Table 1.

Bacterial strains and plasmids used in this study

Strain or plasmidRelevant genotypeSource or reference
E. coli
  DH5α hsdR recA lacZYA φ80 lacZ ΔM15GIBCO BRL
  BL21(DE3)F ompT hsdSB (rB mB ) gal dcm (DE3)Novagen, Inc., Madison, Wis.
P. aeruginosa
  PAKWild-type clinical isolateD. Bradley
  PAK-NPPAKpilA::Tcr 22
  PAK-N1GPAKrpoN::Gmr 9
  PAK-RGPAKfleR::Gmr 21
  PAK-QPAKfleQ::Gmr 3
  MS540PAKfliA::Gmr 28
  PAK-DPAKfliD::Gmr This study
  PAK-NPDPAKpilA::Tcr fliD::Gmr This study
 pUC18 E. coli cloning vector, Ampr GIBCO BRL
 pLysSPlasmid containing the T7 lysozyme geneNovagen
 pGEM3ZSequencing vector, Ampr, lacZα peptidePromega, Madison, Wis.
 pBluescript KS(+) E. coli cloning vector, Ampr Stratagene, Inc.
 pDN19lacΩPromoterlesslacZ oriV oriT Tetr Strr Ω fragment 30
 pPZ375 oriV in pGEM3Z 29
 pG10EpGEM3Z containing a 10-kb EcoRI fragment isolated from the cosmid pRR194This study
 pBS7EApBluescript KS(+) containing a 7-kbEcoRI-ApaI insert including thefliDSorf126orf96 operonThis study
 pBS7EAGpBS7EA with a gentamicin resistance gene inserted into the EcoRV site in the fliD geneThis study
 placΩDpDN19lacΩ with a 495-bpEcoRI-BamHI fragment including the promoter region of the fliD geneThis study
 pPZ375DpPZ375 with a complete fliD geneThis study
 pET15BExpression vector, T7 promoter, His-tag coding sequence, Ampr, pBR322 originNovagen
 pET15BD fliD gene inserted as a PCR product into the NdeI-BamHI sites of pET15BThis study