TABLE 1.

Bacterial strains and plasmids used in this study

Strain or plasmidResistance phenotype or genotype or cloning vectoraDescription (source)
Strains
    E. faecium
        D344RApr Eryr TcrAmpicillin-resistant clinical isolate (9)
        C68Apr Eryr Gmr Tcr VmrClinical isolate; source for amplification of termini and flanking sequences of Tn5382
    E. coli
        DH10BΔM15 ΔlacX74 deoR recA1 araΔ139 Δ(ara leu)7697 galU galK ΔrpsL endA1 nupGTransformation-competent E. coli (Invitrogen)
        BL21(DE3)E. coli B F dcm ompT hsdS(rB mB) gal λ(DE3)Strain used to express Tn916 or Tn5386 integrase or excisase-integrase behind T7lac promoter in pCDF-1b (Novagen)
        CJ236(NEB)FΔ(HindIII)::cat (Tra+ Pil+ Camr)/ung-1 relA1 dut-1 thi-1 spoT1 mcrAStrain used for cloning dUTP-containing PCR product into PCR-XL-TOPO (Invitrogen)
        EPI300F mcrA Δ(mrr-hsdRMS-mcrBC) φ80dlacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara leu)7697 galU galK λ rpsL nupG trfA dhfrStrain used for maintaining copy control of pCC1 plasmids (Epicenter Biotechnologies)
Plasmids
    pUC18AprCloning vector
    pCR-XL-TOPOKmrE. coli vector for cloning PCR products directly (Invitrogen)
    pCC1(Blunt)CmrE. coli F-factor replicon (maintains one copy per cell) with an inducible high-copy oriV origin of replication (Epicenter Biotechnologies)
    pCDF-1bSmr SprReplicon from CloDF13 for protein expression (Novagen)
    pACYC184Cmr TcrCloning vector (10)
    pCWR1324pCC1(blunt)PCR product containing the integrase gene from Tn916
    pCWR1122pCC1(blunt)PCR product containing the integrase gene from Tn5386
    pCWR1121pCDF-1bInducible expression of Tn5386 integrase
    pCWR1323pCDF-1bInducible expression of Tn5386 integrase and excisase
    pCWR1326pCDF-1bInducible expression of Tn916 integrase
    pCWR1327pCDF-1bInducible expression of Tn916 integrase and excisase
    pCWR1109pACYC184bp 1 end and integrase end of Tn5386
    pCWR1110pACYC184bp 1 end and integrase end of Tn916
    pCWR1176pACYC184bp 1 end and integrase end of Tn5382
    pCWR1126pACYC184bp 1 end of Tn916 and integrase end of Tn5386
    pCWR1127pACYC184bp 1 end of Tn5386 and integrase end of Tn916
  • a Apr, ampicillin resistance; Cmr, chloramphenicol resistance; Eryr, erythromycin resistance; Kmr, kanamycin resistance; Smr, streptomycin resistance; Spr, spectinomycin resistance; Tcr, tetracycline resistance. Cloning vectors are specified for plasmids.