Figure 4.

Depletion of CcrM results in ori2 replication/partitioning delays in cells cultivated in minimal medium for prolonged periods of time. (A) Schematic representing the subcellular localization of ori1 and ori2 during the A. tumefaciens cell cycle. (B and C) Demographs showing the subcellular localization of ori1 (B) or ori2 (C) foci as a function of cell size (from images as shown in Supplementary Figure S8). JC2660 (ΔccrM Ptac-ccrM with ori1/ygfp reporter in (B)) or JC2661 (ΔccrM Ptac-ccrM with ori2/ygfp reporter in (C)) cells were cultivated over-night in ATGN ± IPTG. Cultures were then diluted into ATGN ± IPTG and grown exponentially for ∼6.5 h (same medium at all steps). Relative position = 0 corresponds to mid-cell; only cells measuring from 1 to 4 μm-long were included into these demographs; n = number of cells used to construct each demograph. (D) Quantification of ori2 number per cell among cells that are in S-phase (with two ori1). JC2836 (ΔccrM Ptac-ccrM with ori1/mcherry and ori2/ygfp reporters) cells were cultivated over-night in ATGN ± IPTG. Cultures were then diluted into ATGN ± IPTG and grown exponentially for ∼6.5 h (>15 h into ATGN ± IPTG). Phase contrast/YGFP/mCherry images were acquired as shown in Supplementary Figure S9a. Among cells that displayed minimum two ori1 foci (S-phase cells from Supplementary Figure S10), the number of ori2/cell was measured (from minimum 100 cells/condition) and means from three independent experiments were plotted for each condition. Error bars correspond to standard deviations. Student's t-test: ns = P-value > 0.01, ** = P-value < 0.01.