TABLE 1.
Bacterial strains, plasmids, and primers used in this study
| Strain, plasmid, or primer | Relevant information or sequence | Restriction site | Reference and/or source |
|---|---|---|---|
| Escherichia coli | |||
| DH10B | F− araD139 Δ(ara, leu)7697 ΔlacX74 galU galK rpsL deoR ϕ80dlacZΔM15 endA1 nupG recA1 mcrA Δ(mrr hsdRMS mcrBC) | 30 | |
| MB3360 | pET28-TEV; M. tuberculosis murI in NdeI and BamHI in DH10B | This study | |
| Mycobacterium smegmatis | |||
| mc2155 wild type | ept-1 | 31 | |
| KK104-10 | mc2155 ΔmurI::aphA-3 Kanr; selected at 15 mM d-Glu | This study | |
| KK104-11 | mc2155 ΔmurI::aphA-3 Kanr; selected at 30 mM d-Glu | This study | |
| KK104-12 | mc2155 ΔmurI::aphA-3 Kanr; selected at 30 mM d-Glu | This study | |
| KK104-13 | mc2155 ΔmurI::aphA-3 Kanr; selected at 60 mM d-Glu | This study | |
| KK104-16 | mc2155 ΔmurI::aphA-3 Kanr; selected at 125 mM d-Glu | This study | |
| KK104-19 | mc2155 ΔmurI::aphA-3 murI+ Kanr; selected at 125 mM d-Glu | This study | |
| KK104-09 | KK104-10 harboring pKKYL03; Hygr | This study | |
| Plasmids | |||
| pBluescript II SK(+) | E. coli cloning vector; f1 Ori; Ampr | Stratagene | |
| pDM3 | Source of the mycobacterial alr promoter | 18 | |
| pKKYL02 | pPR23 harboring ΔmurI::aphA-3; Kanr Gmr | This study | |
| pKKYL03 | pUHA267 expressing murI; Hygr | This study | |
| pPR23 | E. coli-mycobacterial shuttle vector; oriM(Ts) sacB Gmr | 32 | |
| pUHA267 | E. coli-mycobacterial shuttle vector; Hygr | AgResearch, Wallaceville, New Zealand; 19 | |
| Primers | |||
| MurISM KO-3.1 | 5′-TTTAAAAAGCTTCCGAATTGGATTTGCTGCGA-3′ | HindIII | This study |
| MurISM KO-4.2 | 5′-AGACTAGTGTCTTCTCGTCTGGACACGT-3′ | SpeI | This study |
| MurISM KO-1.3 | 5′-CTCGTTCGACGCGGCCGCCCGCACCCGTGAACTCGTCA-3′ | NotI | This study |
| MurISM KO-2.2 | 5′-AAATTTGAATTCGCGCGGATCTGCGGGATGGT-3′ | EcoRI | This study |
| 5alr2 | 5′-AAATTTCCATGGTGGGGCAGTACTACAACTTC-3′ | KpnI | 18 |
| 3alrpro | 5′-GTGGTCTGCATATGCATAATCTCCGGCGCCCATT-3′ | NdeI | This study |
| MurISM EX-1.0 | 5′-TTTAAACATATGAGCGATCGACTCGCGCC-3′ | NdeI | This study |
| MurISM EX-2.1 | 5′-TTTAAAGGATCCGGTGCCAAGACATGCCCGGT-3′ | BamHI | This study |