TABLE 1.
Strains and plasmids used in this study
| Strains and plasmids | Description | Reference |
|---|---|---|
| E. coli strains | ||
| DH5α | F′ Phi80dlacZ ΔM15 Δ (lacZYA-argF)U169 deoR recA1 endA1 hsdR17(rK-mK+) phoA supE44 λ-thi-1 | Invitrogen |
| TOP10 | F-mcrA Δ (mrr-hsdRMS-mcrBC) ϕ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara-leu) 7697 galU galK rpsL endA1 nupG | Invitrogen |
| Origami B (DE3) | F− ompT hsdSB(rB− mB−) gal dcm lacY1 ahpC (DE3) gor522::Tn10 trxB (KanR, TetR) | Novagen |
| BTH101 | F−, cya-99, araD139, galE15, galK16, rpsL1 (StrR), hsdR2, mcrA1, mcrB1 | Euromedex |
| P. aeruginosa strains | ||
| mPAO1 | Wild-type laboratory strain | 50 |
| mPAO1 pilA::Tn | ISphoA/hah transposon insertion at position 163 of pilA | 50 |
| mPAO1 pilT::Tn | ISphoA/hah transposon insertion at position 885 of pilT | 50 |
| mPAO1 pilE::Tn | ISphoA/hah transposon insertion at position 183 of pilE | 50 |
| mPAO1 pilT::Tn ΔMP ΔMPP::FRT | ISphoA/hah transposon insertion at position 885 of pilT and deletion of fimTUpilVWXY1Y2E and deletion of xcpUVWX replaced with an FRT scar | 10 |
| mPAO1 pilE::Tn pilT::FRT ΔfimU ΔMPP::FRT | ISphoA/hah transposon insertion at position 183 of pilE with FRT insertion in the NruI site of pilT and deletion of fimU and deletion of xcpUVWX replaced with an FRT scar | 10 |
| Plasmids | ||
| pBADGr | pMLBAD with aacC1 gene (gentamicin resistance) disrupting dhfrII (trimethoprim resistance), arabinose inducible | 33 |
| pBADGrpilE | PAO1 pilE cloned in pBADGr, GmR | 7 |
| pBADGrpilE mCherry | PAO1 pilE cloned in pBADGr encoding a C-terminal mCherry fluorescent tag | This study |
| pUT18C | pUC19-derived vector with T18 fragment (residues 225–399) of CyaA under control of lac promoter, MCS at 3′ end of T18 ORF, AmpR | 51 |
| pUT18CpilE | mature PAO1 pilE cloned in pUT18C, AmpR | This study |
| pKT25 | pSU40-derived vector with T25 fragment (residues 1–224) of CyaA under control of a lac promoter; MCS at 3′ end of T25 ORF, KanR | 51 |
| pKT25pilA | Mature PAO1 pilA cloned in pKT25, KanR | 10 |
| pKT25fimU | Mature PAO1 fimU cloned in pKT25, KanR | This study |
| pKT25pilV | Mature PAO1 pilV cloned in pKT25, KanR | 10 |
| pKT25pilW | Mature PAO1 pilW cloned in pKT25, KanR | 10 |
| pKT25pilX | Mature PAO1 pilX cloned in pKT25, KanR | 10 |
| pT18zip | pT18 plasmid with 35-residue leucine zipper cloned into KpnI site, AmpR | 28 |
| pT25zip | pT25 plasmid with 35-residue leucine zipper cloned into KpnI site, CmR | 28 |
| pET151pilE Δ1–28 | PilE (Δ1–28 of mature protein) expression vector with N-terminal His6 and V5 epitope tag | This study |