TABLE 1.
Strains and plasmids used in this study
| Bacterial strain or plasmid | Relevant characteristics | Source or Ref. |
|---|---|---|
| E. coli strains | ||
| DH5α | F−endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15 Δ(lacZYA-argF)U169, hsdR17(rK−mK+), λ− | Invitrogen |
| C2925 | ara-14 leuB6 fhuA31 lacY1 tsx78 glnV44 galK2 galT22 mcrA dcm-6 hisG4 rfbD1 R(zgb210::Tn10) TetSendA1 rspL136 (StrR) dam13::Tn9 (CamR) xylA-5 mtl-1 thi-1 mcrB1 hsdR2 | New England Biolabs |
| SM10 | KmR, thi-1, thr, leu, tonA, lacY, supE, recA::RP4-2-Tc::Mu, pir | 32 |
| M. smegmatis mc2155 | Source of DNA for complementation of Pa5196 mutants | Jun Liu, University of Toronto |
| M. tuberculosis H37Rv | Source of DNA for complementation of Pa5196 mutants | Jun Liu, University of Toronto |
| P. aeruginosa strains | ||
| PAO1 | Group II T4P | Laboratory stock |
| PAO1 NP | Tn5-phoA insertion at base 163 in pilA | 33 |
| Pa5196 | Group IV T4P; rectal isolate | 5 |
| Pa5196 tfpW | EZ::Tn FRT insertion in tfpW | 7 |
| Pa5196 6246::FRT | FRT insertion at position 309 (EcoRV) in PsPA7_6246 | This study |
| Pa5196 6247::FRT | FRT insertion at position 173 (AfeI) in PsPA7_6247 | This study |
| Pa5196 6248::FRT | FRT insertion at position 569 (XhoI) in PsPA7_6248 | This study |
| Pa5196 6249::FRT | FRT insertion at position 105 (NruI) in PsPA7_6249 | This study |
| Pa5196 6250::FRT | FRT insertion at position 336 (BclI) in PsPA7_6250 | This study |
| Pa5196 6251::FRT | FRT insertion at position 422 (EcoRI) in PsPA7_6251 | This study |
| Pa5196 6250::FRT-6251::FRT | FRT insertions at position 336 of PsPA7_6250 and position 422 of PsPA7_6251 | This study |
| Pa5196 pilT::FRT | FRT insertion at position 540 (NruI) in pilT | This study |
| Pa5196 tfpW pilT | FRT insertion at position 540 (NruI) in pilT on the tfpW background | This study |
| Pa5196 PsPA7_6248 pilT | FRT insertion at position 540 (NruI) in pilT on the PsPA7_6248::FRT background | This study |
| PAO1 NP + AWX | PAO1 pilA mutant complemented with pilAIV-tfpW-tfpX from Pa5196 in pBADGr | 10 |
| Plasmids | ||
| pEX18Ap | Carbenicillin-resistant suicide vector used for gene replacement | 34 |
| pFLP2 | Suicide vector encoding Flp recombinase | 34 |
| pPS856 | Source of FRT-flanked gentamicin resistance cassette | 34 |
| pBADGr | Broad host range arabinose-inducible vector used for complementation; gentamicin resistance marker | 35 |
| pUCP20 | Carbenicillin-resistant vector used for complementation; TEM-1 resistance marker | 34 |
| pEX18Ap + 6246::GmFRT | PsPA7_6246 knock-out construct with SmaI-flanked Gm-FRT cassette inserted at position 309 (EcoRV) in PsPA7_6246 | This study |
| pEX18Ap + 6247::GmFRT | PsPA7_6247 knock-out construct with SmaI-flanked Gm-FRT cassette inserted at position 173 (AfeI) in PsPA7_6247 | This study |
| pEX18Ap + 6248::GmFRT | PsPA7_6248 knock-out construct with SstI-flanked Gm-FRT cassette inserted at position 569 (XhoI) in PsPA7_6248 | This study |
| pEX18Ap + 6249::GmFRT | PsPA7_6249 knock-out construct with SmaI-flanked Gm-FRT cassette inserted at position 105 (NruI) in PsPA7_6249 | This study |
| pEX18Ap + 6250::GmFRT | PsPA7_6250 knock-out construct with BamHI-flanked Gm-FRT cassette inserted at position 336 (BclI) in PsPA7_6250 | This study |
| pEX18Ap + 6251::GmFRT | PsPA7_6251 knock-out construct with EcoRI-flanked Gm-FRT cassette inserted at position 422 (EcoRI) in PsPA7_6251 | This study |
| pEX18Ap + pilT::GmFRT | pilT knock-out construct with SmaI-flanked Gm-FRT cassette inserted at position (NruI) in pilT | 10 |
| pBADGr + AWX | Complementation construct carrying the Pa5196 pilAIV, tfpW, and tfpX genes | 3 |
| pBADGr + PsPA7_6248 | Complementation construct carrying the Pa5196 PsPA7_6248 gene | This study |
| pBADGr + PA1416 | Complementation construct carrying the PAO1 PA1416 gene | This study |
| pBADGr + MSMEG6382 | Complementation construct carrying the M. smegmatis MSMEG6392 gene | This study |
| pBADGr + Rv3790 | Complementation construct carrying the M. tuberculosis Rv3790 gene | This study |
| pUCP20 + 6245–6251 | Complementation construct carrying the Pa5196 PsPA7_6245-51 genes, lac promoter | This study |
| pUCP20 + 6247–6251 | Complementation construct carrying the Pa5196 PsPA7_6247-51 genes, lac promoter | This study |
| pUCP20 + 6245–6249 | Complementation construct carrying the Pa5196 PsPA7_6245-49 genes, lac promoter | This study |
| pUCP20 + 6245–6249, 6247::GmFRT | Complementation construct carrying the Pa5196 PsPA7_6245-49 genes, with an insertion of the SmaI-flanked Gm-FRT cassette at position 173 (AfeI) in PsPA7_6247, lac promoter | This study |
| pUCP20 + 6246–6249 | Complementation construct carrying the Pa5196 PsPA7_6246-49 genes, lac promoter | This study |
| pUCP20 + 6246–6249, 6247::GmFRT | Complementation construct carrying the Pa5196 PsPA7_6246-49 genes, with an insertion of the SmaI-flanked Gm-FRT cassette at position 173 (AfeI) in PsPA7_6247, lac promoter | This study |