Asymmetric chromosome segregation and cell division in DNA damage-induced bacterial filaments

Abstract

Faithful propagation of life requires coordination of DNA replication and segregation with cell growth and division. In bacteria, this results in cell size homeostasis and periodicity in replication and division. The situation is perturbed under stress such as DNA damage, which induces filamentation as cell cycle progression is blocked to allow for repair. Mechanisms that release this morphological state for reentry into wild-type growth are unclear. Here we show that damage-induced Escherichia coli filaments divide asymmetrically, producing short daughter cells that tend to be devoid of damage and have wild-type size and growth dynamics. The Min-system primarily determines division site location in the filament, with additional regulation of division completion by chromosome segregation. Collectively, we propose that coordination between chromosome (and specifically terminus) segregation and cell division may result in asymmetric division in damage-induced filaments and facilitate recovery from a stressed state.

FIGURE 1:

Asymmetric division in DNA damage-induced filaments during recovery. (A) Representative time-lapse montage of filamentous cells during recovery. White asterisks indicate divisions occurring toward a cell pole. Scale bar = 5 µm; time in minutes here and in all other images. Images were taken every 2 min. (B) Cell length of two daughter cells generated from a single division in wild-type conditions. Each gray dot represents a single division event (n = 157). The red line plots the expected values if all cells were dividing at their midpoint. (C) Cell length of long daughter (L_D) and short daughter (S_D) generated from a DNA damage-induced filament during recovery. Cells are treated with mitomycin-C (MMC) for 60 min. Each gray dot represents a single division event (n = 531). The red line plots the expected values if all cells were dividing at their midpoint. (D) Location of division is plotted as a function of cell length in filamentous E. coli during recovery from DNA damage treatment (60 min; n = 531). (E) Cell length of a long daughter (L_D) and short daughter (S_D) is tracked over time during damage recovery. Decrease in cell length is indicative of division. (F) Distribution of S_D lengths generated from filaments between 12 and 40 µm long after 30, 60, and 90 min of MMC treatment (n = 142 [30 min], 363 [60 min], 96 [90 min]). (G) As C for cells treated with MMC for 30 min (n = 151). (H) Fate of S_D and L_D during recovery. Cell is classified as recovered if it undergoes midcell division and produces a daughter of wild-type size and filamentous if it continues to filament after division (n = 116 [30 min, L_D], 98 [60 min, L_D], 106 [90 min, L_D], 150 [30 min, S_D], 264 [60 min, S_D], 150 [90 min, S_D]). (I) Number of divisions per cell in 1 h for all durations of damage treatment. As a control, the number of divisions wild-type cells undergo is also shown (n = 50 [wt], 150 [filaments]). (J) Distribution of time between divisions for wild-type (no damage control), damage-induced filament, and S_D during recovery from MMC (n = 148 [wt], 611 [filament], 468 [S_D]).

FIGURE 2:

Division dynamics of damage-induced filaments. (A) Length added to a cell between divisions as a function of the birth length of the cell for MMC-treated cells (n = 452), p value 0.0293, 95% CI [−0.0631 0.1212], Pearson correlation value via MATLAB corrcoef function. (B) Time between divisions for wild-type cells or damage-recovery filaments for all divisions and for cells between first and second, second and third, and so on until the sixth division in filaments during recovery is plotted (n = 148 [wt], 410 [all filaments]). (C) Time between divisions as a function of cell length at birth for MMC-treated cells. Red line shows the relation that would be necessary for the system to be an adder (Eq. 3 in supplementary results), given that cells are growing exponentially with the rates given in Supplemental Figure S2A (n = 458). (D) S_D cell length as a function of the birth length of the cell for MMC-treated cells (n = 457, p value 0.4650, 95%CI [0.3894 0.5343], Pearson correlation value via MATLAB corrcoef function). (E) Distribution of length added and S_D cell length at each division for MMC-treated cells (distributions are significantly different, two-sample Kolmogorov-Smirnov test, p value 1.0721 × 10⁻⁸, n = 452). (F) As A and D for wild-type cells (length added, n = 107, p value 0.0647, 95%CI [−0.1268 0.2514]; length removed, n = 107, p value 0.6057, CI [0.4698 0.7135], Pearson correlation value via MATLAB corrcoef function). (G) Phase profile for a wild-type cell before and after division is plotted. Constriction is marked with an * and divisions are identified during segmentation. (H) As G for a MMC-treated filament.

FIGURE 3:

Role of Min-system in division positioning. (A) Representative time-lapse montage of division in wild-type cells during damage recovery. (B–D) Cell length of long daughter (L_D) and short daughter (S_D) generated from a DNA damage-induced filament during recovery for slmA (n = 144), sulA (n = 246), and minCDE (n = 186) backgrounds, respectively (blue dots; minicells are shown in gray). As a reference, lengths for wild type (n = 137) during recovery are shown in red. The red line plots the expected values if all cells were dividing at their midpoint. (E) Schematic representation of the Min-driven division site rule (figure adapted from Wehrens et al., 2018). Location of division for various filament length bins is shown with the blue band. Precise location of division (relative to cell length) is depicted inside each cell. (F) Distribution of relative position of division for filaments between 12 and 18 µm for 30 or 60 min of damage treatment. Data are not shown for 90 min of treatment as the number of filaments in this length range in 90 min treatment is low. Location of division as determined by the Min-rule is shown as a shaded bar. (G–I) As F for filaments between 18 and 26, 26 and 33, and 33 and 40 µm, respectively. (J) As F for all filaments between 12 and 40 µm. Location of potential midcell division is shown with the shaded bar (n [all] = 135 [30 min], 453 [60 min], 95 [90 min]).

FIGURE 4:

Impact of chromosome and terminus segregation on division regulation. (A) Representative time-lapse montage of division in cells during recovery. Gray: phase; red, HupA-mCherry (chromosome); scale bar = 5 µm; time in minutes. Fluorescence intensity traces for the cell in montage is provided below. Division sites are marked with “*”. (B) Distribution of relative position of division for filaments between 12 and 18, 18 and 26, and 26 and 33 µm is plotted for nucleate and anucleate (from wild-type and matP backgrounds) divisions (n = 398 [nucleate] and 160 [anucleate]). (C) Cell length distribution for wild-type cells (no perturbation) is plotted. Along with this, cell length distribution of S_D during DNA damage recovery is plotted for nucleate cells and anucleate cells. To highlight the distinction between these cell division events and minicell formation, cell length distribution of minicells (from min-deleted cells) is also shown. (n = 1110 [control], 531 [nucleate S_D], 41 [anucleate S_D], 117 [minicells]). (D) Position of least intensity of HupA fluorescence (gaps between chromosomes) plotted as a function of cell length (from one pole to midcell) in recovering MMC or cephalexin-treated filaments. As reference, these data are also shown for wild-type cells with no damage treatment (control; n = 191 [cephalexin], 476 [MMC], 150 [control]). (E) Distribution of time to division after FtsZ, ZapA, and FtsN localization to division site is plotted. Along with this, time to division after segregation of terminus (ter) during recovery after MMC treatment is also shown (n = 103 [FtsZ], 102 [ZapA], 127 [FtsN], 123 [ter]). (F) Percentage of S_D that are anucleate, recover, and filament is plotted for wild type and deletions of slmA, sulA, and matP, during DNA damage recovery (n = 103 [wild type], 105 [slmA], 105 [sulA], 103 [matP]). (G) Time from FtsZ localization to division completion is plotted for divisions that result in nucleated or anucleated S_D cells (n = 231 [nucleate], 71 [anucleate]).