Fig. 3 |. Donachie’s constant-initiation-mass hypothesis breaks down.

During steady-state growth, wild-type bacterial cells initiate DNA replication at all available origins synchronously once per cell cycle. The cell mass per origin at initiation, termed the initiation mass, was hypothesized by Donachie to be a constant independent of growth rate. a, The initiation mass m_i (in units of 10⁻¹⁰ OD₆₀₀ ml, characterized as described in the main text) is plotted against growth rate λ. In contrast to the Donachie hypothesis, it varied by ~50% across different growth rates, reaching a peak value ~5.5 × 10⁻¹⁰ OD₆₀₀ ml at a growth rate 0.7 h⁻¹. The growth rate dependence of the initiation mass is well described by the black curve from $m_i=\frac{m_0}{\ln 2}(\alpha +\beta \lambda )e^{-(\alpha +\beta \lambda )}$. b, Comparison between the data from a (grey circles) and those extracted from Wallden et al.²⁴ (MG1655, red squares, right y axis). For the data from Wallden et al.²⁴, the error bars for initiation volume are set to 10% as they described; the error bars for growth rate represent the s.d. at the single-cell level as they described. The culture temperature for the middle data point (arrow) was 30 °C, while the other two data points and ours were obtained at 37 °C. c, Comparison between the data from a (grey circles) and those extracted from Si et al.⁵ (MG1655, green diamonds, right y axis). The error bars for the data from Si et al.⁵ represent the s.d. of biological replicates. d, Relative DnaA protein concentration exhibits a non-monotonic dependence on growth rate. Relative DnaA protein concentration varied by ~50%, reaching a minimum value at ~0.7 h⁻¹, approximately where the initiation mass reaches a peak. It was quantified by quantitative proteomics and was normalized to the population average of the total protein concentration across growth rates (see Methods). Except for the data points that have already been published elsewhere and described above (the red squares in b and the green diamonds in c), in a–d, data are mean ± s.d.; many of the error bars are smaller than the size of the symbols. Sample sizes and mean values for each symbol are provided in Extended Data Fig. 8.