FIGURE 1.

PknB depletion and PknB over expression affects growth of mycobacteria. A, schematic representation of mc²ΔpknB conditional mutant. The primers used for PCR-based confirmation are depicted as F1 and F2 (forward primers) and R1 and R2 (reverse primers). B, agarose gels showing PCR amplification using the respective genomic DNA of different M. smegmatis strains, with various primer sets as indicated. Expected sizes of PCR amplicons are as follows: for the F1-R1 primer pair: wild type, 0.95 kb; mutants, no amplicon; for the F1-R2 primer pair: wild type, 1.98 kb; mutants, 0.49 kb; for the F2-R2 primer pair: wild type, 2.9 kb; mutants, 1.42 kb. Leftmost lane, gene ruler 1-kb DNA ladder; lane 1, mc²155 (wild type); lane 2, mc²HiT-PknB (merodiploid PknB strain); lane 3, mc²ΔpknB-23 (conditional depletion strain); lane 4, mc²ΔpknB-26 (conditional depletion strain); lane 5, negative conditional depletion strain. C, growth of M. smegmatis mc²155, mc²::pNit-PknB, negative mutant clone, and mc²ΔpknB clones 23 and 26 on 7H10 agar plates containing either no antibiotic, hygromycin only, or hygromycin with ATc inducer. D, all of the cultures were seeded at an initial A₆₀₀ of 0.2 in order to obtain sufficient cell pellets for whole cell lysate (WCL) preparations. WCLs from M. smegmatis mc²155 (mc²155), mc²155 transformed with pNit (mc²::pNit), or pNit-PknB (mc²::PknB) grown for 6 h in the absence or presence of 5 μm IVN inducer and PknB conditional mutant strain M. smegmatisΔpknB (mc²ΔpknB) grown for 6 h in the absence or presence of ATc were subjected to Western blotting with rabbit polyclonal α-PknB and α-GroEL1 antibodies. GroEL1 was used as a loading control to demonstrate equal loading of lysates. E, in vitro growth pattern analysis. All of the cultures were seeded at an initial A₆₀₀ of 0.02. Growth of the different strains was monitored as described under “Experimental Procedures.” F, schematic representation of M. tuberculosis-pptr-pknB (Rv-pptr-B) mutant. G, M. tuberculosis H37Rv (H37Rv), H37Rv transformed with pNit (Rv::pNit), or pNit-PknB (Rv::PknB) grown to A₆₀₀ ∼0.8 were used to seed fresh cultures. Cultures were seeded at an initial A₆₀₀ of 0.2 in order to obtain sufficient cell pellets for WCL preparations. Cultures were grown in the absence or presence of inducer (5 μm IVN) for 4 days. In the case of Rv-pptr-B, freshly seeded cultures (A₆₀₀ of 0.2) were grown in the presence or absence of pristinamycin for 4 days. WCLs were prepared and subjected to Western blotting with α-PknB, α-PknA, and α-GroEL1 (loading control) antibodies. H, in vitro growth pattern analysis of M. tuberculosis H37Rv cells that are either overexpressing PknB or depleted of PknB. All of the cultures were seeded at an initial A₆₀₀ of 0.1. Error bars, S.E.