TABLE 1.
Strains and plasmids used in this study
| Strain, plasmid, or mycobacteriophage | Genotype or descriptiona | Reference or source |
|---|---|---|
| Strains | ||
| E. coli DH5α | F− ϕ80lacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rK− mK+) phoA supE44 λ thi-1 gyrA96 relA1 | Lab collection |
| E. coli TOP10 | F− mcrA Δ(mrr-hsdRMS-mcrBC) ϕ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara-leu)7697 galU galK rpsL str endA1 nupG | Lab collection |
| E. coli BL21(DE3)/pLysS | F− ompT hsdSB(rB− mB−) gal dcm (DE3); contains pLysS | Lab collection |
| E. coli HB101 | F− Δ(gpt-proA)62 leuB6 glnV44 ara-14 galK2 lacY1 Δ(mcrC-mrr) rpsL20 str xyl-5 mtl-1 recA13 thi-1 | Lab collection |
| M. tuberculosis H37Rv | Laboratory strain | ATCC 27294 |
| M. smegmatis mc2155 | Laboratory strain | ATCC 700084 |
| TCZ1441 | M. smegmatis containing pTZ1001 | Lab collection |
| TCZ1820 | E. coli BL21(DE3)/pLysS containing pTZ1168 | This study |
| TCZ2054 | M. smegmatis ΔMSMEG_2695 | This study |
| TCZ2365 | M. smegmatis ΔMSMEG_2695 containing pTZ1227 | This study |
| TCZ2455 | M. smegmatis ΔMSMEG_2695 attB::pTZ1343 containing pTZ1227 | |
| TCZ2469 | M. smegmatis ΔMSMEG_2695 containing pTZ1167 | This study |
| TCZ2654 | M. smegmatis ΔMSMEG_2695 containing pTZ1489 | This study |
| TB284 | M. tuberculosis H37Rv ΔRv2744c | This study |
| TB303 | M. tuberculosis H37Rv ΔRv2744c containing pTZ1227 | This study |
| TB314 | M. tuberculosis H37Rv ΔRv2744c containing pTZ1463 | This study |
| Plasmids | ||
| CT158 | pMV261 containing GFPmut3 gene; Kanr | Eric Rubin |
| pCR2.1-TOPO | 3.9-kb plasmid for cloning PCR products; Ampr Kanr | Thermo Fisher Scientific |
| pET24b | 3.5-kb plasmid allowing N-terminal protein fusion to 6×-His; Ampr | EMD Biosciences |
| pKT25 | 3.4-kb plasmid allowing N-terminal protein fusion to T25 fragment; Kanr | Euromedex |
| pSE100 | TetR-regulated protein expression vector; Hygr | 37 |
| pTZ898 | pYUB854 containing sacB from pYUB870; Kanr | 31 |
| pTZ1001 | pMV261 expressing gfp; Kanr | Lab collection |
| pTZ1167 | pSE100 expressing 3×FLAG-6×His; Hygr | This study |
| pTZ1168 | pET-24b containing Rv2744c coding sequence cloned into NheI-NotI site; Kanr | 31 |
| pTZ1182 | pUT18c expressing Rv2744c coding sequence; Ampr | 31 |
| pTZ1183 | pUT18C containing Rv2744c coding sequence; Ampr | 31 |
| pTZ1185 | pKT25 expressing Rv2744c coding sequence; Kanr | 31 |
| pTZ1227 | pSE100 expressing 3×FLAG-Rv2744c-6×His; Hygr | This study |
| pTZ1231 | pTZ898 containing MS_2695 deletion sequence cloned into XbaI site; Hygr | This study |
| pTZ1246 | pYUB854 containing 1.0-kb fragments upstream and downstream of Rv2744c flanking Hygr cassette; Hygr | This study |
| pTZ1343 | pMV361 expressing tetR; Kanr | This study |
| pTZ1345 | pKT25 expressing Rv2745c (clgR) coding sequence; Ampr | This study |
| pTZ1346 | pUT18c expressing Rv2745c (clgR) coding sequence; Ampr | This study |
| pTZ1364 | pUT18 expressing Rv2745c (clgR) coding sequence; Ampr | This study |
| pTZ1379 | pUT18c expressing Rv2743c coding sequence; Ampr | This study |
| pTZ1380 | pUT18c expressing Rv2743c coding sequence; Ampr | This study |
| pTZ1463 | pSE100 containing Rv2744c-GFPmut3 gene; Hygr | This study |
| pTZ1489 | pSE100 expressing Rv2744c; Hygr | This study |
| pUT18 | 3.0-kb plasmid allowing C-terminal protein fusion to T18 fragment; Ampr | Euromedex |
| pUT18C | 3.0-kb plasmid allowing N-terminal protein fusion to T18 fragment; Ampr | Euromedex |
| pYUB854 | 3.9-kb cosmid for generating gene deletions in Mycobacterium; carries Hygr cassette flanked by multiple cloning sites and λpac site; Hygr | 33 |
| Mycobacteriophages | ||
| phAE87 | Derivative of conditionally replicating mycobacteriophage PH101(ts) | 33 |
| phTZ1246 | phAE87 derivative containing pTZ1246 | This study |
a
Kanr, kanamycin resistant; Ampr, ampicillin resistant; Hygr, hygromycin resistant.