Bacterial strains and plasmids used in this study

Strain or plasmidDescriptionAntibiotic resistancebReference or source
    E. coli strains
        DH5αGeneral cloning strainNoneGibco BRL
        DY380Recombineering strainTetr33
    M. bovis BCG strainsa
        BCGM. bovis BCG Tice, parental strainNoneOrganon
        rBCG30rBCG30 TiceHygr26
        BCG mbtBΔmbtB::Kmr; mycobactin dependentKmrThis study
        rBCG(mbtB)30ΔmbtB::Kmr; mycobactin dependent, carries pNBV1-30 (Hygr)Kmr HygrThis study
        BCG panCDΔpanCD::Aprr; pantothenate auxotrophAprrThis study
        rBCG(panCD)30ΔpanCD::Aprr; pantothenate auxotroph, carries pNBV1-30 (Hygr)Aprr HygrThis study
        BCG trpDΔtrpD::Kmr; l-tryptophan auxotrophKmrThis study
        rBCG(trpD)30ΔtrpD::Kmr; l-tryptophan auxotroph, carries pMTB30 (Hygr)Kmr HygrThis study
    pNBV1-30Expression of 30-kDa antigen in mycobacteria, carries fbpB coding region and endogenous promoterHygr17
    pMTB30Expression of 30-kDa antigen in mycobacteria, carries 4.5-kb genomic fragmentHygr15
    pNBV1-mbtBExpression of mbtB from endogenous mbtB promoterHygrThis study
    pNBV1-panCDExpression of panCD from hsp60 promoterHygrThis study
    pNBV1-trpDExpression of trpD from endogenous trpD promoterHygrThis study
    pEX2Mycobacterial allelic exchange vector derived from pPR27; ori ts sacB codBAHygr18
    phEX1Derivative of phAE87 in which pZErO-2 replaces the pYUB328 cosmidKmrThis study
    phAE87Conditionally replicating mycobacteriophageAmpr2
    pNBV1E. coli-mycobacterial shuttle vectorHygr27
    pZErO-2Cloning vectorKmrInvitrogen
    pUC19-KmrCarries kanamycin resistance cassetteAmpr Kmr50
    pPE207Source of the apramycin resistance geneAprr38
  • a All BCG strains were derived from M. bovis BCG Tice.

  • b The apramycin resistance gene [aacC4, also known as aac(3)IV] also confers resistance to kanamycin and gentamicin.