TABLE 1.
Bacterial strains and plasmids used in this study
| Strain or plasmid | Relevant characteristics | Reference or source |
|---|---|---|
| E. coli strains | ||
| S17-1λpir | λpir lysogen of S17-1, recA RP4 2-Tc::Mu-Km::Tn7 | 47 |
| TOP10 | F−mcrA recA1 araD139 Δ(ara leu) endA1 | Invitrogen |
| EHEC strains | ||
| Sakai Δstx2A | Sakai strain with stx2A deletion | This study |
| Sakai Δure | Urease gene cluster deletion mutant of Sakai Δstx2A | This study |
| Sakai ureD(Q) | Sakai Δstx2A with T742C mutation in ureD, resulting in replacement of amber codon with glutamine in UreD; aadA inserted at attTn7, conferring Smr | This study |
| Sakai ureD(P,Q) | Sakai ureD(Q) with T113C mutation in ureD, resulting in L38P mutation in UreD | This study |
| Sakai tet(Am) | Sakai Δstx2A carrying mutant (Q56amber) Tcr cassette at attTn7 | This study |
| Plasmids | ||
| pSRS1 | Derived from suicide plasmid CVD442 by addition of mcs; contains Apr and sacB | This study |
| pSRS10 | Suicide vector for deletion of stx2A | This study |
| pKD3 | Cmr cassette flanked by FRTa sites | 8 |
| pKD46 | Red recombinase Relper plasmid, RepA101(Ts), Apr | 8 |
| pSRS20 | Suicide vector for construction of Sakai ureD(Q) | This study |
| pGRG24 | Plasmid for transgene insertion of aadA at attTn7 site on chromosome | 29 |
| pACYC184 | Tcr cassette | New England Biolabs |
| pSRS50 | First suicide vector for construction of Sakai ureD(P,Q) | This study |
| pSRS60 | Second suicide vector for construction of Sakai ureD(P,Q) | This study |
| pGFIB-1 | Vector for cloning tRNA for constitutive expression in E. coli, Apr | National Institute of Genetics, Japan (28) |
| pSUPE | pGFIB-1 expressing the supE tRNA | This study |
| pGRG36 | Plasmid for insertion of cloned transgene at attTn7 site on chromosome | 29 |
| pBADHisA | Apr, araBAD promoter based expression vector | Invitrogen |
| pLLamber | pBADHisA expressing 6× His UreD(L,L,amber) | This study |
| pLLQ | pBADHisA expressing 6× His UreD(L,L,Q) | This study |
| pPLQ | pBADHisA expressing 6× His UreD(P,L,Q) | This study |
| pPPQ | pBADHisA expressing 6× His UreD(P,P,Q) | This study |
| pLPQ | pBADHisA expressing 6× His UreD(L,P,Q) | This study |
a
FRT, FLP recombination target.