Fig 2. Cell division cycle timing of the localization of PBP4.

(A) MC4100 cells were grown to steady state in minimal glucose medium at 28°C and immunolabeled with antibodies specific for PBP4. An overlay of a phase contrast and its corresponding fluorescence image is shown. Scale bar equals 2 μm. (B) The potential number of PBP4 molecules in the cell wall area (see results section), calculated from the volume of the cells is plotted as function of the cell division cycle age. The dots are the values for the individual cells and the markers are 5% age bins with 95% confidence. (C) The extra fluorescence at midcell compared to the rest of the cell (FCPlus) is plotted as function of the normalized cell division cycle age. The dots (grey for MC4100 and light pink for the BW25113 ΔdacB strain) are the values measured for the individual cells and the markers with bars (black for the wild-type MC4100, red for the BW25113 ΔdacB) are 5% age bins with 95% confidence. (D) Demographs of the localization of FtsZ, PBP4 and FtsN in the cells sorted according to cell length. The white line indicates the length of the cells. (E) Comparison of the timing of the localization at midcell of FtsZ (black), PBP4 (green) and FtsN (blue) during the cell division cycle age. The FCPlus values are min-max normalized to enable timescale comparison, despite differences in molecule number and antibody affinities. (F) BW25113 wild-type cells were grown to “steady state” in minimal glucose medium at 28°C and immunolabeled with antibodies specific for FtsZ, PBP4 and FtsN. The FCPlus of three independent biological experiments in 5% age bins was determined. The binned FCPlus values were min-max normalized. The average min-max normalized FCPlus values of the three experiments were subsequently plotted as function of the cell division age cycle.