Effect of Wag31 phosphorylation on nascent peptidoglycan biosynthesis. M. smegmatis wag31
deletion mutants containing wild-type Ptet-wag31
was cultured until mid-log phase and incubated with Van-alexa568 (5 μg ml-1) for 20 min at 37°C. Cells were washed with PBS buffer and examined by an Olympus BX51 microscope. To quantify the polar fluorescence intensity, DIC (middle panel) and fluorescence (upper panel) images were superimposed to align cells and fluorescence signals (lower panel), and the average fluorescence density from the poles of approximately 300 cells was determined by using the ImageJ software. Intensity of fluorescence signals relative to that of cells expressing wild-type gfp-wag31 is shown. p-values for the difference (one-tailed, unpaired t-tests): wild-type Wag31Mtb vs. Wag31T73EMtb = 1.1 × 10-4 significant, wild-type Wag31Mtb vs. Wag31T73AMtb = 3.3 × 10-10 significant (significant to p < 0.05). bar, 5 μm.