TABLE 1.

Bacterial strains and plasmidsb

Strain or plasmidRelevant propertiesSource or reference
E. coli
    SM10F tonA21 lacY1 supE44 Mu λ [RP4-2 (Tc::Mu)]Km Tra+43
    Top10F mcrA Δ(mrr-hsdRMS-mcr BC) φ80lacZΔM15 ΔlacX74 deoR rec araD139 Δ(ara-leu)7697 galU galK rpsL endA1 nupGInvitrogen
    recA::RP4::ΔnicchrRP4::Δnic Km DAP1
    C1arep+ ilv+ Nal40
    BL21λ DE3F ompT hsdSB(rB mB) gal dcm (DE3)Invitrogen
E. faecalis
    JH2-2::Tn1549Fus Rif Vm28
Plasmids
    pACYCGmDerivative of pACYC184 in which the cat-tet fragment was replaced by aac(3)-IV; Gm ApraLaboratory
    pCM105pACYCGm Ω250-bp EcoRI-HindIII PCR intergenic fragment orf29-orf30 from Tn1549This study
    pCM105-1pCM105 (C141→T)aThis study
    pCM105-2pCM105 (G152G154→ T152C154)This study
    pCM105-3pCM105 (G159C160→ T159G160)This study
    pCM105-4pCM105 (G194C195→ T194G195)This study
    pCM105-5pCM105 (C216C218→ G216G218)This study
    pCM106pACYCGm Ω28-bp oriTTn1549This study
    pCM107pACYCGm Ω130-bp EcoRI-HindIII PCR part of the PCR intergenic fragment orf29-orf30 from Tn1549This study
    pBAD/HisAExpression vector, araBAD promoter; AmpInvitrogen
    pCM108pBAD/HisA Ω1,328-bp NcoI-XhoI PCR fragment (rlxTn1549)This study
    pUC18GmDerivative of pUC18 in which blaT is replaced by aac(3)-IV; Gm ApraThis study
    pCM109pUC18Gm Ω1,879-bp EcoRI-XbaI PCR fragment (intergenic orf30-orf29 region, orf29, and orf28) of Tn1549This study
    pCM110pUC18Gm Ω2,664-bp EcoRI-XbaI PCR fragment (intergenic region orf30-orf29, orf29, orf28, orfz, and orf27) of Tn1549This study
    pGB2Spc Str15
    pCM111pGB2 Ω372-bp EcoRI-HindIII PCR fragment (mobC) of Tn1549This study
    pET28a(+)His tag expression vector; KmInvitrogen
    pCM112pET28a+ Ω354-bp EcoRI-NdeI PCR fragment (mobC) of Tn1549This study
    pCM113pET28a+ Ω1,325-bp NcoI-XhoI PCR fragment (rlx) of Tn1549This study
    pCM114pET28a+ Ω741-bp NcoI-XhoI PCR fragment (N247rlx) of Tn1549This study
    pCR2.1PCR cloning vector; Amp KmInvitrogen
    pCM115pCR2.1 Ω2,374 bp PCR fragment (intergenic region orf30-orf29 mobC rlx orfz) of Tn1549This study
  • a Sequence was from GenBank accession number AF192329.1, but numbering refers to the orf30 termination codon.

  • b Amp, ampicillin; Apra, apramycin; Fus, fusidic acid; Gm, gentamicin; Km, kanamycin; Nal, nalidixic acid; Rif, rifampin; Str, streptomycin; Vm, vancomycin.