Strategy for targeted knockout of msmeg_6554.

(A) msmeg_6554, a homologue of M.tb irtA in M.smeg was disrupted by introducing hygromycin cassette as described in materials and methods. A 3.3 kb insert at the msmeg_6554 locus was incorporated to create the knockout. (B) PCR amplifications indicating the disruption of msmeg_6554. Lane 2, negative control; lane 3, amplification from M.smeg mc²155 genomic DNA; lane 4, amplification from PCK48hyg vector; lane 5, KO48-SCO, single crossover; lane 6, KO48-DCO, double crossover. (C) RT-PCR of msmeg_6553 on RNA isolated from mc²155▵6554 grown under iron replete (lane 1) and iron depleted (lane 2) conditions.