Fig. 3.

aftD is an essential gene of M. smegmatis mc²155. (A) Evidence for allelic replacement at the aftD locus of M. smegmatis in the presence of a rescue copy of this gene expressed from an episomal plasmid. Allelic exchange mutants were rescued with the aftD gene from M. smegmatis expressed from pCG76MSMEG_0359. Allelic replacement was confirmed by PCR using primers smg0236c.3 and smg0236c.6 (see Material and methods). The WT 4560-bp amplification signal is replaced by a 3541-bp fragment in the mutants due to the 2219-bp AgeI deletion in the aftD gene and insertion of a 1.2 kb-kanamycin resistance cassette. (B) Growth characteristics of WT mc²155 (diamonds) and two independent mc²155ΔMSMEG_0359/pCG76MSMEG_0359 conditional mutants (squares and triangles) in the LB-Kan-Tween80 medium at 30°C (where the rescue plasmid replicates) and 42°C (where the rescue plasmid is lost).