TABLE 1.
Bacterial strains and plasmids used in this study
| Strain or plasmid | Relevant genotype | Source or reference |
|---|---|---|
| Strains | ||
| E. coli | ||
| DH5α | hsdR recA lacZYA φ80 lacZ ΔM15 | GIBCO BRL |
| BL21(DE3) | F−ompT hsdSB (rB− mB−) gal dcm (DE3) | Novagen, Inc., Madison, Wis. |
| P. aeruginosa | ||
| PAK | Wild-type clinical isolate | D. Bradley |
| PAK-NP | PAKpilA::Tcr | 22 |
| PAK-N1G | PAKrpoN::Gmr | 9 |
| PAK-RG | PAKfleR::Gmr | 21 |
| PAK-Q | PAKfleQ::Gmr | 3 |
| MS540 | PAKfliA::Gmr | 28 |
| PAK-D | PAKfliD::Gmr | This study |
| PAK-NPD | PAKpilA::TcrfliD::Gmr | This study |
| Plasmids | ||
| pUC18 | E. coli cloning vector, Ampr | GIBCO BRL |
| pLysS | Plasmid containing the T7 lysozyme gene | Novagen |
| pGEM3Z | Sequencing vector, Ampr, lacZα peptide | Promega, Madison, Wis. |
| pBluescript KS(+) | E. coli cloning vector, Ampr | Stratagene, Inc. |
| pDN19lacΩ | Promoterless lacZ oriV oriT Tetr Strr Ω fragment | 30 |
| pPZ375 | oriV in pGEM3Z | 29 |
| pG10E | pGEM3Z containing a 10-kb EcoRI fragment isolated from the cosmid pRR194 | This study |
| pBS7EA | pBluescript KS(+) containing a 7-kb EcoRI-ApaI insert including the fliDSorf126orf96 operon | This study |
| pBS7EAG | pBS7EA with a gentamicin resistance gene inserted into the EcoRV site in the fliD gene | This study |
| placΩD | pDN19lacΩ with a 495-bp EcoRI-BamHI fragment including the promoter region of the fliD gene | This study |
| pPZ375D | pPZ375 with a complete fliD gene | This study |
| pET15B | Expression vector, T7 promoter, His-tag coding sequence, Ampr, pBR322 origin | Novagen |
| pET15BD | fliD gene inserted as a PCR product into the NdeI-BamHI sites of pET15B | This study |