TABLE 1.
Bacterial strains, plasmids, and primers used in this study
| Strain, plasmid, or primer | Relevant genotype, description, or sequence (5′–3′) | Source or reference |
|---|---|---|
| Strains | ||
| E. coli | ||
| DH5α | F− ϕ80dlacZΔM15 Δ(lacZYA-argF)U169 deoR recA1 endA1 hsdR17(rK− mK+) phoA supE44 λ thi-1 gyrA96 relA1 | Gibco, BRL |
| BL21(DE3) | For inducible T7 expression; F− ompT gal dcm lon hsdSB(rB− mB−) λ(DE3 [lacI lacUV5-T7p07 ind1 sam7 nin5]) [malB+]K-12 (λs) | New England Biolabs, Inc. |
| M. tuberculosis | ||
| H37Rv | Wild type | ATCCa 25618 |
| MHD1428 | Hygr ΔbagAB::hyg | This work |
| MHD1385 | Kanr H37Rv/pMV306.kan | This work |
| MHD1431 | Hygr Kanr ΔbagAB::hyg ppsE/pMV306.kan | This work |
| MHD1432 | Hygr Kanr ΔbagAB::hyg ppsE/pMV-bagAB | This work |
| Plasmids | ||
| pMV306.kan | Kanr; plasmid that integrates at attB site on the M. tuberculosis chromosome | 33 |
| pMV-bagAB | Kanr; pMV306.kan with 2.3-kb bagAB insert including 200 bp of upstream sequence | This work |
| pET24b(+) | Kanr; for production of C-terminal His6 epitope-tagged protein | Novagen |
| pET24b(+)-bagAB | Kanr; for production of BagA-BagB-His6 | This work |
| pET24b(+)-bagABD56N | Kanr; for production of BagA-BagBD56N-His6 | This work |
| pET24b(+)-bagAE58QBD56N | Kanr; for production of BagAE58Q-BagBD56N-His6 | This work |
| pYUB854 | Hygr; allelic exchange vector. | 34 |
| pYUB-ΔbagAB | Hygr; allelic exchange vector with ΔbagAB::hyg | This work |
| pET24b(+)-bagAB | Kanr; for production of BagAB-His6 | This work |
| pET24b(+)-bagABD56N | Kanr; for production of BagABD56N-His6 | This work |
| pET24b(+)-bagAE58QBD56N | Kanr; for production of BagAE58QBD56N-His6 | This work |
| DNA primers | ||
| For cloning bagAB into pET24b(+) | ||
| Rv3679NdeIf1 | GCCATATGGTGGCAACCACATCTAGCGGCGG | |
| Rv3680XhoIr1 | GGCTCGAGGCGGCCCGCTGCGCGGCCCGCATC | |
| For making the ΔbagAB::hyg mutant | ||
| stuI-bagABup-for | CATGAGGCCTTCAACTCGACTTCCACCGGCGCATC | |
| xbaI-bagABup-rev | CGGTTCTAGACATGGCATGCTAGCCCGATGGCCTG | |
| xhoI-bagABdown-for | CATGCTCGAGGCGCTCGCCGACCAGCTCACCACGG | |
| speI-bagABdown-rev | CGGTACTAGTATACAGGTAACACCGATGTGCATGG | |
| For making the bagAB complementation plasmid | ||
| Xbal_BagA_F | GTGTCTAGAGATCTGTTTGGTGGCGTGCGTCAACAGC | |
| SOE_BagAB_F | GTCACACCGAAGACCCTCGATATGG | |
| SOE_BagAB_R | CCATATCGAGGGTCTTCGGTGTGAC | |
| BagB_EcoRV_R | TAAGATATCTCAGCGGCCCGCTGCGC | |
| For sequencing the bagAB locus | ||
| Rv3679seqf2 | CTGTTAGAAGCGCTGCCGGTG | |
| Rv3680seqr2 | CCGAGCACGGTGGACAACGCC | |
| For making the bagAE58Q mutant | ||
| Rv3679_E58Q_F | AGTCCTACTCGTCGAAGTCCAGGGGCGCCAGGGGATTGC | |
| Rv3679_E58Q_R | GCAATCCCCTGGCGCCCCTGGACTTCGACGAGTAGGACT | |
| For making the bagBD56N mutant | ||
| Rv3680_D56N_F | TGTGGTCGTTTTGACGATTAACCCAGCCAAGCGATTGGC | |
| Rv3680_D56N_R | GCCAATCGCTTGGCTGGGTTAATCGTCAAAACGACCACA | |
| For checking the ppsE sequence | ||
| ppsE seq1 | CTAAACCTGAAGTACAGGGAGGATAAGACC | |
| ppsE seq2 | ATGGGCTTCGGCGATGACATCGGCTTGAGC |
a
ATTC, American Type Culture Collection.