TABLE 1.
Bacterial strains and plasmids used in this study
| Strain or plasmida | Relevant characteristicsb | Source or reference |
|---|---|---|
| Strains | ||
| P. aeruginosa | ||
| K767 | PAO1 prototroph (described in reference 48) | K. Poole |
| K1454 | K767 mexR (nalB), upregulated MexAB-OprM | 49 |
| K767-CDA0033 | K767 nfxB, upregulated MexCD-OprJ | This study |
| K767-CDR0026 | K767 overexpressing LpxC | 20 |
| K767-LpxCG208S | K767 engineered to encode LpxCG208S | This study |
| K767-CDJ0011 | K767 engineered to encode LpxCL18V | 20 |
| K767-CDJ0037 | K767 encoding LpxCA214V, selected on compound 3 | This study |
| K767-CDR0061 | K767 fabG (C494T), selected on CHIR-090 | 20 |
| K2732 | PAO1 prototroph | K. Poole |
| K2732-P2 | K2732 FabF1T306A, passage 2, selected on CHIR-090 | This study |
| K2732-P6 | K3732 nfxB stop TGA-Cys; FabGD190G; PA4465N193T, passage 6, selected on CHIR-090 | This study |
| K2732-P13 | K2732 MexRT130P; nfxB stop TGA-Cys; FabGD190G; PA4465N193T, passage 13, selected on CHIR-090 | This study |
| K2733 | K2733 (K2732 ΔmexB ΔmexX ΔmexCD-oprJ ΔmexEF-oprN) | K. Poole |
| K2733-P6a (NC) | K2733 PA4465N193T, passage 6, selected on CHIR-090 | This study |
| K2733-P6b (SC) | K2733 FabGA159V, passage 6, selected on CHIR-090 | This study |
| K2733-P13a (NC) | K2733 FabGA167V; PA4465N193T, passage 13, selected on CHIR-090 | This study |
| K2733-P13b (SC) | K2733 FabGA167V; PA4465N193T, passage 13, selected on CHIR-090 | This study |
| K2733-PA4465N193T | K2732 engineered to encode PA4465N193T | This study |
| PAO1V | PAO1 prototroph | 50 |
| PAO1V-CDJ0042 | PAO1V encoding LpxCA214V, selected on compound 2 | This study |
| K2153 | Clinical isolate, MexXY upregulated, active mexT | 25 |
| K2376 | K2153 mexS in-frame deletion, upregulated MexEF-OprN expression | 51 |
| K2153-CDR0063 | K2153 mexS, upregulated MexEF-OprN expression | 20 |
| E. coli | ||
| Stellar | High transformation efficiency HST08 strain | Clontech |
| Plasmids | ||
| pRK2013 | Helper plasmid | 52 |
| pAK1900 | E. coli-P. aeruginosa shuttle vector; Apr | A. Kropinski |
| pAK-lpxC | pAK1900 with lpxC and 100 bp upstream sequence from K767 | 20 |
| pAK-lpxC WT | pAK1900 with WT lpxC and 100 bp upstream sequence from K767 | 20 |
| pAK-lpxC WT UP | pAK-lpxC WT with C→A substitution 11 bp upstream of lpxC; LpxC upregulated) | 20 |
| pAK-LpxCA214V | pAKlpxC WT altered so that lpxC encodes A214V | This study |
| pAK- LpxCA214V UP | pAK-LpxCA214V with C→A substitution 11 bp upstream of lpxC; LpxCA214V upregulated) | This study |
| pMMB-10 | Gateway adapted pMMB low-copy-number expression vector; Apr Cbr; IPTG inducible | 53 |
| pMM-10-fabF1 | IPTG inducible fabF1 expression vector | This study |
| pMM-10-PA4465 | IPTG inducible PA4465 expression vector | This study |
| pEX18ApGW | Gateway adapted gene replacement vector, Apr Cbr, sacB | 54 |
| pEX18ApGW-lpxC-G622A | Gene replacement vector with lpxC G622A allele | This study |
| pEX18ApGW-PA4465-A578C | Gene replacement vector with PA4465 A578C allele | This study |
a
Additional mutants are summarized in Table S1 in the supplemental material. NC, normal-size colony; SC, small colony.
b
Cbr, carbenicillin resistance; Apr, ampicillin resistance.