Plasmid construction for the constitutive expression of P. aeruginosa genes

All the primers used for plasmid construction are listed in table S2. To construct the plasmid for constitutive expression of gtrS, a ~1.5-kb PCR product covering 72 base pairs (bp) of the gtrS upstream region, the gtrS gene, and 9-bp downstream of gtrS was amplified from MPAO1 genomic DNA using primers gtrS-comp-F (with Hind III site) and gtrS-comp-R (with Bam HI site). For generating pAK1900-gltR (p-gltR), a ~0.77-kb PCR product covering 38 bp of the gltR upstream region, the gltR gene, and 7-bp downstream of gltR was amplified from MPAO1 genomic DNA using primers gltR-comp-F (with Hind III site) and gltR-comp-R (with Kpn I site). For generating pAK1900-gltR-gtrS (p-gltR-gtrS), a ~2.3-kb PCR product covering 38 bp of the gltR upstream region, the gltR and gtrS genes, and 9-bp downstream of gtrS was amplified from MPAO1 genomic DNA using primers gltR-comp-F (with Hind III site) and gtrS-comp-R (with Bam HI site). For generating pAK1900-gltB (p-gltB), a ~1.3-kb PCR product covering 40 bp of the gltB upstream region, the gltB gene, and 15-bp downstream of gltB was amplified from MPAO1 genomic DNA using primers pa3190-comp-F (with Hind III site) and pa3190-comp-R (with Kpn I site). All the products were digested with the indicated enzymes and cloned into pAK1900 (58), and the direction of transcription of the cloned genes is in the same orientation as plac on pAK1900.

To generate pRK415-bfmS, a ~1.4-kb PCR product covering 20 bp of the bfmS upstream region, the bfmS gene, and 15-bp downstream of bfmS was amplified from MPAO1 genomic DNA using primer pair bfmS-comp-F/bfmS-comp-R (Hind III/Bam HI sites). For generating pRK415-gtrS, the primer pair gtrS-comp-F/gtrS-comp-R and MPAO1 genomic DNA were used. All the PCR products were cloned into pRK415 (59), and the direction of transcription of the cloned genes was in the same orientation as the plac promoter on pRK415.

For generating mini-bfmS, mini-gltR-gtrS, mini-gltR, and mini-gtrS, primers pAK1900-mini-F (with Xho I site) and pAK1900-mini-R (with Xba I site) were used. Briefly, a ~1.7-kb PCR product covering the lac promoter of pAK1900 plasmid and the bfmS was amplified from p-bfmS plasmid (table S1) DNA, and the PCR products were cloned into integrated mini-CTX-lacZ vector. For generating mini-gltR-gtrS, a ~2.5-kb PCR product covering the lac promoter of pAK1900 plasmid and the gltR-gtrS was amplified from p-gltR-gtrS plasmid (table S1) DNA. For generating mini-gltR, a ~1-kb PCR product covering the lac promoter of the pAK1900 and the gltR was amplified from p-gltR plasmid (table S1). For generating mini-gtrS, a ~1.8-kb PCR product covering the lac promoter of the pAK1900 and the gtrS was amplified from p-gtrS plasmid DNA (table S1). The PCR products were cloned into integrated mini-CTX-lacZ vector (54). All constructs were sequenced to prevent unwanted mutations.