Figure 6: Impact of mutated MapZextra2 on peptidoglycan-mediated localization of MapZ.
(a) Interaction of MapZextra2 and MapZextra2-Mut with the pneumococcal cell wall. The fraction of MapZextra2 or MapZextra2-Mut unbound (UB) to cell wall and bound to cell wall was detected using a mouse anti-histidine-tag antibody. The experiment was made in triplicate. (b) Localization of peptidoglycan synthesis and MapZ in gfp-mapZ and gfp-mapZ-extra2Mut strains. Images of GFP fluorescence and peptidoglycan synthesis by two consecutive pulse-chase labellings, using the red fluorescent derivative of D-alanine TDL and then the blue fluorescent derivative of D-alanine HADA, are shown. An overlay of the three fluorescent labellings is shown at the bottom of the figure. Scale bars, 2 μm. The maps of fluorescence profiles for GFP-MapZ, HADA and TDL is also shown as in Fig. 5. Scale bars, 3 μm. The diagrams at the bottom of these columns show the relative distribution of fluorescence intensities of GFP-MapZ (green), TDL (red) and HADA (blue) along the cell length normalized to 100%. n indicates the number of cells analysed. (c) Lowest-energy structure obtained from the docking of 10 different peptidoglycan hexamuropeptide structures onto the MapZextra2 lowest-energy structure as described in Methods. All the residues mutated in the MapZextra2Mut were considered as active Ambiguous Interaction Restraints during the HADDOCK minimization protocol and are coloured in red on the protein surface. Peptide stems and the glycosidic chain of the peptidoglycan are coloured in dark green and light green, respectively.
