FIG 5.

PBP2 delocalization does not correlate with VraTSR activation. Strains COL Pvra-sGFP and BCBPM073 (expressing a GFP fluorescent derivative of PBP2) were both incubated in the presence of increasing vancomycin concentrations (Van 0, 1, 5, and 10 μg · mL⁻¹) for 45 min. COL Pvra-sGFP cells were then stained with DNA dye Hoechst 33342 to differentiate the two populations and mixed with BCBPM073 cells just prior to imaging on the same microscope slide. (a) GFP fluorescence of COL Pvra-sGFP cells, showing that VraTSR activation occurs only in the presence of 10 μg · mL⁻¹ of vancomycin. N > 277 cells for each condition in each replicate. (b) PBP2 septal enrichment was evaluated in BCBPM073 cells by measuring the fluorescence ratio of GFP-PBP2 fluorescence at the septum versus at the cell periphery, both corrected for background fluorescence. PBP2 delocalized from the septum at the lowest vancomycin concentration tested. N = between 62 and 90 for each condition in each experiment; experiments performed in duplicate.