TABLE 1.
Plasmids used in this study
| Plasmid | Description or source |
|---|---|
| pYUB509 | The complete coding region for leuD and leuC was generated by PCR of pYUB 516 (8) using primers B1A and B4B. The PCR fragment was digested with SnaB1 and NsiI and cloned into SnaB1/NsiI-digested pYUB469 (see below) to generate pLCD1. Plasmid pLCD1 was digested with SnaB1 and HindIII (partial); the resulting 5.2-kb fragment was treated with Klenow enzyme and cloned into SnaB1/PmlI-digested pYUB503 (see below) to generate pYUB506. The kanamycin resistance gene present in pYUB506 was removed by SpeI and HindIII (partial) digestion and replaced with the hygromycin gene generated by PCR of pMV261-H (a hygromycin-resistant derivative of pMV261 (36), using primers HYG1 and HYG2 to generate pYUB509. |
| pYUB503 | Plasmid pYUB178 (31) containing an EcoRI polylinker with PacI, SnaB1, KpnI, PmlI, and PacI restriction sites cloned into the EcoRI site. The KpnI site of pYUB178 was also destroyed with Klenow enzyme. |
| pYUB469 | A general laboratory plasmid containing the full-length, promoterless E. coli lacZ and firefly fflux genes, cloned in tandem into pBluescript KS (Stratagene, La Jolla, Calif.). |
| pG4697-6 | The integrating plasmid pYUB509 with a 211-bp insertion containing the iniBAC promoter region. The insert consisted of the sequence beginning 211 bp upstream of the first iniBAC open reading frame (iniB) extending to the translational start site (409142–409353). The sequence was generated from M. tuberculosis strain H37Rv genomic DNA by a seminested PCR using primers iigBPT and iigBPIIB followed by a second PCR using primers iigBproxhoT and iigBpro2xhoB, which contain 5′ XhoI sites. The PCR fragment was then inserted into the unique XhoI site of pYUB509 in the correct orientation. |
| pG1697-3 | Same as pG4697-6 but inserted into pYUB509 in the reverse orientation |
| pCV125 | Described in Materials and Methods |
| pG21898-12 | 211-bp iniBAC promoter sequence generated by PCR of pG4697-6 plasmid DNA using primers iniBproEcoT and iniBproSalB, inserted into the integrating plasmid pCV125 between the EcoRI and SalI restriction sites |
| pG21298-1 | pG21898-12 with 18-bp 5′ deletion of the 211-bp fragment, generated by PCR using primers iniBproEcoT20 and iniBproSalB |
| pG20298-2 | pG21898-1 with 42-bp 5′ deletion, generated using primers iniBproEcoT43 and iniBproSalB |
| pG20298-3 | pG21898-1 with 64-bp 5′ deletion, generated using primers iniBproEcoT65 and iniBproSalB |
| pG21298-4 | As in pG21898-1 with 89-bp 5′ deletion, generated using primers iniBproEcoT90 and iniBproSalB |
| pG20298-5 | pG21898-1 with 112-bp 5′ deletion, generated using primers iniBproEcoT113 and iniBproSalB |
| pG1199-6 | pG21898-1 with 133-bp 5′ deletion, generated using primers iniBproEcoT134 and iniBproSalB |
| pG1199-7 | pG21898-1 with 153-bp 5′ deletion, generated using primers iniBproEcoT154 and iniBproSalB |
| pG20298-8 | pG21898-1 with 173-bp 5′ deletion, generated using primers iniBproEcoT175 and iniBproSalB |
| pG20298-9 | pG21898-1 with 188-bp 5′ deletion, generated using primers iniBproEcoT189 and iniBproSalB |
| pG20298-10 | pG21898-1 with 19-bp 3′ deletion, generated using primers iniBproEcoT and iniBproSalB193 |
| pG599-11 | pG21898-1 with 39-bp 3′ deletion, generated using primers iniBproEcoT and iniBproSalB173 |
| pG15499-2 | pG20298-10 with 19-bp 3′ deletion and 19-bp 3′ spacer sequence, generated using primers iniBproEcoT and iniBproSalB193spa |
| pG15499-1 | pG21898-1 with 65-bp 3′ deletion, generated using primers iniBproEcoT and iniBproSalB148 |
| pG7897-4 | XmnI-PvuII fragment of the M. tuberculosis genome containing the iniB and iniA gene, cloned into the PvuII site of pMV261 (36) |
| pKB115 | Described in Materials and Methods |