TABLE 1.
Bacterial strains and plasmids
| Strain or plasmid | Relevant genotype or descriptiona | Reference(s) |
|---|---|---|
| Strains | ||
| H10407 | ETEC serotype O78:H11, LT+ ST+ | 14 |
| MM294 | endA hsdR pro supF | 59 |
| TnphoA.977 | eatA::TnphoA mutant obtained by random H10407 mutagenesis | This study |
| BL21 (DE3) | F−ompT hsdSB (rB− mB−) gal dcm(DE3) | Novagen |
| SM10λpir | thi thr leu tonA lacY supE recA::RP4-2-Tc::Mu | 29, 56 |
| DH5αpir | supE44 ΔlacU 169 (φ80lacZΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 | 57 |
| DH10B T1 | F−mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 deoR recA1 endA1 araD139 Δ(ara leu)7697 galU galK λ−rpsL nupG tonA | 28 |
| LMG194 | F− ΔlacX74 galE thi rpsL ΔphoA (Pvull) Δara-714 leu::Tn10 | Invitrogen |
| Plasmids | ||
| pRT291 | IncP1 TnphoA, Kmr Tcr | 59 |
| pPH1HI | IncP1, tra+ Spr Gmr | 50 |
| pCVD422 | pACYC184-based cloning plasmid with MluI linker inserted into EcoRV site | 10 |
| pRE107 | R6K ori-based suicide plasmid derived from pGP704, Ampr | 11 |
| pSG76-C | R6K ori-based suicide plasmid, Cmr | 48 |
| pPIR-K | pSC101ts-based pir helper plasmid, Kmr | 48 |
| pST76K-Sac | Temperature-sensitive sacB suicide plasmid, Kmr | 48 |
| pT7Blue-3 | PCR cloning vector, Ampr Kmr | Novagen |
| pET-30b(+) | Polyhistidine fusion protein expression vector, Kmr | Novagen |
| pGEM-7zf(+) | Cloning vector, Ampr | Promega |
| pJTH019 | 8,653-bp BspHI fragment containing the eatA-TnphoA junction from strain TnphoA.977 cloned into pCVD422 | This study |
| pJTH023 | 611-bp PCR amplicon internal to eatA gene cloned into EcoRV site of pT7Blue-3 | This study |
| pJTH024 | 647-bp EcoRI fragment from pJTH023 cloned into pRE107 (Ampr) | This study |
| pWY002 | Recombination of pJTH024 in H10407, excision with NotI and religation | This study |
| pJTH025 | 1,483-bp eatA gene fragment cloned into pT7Blue-3 | This study |
| pET30b(+).EatA | Six-histidine-rEatA fusion expression plasmid, Kmr | This study |
| pSP001 | 1,579-bp internal eatA fragment cloned into pT7Blue-3 | This study |
| pSP007 | 1,628-bp SalI-SphI pSP001 fragment cloned into pSG76-C | This study |
| pSP008 | Recombination of pSP007 with native H10407 plasmid, Cmr | This study |
| pSP009 | 4,267-bp amplicon cloned into EcoRV site of pSTBlue-1 | This study |
| pSP010 | 3,253-bp BstXI eatA fragment from pSP009 into pST76KSac | This study |
| PGPS2.1 | 4,490-bp Tn7 (cat) donor plasmid, R6K-g ori Cmr Tcr | NEBb |
| pSP011 | Transposition of Tn7 Cmr cassette into pSP010 eatA fragment | This study |
| pBAD-TOPO | 4,126-bp expression plasmid; arabinose promoter | Invitrogen, 23 |
| pSP014 | 4,185-bp eatA amplicon cloned into pBAD-TOPO | This study |
| pSP018 | pSP014 altered by SDM for Ser267 → Gly267 substitution of EatA | This study |
| pSP019 | pSP014 altered by SDM for His134 → Arg134 substitution of EatA | This study |
| PSP020 | pSP014 altered by SDM for Asp162 → Ala162 substitution of EatA | This study |
a
Ampr, Cmr, Kmr, Spr, and Tcr denote ampicillin, chloramphenicol, kanamycin, spectinomycin, and tetracycline resistance, respectively. SDM, site-directed mutagenesis.
b
NEB, New England Biolabs.