(A) Endogenously expressed CpoB-mCherry localizes to mid-cell during cell division. Left, phase contrast image; right, mCherry fluorescence; scale bar, 2 μm. (B) Localization of CpoB-mCherry as a function of cell cycle progression (cell length), with comparison to other proteins. PBP1B, LpoB, FtsZ, PBP3, and FtsN were visualized by immunofluorescence using specific antibodies. Cell membranes were stained with Bodipy-12. Cell width (diameter) was measured from phase contrast images; constant brightness along the length of the cell indicates constant diameter, and darkening at mid-cell in longer cells indicates constriction. To generate profile maps for each protein, fluorescence intensity profiles (integrated fluorescence as a function of cell length) were derived for >1000 individual cell images and sorted vertically by cell length. Each horizontal line corresponds to a single cell. Y-axis scale indicates % cell-cycle progression based on cell length. Data for FtsZ, PBP3, FtsN, and cell width have been previously published (van der Ploeg et al., 2013) and are used here for comparisons. (C) Average distribution of CpoB-mCherry for different cell-cycle progression age groups. Fluorescence intensity profiles were normalized by length, then averaged and normalized to the peak local brightness for each age group. (D) Relative timing of mid-cell recruitment for each examined protein. Initiation is based on the onset of enriched localization at mid-cell (quantified in Figure 4—figure supplement 3A,B and van der Ploeg et al., 2013). Peak is the point in the division cycle when maximal localization is reached.