TABLE 1.
Strains and plasmids used in this study
| Strain or plasmid | Relevant characteristic(s)a | Reference(s) or source |
|---|---|---|
| Strains | ||
| PA103 | Virulent lung isolate of P. aeruginosa; known type III secreted effector proteins are ExoT and ExoU | 1, 36 |
| PA103pscJ::Tn5 | Tn5 Gmr cassette inserted into pscJ; defective in type III secretion | 29 |
| PA103exoU::Tn5 | Tn5 Gmr cassette inserted into exoU; referred to as mutant 8 in previous publications | 23, 29 |
| PA103ΔT | PA103 with an xylE aacC1 cassette replacing aa 36–348 of exoT; Gmr | This study |
| PA103ΔU | PA103 with an in-frame deletion of aa 330–571 of exoU | This study |
| PA103ΔU+pUCP20 | PA103ΔU transformed with pUCP20; Cbr | This study |
| PA103ΔU+pExoS | PA103ΔU transformed with pUCP20/exoS; Cbr | This study |
| PA103ΔUΔT | PA103ΔU with an xylE aacC1 cassette replacing aa 36–348 of exoT; Gmr | This study |
| PA103ΔUΔT+pUCP20 | PA103ΔUΔT transformed with pUCP20; Gmr Cbr | This study |
| PA103ΔUΔT+pExoS | PA103ΔUΔT transformed with pUCP20/exoS; Gmr Cbr | This study |
| PA103ΔUΔT+pExoT | PA103ΔUΔT 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 lacIqZΔM15 Tn10 (Tetr) Amy Camr] | Stratagene |
| Plasmids | ||
| pAH806 | pBR322 containing a 3.5-kb genomic fragment from PA103 including the exoU gene | 23 |
| pBK151 | pUCP20 with ExoT(R149K) point mutation | This study |
| pBK152 | pEX100T with XmaI fragment from pBK151 | This study |
| pBK162 | pUCP20 with ExoT(R149G) point mutation | This study |
| pBK163 | pEX100T with XmaI fragment from pBK162 | This study |
| pEX100T | Allelic replacement suicide plasmid; Apr (Cbr)bsacB oriT | 45 |
| pEX100T/exoTgent | pEX100T with a 3,055-bp ApaI-NotI fragment of pGEM-T/exoTgent cloned into the SmaI site; Gmr | This study |
| pEX100T/exoU | pEX100T with a 3.2-kb SspI fragment from pAH806 including the exoU gene cloned into the SmaI site | This study |
| pEX100T/ΔexoU | pEX100T/exoU with a 726-bp BclI fragment encoding aa 330–571 of ExoU removed | This study |
| pGEM-T | Vector 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 388 | This study |
| pGEM-T/exoT | pGEM-T containing a 1,494-bp PCR product and bearing the exoT gene from strain PA103 | This study |
| pGEM-T/exoT+ | pGEM-T with a 1,619-bp PCR product and bearing the exoT gene and upstream ExsA binding site from PA103 | This study |
| pGEM-T/exoTgent | pGEM-T/exoT with a 939-bp SmaI fragment encoding ExoT with aa 36–348 replaced with a 2,446-bp SmaI fragment encoding the xylE aaC1 cassette from pX1918GT | This study |
| pUCP20 | P. aeruginosa expression vector; Apr (Cbr)b | 51 |
| pUCP20/exoS | pUCP20 with a 1,570-bp SacI-SphI fragment containing exoS from pGEM-T/exoS | This study |
| pUCP20/exoT | pUCP20 with a 1,666-bp NcoI-NdeI fragment containing exoT from pGEM-T/exoT | This study |
| pX1918GT | Plasmid containing xylE aacC1 insertional 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.