Correlations between polysome and nucleoid dynamics at the single-cell level.

CJW7323 cells were grown in in M9gluCAAT in a microfluidic device. A. Schematic illustrating observable nucleoid segregation events. B. Fluorescence images of RplA-GFP and HupA-mCherry for a representative cell (CJW7323) from birth to division. C. Ensemble kymographs of the average RplA-GFP and HupA-mCherry fluorescence during the cell division cycle (>300,000 segmented cell instances from 4122 complete cell division cycles). D. Two-dimensional projections of the average RplA-GFP and HupA-mCherry fluorescence signals in predivisional cells (4564 cells with two nucleoid objects, from 1907 cell division cycles, 95-100% into the cell division cycle) and their intensity profiles. White arrows indicate RplA-GFP enrichments at the quarter cell positions, while the black arrow indicates the site of cell constriction. E. Plot showing the dynamics of RplA-GFP accumulation and HupA-mCherry depletion at mid-nucleoid (median ± IQR) during the nucleoid cycle (see Figure 1 – Figure supplement 2D). Data from 3240 nucleoid segregation cycles are shown (40 nucleoid cycle bins, 2512-4823 segmented nucleoids per bin). F. Correlation (Spearman ρ = −0.52, p-value < 10−10) between the rate of RplA-GFP accumulation in the middle of the nucleoid and the rate of HupA-mCherry depletion in the same region between the initiation of nucleoid splitting and just before the end of nucleoid splitting (3214 complete nucleoid cycles). The colormap indicates the Gaussian kernel density estimation (KDE). Solid black line indicates the linear-regression fit to the data. G. Percentage of cells that continue to accumulate polysomes in the middle of the cell during four relative time bins (1335 to 1957 cell division cycles per bin) covering the period from the end of nucleoid splitting until cell division. H. Correlation (Spearman ρ = 0.47, p-value < 10−10) between the rate of RplA-GFP accumulation at mid-cell and the rate of distance increase between the sister nucleoids during the first quartile (0-25%) of the period between the end of nucleoid splitting and cell division (1376 cell division cycles). The black markers correspond to nine bins (mean ± SEM, 75 to 177 cell division cycles per bin) within the 5th-95th percentiles of x-axis range. Also shown is the distribution of the cell elongation rates during the same time interval, with the mean and SD shown by the solid and dashed lines, respectively. I. Plot showing the coefficients of a linear mixed-effects model (see eq. 3 in Methods and Figure 1 – figure supplement 3B) for four interval bins between the completion of nucleoid splitting and cell division. The coefficients quantify the relative contribution of polysome accumulation at mid-cell and cell elongation to the rate of sister nucleoid migration . All coefficients are significant (Prob(<|Z|) < 10−9), except for the one marked with an asterisk that is marginally significant (Prob(<|Z|) = 0.02).

Correlation of the extent and relative timing of polysome accumulation with nucleoid segregation at the population and single-cell levels.

A. RplA-mEos2 and DAPI (scaled by the whole cell average) demographs constructed from snapshots of DAPI-stained CJW6768 cells (815 to 2771 cells per demograph) expressing RplA-mEos2 and growing in different nutrient conditions (see Table S1 for abbreviations). The demographs were arranged from smallest (M9mann, top) to biggest average cell area (M9malaCAAT, bottom). Additional demographs for different ribosomal reporters and nutrient conditions are shown in Figure 2 – figure supplement 1. B. Correlation between average polysome accumulation and average nucleoid depletion at mid-cell for all tested strains (Spearman ρall = −0.85, p-value < 10−10) with different ribosomal and nucleoid reporters, and within each strain (−0.75 < Spearman ρstrain < −0.95, p-values < 10−3) across nutrient conditions. A linear regression was fitted to all the data. D. Plot showing the variability in growth rate (GR) across cell division cycles for cells growing in microfluidics in M9gluCAAT (4114 cell division cycles, 9 growth rate bins with 93 to 860 cell division cycles each). E. Ensemble kymographs of the RplA-GFP and HupA-mCherry concentration normalized by the average fluorescence for the slowest, intermediate and fastest growing population bins shown in panel D. F. Plot showing the correlation between the average RplA-GFP accumulation and HupA-mCherry depletion at mid-cell across the growth rate bins (mean ± SD, 93 to 860 cell division cycles per bin) shown in panel D.

Correlations between polysome and nucleoid asymmetries.

A. Distributions of RplA-GFP concentration in the new (grey) and the old (black) pole regions of newborn cells (0-2.5% of the cell division cycle, n = 912 cell division cycles). The histograms were smoothed using Gaussian kernel density estimations. B. Correlation (Spearman ρ = −0.41, p-value < 10−10) between the polar polysome asymmetry and the position of the nucleoid centroid around the cell center of cells at the beginning of the division cycle (0-10%, n = 1179 cell division cycles). The contour plot consists of 9 levels with a lower data density threshold of 25%. The polar polysome profile for cells with correlations indicated by the numbers 1 and 2 are schematically illustrated in the next panel. A linear regression (solid grey line) was fitted to the data. C. Schematic illustrating the effects of the relative polysome abundance between the poles on the position of the nucleoid. D. Average 2D projections of the RplA-GFP and HupA-mCherry concentration (conc.) at different cell division cycle intervals (∼9440 to 47240 cell images per cell division cycle interval from 4122 cell division cycles). The dotted line indicates the boundary between two cell division cycles. E. Density plot comparing the distribution of the HupA-mCherry maximum concentration toward the new pole (gray) to that toward the old pole (black) in newborn cells (0-2.5% of the division cycle, n = 912 cell division cycles). The histograms were smoothed using Gaussian kernel density estimations. F. Correlation (Spearman ρ = 0.52, p-value<10−10) between the nucleoid density asymmetry and the relative availability of polysome-free space between the two cell halves early in the cell division cycle (0-10% of the division cycle, n = 2150 cell division cycles). The contour plot consists of 9 levels with a lower data density threshold of 25%. Values above 1 on the x-axis indicate more polysome-free space toward the new pole, and values below 1 correspond to cells with more polysome-free space toward the old pole. On the y-axis, values above 1 indicate higher DNA density toward the new pole and values below 1 indicate higher DNA density toward the old pole. G. Average 2D projections of newborn cells (0-10% into the cell division cycle) from the lower-left quartile in panel C (region 2, n = 223 cell division cycles) and the upper right quartile in panel C (region 1, n = 557 cell division cycles) and their 1D intensity profiles.

Simulation results of the reaction-diffusion model for different growth rates or nucleoid diffusion rates, and comparison of the nonequilibrium polysome accumulation with freely diffusing particles.

A. 1D simulation of polysome (blue) and nucleoid (red) dynamics during slow growth (growth rate = 0.25 h−1, Dn = 0.001 μm2/s, cell length at birth = 1.84 μm) at different relative cell division cycle timepoints. The simulation was initialized from the equilibrium polysome and nucleoid distribution (at 0%). B. Schematic summarizing how polysomes accumulate in the middle of the elongating nucleoid, causing nucleoid splitting. C. Correlation between the relative timing of nucleoid splitting and the growth rate as captured by our reaction-diffusion model (Dn = 0.001 μm2/sec) across six growth rate bins. The cell and nucleoid lengths for each growth rate bin matched previously published data shown in Figure 4 – figure supplement 1A. D. Deviation between the steady state after infinite relaxation time (dashed curves) and the polysome or nucleoid profiles in newborn cells after one simulation round (solid curves) for increasing nucleoid diffusion constants during fast growth (1 h−1). E. Phase contrast and fluorescence (fluor.) images of two representative single cells (CJW7651). The red circles indicate the position of the mCherry-μNS particle in each cell. F. Two-dimensional average cell projections of the DAPI concentration (conc.) and the RplA-msfGFP concentration, and 2D histogram of the mCherry-μNS particle density for four cell length bins of CJW7651 cells (∼2580 cells per bin) grown in M9gluCAAT and spotted on an agarose pad containing the same medium. Since the cell pole identity cannot be inferred from snapshot images, pole assignment was random. G. Average 1D profiles of the scaled DAPI and RplA-msfGFP concentrations and the mCherry-μNS probability density.

Effects of rifampicin treatment and polysome depletion on nucleoid segregation and compaction.

A. Plot showing the average instantaneous growth rate (mean ± SD shown by the solid black curve and grey shaded region, respectively) of a cell population (n = 2629 cell division cycles) undergoing two rounds of rifampicin treatment in a microfluidic device supplemented with M9gluCAAT. The distribution of the average cell cycle growth rate of unperturbed populations is also shown on the right (n = 4122 cell division cycles from a different microfluidics experiment). The solid horizontal line indicates the average growth rate. B. Plot showing the average distance between nucleoid peaks for a population of cells (squares, 114 cell division cycles) that were born (−112 to −102 min) and divided before the addition of rifampicin, and for a population of cells (circles, 112 cell division cycles) that were born just before (−22 to − 12 min) and divided after the addition of rifampicin. A third-degree polynomial was fitted to the data from the unperturbed population (solid blue curve) and juxtaposed (dashed blue curve) with the data from the interrupted population. C. Average 1D profile and 2D projections of the scaled (divided by the whole cell average concentration) RplA-GFP and HupA-mCherry signals for cells before and after rifampicin addition (n = 112 cell division cycles). The red dashed horizontal lines in the 1D intensity profiles and the white crosses in the 2D profiles mark the nucleoid peaks. D. Plot showing the RplA-GFP accumulation relative to the HupA-mCherry depletion at mid-cell from 0 to 24 min after birth (colormap) for cells that completed their division cycle before the addition of rifampicin (n = 114 cell division cycles) and for cells that were subjected to rifampicin 12 min (n = 112 cell division cycles) or 3 min (n = 99 cell division cycles) after birth. E. Average 1D and 2D scaled RplA-GFP and HupA-mCherry intensity profiles for newborn cells (0 to 10 min after birth) before (left, n = 726 cell division cycles), just after (middle, n = 367 cell division cycles), and much after (right, n = 235 cell division cycles) rifampicin addition.

Effects of ectopic polysome accumulation on nucleoid dynamics.

A. Schematic summarizing the experiment. B. Representative phase contrast and mTagBFP2 fluorescence images at different times after induction with IPTG (100 µM) are shown, next to a plot showing the mTagBFP2 fluorescence of the entire population (mean ± SD, n = 3624 cell trajectories) over time. C. Plot showing how instantaneous growth rate (mean ±SD, n = 3624 mTagBFP2 induction trajectories) decreases following induction of mTagBFP2 synthesis. D-F. Representative kymographs and images of the normalized (divided by the whole cell average) RplA-msfGFP and HupA-mCherry fluorescence signals in cells (CJW7749) born during mTagBFP2 over-expression. F. Phase contrast images are shown to illustrate the formation of inclusion bodies (see also Figure 6 – figure supplement 1). Additional cell examples are shown in Movie S5.

Effects of cell width increase on polysome and nucleoid dynamics.

A. Comparison of the cell width increase during cell growth between CJW7323 cells treated with cephalexin (mean ± SD, 360 cell growth trajectories, 418 to 1511 segmented cells per bin) and cells treated with both cephalexin (50 μg/mL) and A22 (4 μg/mL) (grey, mean ± SD, 309 cell growth trajectories, 51 to 1684 segmented cells per bin). The same cell area bins are compared between the two populations. B. Phase contrast and fluorescence images of a representative cephalexin-treated cell expressing RplA-GFP and HupA-mCherry. C. Same as B but for a short cell growing in the presence of A22 and cephalexin. D. Same as C but for a longer cell. The white arrowheads indicate the polysome bridges that connect polysome accumulations between two DNA-free regions. Additional examples are shown in Movie S6 E. Representative fluorescence images of RplA-GFP and HupA-mCherry in a cell treated with A22 and cephalexin. The dotted lines indicate the representative cross-like polysome accumulation, which forms during the fusion of the polysome accumulation towards the center (see also Movie S6). F. Comparison of the segmented polysome accumulations and nucleoid objects between A22+cephalexin (150 sampled segmented cells from 47 growth trajectories) and cephalexin-treated (150 sampled segmented cells from 100 growth trajectories) cells. The polysome and nucleoid areas per cell were normalized by the population-average statistic from cephalexin-treated cells. All differences between the two populations are statistically significant (Mann-Whitney p-value < 10−10).

Growth medium abbreviation and composition. Abbreviation Composition

Escherichia coli strains used in this study.

Plasmids used in this study.

DNA oligonucleotides used in this study.

Chemicals used in this study.

Software used in this study.

Reproducibility analysis of the dynamic ribosome and nucleoid distributions between microfluidic experiments.

While phase-contrast images were acquired every minute in all microfluidics experiments, two different intervals (1 min and 3 min) were used for fluorescence image acquisition. A. Kymographs showing the RplA-GFP and HupA-mCherry concentration in CJW7323 cells growing in microfluidic channels in M9gluCAAT. Cell and nucleoid contours are shown in each channel using a different color for each cell lineage (from dark purple to bright orange). B-E. Two biological replicate experiments were performed for this strain and nutrient condition using different intervals of fluorescence image acquisition (1 or 3 min). B. Plots showing that the instantaneous and average division cycle growth rates were nearly identical between the two experiments. C. Plots showing the distributions of the indicated fluorescence signals for the 1-min acquisition interval (black) compared to the 3-min frame rate interval (orange) before and after correction. The difference in the excitation power between the two experiments was 50% (240 %ms vs. 360 %ms), which was also reflected in the ratio of their average fluorescence values (1.4 for RplA-GFP and 1.6 for HupA-mCherry). The fluorescence values from the 1-min interval experiment were corrected by multiplying by these ratios, resulting in a near-perfect overlap between fluorescence distributions after correction. D. Plots showing that the scaled (z-score) RplA-GFP and HupA-mCherry intensity profiles from birth to division were almost identical between the two experiments. The intensity profiles are shown for 10 cell division cycle intervals (1489 and 2633 cell division cycles for the 1-min and 3-min interval experiments, respectively). The z-score was calculated for each segmented cell by subtracting the whole cell average fluorescence and then dividing the difference by the standard deviation. Each intensity profile corresponds to the average z-score of all segmented cell instances within the corresponding cell cycle interval. Ε. Plot showing that the average RplA-GFP concentration from birth to division (averages ± SD across 20 cell division cycle bins) remained constant and was virtually identical between the two experiments. F. Representative image of RplA-GFP fluorescence in cells (CJW7323) treated with rifampicin (100 μg/mL). The cells were treated for 45 min, washed, and spotted on an M9gluCAAT agarose pad without antibiotic. The presented snapshot corresponds to the first time point immediately after cell spotting showing diffuse distribution of RplA-GFP signal.

Tracking nucleoid segregation cycles.

Since the timing of nucleoid segregation varied between cell division cycles, the nucleoid segregation cycles were tracked independently of the cell division cycles for each cell lineage to measure the relative timing of RplA-GFP accumulation and HupA-mCherry depletion in the middle of the nucleoid (as shown in Figure 1E). A. Plots showing the frequency of cells with one, two, three, or four detected nucleoid objects across 10 cell division cycle bins from birth (top) to division (bottom). Data from 4122 cell division cycles are shown. B. The polarity of the cells (+/-) and the relative position of the nucleoid mask (toward the new or old pole) was used to track the nucleoid segregation cycle. This strategy was used to identify four groups of nucleoid segregation cycles (group −2, −1, 1 and 2). Group −2 includes nucleoids that were “born” toward the old cell pole in a mother cell with negative (−) polarity and were inherited at the center of a daughter cell with a negative polarity. Group −1 includes nucleoids that were “born” toward the new cell pole in a mother cell with negative (−) polarity and were inherited at the center of a daughter cell with a positive (+) polarity. Group 1 includes nucleoids that were “born” toward the new cell pole in a mother cell with positive (+) polarity and were inherited at the center of a daughter cell with negative (−) polarity. Group 2 includes nucleoids that were “born” toward the old cell pole in a mother cell with positive (+) polarity and were inherited at the center of a daughter cell with positive (+) polarity. C. Plots showing the distributions of nucleoid positions around the cell center for 10 nucleoid segregation cycle bins, from the time a nucleoid was “born” (top) until it split (bottom). These plots show the inheritance of the nucleoids from the quarter cell positions of the mother cells to the middle of their daughters for the four groups of nucleoid segregation cycles. The solid gray line represents the average density of the nucleoid positions for all four groups. Data from 2286 complete nucleoid cycles are shown. D. Schematic that explains the definition of a nucleoid cycle. The nucleoid cycle ranges from the end of a nucleoid splitting event, until the next splitting of the sister nucleoids. It usually extends beyond cell division, into the next cell division cycle.

Correlations used to calculate the relative contribution of polysome accumulation and cell elongation to nucleoid migration.

A. Correlation (Spearman ρ = 0.47, p-value < 10−10) between the rate of RplA-GFP accumulation at mid-cell and the rate of distance increase between the sister nucleoids minus the rate of cell elongation across cells. The colormap corresponds to a Gaussian kernel density estimation. Binned data are also shown (mean ± SEM, 75 to 177 cell division cycles per bin, 9 bins in total) within the 5th-95th percentiles of the x-axis range. B. Scaled correlations (z-scores) between (blue) or (grey) and (y-axis) during four relative time bins (1335 to 1957 cell division cycles per bin) covering the period from the end of nucleoid splitting until cell division. Shown are the Spearman correlations (ρ), all with a p-value below 10−6, except for the one marked with an asterisk (p-value = 0.02).

Examination of the relative timing of the initiation of nucleoid constriction and the accumulation of polysomes at mid-nucleoid.

A. RplA-GFP and HupA-mCherry concentration (fluorescence arbitrary units) images of four representative single cells (CJW7323) growing in M9glyT and their fluorescence intensity profiles along the cell length. B. Demographs of the scaled RplA-GFP and HupA-mCherry concentration from 13554 E. coli cells grown in M9glyT. C. Average 1D intensity profiles of the scaled (divided by the whole cell average) RplA-GFP and HupA-mCherry concentration for 12 bins of cells lengths (>1100 cells per intensity profile). D. Plot showing the scaled RplA-GFP and HupA-mCherry concentration in the middle of the cells for increasing cell length (∼370 cells per bin, 25 bins). Cells longer than 3 μm were excluded to avoid the effects of cell constriction. The arrow and vertical dashed line indicate the minimum cell length bin with an apparent HupA-mCherry depletion at mid-cell, marking the initiation of nucleoid splitting. The error bars indicate mean ± standard error of the mean (SEM).

Demographs of scaled ribosome and nucleoid fluorescence for strains with different ribosome markers and under various nutrient conditions.

Demographs are ordered according to increasing average cell area, which scales with growth rate (Schaechter et al., 1958), from left to right and top to bottom. These demographs were constructed from snapshots of the following strains: A. CJW6768 (RplA-mEos2 ribosomal marker, 747 to 2446 cells per condition), B. CJW6769 (RpsB-mEos2 ribosomal marker, 690 to 3169 cells per condition), C. CJW7020 (RplA-msfGFP ribosomal marker, 657 to 2432 cells per condition) and D. CJW7021 (RpsB-msfGFP ribosomal marker, 788 to 1950 cells per condition), using the cell length as a proxy for the cell division cycle, with the signal intensity profile sorted from the shortest newborn cells to the longest predivisional cells. Note that the demographs for the CJW6768 strain (panel A) in the nutrient conditions M9mann, M9mala, M9malt, M9mannCAAT, M9glyCAAT and M9malaCAAT are not shown here, as they are presented in Figure 2A. The nutrient abbreviations and compositions are explained in Table S1.

Calculation of RplA-msfGFP concentration after cell curvature correction.

A. Average 2D RplA-msfGFP projections for short (2.5 to 3 μm – left), unconstricted cells and long (3.5 and 5.5 μm - right) constricted cells. The average RplA-msfGFP signal from 1000 sampled cells (CJW7651) is shown for each population. B. The 2D cell areas were divided in cylindrical sectors with a height (ℎ) of a single pixel. The number of cylindrical sectors corresponds to the average cell length in pixels (40 and 60 sectors for the unconstricted and constricted cells, respectively). The radius (r) of each cylindrical segment corresponds to half the cell diameter for that specific cell length position. The cylindrical segments at the poles or constriction site have a smaller radius due to the curvature of the cell boundaries. The division of the cell area into cylindrical segments allowed us to calculate the volume for each segment (Vcylinder = π r2 h). The sum of the fluorescence per segment was corrected for the 3D curvature of the cell boundaries by dividing by the volume of the cylinder, either considering the entire cell width or different depths of view narrower than the maximal cell width. C. 1D profiles of the cylinder diameter are also indicative of the cell curvature at the poles and constriction site. D. Comparisons of the 1D average RplA-msfGFP projection between the poles before (dashed lines) or after correcting for the cell curvature (solid lines) for different depths of view (colormap). The uncorrected signal (dashed lines) corresponds to the average fluorescence along each cylindrical segment (mean of pixel intensities). The 1D profiles were normalized by dividing by the maximal RplA-msfGFP concentration at mid-cell. This correction shows that the pronounced decrease of the RplA-msfGFP fluorescence at the poles is the result of the curvature of the cell boundaries. The observed over-correction at the cell poles for larger depths of view (> 8 pixels or px) is likely due to the over-estimation of the cell segmentation mask that was determined based on the phase contrast snapshot images of cells on agarose pads.

Analysis of the polysome and nucleoid asymmetries using fitted Gaussian functions.

A. Example images of RplA-GFP and HupA-mCherry fluorescence signals in a single newborn cell instance. B. Plots showing the Gaussian-function fitting on RplA-GFP and HupA-mCherry intensity profiles from the single cell snapshot shown in panel A. Three Gaussian functions were fitted to the RplA-GFP fluorescence in newborn cells (eq. 11 in Methods), capturing the accumulation of polysomes at mid-cell and the poles. Two Gaussian functions were fitted to the HupA-mCherry fluorescence (eq. 12 in methods) capturing the two lobes of the segregating sister nucleoids. The parameters of the fitted Gaussian functions provided information about the position (μ: mean) and the concentration (A: amplitude) of each fluorescence statistic, as well as the cell length range occupied by it (σ: standard deviation). The Gaussian area (eq. 13 in Methods) corresponds to the abundance of each macromolecule. The RplA-GFP Gaussians were fitted above the cellular background, which presumably corresponds to the uniform fluorescence of free ribosomes or ribosomal subunits. For the HupA-mCherry Gaussian fittings, the cellular background corresponds to the DNA-free cell regions. The Gaussian parameters were also used to describe the polysome asymmetries between the poles (eq. 14 in Methods) as well as the position of the nucleoid around the cell center (eq. 15 in Methods). These statistics were used in Figure 3B. C. Gaussian functions were also fitted to the ensemble average RplA-GFP and HupA-mCherry fluorescence (right) calculated from all the cell segmentation instances of a single cell division cycle, 0-10% from birth to division (left). D. The Gaussian fitting to the ensemble RplA-GFP and HupA-mCherry intensity profiles (concentration in arbitrary fluorescence units) early in the cell division cycle for the 2D projection shown in panel C (right). E. Plots showing the nucleoid and polysome density axial asymmetry determined using the parameters of the fitted Gaussians on the HupA-mCherry and RplA-GFP fluorescence (eq. 16 and eq. 18 in Methods). F. Plots showing the scaled correlations between the polysome Gaussian parameters (μ, Α, and σ for each polysome accumulation) and the nucleoid density asymmetry (eq. 16 in Method; 2103 cell division cycles). Each marker corresponds to the data for a single cell division cycle (0-10% into the cell division cycle) as in panel C (right). A Gaussian kernel density estimation (KDE) was used to illustrate the density of the scatter plots (see Methods). G. Bar graph showing the coefficients of a linear mixed-effects model (see eq. 17 in Methods) used to identify the polysome statistics that contribute to the nucleoid density asymmetry. The highly contributing polysome statistics were combined to a compound polysome statistic (eq. 18 in Methods) that describes the available DNA space between polysome accumulations. This compound statistic correlates with the nucleoid density asymmetry (eq. 16 in Methods) as shown in Figure 3F. The stars indicate the polysome statistics that most significantly correlate with the nucleoid density asymmetry (absolute coefficient above 0.3).

Statistics of polysome accumulation and nucleoid depletion during simulated cell growth.

Polysome and nucleoid dynamics were simulated for six different growth rates. The predivisional polysome and nucleoid profiles of simulated trajectories initialized from the steady state (infinite relaxation time) were used as initial conditions (Dn = 10−3 μm2/s). A. Plot showing cell length at birth increasing with growth rate according to published measurements (Govers et al., 2024). B. Plot showing the dynamics of polysome accumulation (top) and nucleoid depletion (bottom) at mid-cell from birth to division for six simulated growth rates (colormap). A nucleoid depletion threshold of 0.1 arbitrary units (a.u., dashed horizontal line) was used to determine the relative time of nucleoid splitting that is presented in Figure 4C.

Phenotypic effects of transcription inhibition.

Plots showing cellular parameters (instantaneous growth rate, cell area, nucleoid area, and the ratio between nucleoid area and cell area, or NC ratio) affected by the rifampicin treatment. The instantaneous growth rate was estimated using the log-transformed cell area, using a rolling window of 10 min. The black line corresponds to the averages from 1859 cell division cycles and the gray-shaded area indicates the range of one standard deviation around the mean. The two cycles of rifampicin treatment (as shown in Figure 5A) were overlaid for this plot, using the time of rifampicin addition as t = 0 min.

Phenotypic effects of prolonged protein over-expression from plasmids.

Representative phase contrast and fluorescence images of cells (CJW7798) after prolonged induction (100 μM IPTG, for 9 h and 15 min) of mTagBFP2 expression from aT7 promoter on a multi-copy plasmid.

Phenotypic of effects of cell growth under A22 and cephalexin treatment.

Representative phase contrast and fluorescence images of cells (CJW7323) treated with A22 (4 μg/mL) alone or with both A22 (4 μg/mL) and cephalexin (50 μg/mL) for 130 min.