aeruginosa mutants, KG7004 and KG7050, lacking quorum sensing and efflux protein genes were constructed by allele exchange using the plasmids listed in Table 1, as described previously [30, 35, 42]. Construction of P. aeruginosa mutants in this study followed the order: PAO1 to KG7001 with plasI (for deletion of lasI), KG7001 to KG7004 with pAF2071 (for deletion of rhlI), and KG7004 to KG7050 with pMexB (for deletion of mexB), respectively. Construction of QS reporter strains pSQG was

selleck screening library constructed by subcloning a 700-bp EcoRI digested fragment derived from pGreen into the KpnI site of mini-CTX1 [38, 39]. A lasB promoter-gfp translational fusion was constructed by ligating a 591-bp fragment including the region encoding N-terminal ten amino acids of LasB derived from the P. aeruginosa PAO1 chromosome. The resulting plasmid, pSQG003, was mobilized into KG7004 and KG7050 via E. coli S17-1. To accomplish excision, pFLP2, encoding Flp recombinase, was introduced into the P. aeruginosa KG7403 and KG7503

strains containing the lasB promoter-gfp translational fusion constructs by using the high transformation method and previously described procedures [40, 43]. In addition, the multicopy reporter plasmid pMQG003 was constructed. A lasB promoter-gfp translational fusion fragment from pSQG003 was cloned into pME6012 [41]. The lasB promoter-gfp translational fusion fragment Pritelivir solubility dmso was prepared by using PCR with the primers CTX1-F (5′-CGATAGATCTGCCGTCCTTGCTGAATTAGC-3′) and CTX1-R (5′-AACTAGATCTCGCTTTTGAAGCTGATGTGC-3′) containing an engineered restriction site BglII (forward and reverse). This fragment was restricted with BglII, and then ligated to the

BglII site of pME6012. Construction of the plasmids expressing the wild-type and mutant mexB genes in P. aeruginosa The stable E. coli–P. aeruginosa shuttle vector Rebamipide pKTA113 carrying mexB was constructed in three steps. The first mexB fragment amplified by PCR using the chromosomal DNA of P. aeruginosa PAO1 as a template and a pair of primers containing the engineered restriction sites HindIII (5′-ACATAAGCTTATGTCGAAGTTTTTCATTGATAGG -3′) and SalI (5′- GCAATCTAGATTGCCCCTTTTCGACGGACG -3′). Next, mexB fragments were ligated to the multicloning site of pUC18 to yield pYT06. To obtain the MexB expression plasmid, a 3138-bp HindIII-XbaI fragment from pYT06 was ligated to the large HindIII-XbaI fragment of pTO003. The resulting construct containing MexB-6His under the lac promoter shall be referred to as pKTA113 in this paper. To produce mexB mutants, the Stratagene Quickchange site-directed mutagenesis kit (Stratagene) was used according to the manufacturer’s protocol. The Phe136Ala or Asp681Ala substitution was introduced into pYT06, respectively. Then the mutated mexB fragments of the pYT06 mutants were www.selleckchem.com/products/th-302.html subcloned into pTO003.