TABLE 1.
Strains, plasmids, and primers
| Strain, plasmid, or primer | Genotype or description | Source or reference |
|---|---|---|
| Strains | ||
| E. coli | ||
| DH5α | F− φ80 δlacZ ΔM15 endA1 recA1 hsdR17 supE44 thi-1 relA1 Δ(lacZYA-argF) gyrA96 deoR | GIBCO-BRL |
| P. aeruginosa | ||
| PAK | Wild-type clinical isolate | David Bradley |
| PAK1-3 | A spontaneous mutant derivative of PAK, Neor | 31 |
| PAK(aph) | PAK with aph mutated by gentamicin cassette insertion | This study |
| PAK(hpaA) | PAK with hpaA mutated by Ω insertion | This study |
| Plasmids | ||
| pUCP19 | Broad-host-range plasmid, Cbr | 32 |
| pWC001 | 2-kb DNA containing aph insert cloned into pUCP19 | This study |
| pEx18Tc | Sucrose selection suicide delivery plasmid, Tcr | 10 |
| pExaph | pEx18Tc containing 1.7-kb BamHI fragment from pWC001 | This study |
| pExaphG | pExaph with gentamicin cassette inserted at NsiI site disrupting aph gene | This study |
| pDN19lacΩ | A lacZ fusion plasmid vector, IncP, Spr/Smr/Tcr | 30 |
| pCR2.1-TOPO | TA-cloning vector, Apr Kmr | Invitrogen |
| pWC003 | 1.5-kb PCR product (HpaA5-Aph3) of hpaA cloned into pCR2.1-TOPO | This study |
| pWC011 | pDN19lacΩ containing 1.6-kb fragment from pWC003, aph::lacZ fusion | This study |
| pWC012 | pDN19lacΩ containing 1.1-kb fragment from pWC003, hpaA::lacZ fusion | This study |
| pWC013 | pDN19lacΩ containing 1.6-kb fragment from pWC003, PA4121::lacZ fusion with intact hpaA | This study |
| pWW001 | 1.0-kb PCR product (Aph5-Aph3) of aph cloned into pCR2.1-TOPO | This study |
| pWC014 | pDN19lac14 containing 1.1-kb fragment from pWW001, aph::lacZ fusion | This study |
| pWC018 | PA4121::lacZ fusion, without hpaA | This study |
| pEx18Ap | Sucrose selection suicide delivery plasmid, Apr | 10 |
| pExhpaA | pEx18Ap containing XbaI/SacI fragment from pWC003 | This study |
| pWC021 | pExhpaA with its BglII fragment replaced by Ω insertion | This study |
| Primers | ||
| HpaA5 | 5′ CGTTGACGATCACGTAGCCGGCGACAT 3′ | |
| Aph5 | 5′ GAGCGCCAGCCGATCCCCAACATCAAC 3′ | |
| Aph3 | 5′ CTCGCCAGCGGTAGCCGGCAAGGTAGT 3′ |