TABLE 1.
Bacterial strains and plasmids
| Strain or plasmid | Genotypea | Construction, selection, source, or referenceb |
|---|---|---|
| P. aeruginosa strains | ||
| PAM1020 | PAO1 prototroph | 17 |
| PAM1032 | nalB1032 | 17 |
| PAM1033 | nfxB1033 | 17 |
| PAM1034 | nfxC1034 | 17 |
| PAM1262 (K879) | met-9011 amiE200 rpsL pvd-9 mexB::ΩHg | K. Poole |
| PAM1154 | oprM::ΩHg | 17 |
| PAM1014 | nfxC oprM::ΩHg | 17 |
| PAM1610 | nalB1032 ΔmexEF-oprN::ΩHg | 17 |
| PAM1409 | ΔmexCD-oprJ::Gm | 17 |
| PAM1275 | mexB::ΩHg | PAM1020 × (PAM1262); HgCl2c |
| PAM1277 | nfxB1033 mexB::ΩHg | PAM1033 × (PAM1262); HgCl2 |
| PAM1438 | nalB1032 nfxB1438 | Selection on LBA + levofloxacin at 1 μg/ml and ABS at 20 μg/ml from PAM1032 |
| PAM1465 | nfxB1438 mexB::ΩHg | PAM1438 × (PAM1262); HgCl2 |
| PAM1466 | nalB1032 ΔmexCD-oprJ::Gm | PAM1438 × (PAM1409); Gm |
| PAM1278 | nfxC1034 mexB::ΩHg | PAM1034× (PAM1262); HgCl2 |
| PAM2281 | nalB2281 nfxC1034 | Selection on LBA + levofloxacin at 1 μg/ml and EFS at 5 μg/ml from PAM1034 |
| PAM2282 | nalB2282 nfxC1034 | Selection on LBA + levofloxacin at 1 μg/ml and EFS at 5 μg/ml from PAM1034 |
| PAM1610 | ΔmexEF-oprN::ΩHg | 17 |
| PAM2359 | nalB2281 ΔmexEF-oprN::ΩHg | PAM2281 × (PAM1610); HgCl2 |
| PAM2360 | nalB2282 ΔmexEF-oprN::ΩHg | PAM2282 × (PAM1610); HgCl2 |
| PAM2302 | nalB2281 nfxC1034 nfxB2302 | Selection on LBA + levofloxacin at 1 μg/ml, ABS at 20 μg/ml, and EFS at 5 μg/ml from PAM2281 |
| PAM2303 | nalB2281 nfxC1034 nfxB2303 | Selection on LBA + levofloxacin at 1 μg/ml, ABS at 20 μg/ml, and EFS at 5 μg/ml from PAM2282 |
| PAM2387 | oprM::ΩHg nfxC1034 nfxB2303 | PAM2303 × (PAM1014); HgCl2 |
| PAM1064 | mexA-phoA::Tc | 17 |
| PAM1116 | nalB1032 mexA-phoA::Tc | Integration of plasmid pSUP202-mexA-phoA (Tcr Cbr Cmr) in the chromosome of PAM1032; Tcr Cbr |
| PAM2454 | nalB1032 mexA-phoA::Tc oprM::ΩHg | PAM1116 × (PAM1014); HgCl2 |
| PAM2455 | mexA-phoA::Tc oprM::ΩHg | PAM1064 × (PAM1014); HgCl2 |
| PAM1194 | oppA::Tc | Transposon mutagenesis with D171; Tc |
| PAM2386 | nalB1032 oppA::Tc | PAM1032 × (PAM1194); Tc |
| PAM1316 | oprM::ΩHg oppA::Tc | PAM1194 × (PAM1014); HgCl2 |
| PAM2458 | oprM::ΩHg mexA-phoA::Tc oppA::Tc | PAM1194 × (PAM2455); HgCl2 |
| E. coli strains | ||
| ECM1194 (AMS6) | wt | 18 |
| ECM1642 | marR1642 | Selection on LBA + chloramphenicol at 10 μg/ml from ECM1194 |
| ECM1694 | ΔacrAB::Km | ECM1194 × (ECM1343); Km |
| ECM1668 | marR1642 ΔacrAB::Km | ECM1642 × (ECM1343); Km |
| ECM1749 | marR1642 ΔacrAB::Km/pLQ821 | ECM1668/pLQ821; Ap |
| ECM1750 | ΔacrAB::Km/pLQ821 | ECM1694/pLQ821; Ap |
| ECM1735 | marR1642 ΔacrAB::Km/pAL261 | ECM1668/pAL261; Ap |
| ECM1640 | ΔacrAB::Km/pAL261 | ECM1694/pAL261; Ap |
| ECM1748 | wt/pLQ821 | ECM1194/pLQ821; Ap |
| ECM1730 | wt/pAL261 | ECM1194/pAL261; Ap |
| ECM1751 | marR1642/pLQ821 | ECM1642/pLQ821; Ap |
| ECM1754 | marR1642/pAL261 | ECM1642/pAL261; Ap |
| ECM1776 (UTL2) | ΔmdfA::Km | 7 |
| ECM1174 (ZK796) | F− araD139 Δ(argF-lac)205 ptsB5301 ptsF25 relA1 rpsL150 deoC1 flbB tolC::Tn10 | R. Kolter |
| ECM1556 | tolC::Tn10 | ECM1194 × (ECM1174); Tc |
| ECM1816 | tolC::Tn10 ΔmdfA::Km | ECM1556 × (ECM1176); Km |
| ECM1888 | tolC::Tn10 mdfR | Stepwise selection on LBA + chloramphenicol (first at 1 μg/ml, then at 4 μg/ml) from ECM1556 |
| ECM1915 | tolC::Tn10 mdfR ΔmdfA::Km | ECM1888 × (ECM1176); Km |
| ECM1911 | tolC::Tn10 ΔmdfA::Km/pLQ821 | ECM1816/pLQ821; Ap |
| ECM1908 | tolC::Tn10 mdfR/pLQ821 | ECM1888/pLQ821; Ap |
| ECM1955 | tolC::Tn10 mdfR ΔmdfA::Km/pLQ821 | ECM1915/pLQ821; Ap |
| ECM1343 (AG100A) | argE3 thi-1 rpsL xyl mlt Δ(gal uvrB) supE44 ΔacrAB::Km | 31 |
| DH5α | endA hsdR17 supE44 thi-1 recA1 gyrA relA1 Δ(lacZYA-argF)U169 deoR [φ80dlacΔ(lacZ)M15] | 2 |
| S17-1 | thi pro hsdR recA tra+ | 39 |
| Plasmids | ||
| pSUP202-mexA-phoA | pSUP202 (Tcr Cbr CmroriT) carrying the 5′ upstream region of mexA fused to promoterless phoA gene | K. Poole |
| pSE380 | trc promoter expression vector; lacIq Apr | Invitrogen |
| pAL261 | Apr; pSE380 with EcoRI-HindIII PCR fragment containing the mdfA gene | This study |
| pLQ821 | Apr; cmlA | 3 |
| pMarR | Apr; marR | P. Miller |
a
ΩHg, HgCl2 resistance derivative of interposon Ω; Gm, gentamicin resistance; Ap, ampicillin resistance; Cb, carbenicillin resistance; Cm, chloramphenicol resistance; Km, kanamycin resistance; Tc, tetracycline resistance; oriT, origin of transfer from RP4; wt, wild type.
b
ABS and EFS, efflux pump inhibitors specific for the MexAB-OprM and the MexEF-OprN pumps, respectively; LBA, L agar.
c
Description of transduction experiment. The first strain is the recipient; the strain in parentheses is the source of a transducing lysate. Antibiotics used for selection of transductants, transformants, or transconjugants are also shown.