FIGURE 5.

Construction and characterization of M. smegmatis mtrA mutant. (A) Schematic of mtrAB region showing the deletion of nucleotides 126–322 followed by insertion of gentamicin cassette in the mtrA coding region. (B) Southern blot confirming the deletion of mtrA. Genomic DNA was isolated from WT M. smegmatis, one single-crossover (S) and two double-crossovers (D1 and D2), digested with NotI, transferred to Hybond-N+ membrane (GE Healthcare) and probed with mtrA fragment (black bar in A). Bands corresponding to the WT (1306-bp) and mutant (1921-bp) copy were seen in single crossover and those corresponding to WT only or mutant only copy were seen in WT and double crossover strains, respectively. Size of mutant band includes 851-bp gentamicin cassette. (C) The loss of MtrA in ΔmtrA strain was confirmed by immunoblotting with MtrA antibodies. (D) Loss of MtrA leads to filamentation and cell shape defects. Exponential cultures of M. smegmatis WT, ΔmtrA, ΔmtrA Pami::mtrA, and ΔmtrA Pami::mtrA_{Y 102C} were examined by bright field microscopy and imaged as described in the text. (E) qRT analysis of MtrA targets. Expression levels of select genes was measured, normalized to sigA and data presented as fold expression relative to the WT strain. The results shown are average from three independent experiments.