TABLE 5

Primers, plasmids, and strains used in this study

NameaDescriptionSource or reference
Primers
    pETkshA1N-f&rTATCCATGGTGACTACCGAGACAGCCGGC/TATCTCGAGGCTCGGCTGAGCCGGTTCTTTThis study
    pETkshA2N-f&rTATCCATGGTGACCGATATCCGCGAGATCG/TATCTCGAGCGCAGTCGAACCGCGACCGThis study
    pETkshBN-f&rTGGCCATGGTGACGGAGGAACCGCTCGGCA/TCACTCGAGTTCGTCGTAGGTGACTTCCAThis study
    p261kshA1N-f&rCAAGGATCCAGTGACTACCGAGACAGCCGGC/TAGAAGCTTCTACAGGTTTTCCTCGACCTCGAThis study
    p261kshA2N-f&rCAAGGATCCAATGACCGATATCCGCGAGATCG/TAGAAGCTTTTACGCAGTCGAACCGCGACCGThis study
    p261kstD1-f&rTACGGATCCAGTGTTCTACATGACTGCCCAGGA/GCCAAGCTTTCAGGCCTTTCCAGCGAGATGCThis study
    o-p261-f&rTAGGCGAGTGCTAAGAATAACGTTG/TCGTTTTATTTGATGCCTGGCAGTThis study
    16SrRNA-f&rCCTATGTTGCCAGCGGGTTATGC/GCGATTACTAGCGACTCCGACTTCAThis study
    RTqA1-f&rGCGTCCTACTTCGGTCCGTCGTTCA/GTCGATCCGGGTCTTGTGCTTCCAGThis study
    RTqA2-f&rGCCTATCCGACGTACTTCAAGAAC/CACTGCAGGACGAACGAATCATGGThis study
    kshA1V207T-f&rTACCTGCACAACACCGGCCGTCCGGAT/ATCCGGACGGCCGGTGTTGTGCAGGTAThis study
    kshA1P210E-f&rAACGTCGGCCGTGAGGATGTCAACGAC/GTCGTTGACATCCTCACGGCCGACGTTThis study
    kshA1D225R-f&rGAGGCGCACCTGCGTTCCGAGGCGTCC/GGACGCCTCGGAACGCAGGTGCGCCTCThis study
    kshA1N238D-f&rTCGTTCATGATCGACTGGCTGCACAAC/GTTGTGCAGCCAGTCGATCATGAACGAThis study
Plasmids
    pET-28a(+)E. coli expression vector, KanrNovagen
    pET28a-kshA1NExpression plasmid pET28a possessing ORF of kshA1NThis study
    pET28a-kshA1V207TExpression plasmid pET28a possessing ORF of kshA1V207TThis study
    pET28a-kshA1P210EExpression plasmid pET28a possessing orf of kshA1P210EThis study
    pET28a-kshA1D225RExpression plasmid pET28a possessing ORF of kshA1D225RThis study
    pET28a-kshA1N238DExpression plasmid pET28a possessing ORF of kshA1N238DThis study
    pET28a-kshA2NExpression plasmid pET28a possessing ORF of kshA2NThis study
    pET28a-kshBNExpression plasmid pET28a possessing ORF of kshBNThis study
    pMV261Shuttle vector of Mycobacterium and E. coli carrying the heat shock (hsp60) promoter, KanrW. R. Jacobs, Jr.
    p261-16srRNApMV261 carrying the complete ORF of the 16S rRNA gene, for calibration in absolute quantificationThis study
    p261-kshA1NpMV261 derived, possessing kshA1N under the control of the hsp60 promoter for augmentationThis study
    p261-kshA1V207TpMV261 derived, possessing kshA1V207T under the control of the hsp60 promoter for augmentationThis study
    p261-kshA2NpMV261 derived, harboring Phsp60-kshA2N operonThis study
    p261-kstD1pMV261 derived, harboring Phsp60-kstD1 operonThis study
Strains
    M. neoaurum
        ATCC 25795Type strain, designated Mn25795 in this studyATCC
        MutkshA1NMutant of Mn25795 with inactivation of KshA1NThis study
        MutkshA2NMutant of Mn25795 with inactivation of KshA2NThis study
        Strain IMutkshA1NA2N, mutant of Mn25795 with inactivation of KshA1N and KshA2NThis study
        MutkstDNMutant of Mn25795 with inactivation of KstD1, KstD2, and KstD3This study
    E. coli
        BL21-kshA1N&kshA2NRecombinant BL21(DE3) possessing pET28a-kshA1N or pET28a-kshA2NThis study
        BL21-kshBNRecombinant BL21(DE3) possessing pET28a-kshBNThis study
        BL21-kshA1V207TRecombinant BL21(DE3) possessing pET28a-kshA1V207TThis study
        BL21-kshA1P210ERecombinant BL21(DE3) possessing pET28a-kshA1P210EThis study
        BL21-kshA1D225RRecombinant BL21(DE3) possessing pET28a-kshA1D225RThis study
        BL21-kshA1N238DRecombinant BL21(DE3) possessing pET28a-kshA1N238DThis study
    Strain IIAugmented strain containing p261kstD1, derived from strain IThis study
    Strain IIIAugmented strain containing p261kstD1-hsd4A, derived from strain IThis study
    Strain IVAugmented strain containing p261kshA1N, derived from MutkstDNYao et al. (4)
    Strain VAugmented strain containing p261kshA1V207T, derived from MutkstDNThis study
  • a Mn25795 is a soil isolate deposited in the American Type Culture Collection.