Fig. 6. DNA replication is not required for centromere detachment, but is required for the accumulation of PopZ at the new cell pole.

A and B. Defects in stalked pole development do not prevent the detachment of CFP-ParB foci from polar mCherry-PopZ. CFP-ParB (green) and mCherry-PopZ (red) localization is shown in a ΔcpdR background, strain GB559 (A), and in a pleC∷Tn5 background, strain GB566 (B). Arrowheads mark poles where CFP-ParB foci are not colocalized with mCherry-PopZ.

C. DnaA depletion does not prevent the detachment of CFP-ParB foci from polar mCherry-PopZ. Swarmer cells (left panel) isolated from strain GB569 were grown in M2X or M2G media supplemented with 50 µM vanillate for normal growth (top panel) or growth under DnaA depletion (lower panel) respectively. Images were collected after 90 min of growth in liquid media. The diagrams and percentages below each image indicate the percentage of cells containing single foci of mCherry-PopZ and CFP-ParB that overlapped by > 50% (left) or by < 50% (right).

D. Constitutively active CtrA does not prevent the detachment of CFP-ParB from polar mCherry-PopZ. Swarmer cells were isolated from strain GB532 were grown in M2G or M2X media supplemented with 50 µM vanillate for normal growth (top panel) or growth under CtrA D51EΔ3Ω production (lower panel) respectively. Image collection and quantification was as in (C).

E. The detachment of CFP-ParB from PopZ-YFP is maintained after DNA replication is blocked with novobiocin treatment. Swarmer cells isolated from strain GB529 were grown in M2G supplemented with 50 µM vanillate in the absence or presence of 100 µg ml⁻¹ novobiocin, and images were collected and quantified as in (C).

F. Western blots showing the level of mCherry-PopZ in whole cell lysates taken from the experiments in (C) to (E). mCherry-PopZ is partially degraded and runs as a doublet. For each experiment, combined band intensity was normalized with respect to the control.