Growth rate and genome copy number in E. coli.

(A) Illustration of 1N (CRISPRi oriC, CJW7457) and multi-N (CRISPRi ftsZ, CJW7576) cells with different numbers of chromosomes along with representative microscopy images at different time points following CRISPRi induction. Scale bars: 1 µm. (B) Plot showing representative single-cell trajectories of cell area as a function of time for the CRISPRi strains following a block in DNA replication and/or cell division. (C) Plot showing the absolute growth rate as a function of cell area for 1N (32735 datapoints from 1568 cells) and multi-N cells (14006 datapoints from 916 cells) in M9glyCAAT. Lines and shaded areas denote mean ± SD from three experiments. This also applies to the panels below. (D) Absolute and (E) relative growth rate in 1N (32735 datapoints from 1568 cells, CJW7457), multi-N (14006 datapoints from 916 cells, CJW7576), and dnaC2 1N (13933 datapoints from 1043 cells, CJW7374) cells as a function of cell area in M9glyCAAT. (F) Absolute growth rate and (G) relative growth rate in 1N (13933 datapoints from 1043 cells), 2N (6265 datapoints from 295 cells) and >2N (2116 datapoints from 95 cells) dnaC2 (CJW7374) cells as a function of cell area in M9glyCAAT. (H) Absolute and (I) relative growth rate in 1N (50352 from 973 cells, CJW7457) and WT (80269 datapoints from 12544 cells, CJW7339) cells in M9gly. (J) Absolute and (K) relative growth rate in 1N (71736 datapoints from 909 cells, CJW7457) and WT (63367 datapoints from 6880 cells, CJW7339) cells in M9ala. Lines and shaded areas denote mean ± SD from three biological replicates.

Lower ribosome activity explains the reduced growth rate of 1N cells.

(A) RpsB-msfGFP fluorescence concentration in 1N (6542 cells, CJW7478) and multi-N (10537 cells, CJW7564) cells as a function of cell area. Lines and shaded areas denote mean ± SD from three experiments. (B) Relative protein concentration of different ribosomal proteins in 1N (SJ_XTL676) and multi-N (SJ_XTL229) cells by TMT-MS. 1N-rich cells were collected 0, 120, 180, 240 and 300 min after addition of 0.2% arabinose, while multi-N cells were collected after 0, 60, and 120 min of induction. Blue and cyan represent two independent experiments. Only proteins with at least four peptide measurements are plotted. (C) Apparent diffusion coefficients (Da) of JF549-labeled RspB-HaloTag in WT (32,410 tracks from 771 cells, CJW7528), 1N (848,367 tracks from 2478 cells, CJW7529) and multi-N cells (107,095 tracks from 1139 cells, CJW7530). Only tracks of length ≥9 displacements are included. 1N cells are color-binned according to their cell area while multi-N cells contain aggregated data for ∼2-10 µm2 cell areas. (D) Da in WT cells fitted by a three-state Gaussian mixture model (GMM): 77 ± 1%, 20 ± 1%, and 3.2 ± 0.5% (±SEM) of the ribosome population, from the slowest moving to the fastest moving (32,410 tracks from 771 cells). (E) Example WT and 1N cells where (red, slow-moving) active and (gray, fast-moving) inactive ribosomes are classified according to the GMM. (F) Active (slow-moving) ribosome fraction in individual WT (237 cells) and 1N (2453 cells) cells as a function of cell area. Only cells with ≥50 tracks are included. Lines and shaded areas denote mean and 95% confidence interval (CI) of the mean from bootstrapping. (G) Same as (F) but for WT (237 cells) and multi-N (683 cells) cells. (H) Absolute growth rate of 1N and multi-N cells (Figure 1C) as a function of cell area was overlaid with the total active ribosome amount (calculated from Fig. 2A, F and G). Lines and shaded areas denote mean and 95% CI of the mean from bootstrapping.

RNAP activity is reduced in 1N cells.

(A) RpoC-YFP fluorescence concentration in 1N (3580 cells, CJW7477) and multi-N (5554 cells, CJW7563) cells as a function of cell area. Lines and shaded areas denote mean ± SD from 3 experiments. (B) Relative protein concentration of core RNAP subunits and σ70 in 1N-rich (SJ_XTL676) and multi-N (SJ_XTL229) cells by TMT-MS. 1N-rich cells were collected 0, 120, 180, 240 and 300 min after addition of 0.2% L-arabinose, while multi-N cells were collected after 0, 60, and 120 min of induction. (C) Apparent diffusion coefficients of JF549-labeled RpoC-HaloTag in WT (91,280 tracks from 1000 cells, CJW7519), 1N (175,884 tracks from 1219 cells, CJW7520) and multi-N cells (186,951 tracks from 1040 cells, CJW7527). Only tracks of length ≥9 displacements are included. 1N cells are binned according to cell area while multi-N cells contain aggregated data for ∼2-15 µm2 cell areas. (D) Da in WT cells fitted by a 3-state GMM: 49 ± 4%, 49 ± 4%, and 2 ± 0.1% (±SEM) of the RNAP population, from the slowest moving to the fastest moving (91,280 tracks from 1,000 cells). (E) Example WT and 1N cells where (red, slow-moving) active and (gray, fast-moving) inactive RNAPs are classified according to the GMM. (F) Active RNAP fraction in individual WT (854 cells) and 1N (1024 cells) cells as a function of cell area. Only cells with at least 50 tracks are included. Lines and shaded areas denote mean ± 95% CI of the mean from bootstrapping (3 experiments). (G) Same as (F) but for WT (854 cells) and multi-N (924 cells) cells. (H) Total amount of active RNAP in WT, 1N and multi-N cells as a function of cell area (calculated from Fig. 3A, F and G). Also, shown is a linear fit to multi-N data (f(x) = 4.16*104*x, R2 0.98). Lines and shaded areas denote mean and 95% CI of the mean from bootstrapping. All microscopy data are from three biological replicates.

Model parameter description

Initial and optimized model parameters

Mathematical modeling and RNASelect concentration measurements in 1N and multi-N cells.

(A-C) Plots comparing simulation results of model A (solid lines) with experimental data points (dots) and averages (open squares) in the M9glyCAAT condition. The multi-N and 1N cells are indicated as blue and yellow, respectively: (A) The relation between the absolute growth rate and cell area (A). (B) The relation between the active RNAP fraction and cell area. (C) The relation between the active ribosome fraction and cell area. (D) A two-dimensional diagram showing how the fractions of active RNAPs and ribosomes change with DNA concentration (colored from blue to yellow). Experimental data (with 2D error bars) from multi-N and 1N cells were combined and shown in the same plot. Simulated results (filled dots) are based on model A. (E) Plot showing the effect of DNA limitation using the ODE model A, with respect to the decay of DNA concentration, mRNA concentration and relative growth rate in 1N cells. Each quantity was normalized to their value at normal cell size (cell area = 2.5 μm2). (F) The phase contrast (top), HU-mCherry (middle) and SYTO RNASelect fluorescence (bottom) images of two representative cells from a mixed population of 1N (CRISPRi oriC, CJW7457) and multi-N (CRISPRi ftsZ, CJW7576) cells. The purple and orange ticks In the SYTO RNASelect colorbar correspond to the average fluorescence (concentration proxy) of the multi-N and the 1N cell respectively. (G) SYTO RNASelect concentration distributions for 1N and multi-N cells (3077 cells for each population from five biological replicates). (H) The average 1N/multi-N SYTO RNASelect concentration ratio (gray bar) calculated from five biological replicates (white circles). (I) RNASelect concentration ratio as a function of cell area (Mean +/− SD from five biological replicates). A single exponential decay function was fitted to the average ratios (R2 = 97%). All RpoC-HaloTag-JF549 concentration comparisons or ratio calculations were performed for equal numbers of 1N and multi-N cells and overlapping cell area distributions (Figure 4 – supplement 4A).

Proteome and transcriptome remodeling in 1N-rich cells.

(A) Schematic explaining the calculation of the protein slopes, which describes the scaling of the relative protein concentration with cell area. (B) Plot showing the protein scaling (average slopes from 2 reproducible biological replicates, see also Figure 5 – supplement 1A-B) in 1N (x-axis) and multi-N (y-axis) cells across the proteome (2360 proteins). The colormaps correspond to a Gaussian kernel density estimation (KDE). (C) Plot showing the first principal component (PC1, which explains 69% of the total variance considering both 1N-rich and multi-N cells) used to reduce the dimensionality of the relative protein concentration during cell growth. The x-axis corresponds to the log-transformed cell area, whereas the marker size shows the cell area increase in linear scale. (D) Relationship between the absolute distance of a gene from oriC and the slope of the protein it encodes. Data from 2268 proteins are shown (grey markers), as well as binned data in six gene distance bins (open circles, average +/− SEM, ∼370 proteins per bin). For the multi-N cells, the Spearman correlation was not significant (ρ = NS, p-value > 0.01), whereas for the 1N cells, a significant Spearman correlation (ρ = 0.23, p-value < 10-10) for genes within 1.35 Mbps of oriC (below the mid-point of the 4th gene distance bin). (E) Relationship between the distance of a gene from oriC and the ion intensity of its encoded protein. The protein ion intensity was divided by the protein sequence length and the quotient was log-transformed. Data from 2258 proteins are shown (grey markers) as well as binned data (open circles, average +/− SEM, ∼370 proteins per bin). The spearman correlation is not significant (ρ = NS, p-value > 0.01). (F) Correlation between average protein and mRNA slopes across the genome (2324 genes).

Strains used in this study

. The abbreviations kan, cat, and spec refer to gene cassette insertions conferring resistance to kanamycin, chloramphenicol, and spectinomycin, respectively. These insertions are flanked by Flp site-specific recombination sites (frt) that allow the removal of the insertion using Flp recombinase from plasmid pCP20 (Cherepanov and Wackernagel, 1995).

Oligonucleotides used in this study.

Relationships between growth rate and cell volume across several growth conditions and cell types of different ploidy.

(A) Cell width in WT (59072 datapoints from 7640 cells, CJW7339), 1N (41313 datapoints from 1585 cells, CJW7477), and multi-N (18489 datapoints from 915 cells, CJW7563) cells in M9GlyCAAT as a function of cell area. Lines and shaded areas denote mean ± SD from 3 biological replicates. (B) Absolute and (C) relative growth rate based on cell volume in 1N (32735 datapoints from 1568 cells, CJW7457), multi-N (14006 datapoints from 916 cells, CJW7576), and dnaC2 1N (13933 datapoints from 1043 cells, CJW7374) cells as a function of cell volume in M9glyCAAT. Lines and shaded areas denote mean ± SD from three experiments. The growth rates are based on volume in this figure. (D) Absolute and (E) relative growth rate in M9glyCAAT in 1N (32735 datapoints from 1568 cells, CJW7457), multi-N (14006 datapoints from 916 cells, CJW7576) and WT (19495 datapoints from 7440 cells, CJW7339) cells as a function of cell area. (F) Absolute growth rate and (G) relative growth rate in 1N (13933 datapoints from 1043 cells), 2N (6265 datapoints from 295 cells) and >2N (2116 datapoints from 95 cells) dnaC2 (CJW7374) cells as a function of cell area in M9glyCAAT. (H) Absolute and (I) relative growth rate in 1N (50352 from 973 cells, CJW7457) and WT (80269 datapoints from 12544 cells, CJW7339) cells in M9gly. (J) Absolute and (K) relative growth rate in 1N (71736 datapoints from 909 cells, CJW7457) and WT (63367 datapoints from 6880 cells, CJW7339) cells in M9ala. Lines and shaded areas denote mean ± SD from three biological replicates.

Relationships between growth rate and cell area in cell types of different ploidy.

(A) Absolute and (B) relative growth rate growth rate in M9glyCAAT in 1N (32735 datapoints from 1568 cells, CJW7457), multi-N (14006 datapoints from 916 cells, CJW7576) and WT (19495 datapoints from 7440 cells, CJW7339) cells as a function of cell area. Lines and shaded areas denote mean ± SD from three biological replicates.

Characterization of ploidy in different cell types.

(A) Representative microscopy images of CRISPRi oriC cells expressing HU-CFP and ParB-mCherry with parS site at ori1 (CJW7517) in M9glyCAAT 210 min after addition of 0.2% L-arabinose. Scale bar: 1 µm. (B) Representative microscopy images of WT (top) and dnaC2 (bottom) cells expressing HU-mCherry growing under microscope observation in M9glyCAAT. DNA replication in dnaC2 cells was blocked by growing cells at 37°C for 145 min. Arrows indicate dnaC2 cells with one (1N) or two (2N) nucleoids. Scale bar: 1 µm. (C) Graph showing the percentage of dnaC2 cells (CJW7374) with 1, 2 or >2 nucleoids after growth at 37°C. Shown are aggregated data from three biological replicates.

Determination of absolute growth rate of ppGpp0 (CJW7518) and ΔrecA (CJW7522) cells grown in M9glyCAAT as a function of cell area.

Lines and shaded areas denote mean ± SD from two biological replicates. Also shown are the data for 1N (CJW7457) and multi-N (CJW7576) cells from Figure 1C.

Validation of stable growth conditions under microscope observation.

(A) Plot showin the absolute growth rate of 1N (CJW7457) cells grown 90 min in a liquid M9glyCAAT culture in the presence of 0.2% L-arabinose and then transferred to an agarose pad and imaged for 2 h. The data for 1N (CJW7457) and multi-N (CJW7576) cells presented in Figure 1C are also shown for comparison; these cells were grown on an agarose pad for 6 h. Lines and shaded areas denote mean ± SD from two biological replicates. (B) Plot showing the relative growth rate of WT (CJW7339) cells in M9glyCAAT grown after placing them on an agarose pad. Lines and shaded areas denote mean ± SD from three biological replicates.

DNA-dependent growth in Caulobacter crescentus.

(A) Plot showing the absolute and (B) relative growth rate of 1N (DnaA depletion, strain CJW4823, 87 cells) and multi-N (FtsZ depletion, strain CJW3673, 181 cells) C. crescentus as a function of cell area. Lines and shaded areas denote mean ± SD from three biological replicates. (C) Example images of 1N and multi-N C. crescentus cells with ploidy distinguished by the number of ParB-eCFP foci. Scale bar: 2 µm.

Determination of RpsB-msfGFP concentration in 1N cells.

Plot showing the RpsB-msfGFP fluorescence concentration in 1N (CJW7478) cells grown in (A) M9gly (from three experiments) or (B) M9ala (from two experiments) as a function of cell area. Lines and shaded areas denote mean ± SD between the biological replicates.

Diffusive characteristics of labeled ribosomes in rifampicin-treated WT cells.

(A) Plot showing the probability density of apparent diffusion coefficients (Da) of JF549-labeled RpsB-HaloTag in WT cells (CJW7528) treated with 200 µg/mL rifampicin for 30 min. Only tracks of length ≥10 are included. Also shown is Da fitted by three-state Gaussian mixture model (GMM) (mean ± SEM). Data are from three biological replicates.

Diffusive characteristics of labeled ribosomes in 1N cells as a function of cell area.

Plots showing the probability density of apparent diffusion coefficients (Da) of JF549-labeled RpsB-HaloTag in 1N cells (CJW7529) in M9glyCAAT at different cell areas. Also shown is Da fitted by three-state Gaussian mixture model (GMM). Only tracks of length ≥10 are included. Data are from three biological replicates.

Characterization of ribosome diffusion and active fraction in poor media conditions.

(A) Plot showing the probability density of apparent diffusion coefficients (Da) of JF549-labeled RpsB-HaloTag across cell areas for WT (CJW7528) and 1N (CJW7529) cells grown in M9gly. Only tracks of length ≥10 are included. 1N cells were binned according to cell area. (B) Same as (A) for cells grown in M9ala. (C) Plot showing probability density of Da of JF549-labeled RpsB-HaloTag in WT cells (CJW7528) grown M9gly. Only tracks of length ≥10 are included. Also shown is Da fitted by three-state Gaussian mixture model (GMM) (mean ± SEM). (D) Same as (C) but for cells grown in M9ala. (E) Plot showing the fraction of active ribosomes as a function of cell area for individual WT (CJW7528) and 1N (CJW7529) cells (dots) grown in M9gly. Only cells with ≥50 tracks are included. Shaded areas denote 95% confidence interval (CI) of the mean from bootstrapping. (F) same as (E) but for cells grown in M9ala. (G) Plot showing the total active ribosome amount (left y-axis) and the absolute growth rate (right y-axis) of 1N and multi-N cells (from Figure 1C) grown in M9gly as a function of cell area. The total amount of active ribosomes was calculated by multiplying the total amount of ribosomes by the fraction of active ribosomes. Also, shown is a linear fit to WT data (f(x) = 2.99*104*x, R2 0.97). Lines and shaded areas denote mean and 95% CI of the mean from bootstrapping. All data are from three biological replicates. (H) Same as (G) but for cells grown in M9Ala. Here the linear fit to the WT data is f(x) = 1.90*104*x, R2 0.99.

Determination of RpoC-YFP concentration in 1N cells.

(A) Plot showing the RpoC-YFP fluorescence concentration in 1N (CJW7477) cells grown in M9gly (three experiments) as a function of cell area. Lines and shaded areas denote mean ± SD between biological replicates. (B) Same as (A) but for 1N cells grown in M9ala (three experiments).

Diffusive characteristics of labeled RNAPs in rifampicin-treated WT cells.

Plot showing the probability density of apparent diffusion coefficients (Da) of JF549-labeled RpoC-HaloTag in WT cells (CJW7519) treated with 200 µg/mL rifampicin for 30 min. Also shown is Da fitted by three-state Gaussian mixture model (GMM) (mean ± SEM). Only tracks of length ≥10 are included. Data are from three biological replicates.

Diffusive characteristics of labeled RNAPs in 1N cells of different cell areas.

Plots showing the apparent diffusion coefficients (Da) of JF549-labeled RpoC-HaloTag in 1N cells (CJW7520) of different cell areas. Cells were grown in M9glyCAAT. Also shown is Da fitted by three-state Gaussian mixture model (GMM). Only tracks of length ≥10 are included. Data is from three biological replicates.

Characterization of RNAP diffusion and active fraction in poor media conditions.

(A) Plot showing the probability densities of apparent diffusion coefficients (Da) of JF549-labeled RpoC-HaloTag in WT (CJW7519) and 1N (CJW7520) cells grown in M9gly. Only tracks of length ≥10 are included. 1N cells were binned according to cell area. (B) Same as (A) but for cells grown in M9ala. (C) Plot showing the probability density of Da of JF549-labeled RpoC-HaloTag in WT cells (CJW7519) grown in M9gly. Also shown is Da fitted by three-state Gaussian mixture model (GMM) (mean ± SEM). (D) Same as (C) but for cells grown in M9ala. (E) Plots showing the fraction of active RNAPs in individual WT (CJW7519) and 1N (CJW7520) cells (dots) grown in M9gly as a function of cell area. Only cells with ≥50 tracks are included. (F) Same as (E) but for cells grown in M9ala. (G) Plot showing the total amount of active RNAPs was calculated by multiplying the total amount of RNAPs by the fraction of active RNAPs in WT and 1N cells grown in M9gly as a function of cell area. Also, shown is a linear fit to WT data (f(x) = 3.99*104*x, R2 0.90). Shaded areas denote 95% confidence interval (CI) of the mean from bootstrapping. All data are from three biological replicates. (H) Same as (G) but for cells grown in M9ala. Here, the linear fit for WT data is f(x) = 3.21*104*x, R2 0.95.

Determination of the relative Rsd concentration in 1N-rich and multi-N cells as a function of cell area.

Plots showing the relative protein concentration of Rsd in 1N-rich (SJ_XTL676) and multi-N (SJ_XTL229) cells as determined by TMT-MS. 1N-rich cells grown in M9glyCAAT were collected 0, 120, 180, 240 and 300 min, while multi-N cells were collected 0, 60, and 120 min after 0.2% L-arabinose induction of CRISPRi. Different shades of gray represent two independent experiments.

Comparison between experimental results from the M9glyCAAT condition and simulation results using model B.

(A-C) Plots comparing simulation results of model B (solid lines) with experimental data (dots) and averages (open squares) in the M9glyCAAT condition. The multi-N and 1N cells are indicated as blue and yellow, respectively: (A) The relation between the absolute growth rate and cell area (A). (B) The relation between the active RNAP fraction and cell area. (C) The relation between the active ribosome fraction and cell area. (D) A two-dimensional diagram showing how the fractions of active RNAPs and ribosomes change with DNA concentration (colored from blue to yellow). Experimental data (with 2D error bars) from multi-N and 1N cells were combined and shown in the same plot.

Comparison between experimental results from the M9gly and M9ala conditions and simulation results using model A.

(A-C) Plots comparing simulation results of model A (solid lines) with experimental data (dots) and averages (open squares) in the M9gly condition. The wild-type and 1N cells are indicated as black and yellow, respectively. (D-F) Plots comparing simulation results of model A (solid lines) with experimental data (dots) and averages (open squares) in the M9ala condition. The wild-type and 1N cells are indicated as black and yellow, respectively. (A,D) The relation between the absolute growth rate and cell area (A). (B,E) The relation between the active RNAP fraction and cell area. (C,F) The relation between the active ribosome fraction and cell area.

Model A-based simulations examining the effects of varying the rates in either mRNA synthesis or mRNA degradation on the relative growth rate of 1N cells as a function of cell area.

(A) Plot showing the decay of DNA concentration (black) and of the relative growth rate (blue) in 1N cells when the rate of bulk mRNA synthesis rate r1 increase or decreases by ten-fold. Each quantity was normalized to its value at normal cell size (cell area = 2.5 μm2). (B) Same as (A) except mRNA degradation rate, increasing or decreasing by ten-fold.

Cell sampling to match cell size distribution in mixed populations of 1N and multi-N cells.

Comparison of the cell area distributions of 1N and multi-N cells before and after random sampling. Single cells were sampled for each biological replicate and cell area bin such that the area distributions and numbers of 1N and multi-N cells overlapped. Bins with less than 50 cells from either population were removed from the analysis. Cell area distributions from mixed CJW7457 (CRISPRi oriC) and CJW7576 (CRISPRi ftsZ) populations stained with SYTO RNASelect (aggregated data from five biological replicates)

Comparison of RpoC-HaloTag-JF549 labelling between 1N and multi-N cells.

(A) Phase contrast (left) and RpoC-HaloTag-JF549 fluorescence (right) images of two representative cells from a mixed population of 1N (CRISPRi oriC, CJW7520) and multi-N (CRISPRi ftsZ, CJW7527) cells. The blue and orange ticks In the RpoC-HaloTag-JF549 color bar correspond to the average fluorescence (concentration proxy) of the multi-N and the 1N cell, respectively. (B) Same as Figure 4– figure supplement 4, except for CJW7520 (CRISPRi oriC) and CJW7527 (CRISPRi ftsZ) cells in which RpoC-HaloTag was stained with the JF549 dye (aggregated data from two biological replicates). (C) Distributions of RpoC-HaloTag-JF549 concentration for 1N and multi-N cells (748 cells for each population from two biological replicates). (D) Average 1N/multi-N RpoC-HaloTag-JF549 concentration ratio (gray bar) calculated from two biological replicates (white circles) after sampling the same number of cells per biological replicate and cell area bin for each population (see Materials and Methods). (E) Plot showing the concentration ratio of fluorescently labeled RpoC derivatives between 1N and multi-N cells versus the cell area. Squares show mean ± full range of RpoC-HaloTag-JF549 signal concentration ratios from two biological replicates, whereas grey circles indicate the mean signal concentration ratio of RpoC-YFP (from data shown in Figure 3A) for comparison. A linear regression (red dashed line) was fitted to the average RpoC-HaloTag-JF549 ratios. A cell area of ∼2.8 μm2 corresponds to a 1N/multi-N RpoC-HaloTag-JF549 concentration ratio equal to 1.

Comparison of protein and mRNA scaling between biological replicates.

(A) Correlation of protein slopes across the proteome (2360 proteins) between two biological replicates for 1N cells. The indicated Spearman correlation (ρ) is significant (p-value < 10-10. (B) Same as (A) but for multi-N cells. (C) Correlation of RNA slopes across the genome (3446 mRNAs) between two biological replicates for 1N cells. The indicated Spearman correlation shown (ρ) is significant (p-value < 10-10).

Protein slopes relative to protein ion intensity for 1N-rich cells.

The summed ion intensity of each protein was divided by the protein sequence length and the quotient was log-transformed. The locations of selected proteins are annotated.