TABLE 2.
Plasmids used in this study
| Plasmid | Descriptiona | Source or reference |
|---|---|---|
| pGEM-T | Cloning vector; Apr | Promega |
| pBS-SK (pBluescriptSK) | Cloning vector; Apr | Stratagene |
| pUCP-SK | P. aeruginosa-E. coli shuttle vector; Apr (Cbr) | 52 |
| mini-CTX-2 | Contains attP site for integration at chromosomal attB site; Tcr | 21 |
| pFLP2 | Source of inducible flp recombinase; Apr (Cbr) | 20 |
| pMLD4 | 3.5-kb NotI-NsiI fragment containing retS under control of its own promoter from cosmid pM0013207 into NotI-PstI of pUCP-SK; Apr (Cbr) | 29 |
| pRetS | pMLD4 derivative in which the EcoRI-XhoI region of the multiple cloning site has been removed; Apr (Cbr) | This study |
| pRetS-BB2 | pMLD4 derivative carrying C-terminal BB2-tagged retS under the control of its own promoter; Apr (Cbr) | 29 |
| Mini-CTX-2-retS | 3.5-kb NotI-EcoRI fragment from pMLD4 in NotI-EcoRI of mini-CTX-2; Tcr | 29 |
| pRetS-SK | 1.0-kb SalI-EcoRV fragment of pRetS containing aa 152 to 500 of RetS into SalI-EcoRV of pBS-SK; Apr | This study |
| pRetS-SK H424Q | pRetS-SK containing H424Q point mutation; Apr | This study |
| pRetS-R1 | 2.7-kb NotI-BamHI fragment of pRetS containing 259 bp upstream and aa 1 to 810 of RetS into NotI-BamHI of pBS-SK; Apr | This study |
| pRetS-R1 D664A D665A | pRetS-R1 containing D664A D665A point mutations; Apr | This study |
| pRetS-R1 D713A | pRetS-R1 containing D713A point mutation; Apr | This study |
| pRetS-R2 | 1.8-kb EcoRV-KpnI fragment from pRetS containing aa 498 to 942 and 457 bp downstream of RetS into EcoRV-KpnI of pBS-SK; Apr | This study |
| pRetS-R2 E814A D815A | pRetS-R2 containing E814A D815A point mutations; Apr | This study |
| pRetS-R2 D858A | pRetS-R2 containing D858A point mutation; Apr | This study |
| pRetS H424Q | 1.0-kb SalI-EcoRV fragment of pRetS-SK H424Q into SalI-EcoRV of pRetS; Apr (Cbr) | This study |
| pRetS D664A D665A | 2.7-kb NotI-BamHI fragment of pRetS-R1 D664A D665A into NotI-BamHI of pRetS; Apr (Cbr) | This study |
| pRetS D713A | 2.7-kb NotI-BamHI fragment of pRetS-R1 D713A into NotI-BamHI of pRetS; Apr (Cbr) | This study |
| pRetS E814A D815A | 1.8-kb EcoRV-KpnI fragment of pRetS-R2 E814A D815A into EcoRV-KpnI of pRetS; Apr (Cbr) | This study |
| pRetS D858A | 1.8-kb EcoRV-KpnI fragment of pRetS-R2 D858A into EcoRV-KpnI of pRetS; Apr (Cbr) | This study |
| pRetS H424Q D713A | 1.0-kb SalI-EcoRV fragment of pRetS H424Q into SalI-EcoRV of pRetS D713A; Apr (Cbr) | This study |
| pRetS H424Q D858A | 1.0-kb SalI-EcoRV fragment of pRetS H424Q into SalI-EcoRV of pRetS D858A; Apr (Cbr) | This study |
| pRetS D713A D858A | 855-bp BamHI-KpnI fragment of pRetS D858A into BamHI-KpnI of pRetS D713A; Apr (Cbr) | This study |
| pRetS H424Q D713A D858A | 1.0-kb SalI-EcoRV fragment of pRetS H424Q into SalI-EcoRV of pRetS D713A D858A; Apr (Cbr) | This study |
| pRetS H424Q-BB2 | 2.7-kb NotI-BamHI fragment of pRetS H424Q into NotI-BamHI of pRetS-BB2; Apr (Cbr) | This study |
| pRetS D713A-BB2 | 2.7-kb NotI-BamHI fragment of pRetS D713A into NotI-BamHI of pRetS-BB2; Apr (Cbr) | This study |
| pRetS D858A-BB2 | 330-bp BamHI-MfeI fragment of pRetS D858A into BamHI-MfeI of pRetS-BB2; Apr (Cbr) | This study |
| mini-CTX-2-retS H424Q | 3.5-kb NotI-KpnI fragment of pRetS H424Q into NotI-KpnI of mini-CTX-2; Tcr | This study |
| mini-CTX-2-retS D664A D665A | 3.5-kb NotI-KpnI fragment of pRetS D664A D665A into NotI-KpnI of mini-CTX-2; Tcr | This study |
| mini-CTX-2-retS D713A | 3.5-kb NotI-KpnI fragment of pRetS D713A into NotI-KpnI of mini-CTX-2; Tcr | This study |
| mini-CTX-2-retS E814A D815A | 3.5-kb NotI-KpnI fragment of pRetS E814A D815A into NotI-KpnI of mini-CTX-2; Tcr | This study |
| mini-CTX-2-retS D858A | 3.5-kb NotI-KpnI fragment of pRetS D858A into NotI-KpnI of mini-CTX-2; Tcr | This study |
| mini-CTX-2-retS H424Q D713A | 3.5-kb NotI-KpnI fragment of pRetS H424Q D713A into NotI-KpnI of mini-CTX-2; Tcr | This study |
| mini-CTX-2-retS H424Q D858A | 3.5-kb NotI-KpnI fragment of pRetS H424Q D858A into NotI-KpnI of mini-CTX-2; Tcr | This study |
| mini-CTX-2-retS D713A D858A | 3.5-kb NotI-KpnI fragment of pRetS D713A D858A into NotI-KpnI of mini-CTX-2; Tcr | This study |
| mini-CTX-2-retS H424A D713A D858A | 3.5-kb NotI-KpnI fragment of pRetS H424Q D713A D858A into NotI-KpnI of mini-CTX-2; Tcr | This study |
| pKO-37-185 | 367-bp NotI-SpeI fragment (promoter and aa 1 to 36) and 939-bp SpeI-EcoRV fragment (aa 186 to 500) of RetS cloned in tandem into NotI-EcoRV of pBS-SK; Apr | This study |
| pKO-37-360 | 367-bp NotI-SpeI fragment (promoter and aa 1 to 36) and 414-bp SpeI-EcoRV fragment (aa 361 to 500) of RetS cloned in tandem into NotI-EcoRV of pBS-SK; Apr | This study |
| pRetS Δ37-185 | 1.3-kb NotI-EcoRV fragment of pKO-37-185 into NotI-EcoRV of pRetS; Apr (Cbr) | This study |
| pRetS Δ37-360 | 780-bp NotI-EcoRV fragment of pKO-37-360 into NotI-EcoRV of pRetS; Apr (Cbr) | This study |
| pRetS Δ37-185-BB2 | 160-bp MfeI-KpnI fragment of pRetS-BB2 into MfeI-KpnI of pRetS Δ37-185; Apr (Cbr) | This study |
| pRetS Δ37-360-BB2 | 160-bp MfeI-KpnI fragment of pRetS-BB2 into MfeI-KpnI of pRetS Δ37-360; Apr (Cbr) | This study |
| mini-CTX-2-retS Δ37-185 | 3.0-kb NotI-KpnI fragment of pRetS Δ37-185 into NotI-KpnI of mini-CTX-2; Tcr | This study |
| mini-CTX-2-retS Δ37-360 | 2.5-kb NotI-KpnI fragment of pRetS Δ37-360 into NotI-KpnI of mini-CTX-2; Tcr | This study |
a
Abbreviations: Ap, ampicillin; Cb, carbenicillin; Cm, chloramphenicol; Gm, gentamicin; Km, kanamycin; Tc, tetracycline; aa, amino acids.