TABLE 1.

Strains and plasmids used

Strain or plasmidRelevant genotype or descriptionaReference or source
Bacterial strains
    H10407ETEC serotype 078:H11, LT+LST+29
    H10407-PDerivative of H10407 cured of 92-kb pCS1 plasmid
    H10407-SSpontaneous Smr derivative of H10407
    jf1181A10407 (pTnphoA.Ts) AmprThis study
    jf1289Isogenic etpA deletion mutantThis study
    jf1358etpC deletion mutant generated with cassette from pKD13, KmrThis study
    jf1370etpC deletion following curing of Kmr cassette using pCP20 helper plasmidThis study
    NovaBlueendA1 HsdR17 (rk12 mk12) supE44 thi-1 recA1 gyrA96 re1A1 lac F′ [proA+B+ lacI ZΔM15::Tn10 (Tcr)]Novagen
    DH10BT1F mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 deoR recA1: endA1 araD139 Δ(ara leu)7697 galU galkλrpsL nupG tonA48
    LMG194FΔlacZX74 galE thi rpsL ΔphoA (PvuII) Δara-714 leu::Tn10Invitrogen
    SM10λpirthi thr leu tonA lacY supE recA::RP4-2-Tc::Mu51
    BL21(DE3)F ompT hsdSp (rpmp) gal dcm (DE3)Novagen
Plasmids
    pSTBlue-13,851-bp PCR cloning plasmid, Ampr KmrNovagen
    pT7Blue-33,821-bp PCR cloning plasmid, Ampr KmrNovagen
    pFLAG-CTC5,348-bp expression plasmid for construction of C-terminal FLAG epitope fusions, AmprSigma
    pGPS1.1Tn7 transprimer donor plasmid, KmrNEBb
    pGPS2.1Tn7 transprimer donor plasmid, CmrNEB
    pBAD/Myc-His AArabinose-inducible expression plasmidInvitrogen
    pGEM-5zf (+)Cloning vector, amprPromega
    pST76-ATemperature-sensitive suicide plasmid, Ampr70
    pmini-OphoA3,362-bp self-cloning phoA minitransposon plasmid, Gmr10
    pET30a (+)5,422-bp His tag expression plasmid, KmrNovagen
    pJY0053,257-bp etpA fragment cloned in frame with polyhistidine tag on pET30a (+)This study
    pJFF0253,077-bp amplicon from pST76-A cloned into pSTBlue-1This study
    pTnphoA.ts2,983-bp BstEII/SacI fragment from pJFF025 cloned into corresponding sites on pmini-OphoA, yielding a Tn5/′phoA/Ampr-based plasposon with temperature-sensitive origin of replicationThis study
    pCVD442pir-dependent SacB+ suicide vector, Ampr25
    pJML00112-kb plasmid obtained by MluI digestion religation of genomic DNA from TnphoA.ts mutant 220This study
    pJY0012,400-bp PCR amplicon containing etpA in-frame deletion cloned into pT7Blue-3This study
    pJMF10011,819-bp PCR amplicon containing etpB cloned into EcoRV site of pT7Blue-3This study
    pJMF10021,811-bp HindIII-EcoRI etpB fragment from pJMF1001 cloned into pFLAG-CTCThis study
    pJY0175,317-bp amplicon containing etpA cloned into pT7Blue-3This study
    pJY019etpBAC locus cloned onto pBAD/Myc-His AThis study
    pJY0282,112-bp SphI fragement from pJY001 cloned into corresponding site of pCVD442This study
    pJY0355,307-bp EcoRI-BglII fragment from pJY017 cloned into pFLAG-CTC, yielding etpA-FLAG fusionThis study
    pJY0391,913-bp etpC HindIII-BglII fragment cloned into pFLAG-CTCThis study
    pJY040etpC cloned in frame with FLAG epitope on pFLAG-CTCThis study
    pKD46Temperature-sensitive λ-Red recombinase expression helper plasmid, Ampr22
    pKD13Template plasmid for construction of gene disruption cassette22
    pCP20FLP recombinase helper plasmid22
  • a Ampr, Kmr, Cmr, Gmr, and Smr, ampicillin, kanamycin, chloramphenicol, gentamicin, and streptomycin resistance, respectively; LT, heat-labile enterotoxin; ST, heat-stable enterotoxin.

  • b NEB, New England Biolabs.