Table 2.
Bacterial strains and plasmids.
| Bacterial strain or plasmid | Relevant characteristics | Source |
|---|---|---|
| E. coli XL1 blue | recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′ proAB lacIqZΔM15 Tn10(Tcr)] | Stratagene |
| E. coli S17-1 | Used for mating constructs into P. aeruginosa; thi pro hsdR recA RP4-2 (Tc::Mu) (Km::Tn7) | Simon et al. (1983) |
| E. coli TOP10 | F−mcrAΔ(mrr-hsdRMS-mcrBC) Φ 80lacZΔM15ΔlacX74 recA1 araD139 galU galKΔ(ara-leu)7697rpsL(Strr) endA1 nupG | Invitrogen |
| E. coli BL21 (DE3) pLysS | F−dcm ompT hsdS(rB−mB−) gal λ(DE3) [pLysS Cmr] | Stratagene |
| PA103 | Virulent lung isolate of P. aeruginosa, known type III-secreted effector proteins are ExoT and ExoU | Liu (1966) |
| ΔfimX | PA103 containing an in-frame deletion of aa 15–685 of fimX ORF | This study |
| ΔfimX attB::FimX | FimX under control of its own promoter integrated at attB site of ΔfimX | This study |
| ΔfimX attB::FimXΔREC | FimXΔREC under control of its own promoter integrated at attB site of ΔfimX | This study |
| ΔfimX attB::FimXΔGGDEF | FimXΔGGDEF under control of its own promoter integrated at attB site of ΔfimX | This study |
| ΔfimX attB::FimXΔEAL | FimXΔEAL under control of its own promoter integrated at attB site of ΔfimX | This study |
| ΔUT | PA103 containing in-frame deletions of exoU ORF (aa 330–571) and exoT ORF (aa 36–348) | Garrity-Ryan et al. (2000) |
| ΔUTΔfimX | ΔfimX containing an in-frame deletion of aa 15–685 of fimX ORF | This study |
| ΔpilA | Gmr cassette inserted into pilA in PA103; Gmr | Whitchurch et al. (2005) |
| pGEM-T Easy | E. coli TA cloning vector; Apr | Promega |
| pBluescript-KS | E. coli cloning vector; Apr | Stratagene |
| tdimer2 | RFP dimer cloned in pBluescript-KS | C. Jacobs-Wagner (Yale) |
| pMO012502 | pLA2917 cosmid from PAO1 library containing PA4959 ORF; Tcr | Huang et al. (2000) |
| pUCP-SK | P. aeruginosa—E. coli shuttle vector; Apr(Cbr) | Watson et al. (1996) |
| mini-CTX-2 | Contains attP site for integration at the attB site of the P. aeruginosa chromosome; Tcr | Hoang et al. (1998) |
| pFLP2 | Source of inducible Flp recombinase; Apr(Cbr) | Hoang et al. (1998) |
| pEX100T | Allelic replacement suicide plasmid; Apr(Cbr) sacB oriT | Schweizer and Hoang (1995) |
| pKO–FimX | N- and C-terminal regions flanking FimX ORF cloned in tandem in pEX100T; Apr(Cbr) | This study |
| pFimX (pMLD9) | 2.9 kb BamHI-MfeI fragment of pMO012502 cloned into pUCP-SK; Apr(Cbr) | This study |
| pMLD9x | pFimX with altered MCS; Apr(Cbr) | This study |
| pFimXΔREC | pFimX with sequences encoding aa 67–147 deleted; Apr(Cbr) | This study |
| pFimXΔGGDEF | pFimX with sequences encoding aa 253–443 deleted; Apr(Cbr) | This study |
| pFimXΔEAL | pFimX with stop codon inserted after aa 444; Apr(Cbr) | This study |
| pFimX(GDSIF→AASIF) | pFimX with point mutations G346A D347A; Apr(Cbr) | This study |
| pFimX(EVL→AAA) | pFimX with point mutations E475A V476A L477A; Apr(Cbr) | This study |
| pBB2–FimX | pFimX with BB2 epitope tag inserted after initiating methionine of FimX; Apr(Cbr) | This study |
| pBB2–FimXΔREC | pFimXΔREC with BB2 epitope tag inserted after initiating methionine of FimX; Apr(Cbr) | This study |
| pBB2–FimXΔGGDEF | pFimXΔGGDEF with BB2 epitope tag inserted after initiating methionine of FimX; Apr(Cbr) | This study |
| pBB2–FimΔEAL | pFimXΔEAL with BB2 epitope tag inserted after initiating methionine of FimX; Apr(Cbr) | This study |
| pBB2–FimX(GDSIF→AASIF) | pFimX(GDSIF→AASIF) with BB2 epitope tag inserted after initiating methionine of FimX; Apr(Cbr) | This study |
| pBB2–FimX(EVL→AAA) | pFimX(EVL→AAA) with BB2 epitope tag inserted after initiating methionine of FimX; Apr(Cbr) | This study |
| pRFP | SacI–HindIII fragment of tdimer2 subcloned into pUCP-SK(RFP under control of lac promoter); Apr(Cbr) | This study |
| pRFP–FimX | pFimX with N-terminal RFP dimer fusion; Apr(Cbr) | This study |
| pRFP–FimXΔREC | pFimXΔREC with N-terminal RFP dimer fusion; Apr(Cbr) | This study |
| pRFP–FimX(GDSIF→AASIF) | pFimX(GDSIF→AASIF) with N-terminal RFP dimer fusion; Apr(Cbr) | This study |
| pRFP–FimX(EVL→AAA) | pFimX(EVL→AAA) with N-terminal RFP dimer fusion; Apr(Cbr) | This study |
| pBAD/His | Apr expression plasmid | Invitrogen |
| pET11 | Apr expression plasmid | Stratagene |
| pET21 | Apr expression plasmid | Stratagene |
| pHis6–FimX | pBAD/His; FimX, N-terminal His6 tag | This study |
| pHis6–FimX(GDSIF→AASIF) | pBAD/His; FimX, N-terminal His6 tag | This study |
| pHis6–FimX(EVL→AAA) | pBAD/His; FimX, N-terminal His6 tag | This study |
| pCC2 | pET11; pleD, C-terminal His6 tag | Paul et al. (2004) |
| pRP89 | pET11; pleD*, C-terminal His6 tag | Paul et al. (2004) |
| pMC24 | pET21; CC3396, C-terminal His6 tag | Christen et al. (2005) |
Ap, ampicillin; Cb, carbenicillin; Gm, gentamicin; Km, kanamycin; Tc, tetracycline; Cm, chloramphenicol; Str, streptomycin.