TABLE 1.
Bacterial strains and plasmids
| Strain or plasmid | Genotype, phenotype or properties | Reference or source |
|---|---|---|
| E. coli | ||
| JM109 | endA1 recA1 gyrA96 thi-1 hsdR17(rK− mK+) relA1 supE44 Δ(lac-proAB) [F′ traD36 proAB lacIqZΔM15] | 51 |
| ONE Shot Mach 1 T1 | F− φ80(lacZ)ΔM15 ΔlacX74 hsdR(rK− mK−) ΔrecA1398 endA1 tonA (confers resistance to phage T1); chemically competent | Invitrogen |
| SubCloning efficiency DH5α competent cells | F− φ80lacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rK− mK+) phoA supE44 thi-1 gyrA96 relA1 λ− | Invitrogen |
| SM10 | thi-1 thr leu tonA lacy supE recA RP4-2-Tc::Mu Kmr | 44 |
| P. aeruginosa | ||
| O5 | Wild-type strain PAO1; IATS serotype O5; A+ B+ | 17 |
| O2 | Wild-type strain 5934; IATS serotype O2 (ATCC 33349); A+ B+ | 33 |
| O16 | Wild-type strain 170003; IATS serotype O16 (ATCC 33363); A− B+ | 33 |
| O5-wzyα-GmR | O5 wzyα::Gmr; O5 mutant with a gentamicin cassette inserted into the wzyα gene | 14 |
| O16-wzyβ-GmR | O16 wzyβ::Gmr; O16 mutant with a gentamicin cassette inserted into the wzyβ gene | This study |
| O5-wzyα-GmR-27 | O5-wzyα mutant transformed with the pUCP27 plasmid | This study |
| O5-wzyα-GmR-comp-O5wzyα | O5-wzyα mutant complemented with pUCP27 containing wzyα from O5 | This study |
| O5-wzyα-GmR-comp-O2wzyα | O5-wzyα mutant complemented with pUCP27 containing wzyα from O2 | This study |
| O5-wzyα-GmR-comp-O16wzyα | O5-wzyα mutant complemented with pUCP27 containing wzyα from O16 | This study |
| O5-wzyα-GmR-comp-O2wzyβ | O5-wzyα mutant complemented with pUCP27 containing wzyβ from O2 | This study |
| O16-wzyβ-GmR-27 | O16 wzyβ::Gmr mutant transformed with the pUCP27 plasmid | This study |
| O16-wzyβ-GmR-comp-O2wzyα | O16 wzyβ::Gmr mutant complemented with pUCP27 containing wzyα from O2 | This study |
| O16-wzyβ-GmR-comp-O2wzyβ | O16 wzyβ::Gmr mutant complemented with pUCP27 containing wzyβ from O2 | This study |
| Plasmid | ||
| pEX18Ap | Apr/CbroriT+sacB; gene replacement vector with multiple cloning site from pUC18 | 20 |
| pEX18Ap-O2wzyβ | pEX18Ap with wzyβ inserted into SacI and PstI sites | This study |
| pEX18Ap-O16 wzyβ-GmR | pEX18Ap with wzyβ with gentamicin cassette inserted in the EcoRI site of wzyβ | This study |
| pPS856 | Apr Gmr; 0.83-kb blunt-ended SacI fragment from pUCGM ligated into the EcoRV site of pPS854 | 20 |
| pQE80 | 4.8-kb histidine-tagged expression vector with RBS; Apr | QIAGEN |
| pUCP27 | 4.9-kb pUC18-based broad-host-range vectors; Tcr | 50 |
| pUCP27-RBS-O5wzyα | wzyα from O5 with RBS from pQE80 cloned into EcoRI and PstI sites of pUCP27 | This study |
| pUCP27-RBS-O2wzyα | wzyα from O2 with RBS from pQE80 cloned into EcoRI and PstI sites of pUCP27 | This study |
| pUCP27-RBS-O16wzyα | wzyα from O16 with RBS from pQE80 cloned into EcoRI and PstI sites of pUCP27 | This study |
| pUCP27-O2wzyβ | wzyβ from O2 with RBS from pQE80 cloned into EcoRI and PstI sites of pUCP27 | This study |
| TOPO-O2wzyβ-topfrag | 5′ PCR fragment of O2 wzyβ cloned into pCR-Blunt II-TOPO vector | This study |
| TOPO-O16wzyβ-topfrag | 5′ PCR fragment of O16 wzyβ cloned into pCR-Blunt II-TOPO vector | This study |
| TOPO-O2wzyβ-bottomfrag | 3′ PCR fragment of O2 wzyβ cloned into pCR-Blunt II-TOPO vector | This study |
| TOPO-O16wzyβ-bottomfrag | 3′ PCR fragment of O16 wzyβ cloned into pCR-Blunt II-TOPO vector | This study |
| TOPO-O2iap | iap from P. aeruginosa O2 cloned into pCR-Blunt II-TOPO vector | This study |
| TOPO-O16iap | iap from P. aeruginosa O16 cloned into pCR-Blunt II-TOPO vector | This study |