Fig. 1: Measurement of growth characteristics of Mtb (video set A).

a, Schematic of growth parameters derived from Mtb across generations by time-lapse imaging. Cells loaded into the device (first generation) establish the pole age for the next generations but are excluded from analysis owing to incomplete cell cycle observation. In the second and subsequent generations, all growth features are determined, including Lb, DNA replication initiation (Li), Ld, interdivision time (Td), growth rate (λ), elongation speed \(\left(\frac{{{\rm{d}}L}}{{{\rm{d}}t}}\right)\) and identification of old and new poles. In the third (and subsequent) generations, we identify accelerators (acc) (n = 173) and alternators (alt) (n = 130). In baseline experiments (video set A), cell cycle timing at the single-cell level is determined using an SSB–GFP reporter strain of Mtb (total cell n = 363). b, Determination of cell cycle state using SSB–GFP. SSB binds to replication forks^81,82, with green foci indicating ongoing chromosome replication (C period); there were no visible foci before and after replication (B and D periods, respectively). The yellow arrows highlight the foci. Scale bars = 2 µm. c, Single-cell traces of SSB–GFP localization with hourly timepoints corresponding to b. The distance from the x-axis to the black circle represents cell length. The green circles indicate SSB–GFP foci localization along the cell body. The asterisks correspond to the images shown in b. d, Annotation of the cell cycle state in a cell with an E period, in which a small population (11%) initiates replication before division (foci positive)²⁵. Daughter cells inheriting the foci enter directly into the C period. The yellow arrows highlight the foci, and the white dashed lines outline some individual Mtb cells. Scale bars = 2 µm. e, Single-cell SSB–GFP traces with hourly timepoints corresponding to d. The green circles indicate SSB–GFP foci. The asterisks correspond to the images shown in d. Length in c and e is unitless. f,g, Cell cycle timing in Mtb (f) and M. smegmatis (g). The average time and proportion of each cell cycle period are shown (B: pre-replication, C: DNA replication, D: post-replication, E: new DNA replication after a D period but before division). The M. smegmatis data are from a previous study²⁵.