185. This mutant strain is designated MGL01. To confirmed constitutive expression of the mexEF-oprN operon in MGL01, posttranscriptional status of mexE mRNA was assessed by qRT-PCR. Compared to the wild-type strain PA14, transcription of the mexE gene is increased 21- and 12-fold at OD600 of 1.4 and 4.0, respectively. The fold change was calculated using the 22DDCt method. The nadB gene was used as a housekeeping control. To complement the mexS2 mutation, the mexS gene was amplified using the inMexT-F and PA14_32440-R primers and MedChemExpress PG 490 cloned into the pGEM-T easy vector. The NotI fragment of the pGEM-T easy::mexS construct was then cloned into the NotI restriction site of the mini-CTX1 plasmid, giving pML01. This construct was then introduced into competent E. coli SM10lpir for mobilization into P. aeruginosa MGL01 by conjugation, to give MGL01. Construction of the mexS2 mexE2 double mutant. Phusion High fidelity DNA polymerase was used for all DNA amplifications. A genomic fragment containing the mexE gene was amplified using primers pmexE-F and mexE-R2 and cloned into the pGEM-T easy vector. An inframe deletion within the mexE gene was performed by PCR using the outward primers mexE59Ext-kpnI and mexE39Ext-kpnI, both containing a 59-end KpnI restriction site. The KpnI digest of the PCR product was circularized using T4 ligase. Then, the SacI-SphI fragment from the resulting construct was inserted into the suicidal vector pEX18Ap cut with the same enzymes, to generate pML02. pML02 was mobilized into PA14 mexS::Tn5 using the E. coli SM10lpir donor strain. Merodiploids were selected on TSB 9 September 2011 | Volume 6 | Issue 9 | e24310 MexEF-OprN Exports HHQ agar containing carbenicillin and tetracycline and then DmexE mutants were resolved on plates containing 7% sucrose. The mexS2 mexE2 double mutant is designated strain MGL03. The PA14 mexE2 single mutant was generated using the same strategy. Construction of chromosomal pqsH-lacZ transcriptional reporter. The pqsH promoter region was amplified using the ppqsH-F and ppqsH-R primers, which contain “2987739 the restriction sites KpnI and BamHI, respectively. The resulting 512 pb fragment was cloned into the corresponding sites of the mini-CTX-lacZ plasmid, giving pML03, which was then introduced into competent E. coli SM10lpir for mobilization into P. aeruginosa PA14 and MGL01 by conjugation, to give PA14 and MGL01, respectively. Clones carrying chromosomal insertion of the mini-CTX constructions were selected on tetracycline TSB plates. The tetracycline resistance cassette was excised from the chromosome using the flipase expressed from plasmid pFLP2. Construction of constitutively expressed pqsH gene. The pqsH gene was amplified using primers pqsH-sacI-F and the pqsHHindIII-R. The resulting 1149 bp fragment was cloned into the SacI and HindIII restriction sites of the pUCP26 plasmid, giving pML04. b-galactosidase assay. b-galactosidase activity “8496905 was measured as described previously by Miller with slight modifications. Briefly, cells were grown in TSB to various cell densities and then diluted in Z buffer. Cells 
were permeabilized by the addition of one drop of 0.1% SDS and two drops of chloroform. Then, 200 mL of ONPG 4 mg/ml was added to each reaction. Color development was monitored at 420 nm and b-galactosidase activity was expressed in Miller units, calculated as follows: 1,0006OD420/T 6V 6OD600. Detection and measurements of HAQs by LC/MS. Except when specified, 300 mL culture samples were take