TABLE 1.
Bacterial strains, plasmids, and primers
| Strain, plasmid, or primer | Genotype, description, or sequencea | Source or reference |
|---|---|---|
| Strains | ||
| E. coli | ||
| DH5α | F− Φ80dlacZΔM15 Δ(lacZYA-argF)U169 deoR recA1 endA1 hsdR17(rK− mK+) | Gibco, BRL |
| ER2566 | F− λ− fhuA2 [lon] ompT lacZ::T7 gene1 gal sulA11 Δ(mcrC-mrr)114::IS10 R(mcr-73::miniTn10)2 R(zgb-210::Tn10) (Tets) endA1 [dcm] | 44 |
| BW25113 | Ampr(Ts); F− DE(araD-araB)567 lacZ4787(Δ)::rrnB-3 LAM− rph-1 DE(rhaD-rhaB)568 hsdR514 | 35 |
| M. tuberculosis | ||
| H37Rv | WT; ATCC 25618 | American Type Culture Collection |
| MHD5 | H37Rv mpa::MycoMarT7 Kanr | 45 |
| MHD802 | H37Rv ΔprcBA::hyg Hygr | 46 |
| MHD920 | H37Rv L5 attB::(Rv3781-Rv3783) Kanr | 9 |
| MHD932 | MHD920 ΔpafE::hyg Kanr Hygr | 9 |
| MHD980 | MHD932/pAJF381 Kanr Hygr Strepr | 9 |
| MHD1091 | MHD932/pAJF381-pafEΔ155–164 Kanr Hygr Strepr | 12 |
| MHD1160 | MHD980 hspRP40T Kanr Hygr Strepr | This study |
| MHD1202 | MHD980 hspRV38A Kanr Hygr Strepr | This study |
| MHD1203 | MHD980 hspRQ27H Kanr Hygr Strepr | This study |
| MHD1256 | MHD932/pSYMP-Strep-PPE12 hspRV38A Kanr Hygr Strepr | This study |
| MHD1257 | MHD932/pSYMP-Strep-PPE12 hspRV38A Kanr Hygr Strepr | This study |
| MHD1258 | MHD932/pSYMP-Strep-PE23 hspRV38A Kanr Hygr Strepr | This study |
| MHD1259 | MHD932/pSYMP-Strep-cyp136 hspRV38A Kanr Hygr Strepr | This study |
| MHD1260 | MHD932/pSYMP-Strep-cyp136 hspRR49H Kanr Hygr Strepr | This study |
| MHD1261 | MHD932/pSYMP-Strep-Rv3480c hspRR49H Kanr Hygr Strepr | This study |
| MHD1262 | MHD932/pSYMP-Strep-Rv3480c hspRR49H Kanr Hygr Strepr | This study |
| MHD1263 | MHD932/pSYMP-Strep-vapC47 hspRV38A Kanr Hygr Strepr | This study |
| MHD1264 | MHD932/pSYMP-Strep-vapC47 hspRM23T Kanr Hygr Strepr | This study |
| MHD1265 | MHD932/pSYMP-Strep-plcB hspRS90P Kanr Hygr Strepr | This study |
| MHD1266 | MHD932/pSYMP-Strep-plcB hspRV38A Kanr Hygr Strepr | This study |
| MHD1267 | MHD932/pSYMP-Strep-Rv2927c hspRV38A Kanr Hygr Strepr | This study |
| MHD1268 | MHD932/pSYMP-Strep-Rv2927c Rv0260cC422T tufC1168T Kanr Hygr Strepr | This study |
| Plasmids | ||
| pET-32a(+) | Ampr; for inducible production of recombinant protein in E. coli | Novagen |
| pET-32a(+)-HspR-G4-His | Ampr; for purification of recombinant His6-HspR | 9 |
| pET-32a(+)-HspRP40T-G4-His | Ampr; for purification of recombinant His6-HspRP40T | This study |
| pKD46 | Ampr(Ts); for arabinose-inducible production of Red recombinase in E. coli | 35 |
| pAJF381 | Strepr; for integration into the M. tuberculosis Tweety attB site | Gift from M. Glickman |
| pSYMP-Strep-tatA | Strepr; episomal plasmid for overexpression of hspR in M. tuberculosis using the constitutive hsp60 promoter | This study |
| pSYMP-Strep-hspR | Strepr; for overproduction of HspR in M. tuberculosis | This study |
| pSYMP-Strep-hspRP40T | Strepr; for overproduction of HspRP40T in M. tuberculosis | This study |
| pSYMP-Strep-PPE12 | Strepr; for overexpression of PPE12-FLAG in M. tuberculosis | This study |
| pSYMP-Strep-PE23 | Strepr; for overexpression of PE23-FLAG in M. tuberculosis | This study |
| pSYMP-Strep-cyp136 | Strepr; for overexpression of cyp136-FLAG in M. tuberculosis | This study |
| pSYMP-Strep-Rv3480c | Strepr; for overexpression of Rv3480c-FLAG in M. tuberculosis | This study |
| pSYMP-Strep-vapC47 | Strepr; for overexpression of vapC47-FLAG in M. tuberculosis | This study |
| pSYMP-Strep-plcB | Strepr; for overexpression of plcB-FLAG in M. tuberculosis | This study |
| pSYMP-Strep-Rv2927c | Strepr; for overexpression of Rv2927c-FLAG in M. tuberculosis | This study |
| Primers | ||
| hspR F NdeI | TTACATATGGCGAAGAACCCAAAGGACGGGGAATCC | |
| hspR R BamHI | TATGGATCCTCACCGGCGCGGTTTCCAGACG | |
| hspR 4G-His R BamHI | TATGGATCCTCAATGATGATGATGATGATGTCCTCCTCCTCCCCGGCGCGGTTTCCAGACG | |
| hspRP40T SOE R | GAGGTGCGCCGCGTGCTGACCAACCCAAGAC | |
| hspRP40T SOE F | GTCTTGGGTTGGTCAGCACGCGGCGCACCTC | |
| dnaK qPCR F | CAACTCCGTCGTCTCGGTTC | |
| dnaK qPCR R | GCGGGCGAACGCGACAATTG | |
| clpB qPCR F | GCTAACCGCGGCGTTACAG | |
| clpB qPCR R | AGTAGCGGTGCGGCGATAC | |
| dlaT qPCR F | CGGGGCACCACCCAGAAG | |
| dlaT qPCR R | CGCCCGCGCCAGACCGGC | |
| dnaK EMSA Fwd IR700 | /5IRD700/GCACGACCAGCGTTAGCATGCTCAG | |
| dnaK EMSA Rev | GTAGAGCTTCTTTCCTGCACTATGATGAG | |
| pSYMP-Strep Fwd | ACCGGCCGTGCGGAATTAAGCCGGCCCGTACCCTGTGAATAGAGGTCCGCTATGCGCTCACGCAACTGGTCCAG | |
| pSYMP-Strep Rev | CAGAGGAACTGCGCCAGTTCCTCCGGATCGGTGAAGCCGGAGAGATCCAGTTATTTGCCGACTACCTTGGTGATC | |
| PPE12_pSYMP_F_NdeI | GAAGGAGATATACATATGGTCGGTTTCGCGTGGTTGC | |
| PPE12-4G-FLAG R HindIII | ATCAAGCTTCTACTTGTCATCGTCATCCTTGTAATCTCCTCCTCCTCCGCGGTGGAAAAAGCCCGACACG | |
| PE23_pSYMP_F_NdeI | GAAGGAGATATACATATGCAGTTCCTGAGCGTGATTCC | |
| PE23-4G-FLAG R HindIII | ATCAAGCTTCTACTTGTCATCGTCATCCTTGTAATCTCCTCCTCCTCCGGAGATCAGCACCGTGACAGC | |
| cyp136_pSYMP_F_NdeI | GAAGGAGATATACATATGGCGACGATCCACCCCCCGGCATACC | |
| cyp136-4G-FLAG R HindIII | ATCAAGCTTCTACTTGTCATCGTCATCCTTGTAATCTCCTCCTCCTCCCCTGGGACGCAGCACGATCG | |
| Rv3480c_pSYMP_F_NdeI | GAAGGAGATATACATATGAGCCAGACGGCCCGGCGGTTGG | |
| Rv3480c-4G-FLAG Rev HindIII | ATCAAGCTTTCACTTGTCATCGTCATCCTTGTAATCTCCTCCTCCTCCGGAACCGAGGCCCGCGGCGCCGTCAG | |
| vapC47_pSYMP_F_NdeI | GAAGGAGATATACATATGATCTATATGGACACCTCGGCCCTGAC | |
| vapC47-4G-FLAG R HindIII | ATCAAGCTTTCACTTGTCATCGTCATCCTTGTAATCTCCTCCTCCTCCCCGGACTGCGCCGGGTGAG | |
| plcB F NdeI | TTACATATGACCCGCCGACAATTTTTTGC | |
| plcB-4G-FLAG R HindIII | ATCAAGCTTTCACTTGTCATCGTCATCCTTGTAATCTCCTCCTCCTCCACAGAGACCGCTGGGAATCC | |
| Rv2927c F NdeI | TTACATATGTACCGAGTCTTTGAAGCGCTGGAC | |
| Rv2927c-4G-FLAG R HindIII | ATCAAGCTTTTACTTGTCATCGTCATCCTTGTAATCTCCTCCTCCTCCGCGCACCGCGTAGTCGTGA |
a
Tets, tetracycline susceptible; Ts, temperature sensitive; Ampr, ampicillin resistance; Kanr, kanamycin resistance; Hygr, hygromycin resistance; Strepr, streptomycin resistance. /5IRD700/ indicates a 5′ infrared dye on the primer that emits at 700 nm.