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. 2003 Nov;71(11):6124–6131. doi: 10.1128/IAI.71.11.6124-6131.2003

FIG. 2.

FIG. 2.

Targeted knockout of RNR-encoding genes in M. tuberculosis. (A) Schematic representation of the inactivated nrdF2 allele showing the site of insertion of the hygromycin cassette, the position of the 1,126-bp BamHI-BglII probe (striped box) used for Southern blot analysis, and the extent of homologous DNA used in the knockout construct. (B) Southern blot analysis of nrdF2 recombinant strains. Chromosomal DNA from wild-type M. tuberculosis strain H37Rv (WT), a single-crossover recombinant carrying tandem copies of the inserted and wild-type alleles (SC), a single-crossover recombinant carrying a functional nrdF2 gene integrated at the attB locus (SCc), and a double-crossover recombinant with a crossover at the chromosomal locus of the nrdF2 allele arising from an SCc background (DCc) were digested with BamHI and probed with the BamHI-BglII probe containing the 5′ portion of the nrdF2 gene. (C) Schematic representation of the inactivated ΔnrdZ allele showing the 814-bp SalI deletion and the positions of the 2,011-bp SalI-PvuII probe (striped box) and the MluI sites used for Southern blot analysis. (D) Southern blot analysis of five ΔnrdZ allele replacement strains. Chromosomal DNA from wild-type M. tuberculosis H37Rv (WT) and five ΔnrdZ isolates were digested with MluI and probed with the SalI-PvuII probe shown in panel C. Abbreviations: Bg, BglII; H, HindIII; S, SalI; M, MluI; Pv, PvuII.