2 hr. Cells were harvested by centrifugation at 4uC and pellets were resuspended in 6 ml buffer A containing 5 mM imidazole and sonicated. Pentachlorophenol Induction of mexAB-oprM Following centrifugation for 60 min at 160006g, the NalC-Hiscontaining supernatant was mixed with 500 ml of Ni-NTA Agarose resin equilibrated with 10 ml buffer A containing 5 mM imidazole and incubated, with shaking, for 10 min. The resin was subsequently pelleted by centrifugation and washed twice with 10 ml buffer A containing 5 mM imidazole and once with 2 ml buffer A containing 5 mM imidazole, the resin again being centrifuged after each wash. Bound protein was eluted stepwise with 500 ml buffer A containing increasing amounts of imidazole; at each step the resin was incubated with PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22189475 shaking at room temperature and centrifuged as above. The NalC-His protein was recovered in the supernatant following elution with buffer A containing 250 mM imidazole. Protein concentration was determined using the BCA Protein Assay Kit, and purified protein was stored at 220uC in 20% glycerol. Electromobility shift assay The binding of purified NalC and MexR to PCR-amplified target DNAs was assessed using the electromobility shift assay as described previously. Briefly, 50 ng target DNA was incubated with purified NalC or MexR for 20 min at room temperature in a 15 ml reaction mixture containing 16 binding buffer. Following the addition of EMSA gel-loading solution, mixtures were CX4945 biological activity separated by electrophoresis on a non-denaturing 8% polyacrylamide gel in 0.5 TBE buffer and gels were stained with 16SYBR Green EMSA nucleic acid stain. DNA was then visualized using digital photography with a S6656 SYPRO photographic filter. NalC target DNAs included the entire nalC-PA3720 intergenic region, ca. 100 bp fragments corresponding to the PA3720-distal and -proximal halves of this region as well as shorter oligonucleotides correspond