Control of Bacillus subtilis Replication Initiation during Physiological Transitions and Perturbations

Abstract

Bacillus subtilis and Escherichia coli are evolutionarily divergent model organisms whose analysis has enabled elucidation of fundamental differences between Gram-positive and Gram-negative bacteria, respectively. Despite their differences in cell cycle control at the molecular level, the two organisms follow the same phenomenological principle, known as the adder principle, for cell size homeostasis. We thus asked to what extent B. subtilis and E. coli share common physiological principles in coordinating growth and the cell cycle. We measured physiological parameters of B. subtilis under various steady-state growth conditions with and without translation inhibition at both the population and single-cell levels. These experiments revealed core physiological principles shared between B. subtilis and E. coli Specifically, both organisms maintain an invariant cell size per replication origin at initiation, under all steady-state conditions, and even during nutrient shifts at the single-cell level. Furthermore, the two organisms also inherit the same "hierarchy" of physiological parameters. On the basis of these findings, we suggest that the basic principles of coordination between growth and the cell cycle in bacteria may have been established early in evolutionary history.IMPORTANCE High-throughput, quantitative approaches have enabled the discovery of fundamental principles describing bacterial physiology. These principles provide a foundation for predicting the behavior of biological systems, a widely held aspiration. However, these approaches are often exclusively applied to the best-known model organism, E. coli In this report, we investigate to what extent quantitative principles discovered in Gram-negative E. coli are applicable to Gram-positive B. subtilis We found that these two extremely divergent bacterial species employ deeply similar strategies in order to coordinate growth, cell size, and the cell cycle. These similarities mean that the quantitative physiological principles described here can likely provide a beachhead for others who wish to understand additional, less-studied prokaryotes.

Keywords: cell cycle; cell size; replication initiation; single cell.

FIG 1

Population and single-cell methods to achieve steady-state growth. (A) Turbidostat experimental method and validation. (Top left) In the multiplex turbidostat vial, the culture volume was maintained at a constant level and the cell concentration was monitored and adjusted automatically by infusing fresh medium. Aerobic conditions were ensured via bubbling and stirring. (Top right) Growth rate measurements were consistent between 5 and 20 doublings, and cell length distributions were reproducible at sample collection. Data shown are from 4 repeats in succinate with 2.7 μM chloramphenicol (cam). (Bottom) A representative growth curve showing the timing of the addition of chloramphenicol, dilution events, and sample collection. Each dilution occurred when the culture reached an OD₆₀₀ of 0.2 and was diluted to 0.05, allowing for two doublings during the growth interval. (B) Overview of population growth conditions and measurements. Growth media and their abbreviations represent 5 different nutrient conditions, all of which are based on S7₅₀. Glycerol-rich, mannose, and succinate media were selected for translation inhibition experiments (succinate results are shown here). Representative growth curves (final 8 doublings), average doubling time (τ), and representative crops of images used for population sizing are shown for each condition. (C) Single-cell experiments performed with the mother machine. A representative image showing cell-containing traps is shown. The fluorescent signal represents DnaN-mGFPmut2 (see Fig. 3 and Materials and Methods). The growth in length (black lines) and division (dotted vertical lines) of a single mother cell is shown over 8 h. Average growth rate data (solid gray lines) from the single-cell measurements (gray scatter points) represent steady-state conditions over the course of the experiment. Data shown are from mannose conditions. Data from additional measurements performed under all conditions are presented in Fig. S1.

FIG 2

Population cell size and C period measurements in B. subtilis and E. coli. (A) Cell size increases with respect to growth rate in B. subtilis and E. coli under conditions of nutrient limitation. The relationship is not as clearly exponential for B. subtilis as it is for E. coli (dotted lines are linear regression fits of logarithm-transformed data, the dashed line is a linear regression fit). Representative images of cells during division show change in aspect ratio as a function of growth rate. Length and width measurements are presented in Fig. S2. (B) C period measurements with respect to growth rate in B. subtilis and E. coli under conditions of nutrient limitation. For E. coli, C is approximately constant at 39 min (horizontal dotted line) for doubling times faster than 60 min (λ = 0.69). The two-sided P value to reject the null hypothesis that the slope of C on growth rate is zero is 0.55 using the Wald test. The constancy is less clear for B. subtilis, with a P value of 0.13 for the same test. Though lower, it is still not enough to reject the null hypothesis that the slope is zero. However, single-cell data show that C + D is proportional to generation time (Fig. S5). B. subtilis growth media are colored as described for Fig. 1B, with additional LB data in gray. The E. coli data are from previously published work (3). Red, synthetic rich; orange, glucose with 12 amino acids; yellow; glucose with 6 amino acids; green, glucose; blue, glycerol. Additional conditions are indicated in gray.

FIG 3

Single-cell growth and cell cycle progression in B. subtilis. (A) Typical cell cycle progression of B. subtilis in media mediating slower growth. All panels show the same two cells. (Top) Chromosome configuration and key cell events. (Middle) Fluorescent images of DnaN-mGFPmut signal. Gray outlines are from the segmented phase-contrast image. Purple and blue backgrounds indicate C and D periods corresponding to the second division. (Bottom) Processed image data represented as a cell lineage trace. Cell length and division are indicated by the solid and dotted black lines, respectively. Vertical green bars represent the DnaN-mGFPmut2 signal summed along the long axis of the cell, with white circles showing focus positions. The single-cell initiation size, C period, and D period are determined manually from these traces. (B) Typical cell cycle progression of B. subtilis in media mediating faster growth. (C) Ensemble method to determine cell cycle parameters. (Left) In succinate, a theoretical cell is born with one pair of replisomes. It may briefly contain no active replisomes upon termination and then contain two pairs of replisomes as the two complete chromosomes begin replication. The length to which the numbers of replisome pairs increases corresponds to the initiation size. Across all cells, the average number of replisome pairs transitions from one to two at the population’s initiation size. The average initiation length (L_i) as determined from the cell traces (dashed purple line) agrees with the ensemble estimate (solid purple line). The average birth length (L_b) and division length (L_d) of the population are shown as dotted vertical lines. (Right) In glycerol-rich media, cells transition from two to four pairs of replisomes. Images of ensembles under all conditions are shown in Fig. S4.

FIG 4

Initiation size is invariant in B. subtilis during steady-state growth. (A) Single-cell initiation size per ori s_i is condition independent. Division size (circles) changes dramatically under both nutrient limitation and translational inhibition conditions. The corresponding initiation size per ori (diamonds) collapses onto a constant value (s_i) across all conditions (dotted horizontal black line). This holds for both single cells (scatter points) and population averages (solid symbols). Growth media are colored as described for Fig. 1B, with the amount of chloramphenicol indicated (0 μM chloramphenicol with empty symbols; lines connect the same growth media with and without chloramphenicol). (B) C + D values are condition dependent and increase with generation time. (Top) Increases in generation time under conditions of nutrient limitation cause an increase in the population average C + D (squares), while initiation size changes only minimally. (Bottom) A similar pattern is seen under conditions of translational inhibition. For both plots, measured parameters are compared to the condition mediating fastest growth. Single-cell C + D data presented in Fig. S5 and Fig. S8.

FIG 5

Initiation size is invariant in B. subtilis during the shift down. (A) Behavior of single cells undergoing the shift down from succinate-rich to succinate-minimal conditions at time zero. Upon the shift down, cells pause growth and initiation for 1 to 2 h. The medium shift is achieved via a Y valve upstream of the mother machine inlet. (Top) Fluorescent images of cell lineage during the shift down. (Bottom) Representative traces of 3 lineages. Representation is as described for Fig. 3, with the density of the vertical green bars representing the intensity of the DnaN-mGFPmut2 signal along the long axis of the cell. (B) Population average behavior during the shift down. Growth rate data represent the instantaneous elongation rate (6-min time step). Initiation size per ori is plotted against the initiation time. Each measurement is normalized to the corresponding mean in the 4 h before the shift down. Lines connect the 12-min binned mean, and error bars represent standard errors of the means. The minimum bin size is 5. The dashed-line portion of the line representing average initiation size signifies a gap in initiation events after the shift down. n = 3,160 cells (752 with initiation size). The entire time course showing the shift up and the shift down is available in Fig. S6.

FIG 6

B. subtilis and E. coli share the same hierarchy of physiological parameters. (A) Single-cell physiological parameter definitions as determined from time-lapse images. The cells were B. subtilis growing in mannose (τ = 38 min). The fluorescent signal represents DnaN-mGFPmut2, and the gray outlines are from the segmented phase-contrast image. The picture interval was 3 min. (B) B. subtilis parameter distributions are shown in order of ascending coefficient of variation (CV) values. Parameters are normalized to their means. The range of CVs for each parameter is shown below the distributions. Note that length-based parameters are shown here; their volume equivalents have slightly higher CVs due to variability in width. (C) Distribution of the same measurements in E. coli displaying the same CV hierarchy. Data representing E. coli NCM3722 grown in MOPS arginine (arg), glucose (glc), and glucose plus 11 amino acids (glc 11 AA) are from previously published work (7).

FIG 7

B. subtilis and E. coli comparative summary.