Table 1.

Bacterial strains and plasmids used in this study

Strain or plasmidRelevant properties or genotypeaSource or reference
Strains
E. coli
  BL21(DE3) hsd-5 gal (λ cIts857 indl Sam7 nin5 lacUV5-T7 gene1) 33
  BL21(DE3) pLysSpLysS (Cmr) 33
  DH5α supE44 ΔlacU169 (φ80lacZΔM15) hsdR17 recA1 endA1gyrA96 22
thi-1 relA1
  SM10 thi thr leu tonA lacY supE recA::Rp4-2Tc::Mu Km 39
P. aeruginosa
  PA103Wild type,exoU + exoT+exoS 5, 8
  PAKWild type, exoU exoT+ exoS + 5, 8
  PA103ΔexoU exoU nonpolar mutantThis study
  PA103exoU::Tn5Tc exoUpolar mutant, mini-Tn5Tc insertion 5
Plasmids
 pUC18, pUC19Wild type, Apr 22
 pUCP19Wild type,E. coli-P. aeruginosa shuttle vector, Apr 28
 pUC18exoU656 Contains the exoU promoter and 656 aa of ExoUThis study
 pET16bN-terminal His tag fusion expression vector, Apr Novagen
 pET23bC-terminal His tag fusion expression vector, Apr Novagen
 pET16bΔEcoRIDeletion of EcoRI site of pET16bJ. T. Barbieri
 pETexoS pET16bΔEcoRI encoding His-ExoS 20
 pETexoU656 pET16bΔEcoRI encoding 656 aa of His-ExoUThis study
 pETexoU pET16bΔEcoRI encoding 687 aa of His-ExoU,This study
 pETexoUspcU pET16bΔEcoRI encoding His-ExoU and SpcUThis study
 pET23exoUspcU pET23b encoding ExoU and SpcU-HisThis study
 pET23Δ3–123exoUspcU pET23b encoding an in-frame deletion (Δ3–123) of ExoU and SpcU-HisThis study
 pUCPNexoU pUCP19 encoding only ExoU with the native promoter and stop sequencesThis study
 pUCPexoUEcoRVpUCP19 containingexoUspcU, as a 2.8-kb EcoRV fragmentThis study
 pUCPexoU(Bam6.5)pUCP19 containing a 6.5-kbBamHI fragment with exoU and flanking downstream sequences 5
 pNOT19Allelic replacement vector 29
 pNOTexoU pNOT19 containing a 6.5-kb fragment with exoU and additional downstream sequencesThis study
 pNOTΔexoU pNOT19 containing an in-frame deletion of aa 3–674 of ExoUThis study
 pMOB3pHSS21; Kmr Cmr sacBR oriT as aNotI cartridge 29
 pMOBΔexoU pNOTΔexoU with theNotI cartridge from pMOB3This study
  • a aa, amino acids.