Growth consequences of the inhomogeneous organization of the bacterial cytoplasm

Reviewer #1 (Public Review):

Summary:

The manuscript by van Heerden et al. reports growth rate variations in the cell cycle of E. coli and links this variation to uneven ribosome concentrations in the cell at birth that arise from an uneven division of cell volumes between the daughter cells. The authors propose a model to explain the experimental data, whose main premises are the exclusion of ribosomes from the nucleoid volume and a linear dependence of the growth rate on ribosome concentration in the cell.

Strengths:

(1) The manuscript highlights an interesting aspect of growth rate variability in bacteria and proposes a mechanism for how this variation is homeostatically corrected.

(2) A sophisticated modeling to explain the experimental data.

Weaknesses:

(1) The experiments lack controls. A partially functional label (L9-mCherry) can make ribosomes much more limiting for growth than are not labeled ribosomes.

(2) The large variation of interdivision times 72-89 min in repeat experiments in Glc is problematic. Some parameters in the measurements related to cell growth appear not properly controlled. It is problematic for a work that aims to establish a new universal behavior related to cell growth.

(3) The authors have not provided convincing evidence that cells in their experiment grow in a steady state.

1. The findings are over-generalized. The existing data show the effects only at some growth rates, but the findings are presented as a new universal principle.

2. The model relies on many assumptions that are not clearly brought out and the choice of model parameters is questionable (in some cases, the parameters seem to contradict well-established experimental data, including the one from the experiments from the very same work). Small changes in parameters and various approximations can have large effects on the model's outcomes; without understanding these responses, the model has a rather limited value.

3. There appears to be a qualitative discrepancy between the model and the experimental data in Glc (the main condition studied). The model predicts that the cells born large have a specific elongation rate that is smaller than the average growth rate of cells, but it grows in time at the beginning of the cell cycle, while the experiments show a decreasing growth rate (Figure 1C, SI Figure S2).

Reviewer #2 (Public Review):

Summary:

This work demonstrates that when E. coli cells divide, and division is not quite symmetric, the smaller cell has a higher growth rate than the larger cell at the beginning, but not the end, of the cell cycle. The authors then demonstrate that smaller cells have a higher ribosome concentration than larger cells, which is consistent with the idea that the two cells receive roughly equal numbers of ribosomes at division because, as they also observe, ribosomes are excluded by the nucleoid from the middle of the mother cell. The experimental observations are reproduced by a mathematical model that assumes growth is driven by ribosome concentration, with contributions from metabolism and active feedback.

Strengths:

The work provides strong evidence in support of the growing consensus that cells correct size fluctuations by modulating growth rate, within a cell cycle and on a single-cell basis. It also offers a plausible explanation for the correction mechanism by showing that ribosomes are excluded from the middle of a mother cell and have a higher concentration in the smaller daughter cell. The work is clearly written and benefits from a strong coupling between the experimental and modeling results. It provides a solid contribution to the field of single-cell bacterial growth control.

Weaknesses:

Although the results strongly suggest it, the work does not explicitly demonstrate (e.g. by direct perturbation) that higher ribosome concentration is the cause of the higher growth rate. Also, it is unclear why an active compensation mechanism is needed in the model, i.e., why size-dependent growth (via ribosome concentration) does not correct growth rate perturbations within a cell cycle automatically.