FIG 5.
Loss of MtrB or depletion of FtsI increases phosphorylation of Wag31. (A) M. smegmatis WT, the ΔmtrB strain, FtsI-depleted cultures (FtsI depletion for 12 h), and the ΔmtrB Pami::mtrB, ΔmtrB Pami::mtrAY102C (where mtrAY102C is the Y-to-C change at position 102 encoded by mtrA) and ΔmtrB Pami::mtrBH305Y strains were grown as described in the legends to Fig. 3 and 4. The complemented ΔmtrB strains were grown with 0.2% acetamide. Wag31∼P/Wag31 ratios were determined by immunoblotting with anti-phospho-Ser/Thr and anti-Wag31 antibodies. Whole-cell lysates (5 μg protein) from the above strains were resolved by SDS-PAGE in 12% gels, and immunoblotting was performed with anti-phospho-Ser/Thr antibodies. The blots were then stripped and reprobed with anti-Wag31 antibodies. Wag31∼P/Wag31 (anti-phospho-Ser/Thr and anti-Wag31) ratios for various strains were normalized to those in the WT and plotted (B). The data shown are represented as the averages ± standard errors from three independent experiments. *, P < 0.05. (C) The Wag31 levels in the indicated strains were measured by immunoblotting, as previously described (36), and normalized to SigA levels. The data are represented as the means ± standard errors from three independent experiments.