Table 1.
Strains and plasmids used
| Strain or plasmid | Source or description | Reference or source |
|---|---|---|
| E. coli Top10F′ | Invitrogen Inc. | |
| M. tuberculosis H37Rv | Lab stock | |
| M. smegmatis | ||
| mC2155 | Lab stock | |
| ΔcrgA | ΔcrgA Gentr | 29 |
| Δwag31 | Δwag31 Hygr | 20 |
| ΔcwsA | ΔcwsA Gentr | This study |
| DKO | ΔcrgA ΔcwsA Gentr Zeor | This study |
| Plasmid | ||
| pMV306 | Integration proficient E. coli-Mycobacterium shuttle vector, Kmr | 32 |
| pLR56 | E. coli-Mycobacterium shuttle vector, integrating, with tet promoter and tet repressor, Hygr | 13 |
| pLR52 | E. coli-Mycobacterium shuttle vector, replicating, with tet promoter and tet repressor, Hygr | 13 |
| pMG101 | E. coli-Mycobacterium shuttle vector, integrating, with amidase promoter, Hygr | This study |
| pMG103 | E. coli-Mycobacterium shuttle vector, integrating, with amidase promoter, Kmr | 13 |
| pGOAL17 | Carrying 6.1-kb PacI cassette, Kmr | 26 |
| pET-19b | E. coli expression vector allowing fusions to N-terminal 6× histidine tag (10× His), Ampr | Novagen |
| pET29b | E. coli expression vector allowing fusions to C-terminal 6× histidine tag (10× His), Kmr | |
| pTBSG | E. coli expression vector allowing fusions to N-terminal 6× histidine tag (6× His), Ampr | 30 |
| pTBMalE | E. coli expression vector allowing fusions to N-terminal maltose binding protein, Ampr | 30 |
| pKT25 | E. coli expression vector allowing fusions to C terminus of the T25 fragment of cyaA, Kmr | 21 |
| pUT18C | E. coli expression vector allowing fusions to C terminus of the T18 fragment of cyaA, Ampr | 21 |
| pKNT25 | E. coli expression vector allowing fusions to N terminus of the T25 fragment of cyaA, Kmr | 21 |
| pUT18 | E. coli expression vector allowing fusions to N terminus of the T18 fragment of cyaA, Ampr | 21 |
| pTBSG-8c | M. tuberculosis cwsA cloned in pTBSG, Ampr | 30 |
| pET29b-11c | M. tuberculosis crgA cloned in pET29b, Kmr | 22 |
| pTBMalE-11c | M. tuberculosis crgA cloned in pTBMalE, Ampr | 30 |
| pTBSG-M2 | Tobacco etch virus M2-coding region in pTBSG, Ampr | 30 |
| pPP7 | Pami::gfp-cwsA in pJFR19, Hygr | This study |
| pNM9 | CwsAsol (aa 1–98)-coding region in pLR52, Hygr | This study |
| pNM10 | PcwsA::cwsAsmeg in pMV306K, Kmr | This study |
| pPP17 | Pami::gfp-cwsAsol in pMG103, Hygr | This study |
| pPP30 | crgATB-cfp cloned in pLR52 vector, Hygr | 29 |
| pPP48 | crgATB cloned in pUT18C vector, Ampr | 29 |
| pPP53 | crgATB in pKT25 vector, Kmr | 29 |
| pPP46 | cwsATB cloned in pUT18C vector, Ampr | This study |
| pPP47 | cwsATB in pKT25 vector, Kmr | This study |
| pPP64 | cwsAsol in pUT18, Ampr | This study |
| pPP65 | cwsAsol in pKNT25 vector, Kmr | This study |
| pPP79 | Pami::gfp-ftsI in pMG103 | 29 |
| pRD65 | wag31 in pUT18 vector, Ampr | This study |
| pRD57 | wag31 in pKNT25 vector, Kmr | This study |
| pKS34 | Coding region for Wag31 C-terminal aa 61–260 cloned in pUT18, Ampr | This study |
| pKS35 | Coding region for Wag31 C-terminal aa 61–260 cloned in pKNT25, Kmr | This study |
| pEB6 | wag31-mCherry fusion cloned in pMG103, Kmr | This study |
| pEB16 | wag31-mCherry fusion cloned in pMG101, Hygr | This study |
| pJFR79 | ftsZsmeg-gfp in pJAM2, Kmr | 31 |
| pPP105a | MSMEG_0023 (cwsAsmeg) with flanking sequences (417 bp upstream and 600 bp downstream) cloned in PstI-NdeI sites of pGEM-T Easy, Ampr | This study |
| pPP105 | Blunt-ended gentamicin resistance cassette inserted in HincII site (37 bp from start codon) of cwsA-coding region in pPP105a, Ampr Gentr | This study |
| pPP106 | 6.1-kb PacI cassette from pGOAL17 inserted in pPP105, Ampr Gentr Sucr lacZ | This study |
| pPP115 | Blunt-ended zeocin resistance cassette inserted in HincII site (37 bp from start codon) of cwsA-coding region in pPP105a, Ampr Zeor | This study |
| pPP116 | 6.1-kb PacI cassette from pGOAL17 inserted in pPP115, Ampr Gentr Sucr lacZ | This study |