Table 1.
Strains and plasmids used
| Strain or plasmid | Characteristic(s) or sequence | Source or reference |
|---|---|---|
| E. coli strains | ||
| DH5α | fhuA2 Δ(argF-lacZ)U169 phoA glnV44 ϕ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17 | Lab strain |
| SM10 | thi thr leu tonA lacY supE recA::RP4-2-Tc::Mu Km λpir | Lab strain |
| P. aeruginosa strains | ||
| PA14 | Virulent burn wound isolate of P. aeruginosa | 33 |
| PA14ΔkinB | PA14 ΔkinB (1.7-kb in-frame deletion of kinB) | 7 |
| PA14ΔalgB | ΔalgB PA14 (1.3-kb in-frame deletion of algB) | 7 |
| PA14ΔalgBΔkinB | PA14 ΔalgB ΔkinB (3.1 kb in-frame deletion of algB and kinB) | This study |
| PA14ΔalgU | PA14 ΔalgU (567 bp in-frame deletion of algU) | This study |
| PA14ΔalgUΔkinB | PA14 ΔalgU ΔkinB (1.7-kb in-frame deletion of kinB and 567-bp in-frame deletion of algU) | This study |
| PA14ΔalgR | PA14 ΔalgR (746-bp in-frame deletion of algR) | This study |
| PA14ΔalgRΔkinB | PA14 ΔalgR ΔkinB (1.7-kb in-frame deletion of kinB and a 746-bp in-frame deletion of algR) | This study |
| PA14ΔlasR | PA14 ΔlasR | 7 |
| PA14ΔkinB(+pKinB) | PA14ΔkinB with plasmid pKinB | 7 |
| PA14ΔkinB(+pKinBH385A) | PA14ΔkinB with plasmid pKinBH385A | 7 |
| PA14ΔkinB(+pKinBP390S) | PA14ΔkinB with plasmid pKinBP390S | This study |
| PA14ΔkinB(+KinBint) | PA14 ΔkinB with Plac-kinB integrated at the attB site | This study |
| PA14ΔkinB(+KinBP390Sint) | PA14ΔkinB with Plac-kinBP390S integrated at the attB site | This study |
| PA14ΔalgBΔkinB(+pAlgB) | PA14ΔalgBΔkinB with plasmid pAlgB | This study |
| PA14ΔalgBΔkinB(+pAlgBD59N) | PA14ΔalgBΔkinB with plasmid pAlgBD59N | This study |
| PA14ΔalgUΔkinB(+pAlgU) | PA14ΔalgUΔkinB with plasmid pAlgU | This study |
| Plasmids | ||
| pEXG2-attP | pEXG2-M13 with attP cassette | 7 |
| pNSC5 | pEXG2-attP with 5′ algB flanks and 3′ kinB flanks from PA14 cloned in. Used to generate ΔalgB ΔkinB PA14 | This study |
| pNSC6 | pEXG2-attP with algU flanks from PA14 cloned in. Used to generate ΔalgU PA14 and ΔalgU ΔkinB PA14 | This study |
| pNSC7 | pEXG2-attP with algR flanks from PA14 cloned in; used to generate ΔalgR ΔkinB PA14 | This study |
| pHERD20T | pUCP20T Plac replaced by a 1.3-kb AflII-EcoRI fragment of the araC-PBAD cassette (5,087bp) | 7 |
| pKinB | pHERD20T with the kinB gene from PA14 at the KpnI/HindIII site | 7 |
| pAlgB | pHERD20T with the algB gene from PA14 at the EcoRI/HindIII site; the algB gene was amplified using primers NSC14D and NSC15D | This study |
| pAlgU | pHERD20T with the algU gene from PA14 at the EcoRI/HindIII site; the algU gene was amplified using primers NSC12 and NSC13 | This study |
| pKinBH385A | pKinB with H385A | 7 |
| pKinBP390S | pKinB with P390S | This study |
| pAlgBD59N | pAlgB with D59N | This study |
| pHisAlgBD59N | pHERD20T with His-tagged algBD59N cloned at the XbaI/HindIII sites; algBD59N was amplified from pAlgBD59N with AC672 and NSC107, allowing insertion of an N-terminal His tag and XbaI/HindIII sites | This study |
| pSM95 | Plasmid that expresses a His-tagged carboxyl-terminal fragment of KinB (HC-KinB) | 26 (Ohman lab) |
| pSM95P390S | Plasmid that expresses a mutated His-tagged carboxyl terminal fragment of KinB (HC-KinB); proline 390 is mutated to serine | This study |
| pDJW403 | Plasmid that expresses a His-tagged AlgB | 25 (Wozniak lab) |
| pQF50 | pQF50 plasmid | 12 (Rahme lab) |
| pQF50algU | Plasmid containing PalgU-lacZ fusion | This study |
| pUCP18 | E. coli-P. aeruginosa shuttle vector | 38 (Rahme lab) |
| pUCP18-KinB | pUCP18 with the kinB gene from PA14 at the XbaI/HindIII site | This study |
| Mini-CTX1 | Integration-proficient plasmid for P. aeruginosa | 17 (lab stock) |
| Mini-CTX1-KinB | Mini-CTX1 with Plac-kinB from pUCP18-KinB at the BamHI/HindIII site | This study |
| Mini-CTX1-KinBP390S | Mini-CTX1-KinB with P390S | This study |