Fig. 9

Dynamics of DNA replication in H. neptunium. a Quantification of S-phase cells at different stages of the developmental cycle. Exponentially growing wild-type cells were exposed to a pulse of the nucleotide analog EdU. After fixation and derivatization of the incorporated EdU with the red fluorescent dye Alexa 954, images were taken and the proportion of cells showing a fluorescence signal was determined for each of the indicated categories. Data represent the average of three experiments. b Localization of newly replicated DNA in H. neptunium. Strain KH22 (parB-yfp) was labeled with EdU as described in (a). Shown are representative images of cells at different developmental stages. Bar: 3 μm. c Time-lapse series showing the movement of the replisome along the two chromosomal arms. Cells of strain RP4 (dnaN-venus) were grown in MB medium, transferred to a 25% MB-agarose pad, and imaged at 5 min intervals. Only selected frames are presented. The full time-lapse series is shown in Supplementary Movie 1. Bar: 2 µm. d Quantitative analysis of replisome movement. The graph indicates the relative subcellular locations of the DnaN-Venus signals in the different frames of Supplementary Movie 1, with 0% indicating the old (previously flagellated) pole and 100% the future stalked pole of the mother cell. For frames in which the two replication forks were clearly separated, the positions of both DnaN-Venus foci are given. e Colocalization of the replisome with the ParB·parS complex at the beginning of S-phase. Strain JR47 (dnaN-venus parB-cerulean) was grown in MB medium, transferred to a 25% MB-agarose pad, and imaged at 5 min intervals. Bar: 2 µm. f Time-lapse series showing the dynamics of ori and replisome movement towards the end of S-phase. Cells of strain JR47 (dnaN-venus parB-cerulean) were grown in MB medium, transferred to a 25% MB-agarose pad, and imaged at 15 min intervals. Bar: 2 µm. g Quantification of the total replication time (n = 50 cells) and the interval between the start of the second segregation step and replisome disassembly (n = 30 cells). The data were obtained by analysis of the time-lapse series described in (d) and (f) and represented as box plots. The horizontal line indicates the median, the box the interquartile range, and the whiskers the 5th and 95th percentiles