TABLE 1 .
Strains, plasmids, and primers used in this study
| Strain, plasmid, or primer | Phenotype, genotype, or sequence | Source or reference |
|---|---|---|
| M. tuberculosis strains | ||
| H37Rv | WT | ATCC 25618 |
| MHD18 | Hygr WT/pMV306 | 12 |
| MHD22 | Kanr Hygr mpa::ΦMycoMarT7/pMV306 | 12 |
| MHD23 | Kanr Hygr mpa::ΦMycoMarT7/pMV-mpa | 12 |
| MHD62 | Kanr Hygr pafA::ΦMycoMarT7/pMV306 | 16 |
| MHD63 | Kanr Hygr pafA::ΦMycoMarT7/pMV-pafA | 16 |
| MHD131 | Kanr lpqS::ΦMycoMarT7 (transposon inserted in codon 44) | This work |
| MHD188 | Kanr Rv2963::ΦMycoMarT7 (transposon inserted in codon 351) | This work |
| MHD696 | Kanr socA::ΦMycoMarT7 (transposon inserted in codon 22) | This work |
| MHD701 | Hygr ΔmymT::hyg | 15 |
| MHD702 | Kanr Hygr ΔmymT::hyg/pMV306.kan-mymT | 15 |
| MHD752 | Hygr ΔmmcO::hyg | This work |
| MHD764 | Kanr Hygr lpqS::ΦMycoMarT7 ΔmmcO::hyg | This work |
| MHD794 | Kanr WT/pMV306.kan | This work |
| MHD795 | Kanr Hygr ΔmmcO::hyg/pMV306.kan | This work |
| MHD796 | Kanr Hygr ΔmmcO::hyg/pMV306.kan-mmcO | This work |
| MHD840 | Kanr Hygr ΔmymT::hyg ΔmmcO::kan | This work |
| MHD867 | Kanr Hygr ΔmymT::hyg ΔmmcO::kan/pMVstrep-mmcO mymT | This work |
| CDC1551 | WT | W. Bishai lab collection |
| MHD557 | Kanr ricR::ΦMycoMarT7 | 14 |
| MHD583 | Hygr WT/pMV306 | 11 |
| MHD589 | Kanr Hygr ricR::ΦMycoMarT7/pMV306 | 11 |
| MHD590 | Kanr Hygr ricR::ΦMycoMarT7/pMV-ricR | 11 |
| MHD694 | Kanr Hygr ricR::ΦMycoMarT7/pMV-ricRC38A | This work |
| MHD707 | Kanr Hygr ricR::ΦMycoMarT7/pMV-ricRpc-ricR | This work |
| MHD708 | Kanr Hygr ricR::ΦMycoMarT7/pMV-ricRpc-ricRC38A | This work |
| MHD755 | Hygr ΔmmcO::hyg | This work |
| E. coli strains | ||
| DH5α | F− ϕ80dlacZΔM15 Δ(lacZYA-argF)U169 deoR recA1 endA1 hsdR17 (rK− mK+) phoA supE44 λ-thi-1 gyrA96 relA1 | 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] | 35 |
| Plasmids | ||
| pET24b(+) | Kanr, for production of C-terminally His6 epitope-tagged protein | Novagen |
| pET24b(+)-mmcO | Kanr, for production of MmcO-His6 in E. coli | This work |
| pET24b(+)-ricR | Kanr, for production of untagged RicR in E. coli | 11 |
| pET24b(+)-ricRC38A | Kanr, for production of untagged RicRC38A in E. coli | This work |
| pMV306.kan | Kanr, integrates at attB site in mycobacterial chromosome | 36 |
| pMV306 | Hygr, integrates at attB site in mycobacterial chromosome | 36 |
| pMV306.strep | Strepr, integrates at attB site in mycobacterial chromosome | Gift from J. McKinney lab |
| pMV306.kan-mmcO | Kanr, for complementation of mmcO mutant | This work |
| pMV-ricR | Hygr, for complementation of ricR mutant | 11 |
| pMV-pc-ricR | Hygr, WT ricR expressed from mutated ricRp (see text) | This work |
| pMV-pc-ricRC38A | Hygr, ricRC38A allele expressed from mutated ricRp | This work |
| pMVstrep-mmcO mymT | Strepr, for complementation of mmcO mymT double mutant | This work |
| pYUB854 | Hygr, allelic-exchange vector | 32 |
| pYUB854.kan | Kanr, allelic-exchange vector | This work |
| pYUB854-mmcO | Hygr, M. tuberculosis ΔmmcO::hyg (deletion-disruption plasmid) | This work |
| pYUB854.kan-mmcO | Kanr, M. tuberculosis ΔmmcO::kan (deletion-disruption plasmid) | This work |
| Primers | ||
| NdeI Rv0846c F1 | GGCATATGCCCGAGCTGGCCACGAGCGGTAAC | |
| XhoI Rv0846c R1 | GGCTCGAGCAGAATGTAGTCCAGGCGGGTCGC | |
| Rv0846cF2seq | GGCACCGAGCCCGCGACTGCGAACATC | |
| Rv0846cR2seq | CCCGGCCAGCGCGATGCGGAACGCGGT | |
| KpnI 846cMutantF1 | GAGGGTACCTATCTGCGGGTTGGAGGTGATGCTTGTTG | |
| XbaI 846cMutantR1 | GTTACCGCTCGTGGCTCTAGACAGCTCGGGCATCGATC | |
| HindIII 846cMutantF2 | ACCCGCCTGGACAAGCTTTACATTCTGTGACAGGCGG | |
| SpeI 846cMutantR2 | TCAGGAGCTCATCGAGTTACTAGTGGATGCCGTAACC | |
| deltammcOF | AGGAGTGACTTGATATCCCTCCGGG | |
| deltammcOR | ATTGCGGAAGCCATTCACGATGGAC | |
| HindIIImmcOF2 | CAAAGCTTACGTGCCCGCTTTCCACGTGGCCC | |
| mmcOcompHpaIR | CTGTTAACTCACAGAATGTAGTCCAGGCGGGTC | |
| C38A SOE F | GTACGCCATTGACGTTCTGACC | |
| C38A SOE R | GGTCAGAACGTCAATGGCGTAC | |
| F1ricRpromsoe | CCGATACCCCGCTGTTGTACAAGATATGAT | |
| R1ricRpromsoe | ATCATATCTTGTACAACAGCGGGGTATCGG | |
| Rv0190 comp F-HindIII | GACAAGCTTCATTGTTCAAGTATGCGGCCCAAG | |
| Rv0190 comp R-KpnI | GACGGTACCTCAGGAACGAACCAGGCGCGCG | |
| Rv0190 F NdeI | GACCATATGACAGCAGCACACGGCTACAC | |
| Rv0190 Rev. Stop EcoRI | GACGAATTCTCAGGAACGAACCAGGCGCGCGATTG | |
| lpqS affinity F-BIO | BioTEG-ATCGCTCCTCGTCTGGATTT | |
| lpqS affinity R | AGCGCGACCGCGACAATC | |
| AgeI pYUBhygtokanfor | CAACCGGTCCCTCCCAAGGACACTGAGTCCTAAAG | |
| NcoI pYUBhygtokanrev | GTCCATGGTTAGAAAAACTCATCGAGCATCAAATG | |
| HpaI-mymT-for | GCGGTTAACGGGCGGTTGGGTTGCTGG | |
| MfeI-mymT-rev | GGCAATTGATAGGTCTACTTGACCGGGGCC |