Generation of M. smegmatis smc mutant. (A) Southern blot analysis. Genomic DNA from M. smegmatis wild type (lane 1), smc mutant (lane 2), and the smc single-crossover mutant (lane 3) was digested with BamHI and probed with a 685-bp PvuII gene fragment containing the 5′ flanking region of the smc gene. The presence of a single 3.8-kbp fragment in the mutant strain instead of a 7.3-kbp fragment observed in the parental strain demonstrates successful deletion of smc coding sequences. (B) Schematic illustration of the smc locus and the Southern blot strategy. Shown are the genomic organization of the wild type (wt), the knockout vector that contains the deletion allele smc::hyg (vector), the single-crossover genotype (sco), and the mutated genomic smc region in the knockout mutant (ko). Fragments detected by the probe specific for the 5′ flanking region are shown in bold letters.

Analysis of M. smegmatis smc mutant strain. (A) Growth of M. smegmatis wild-type strain (solid lines) and smc mutant strain (dashed lines) at various temperatures. OD₆₀₀, optical density at 600 nm. (B) Survival following exposure to ionizing radiation. (C) Survival following exposure to UV.

Long-term survival of M. smegmatis wild-type strain (solid line) and smc mutant strain (dashed line).

Generation of M. tuberculosis smc mutant. Genomic DNA from M. tuberculosis wild-type (lane 1) and smc mutant (lane 2) strains was digested with BamHI and probed with a 935-bp NdeI gene fragment containing the 5′ flanking region of the smc gene. The presence of a single 4.3-kbp fragment instead of an 11.1-kbp fragment as seen in the parental strain demonstrates successful deletion of smc coding sequences.