Fig. 1: FROS reveals number of origins across cell cycle stages.

A Schematic of the single-locus FROS system used in this study. A sequence encoding the TetR repressor fused to the mNeonGreen (mNG) fluorescent protein, under the control of a cadmium-inducible promoter with leaky expression, was integrated in the spa locus (right). TetR-mNG binds to an array of 48 tetO operator sequences introduced at the locus of interest (left). B Bar chart representing the relative frequency of cells of the JE2_FROS^Ori_DnaN-Halo strain with 1-4 Ori foci in P1 (yellow, no septum) or P2/P3 (blue, incomplete and complete septum) cell cycle stages. Data are from three biological replicates (n = 100 each). C Bar chart showing the relative frequency of P1 and P2/P3 cells of the JE2_FROS^Ori_DnaN-Halo strain with Ori and replisome (visualized using DnaN as a proxy) either colocalizing (yellow) or not (blue). Data are from three biological replicates (n = 100 each). Examples of P1 cells with Ori-DnaN colocalization (yellow arrows) or no colocalization are shown on the right side; DnaN-Halo signal is shown in red and TetR-mNG (Ori) in green. Scale bar: 1 µm. D Classification of newborn cells by Ori number and Ori /replisome colocalization. Left, brightfield and fluorescence microscopy images of a representative cell from JE2_FROS^Ori_DnaN-Halo strain that underwent division between frame 0 and frame +1, showing two newborn cells with two Ori each in the latter frame (3 min interval between frames). JF549-labelled DnaN-Halo signal is shown in red and TetR-mNG (Ori) signal in green, scale bar: 1 µm. Right, bar chart showing the relative frequency of each class of newborn cells, categorized by Ori number, replisome assembly, and Ori/replisome colocalization (Y, yes; N, no). Data are from three biological replicates (n = 36, 36, 30). For panels B, C and D error bars indicate the standard deviation and are centered on the mean; individual data points are shown as colored circles; source data are provided as a Source Data file.