FIG. 2.

Deletion strategy and Southern blot analysis. (A) Schematic diagram of the ung locus. The organizations of the wild-type region, the knockout vector that contains the ung deletion allele, the 3′ single-crossover region, and the mutated genomic ung region in the knockout mutant are shown. Probe locations and BamHI fragments detected by the probe are indicated. (B) Southern blot analysis of the Δung strain. Genomic DNA from parental strain M. smegmatis mc²155 (lane 1), the 3′ single-crossover mutant (lane 2), the Δung mutant (lane 3), and the Δung Δ5031 double-knockout mutant (lane 4) were digested with BamHI and probed with a 1.2-kb ung gene fragment. The presence of a single 4.6-kb fragment in Δung and Δung Δ5031 mutants instead of the single 2.7-kb fragment in the parental strain demonstrates that there is inactivation of ung and that the wild-type allele is not present. (C) Schematic diagram of the M. smegmatis udgB locus. The organizations of the wild-type region, the knockout vector that contains the udgB deletion allele, the 5′ single-crossover region, and the mutated genomic udgB region in the knockout mutant are shown. Probe locations and SexAI fragments detected by the probe are indicated. (D) Southern blot analysis of the Δ5031 strain. Inactivation of the M. smegmatis udgB gene and the absence of the wild-type allele are indicated by the presence of a single 2.4-kb fragment in the Δ5031 (lane 3) and Δung Δ5031 (lane 4) mutants compared to the single 2.8-kb fragment in the parental strain (lane 1) when preparations were probed with a 340-bp M. smegmatis udgB gene fragment. The results for a 5′ single crossover are shown in lane 2. Abbreviations: B, BamHI restriction site; S, SexAI restriction site; wt, wild type; ko, knockout; sco, single crossover.