Fig. 2

Two-step segregation of the sister ori regions. a Time-lapse series showing the localization of ParB-YFP over the course of the cell cycle. H. neptunium KH22 (parB-yfp) was grown in MB medium, transferred to an MB-agarose pad, and imaged at 30 min intervals. Shown are overlays of differential interference contrast (DIC) and fluorescence micrographs as well as schematics of the fluorescence patterns observed. Bar: 3 µm. b Demographic analysis of ParB-YFP localization in swarmer (left), stalked (middle), and budding (right) cells of strain KH22. The fluorescence intensity profiles obtained for each cell type were sorted according to cell length and stacked on each other, with the shortest cell shown at the top and the longest cell shown at the bottom. Note that due to the variable length of the stalk, the total cell length only roughly corresponds to the developmental state of the cell. c Time-lapse series showing the movement of ParB-YFP through the stalk of strain KH22. Cells were imaged at 1 min intervals. Shown are overlays of DIC and fluorescence images. Bar: 3 µm. d Relative size of the bud at the onset of the second segregation step. Strain KH22 was imaged at 5 min intervals. At the first time point at which ParB-YFP had left the stalk base to migrate toward the bud, the widths of the mother cell and bud compartments were measured. The graphs show the relative width of the bud plotted versus the absolute width of the mother cell (n = 57). The average of the data is 60 ± 7%, corresponding to a total width of 0.61 ± 0.08 µm. e Time-lapse series following ori translocation through the elongated stalk of a phosphate-starved cell. Strain KH22 was grown in minimal medium lacking phosphate for 22 h. Subsequently, MB medium was added to a final concentration of 20% and growth was continued for 40 min. After transfer of the cells onto an MB-agarose pad, images were taken at 1 min intervals. Shown are overlays of DIC and fluorescence micrographs. Bar: 3 µm