Table 1.

Strains and plasmids used in this study

Strain or plasmidRelevant characteristic(s)aReference(s) or source
Strains
 PA103Virulent lung isolate of P. aeruginosa; known type III secreted effector proteins are ExoT and ExoU 1,36
 PA103pscJ::Tn5 Tn5Gmr cassette inserted into pscJ; defective in type III secretion 29
 PA103exoU::Tn5 Tn5Gmr cassette inserted into exoU; referred to as mutant 8 in previous publications 23, 29
 PA103ΔTPA103 with an xylE aacC1 cassette replacing aa 36–348 of exoT; Gmr This study
 PA103ΔUPA103 with an in-frame deletion of aa 330–571 of exoU This study
 PA103ΔU+pUCP20PA103ΔU transformed with pUCP20; Cbr This study
 PA103ΔU+pExoSPA103ΔU transformed with pUCP20/exoS; Cbr This study
 PA103ΔUΔTPA103ΔU with an xylE aacC1cassette replacing aa 36–348 of exoT; Gmr This study
 PA103ΔUΔT+pUCP20PA103ΔUΔT transformed with pUCP20; Gmr Cbr This study
 PA103ΔUΔT+pExoSPA103ΔUΔT transformed with pUCP20/exoS; Gmr Cbr This study
 PA103ΔUΔT+pExoTPA103ΔUΔT transformed with pUCP20/exoT; Gmr Cbr This study
 PA103ΔUΔT+pExoT(R149G)PA103ΔUΔT transformed with pBK162; Gmr Cbr This study
 PA103ΔUΔT+pExoT(R149K)PA103ΔUΔT transformed with pBK151; Gmr Cbr This study
 PA103ΔU/T(R149G)PA103ΔU with a point mutation in ExoT(R149G)This study
 PA103ΔU/T(R149K)PA103ΔU with a point mutation in ExoT(R149K)This study
 388 P. aeruginosa laboratory strain; known type III secreted effector proteins are ExoS, ExoT, and ExoY 2, 25
 S17.1 E. coli strain used for mating constructs into P. aeruginosa; thi pro hsdR recA RP4-2 (Tet::Mu) (Km::Tn7) 46
 XL2-Blue E. coli strain used for cloning;recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′proAB lacI q ZΔM15 Tn10(Tetr) Amy Camr]Stratagene
Plasmids
 pAH806pBR322 containing a 3.5-kb genomic fragment from PA103 including the exoUgene 23
 pBK151pUCP20 with ExoT(R149K) point mutationThis study
 pBK152pEX100T with XmaI fragment from pBK151This study
 pBK162pUCP20 with ExoT(R149G) point mutationThis study
 pBK163pEX100T withXmaI fragment from pBK162This study
 pEX100TAllelic replacement suicide plasmid; Apr (Cbr)b sacB oriT 45
 pEX100T/exoTgentpEX100T with a 3,055-bpApaI-NotI fragment of pGEM-T/exoTgent cloned into the SmaI site; Gmr This study
 pEX100T/exoU pEX100T with a 3.2-kbSspI fragment from pAH806 including the exoU gene cloned into the SmaI siteThis study
 pEX100T/ΔexoU pEX100T/exoUwith a 726-bp BclI fragment encoding aa 330–571 of ExoU removedThis study
 pGEM-TVector for cloning PCR products; Apr Promega
 pGEM-T/exoS pGEM-T containing a 1,503-bp PCR product and bearing the exoS gene and upstream ExsA binding site from strain 388This study
 pGEM-T/exoT pGEM-T containing a 1,494-bp PCR product and bearing the exoT gene from strain PA103This study
 pGEM-T/exoT + pGEM-T with a 1,619-bp PCR product and bearing the exoT gene and upstream ExsA binding site from PA103This study
 pGEM-T/exoTgentpGEM-T/exoT with a 939-bp SmaI fragment encoding ExoT with aa 36–348 replaced with a 2,446-bp SmaI fragment encoding thexylE aaC1 cassette from pX1918GTThis study
 pUCP20 P. aeruginosa expression vector; Apr(Cbr)b 51
 pUCP20/exoS pUCP20 with a 1,570-bpSacI-SphI fragment containing exoSfrom pGEM-T/exoS This study
 pUCP20/exoT pUCP20 with a 1,666-bpNcoI-NdeI fragment containing exoTfrom pGEM-T/exoT This study
 pX1918GTPlasmid containing xylE aacC1insertional Gmr cassette; Apr 45
  • a aa, amino acids; Tetr, tetracycline resistance; Camr, chloramphenicol resistance; Apr, ampicillin resistance.

  • b Apr in E. coli; Cbr in P. aeruginosa.