Figure 4

Localization of Wag31 and nascent peptidoglycan biosynthesis in the presence or absence of pknB _Mtb -overexpression. Early-log phase cells of M. smegmatis (KMS4) containing pCK314 were divided into two flasks, and pknB _Mtb was expressed in one of the flasks for 2 hr by adding 0.1% of acetamide. Subsequently, both cultures were incubated with tetracycline (20 ng ml^-1) for 2 hr to express gfp-wag31 _Mtb and examined by a florescent microscope. For vancomycin staining, Van-Alexa568 (5 μg/ml) was added to each culture at the end of tetracycline induction and further incubated for 20 min before examining by a fluorescence microscope. Average GFP and Van-Alex568 intensity from cells with pknB _Mtb -overexpression relative to that of cells without pknB _Mtb -overexpression are shown at the bottom of each panel. (p-value for the difference in GFP signals = 1.63 × 10^-11, and for Van-Alexa568 signals = 1.82 × 10^-7). Phosphorylation of GFP-Wag31 by pknB _Mtb -overexpression is shown at the bottom panel. 200 μg of total protein was used for 2-D PAGE and Western blot analysis with a phospho-(S/T)Q antibody, which was then stripped before conducting a subsequent Western blot with a GFP antibody. bar, 5 μm.