TABLE 1.
Strains and plasmids used in this study
| Strain or plasmid | Description | Reference and/or source |
|---|---|---|
| Strains | ||
| E. coli XL10-GOLD | Tetr Δ(mcrA)183183 Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac Hte [F′ proAB lacIqZ ΔM15 Tn10 (Tetr) Amy Camr] | Stratagene |
| M. smegmatis mc2155 | High-transformation mutant of M. smegmatis mc26 | 40 |
| M. smegmatis GPM258 | mc2155(pBUN246); overproducing Ald | This study |
| M. smegmatis GPM267 | mc2155Δald::aph; ald null mutant | This study |
| M. smegmatis GPM268 | mc2155Δald::aph; ald null mutant | This study |
| M. smegmatis GPM269 | mc2155Δald::aph; ald null mutant | This study |
| M. smegmatis GPM270 | mc2155Δald::aph; ald null mutant | This study |
| M. smegmatis GPM276 | GPM267(pBUN279); M. smegmatis ald mutant complemented with the wild-type ald gene integrated at the mycobacteriophage L5 attB site | This study |
| M. smegmatis GPM282 | mc2155(pBUN286a); wild-type M. smegmatis carrying an additional copy of the ald promoter at the mycobacteriophage L5 attB site | This study |
| Plasmids | ||
| pBluescript II KS+ | Standard E. coli cloning vector | Stratagene |
| pCR2.1 | TA cloning vector | Invitrogen |
| pUC4K | Plasmid containing an excisable aminoglycoside 3′-phosphotransferase gene from Tn903 | Amersham Pharmacia (28) |
| pMV262 | E. coli-Mycobacterium shuttle plasmid | MedImmune (41) |
| pPR27 | E. coli-Mycobacterium shuttle plasmid with sacB and the temperature-sensitive mycobacterial ori | 31 |
| pYUB178 | E. coli-Mycobacterium integration-proficient vector; integrates at the mycobacteriophage L5 attB site | 29 |
| pYUB412 | E. coli-Mycobacterium integration-proficient vector; integrates at the mycobacteriophage L5 attB site | 30 |
| pBUN246 | Recombinant plasmid isolated from an M. smegmatis genomic library which hybridized with the M. tuberculosis ald gene; 8.0-kb insert | This study |
| pBUN253 | pCR2.1 with 1.5-kb PCR fragment containing complete M. smegmatis ald gene amplified with SMALDCF1 and SMALDCR | This study |
| pBUN261 | pCR2.1 with 0.4-kb PCR fragment containing the M. smegmatis ald promoter amplified with SMALDCF1 and SMALDPE | This study |
| pBUN268 | pBluescript II KS+ with 1.5-kb EcoRI fragment from pBUN253 in the EcoRI site | This study |
| pBUN270a | pBUN268 carrying the 1.2-kb blunted EcoRI fragment of pUC4K containing aph gene for the 0.7-kb NruI fragment internal of the ald gene | This study |
| pBUN272 | pPR27 with 2.2-kb EcoRV/XbaI fragment from pBUN270a in the BamHI (polished)/XbaI site | This study |
| pBUN279 | pYUB412 with 1.5-kb EcoRI fragment from pBUN253 in the EcoRI site | This study |
| pBUN286a | pYUB178 with 0.4-kb EcoRI fragment from pBUN261 in the EcoRI site | This study |