Fig. 4.

Synthesis of teichoic acids, and not TagO protein itself, is required for PBP4 recruitment to the division septum. (A) Quantification of septum versus lateral membrane fluorescence (fluorescence ratios, FR) and fluorescence microscopy images for PBP4–YFP protein fusion in ΔtagO mutants complemented with wild-type TagO protein (RNΔtagOPBP4YFPptagO^wt) or with different TagO mutants (RNΔtagOPBP4YFPptagO^{D87A, D88A, D87A/D88A, G152A, and N198A}). Quantification was performed in 100 cells that displayed closed septa for each strain. Horizontal lines correspond to average FR values. FR values over 2 indicate septal localization, and FR values equal to or under 2 indicate that a protein is dispersed over the cell surface. p values < 10⁻⁷. Scale bar: 1 μm. (B) WTAs were isolated from RNΔtagOptagO and RNΔtagOptagO^{D87A, D88A, D87A/D88A, G152A, and N198A} and analyzed by native PAGE stained with alcian blue/silver stain. Mutations of the aspartic acids and glycine residues led to a decrease or absence of the WTAs. (C) Comparison between the levels of WTA and the degree of PBP4 localization to the division septa (calculated as described in Materials and Methods) indicates a strong correlation between the amount of WTA present in the cell and the ability of PBP4 to localize at the septum. (D) Fluorescence microscopy images of RNTagO^wtGFP and RNTagO^G152AGFP showing that the TagO^G152A–GFP fusion localizes to the division septum, similarly to the GFP fusion to the wild-type TagO protein. Scale bar: 1 μm.