Fig 1.

Disruption of M. smegmatis lgt (MSMEG_3222). (Left) Genomic DNAs from M. smegmatis (lane 1), lgt single-crossover (5′sco) mutant (lane 2), Δlgt mutant (lane 3), and Δlgt-lgt_Mtb mutant (lane 4; lgt_Mtb indicates lgt from M. tuberculosis) were digested with XcmI and probed with a 967-bp ApaI lgt gene fragment. The presence of a single 5.6-kbp fragment in the Δlgt knockout strain compared to the single 5.4-kbp fragment in the parental strain demonstrates inactivation of lgt. The shift in fragment size in the Δlgt knockout strain results from replacement of a 959-bp lgt fragment with a 1.2-kbp kanamycin resistance cassette. (Right) Genomic DNAs of M. smegmatis (lane 5), lgt-5′ single-crossover mutant (lane 6), Δlgt mutant (lane 7), and Δlgt-lgt_Mtb mutant (lane 8) were digested with BamHI and probed with a 491-bp SacI M. tuberculosis lgt gene fragment to demonstrate complementation. Complementation is indicated by a hybridization signal with genomic DNA derived from strain Δlgt-lgt_Mtb (the M. tuberculosis lgt probe does not hybridize with M. smegmatis lgt).