FIGURE 3.

Depletion of PstP in M. smegmatis leads to poor cell survival. A, schematic depiction of generation of gene replacement mutant using the recombineering method. Primers used for screening the mutant are shown. B, agarose gels showing PCR products obtained using different primer pairs with genomic DNA obtained from mc² (lane 1) and putative mc²-cd-pstP mutant (lane 2). F3-R3 amplifying a region of the rodA gene (∼1.5 kb) was used as a control. F1-R1, ∼1.5-kb fragment for mc² and no amplicon expected for mc²-cd-pstP. F2-R2, no amplicon expected for mc² and ∼1.1 kb for mc²-cd-pstP. C, mc², mc²::pKirT-pstP, and mc²-cd-pstP cultures were grown until A₆₀₀ reached ∼0.6, and the cultures were diluted to an A₆₀₀ of 0.1. Diluted cultures were streaked on 7H10 agar plates not containing or containing 50 ng/ml ATc. D, mc² and mc²-cd-pstP cultures were initiated at an A₆₀₀ of 0.1 and grown in the absence or presence of ATc for 6, 9, or 12 h. Whole cell lysates were isolated, resolved on SDS-PAGE, transferred to nitrocellulose membrane, and probed with α-PstP, α-PknB, and α-GroEL1 antibodies. E, mc² and mc²-cd-pstP cultures were initiated at an initial A₆₀₀ of 0.02 and grown in the absence or presence of ATc for 30 h. The experiment was performed in triplicates, and the error bar represents S.E. F, cfu were enumerated at 0 and 30 h for both mc² and mc²-cd-pstP in the presence and absence of ATc. cfu data were analyzed and plotted as mean with S.D. (error bars) using GraphPad Prism version 6, and significance was calculated using two-way ANOVA with p < 0.0001 (****).