Fig 1.
Replication of the E. coli chromosome.
Top: Bacterial chromosome depicting the origin of replication (oriC, ●) and terminus (ter, ■). After birth, replication initiation yields a single replication bubble, a replication state termed “single fork replication” (- - -). When a second initiation event occurs before termination of the prior round, “multifork replication” occurs (—). Active origins and newly synthesized DNA are indicated with colors corresponding to replication state. Bottom: In the Cooper–Helmstetter model, as nutrient-imposed growth rate increases, the C period length decreases until it reaches a plateau during fast growth.
Fig 2.
Deterministic paradigm vs. stochastic nature of cell cycle timescales.
(A) The Cooper–Helmstetter model assumes that all cells within a given condition follow the population average B, C, and D periods. (B) Relative B, C, and D periods are shown over multiple consecutive replication cycles for 2 cell lineages grown on MOPS glucose (based on data from Si and colleagues). Significant differences between replication cycles necessitate a new theory accounting for stochasticity.
Fig 3.
Visual representations of the timers τi, τC, and τd in a cell undergoing single fork (A) and multifork (B) replication. These stochastic timers represent single-cell parameters. During multifork replication, C periods extend beyond a single division cycle. This overlap is indicated by extended dotted lines. Color schemes match Fig 2.
Fig 4.
Flow chart depicting the possible replication cycles depending on the order of initiation, termination, and division events.
N represents the number of chromosome copies present in the cell and is equal to the number of termini (ter, ■). The 2-fork cycle (blue) forms 2 forks upon initiation at oriC, ●, then replication is completed leaving 0 forks. The 4-fork cycle (magenta) progresses from 4 forks at initiation to 2 forks after division to termination. The 6-fork cycle (teal) progresses from 6 forks at initiation to 4 forks after termination of previous replication to 2 forks after division to 6 forks after new round of initiation. Further configurations with 8 forks or more are relevant only at growth rates faster than available in this dataset.
Fig 5.
Comparing model predictions to experimental data.
Model simulations (dashed lines) are compared with the experimentally obtained distributions (solid lines) of number of forks after initiation at different single-cell growth rates for the following growth conditions: (A) M9 acetate, (B) MOPS glucose, and (C) MOPS glycerol 11aa. Colors represent different number of forks after initiation: 2 (blue), 4 (magenta), and 6 (teal). There is a close match between model predictions and experimental data, indicating that the presence of different numbers of forks and both single and multifork replication within the same growth condition is purely a consequence of the inherent stochasticity in the 3 time periods governing the replication cycle (the C period, the inter-initiation period, and the time to division), each of which depends solely on single-cell growth rate for a given growth condition.
Fig 6.
Determining dominant fork numbers from nutrient-imposed population growth rate.
Our expectation for the trends in ,
, and
(representing the population mean values related to the stochastic timers τi, τC, and τd) as a function of nutrient-imposed population growth rate. We expect mean τC and τd to flatten as growth rate goes to infinity, while mean τi (equal to mass doubling time) approaches zero. Fork numbers at different population growth rates are determined by the relative order of these timers, as shown.
Fig 7.
Two hypothesized models for C period decrease as growth rate increases.
(A) Titration. (B) Fork spacing. Ovals represent active replisomes, triangles represent accessory replisome components and dNTP substrates, arrows represent relative replisome speed, and red clouds around the replisome represent steric repulsion and topological changes that alter replisome kinetics.
Fig 8.
Both inter-initiation time and C period are independent of fork number.
(A) Inter-initiation time (τi) and (B) C period (τC) is plotted against single-cell growth rate (k) for an intermediate growth condition (medium 4). Mean and SD of binned C periods and the spread of data points are plotted separately for ● 4-fork (single fork) and ● 6-fork (multifork) data, based on the fork number observed just after initiation.