TABLE 1.
Strains, plasmids, and primers
| Strain, plasmid, or primer | Relevant information | Source or reference |
|---|---|---|
| E. coli strains | ||
| DH5α | hsdR recA lacZYA φ80 lacZΔM15 | GIBCO-BRL |
| XL1 Blue | recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′ proAB lac1qZΔM15 Tn10 (Tetr)] | Stratagene |
| S17-1 | RP4-2 Tc::Mu Km::Tn7 Tpr Smr Pro Res− Mod+ | 27 |
| P. aeruginosa strains | ||
| PAK | Wild-type clinical isolate | D. Bradley |
| PAK fleQp-lacZ | PAK with fleQp-lacZ in the attB site | This study |
| PAK fleQpmutV-lacZ | PAK with fleQpmutV-lacZ in the attB site | This study |
| PAK lacZ | PAK with lacZ in the attB site | This study |
| PAO1 | Wild type | 14 |
| PAO fleQp-lacZ | PAO with fleQp-lacZ in the attB site | This study |
| PAO fleQpmutV-lacZ | PAO with fleQpmutV-lacZ in the attB site | This study |
| PAO lacZ | PAO with lacZ in the attB site | This study |
| PA103 | Prototroph, exotoxin A hyperproducer, elastase deficient | 18 |
| PAO9001 | Δvfr::Gentr derivative of strain PAO1 | 2 |
| PAO9001 fleQp-lacZ | PAO9001 with fleQp-lacZ in the attB site | This study |
| Plasmids | ||
| pMMB66HE/EH | Broad-host-range tacP expression vector, lac1q Apr Cbr | 11 |
| pWNP28 (ptac917Δp) | pMMB66HE carrying the 0.9-kb NarI fragment containing vfr region from positions −77 to +999, Apr Cbr | 2 |
| pDN19lacΩ | Promoterless lacZ oriV oriT Tetr StrrΩ fragment | 7 |
| placΩQ | pDN19lacΩ containing the fleQ promoter region as a 720-bp EcoRI-BamHI insert | 3 |
| placΩQmutS8 | placΩQ with 8 bp in the −10 region of the fleQ promoter mutated | This study |
| placΩQmutS4 | placΩQ with 4 bp in the −10 region of the fleQ promoter mutated | This study |
| pBluescript II/KS(+) | General-purpose cloning vector, Apr | Stratagene |
| pBS-fleQp | pBluescript II/KS(+) containing the fleQ promoter region as a 720-bp EcoRI-BamHI nsert | This study |
| pBS-fleQpmutV | pBS-fleQp with an ACA→CGC mutation in the Vfr binding site | This study |
| mini-CTXlacZ | Integration-proficient vector for single-copy chromosomal lacZ fusion | 4 |
| CTX fleQp-lacZ | Insert from pBS-fleQp cloned into mini-CTXlacZ | This study |
| CTX fleQpmutV-lacZ | Insert from pBS-fleQpmutV cloned into mini-CTXlacZ | This study |
| pFLP2 | Plasmid encoding the FLP recombinase, Cbr | 4 |
| Primersa | ||
| fleQ2F | 5′ ATCGGTCGACTTTTTTATTGCCTCT 3′ | |
| fleQ2R | 5′ CGATCTCGAGATCGAATGGGTCTCGCTC 3′ | |
| fleQ1F | 5′ CAGCAAAAAAAACACGCC 3′ | |
| fleQ1R | 5′ GCCTATTTCTAATAAGCAGCC 3′ | |
| Qpmut1 | 5′ CCGCCATAAATTGACTAATCGTTCCGCTTTGACTTAACTAGTGGCC 3′ | |
| Qpmutcom2 | 5′ GGCCACTAGTTAAGTCAAAGCGGAACGATTAGTCAATTTTATGGCGTG 3′ | |
| Qp70mut1 | 5′ GAAACAGCAAAAAAAACACGCCGCGCGCGCGACTAATCGTTCACATTTGACTTA 3′ | |
| Qp70mutcom2 | 5′ TAAGTCAAATGTGAACGATTAGTCGCGCGCGCGGCGTGTTTTTTTTGCTGTTTC 3′ | |
| Qp70mut3 | 5′ CAGCAAAAAAAACACGCCATAGCGCTGACTAATCGTTCACATTTGAC 3′ | |
| Qp70mutcom4 | 5′ GTCGTTTTTTTTGTGCGGTATCGCGACTGATTAGCAAGTGTAAACTG 3′ | |
| RT2 | 5′ CAGCTGCCTTGCATCGAATGGGTCTCGC 3′ |
a
Bases mutated in the primers are underlined.