TABLE 5.
Primers, plasmids, and strains used in this study
| Namea | Description | Source or reference |
|---|---|---|
| Primers | ||
| pETkshA1N-f&r | TATCCATGGTGACTACCGAGACAGCCGGC/TATCTCGAGGCTCGGCTGAGCCGGTTCTTT | This study |
| pETkshA2N-f&r | TATCCATGGTGACCGATATCCGCGAGATCG/TATCTCGAGCGCAGTCGAACCGCGACCG | This study |
| pETkshBN-f&r | TGGCCATGGTGACGGAGGAACCGCTCGGCA/TCACTCGAGTTCGTCGTAGGTGACTTCCA | This study |
| p261kshA1N-f&r | CAAGGATCCAGTGACTACCGAGACAGCCGGC/TAGAAGCTTCTACAGGTTTTCCTCGACCTCGA | This study |
| p261kshA2N-f&r | CAAGGATCCAATGACCGATATCCGCGAGATCG/TAGAAGCTTTTACGCAGTCGAACCGCGACCG | This study |
| p261kstD1-f&r | TACGGATCCAGTGTTCTACATGACTGCCCAGGA/GCCAAGCTTTCAGGCCTTTCCAGCGAGATGC | This study |
| o-p261-f&r | TAGGCGAGTGCTAAGAATAACGTTG/TCGTTTTATTTGATGCCTGGCAGT | This study |
| 16SrRNA-f&r | CCTATGTTGCCAGCGGGTTATGC/GCGATTACTAGCGACTCCGACTTCA | This study |
| RTqA1-f&r | GCGTCCTACTTCGGTCCGTCGTTCA/GTCGATCCGGGTCTTGTGCTTCCAG | This study |
| RTqA2-f&r | GCCTATCCGACGTACTTCAAGAAC/CACTGCAGGACGAACGAATCATGG | This study |
| kshA1V207T-f&r | TACCTGCACAACACCGGCCGTCCGGAT/ATCCGGACGGCCGGTGTTGTGCAGGTA | This study |
| kshA1P210E-f&r | AACGTCGGCCGTGAGGATGTCAACGAC/GTCGTTGACATCCTCACGGCCGACGTT | This study |
| kshA1D225R-f&r | GAGGCGCACCTGCGTTCCGAGGCGTCC/GGACGCCTCGGAACGCAGGTGCGCCTC | This study |
| kshA1N238D-f&r | TCGTTCATGATCGACTGGCTGCACAAC/GTTGTGCAGCCAGTCGATCATGAACGA | This study |
| Plasmids | ||
| pET-28a(+) | E. coli expression vector, Kanr | Novagen |
| pET28a-kshA1N | Expression plasmid pET28a possessing ORF of kshA1N | This study |
| pET28a-kshA1V207T | Expression plasmid pET28a possessing ORF of kshA1V207T | This study |
| pET28a-kshA1P210E | Expression plasmid pET28a possessing orf of kshA1P210E | This study |
| pET28a-kshA1D225R | Expression plasmid pET28a possessing ORF of kshA1D225R | This study |
| pET28a-kshA1N238D | Expression plasmid pET28a possessing ORF of kshA1N238D | This study |
| pET28a-kshA2N | Expression plasmid pET28a possessing ORF of kshA2N | This study |
| pET28a-kshBN | Expression plasmid pET28a possessing ORF of kshBN | This study |
| pMV261 | Shuttle vector of Mycobacterium and E. coli carrying the heat shock (hsp60) promoter, Kanr | W. R. Jacobs, Jr. |
| p261-16srRNA | pMV261 carrying the complete ORF of the 16S rRNA gene, for calibration in absolute quantification | This study |
| p261-kshA1N | pMV261 derived, possessing kshA1N under the control of the hsp60 promoter for augmentation | This study |
| p261-kshA1V207T | pMV261 derived, possessing kshA1V207T under the control of the hsp60 promoter for augmentation | This study |
| p261-kshA2N | pMV261 derived, harboring Phsp60-kshA2N operon | This study |
| p261-kstD1 | pMV261 derived, harboring Phsp60-kstD1 operon | This study |
| Strains | ||
| M. neoaurum | ||
| ATCC 25795 | Type strain, designated Mn25795 in this study | ATCC |
| MutkshA1N | Mutant of Mn25795 with inactivation of KshA1N | This study |
| MutkshA2N | Mutant of Mn25795 with inactivation of KshA2N | This study |
| Strain I | MutkshA1NA2N, mutant of Mn25795 with inactivation of KshA1N and KshA2N | This study |
| MutkstDN | Mutant of Mn25795 with inactivation of KstD1, KstD2, and KstD3 | This study |
| E. coli | ||
| BL21-kshA1N&kshA2N | Recombinant BL21(DE3) possessing pET28a-kshA1N or pET28a-kshA2N | This study |
| BL21-kshBN | Recombinant BL21(DE3) possessing pET28a-kshBN | This study |
| BL21-kshA1V207T | Recombinant BL21(DE3) possessing pET28a-kshA1V207T | This study |
| BL21-kshA1P210E | Recombinant BL21(DE3) possessing pET28a-kshA1P210E | This study |
| BL21-kshA1D225R | Recombinant BL21(DE3) possessing pET28a-kshA1D225R | This study |
| BL21-kshA1N238D | Recombinant BL21(DE3) possessing pET28a-kshA1N238D | This study |
| Strain II | Augmented strain containing p261kstD1, derived from strain I | This study |
| Strain III | Augmented strain containing p261kstD1-hsd4A, derived from strain I | This study |
| Strain IV | Augmented strain containing p261kshA1N, derived from MutkstDN | Yao et al. (4) |
| Strain V | Augmented strain containing p261kshA1V207T, derived from MutkstDN | This study |
a
Mn25795 is a soil isolate deposited in the American Type Culture Collection.