Principles of bacterial cell-size determination revealed by cell-wall synthesis perturbations

Abstract

Although bacterial cell morphology is tightly controlled, the principles of size regulation remain elusive. In Escherichia coli, perturbation of cell-wall synthesis often results in similar morphologies, making it difficult to deconvolve the complex genotype-phenotype relationships underlying morphogenesis. Here we modulated cell width through heterologous expression of sequences encoding the essential enzyme PBP2 and through sublethal treatments with drugs that inhibit PBP2 and the MreB cytoskeleton. We quantified the biochemical and biophysical properties of the cell wall across a wide range of cell sizes. We find that, although cell-wall chemical composition is unaltered, MreB dynamics, cell twisting, and cellular mechanics exhibit systematic large-scale changes consistent with altered chirality and a more isotropic cell wall. This multiscale analysis enabled identification of distinct roles for MreB and PBP2, despite having similar morphological effects when depleted. Altogether, our results highlight the robustness of cell-wall synthesis and physical principles dictating cell-size control.

Figure 1. Heterologous sequences can complement the essential PBP2 functions in an E. coli MG1655 mrdA knockout

A) Schematic of experimental protocol. Heterologous mrdA genes from several species were introduced on an inducible plasmid in an E. coli MG1655 ΔmrdA strain. Since PBP2 is essential, only heterologous mrdA plasmids that successfully complement the native PBP2 function allow proliferation. B) Phase-contrast images of complementing heterologous mrdA strains (see also Fig. S1 for non-complementing strains). Complementing strains show changes in width (C) but no large differences in length distributions (D). In (C) and (D), boxes define the mean (central bar) and standard deviation (box edges) across 3 separate experiments. Dots represent single-cell measurements (n=906-10902 cells). E) Ec mrdA suppresses the effect of Vc mrdA when the two genes are co-expressed at high levels. Cells expressing Ec and Vc mrdA controlled by inducible and constitutive promoters, respectively, exhibit a range of stable widths dependent on the level of Ec PBP2. Error bars represent standard deviations from the mean. In (B) and (E), cell width and length closely match the population means. Scale bars: 2 μm.

Figure 2. Quantification of PG content in heterologous and under-expressed E. coli mrdA strains

UPLC of PG isolated from cells reveals no significant differences in overall crosslinking levels, average strand length, or relative concentrations of particular chemical species (Experimental Procedures, Tables S2). Data are represented as mean ± standard deviation over 3 separate experiments. The legend color scheme is consistent with that of Fig. 1.

Figure 3. MreB dynamics are affected by cell width

A) Cells with MreB^sw-sfGFP as the sole copy of mreB were imaged via TIRF microscopy to quantify (B) MreB angle, (C) speed, and (D) fraction of processive MreB molecules (Experimental Procedures). Data in (B-D) are mean ± standard error (n = 84-185 cells). B) MreB angle is not affected by mecillinam treatment but does exhibit a decrease in average angle and increase in the width of the distribution of angles in both A22-treated and Ec/Vc cells. C) MreB speed decreases under mecillinam treatment and in Ec/Vc cells but is relatively constant under A22 treatment. D) In all cases, larger width is coupled to a decreased fraction of processive MreB. See Fig. S2 for detailed MreB dynamics.

Figure 4. Cell twisting properties change with cell width

A) Cell surfaces were uniformly labeled with flWGA, washed, and imaged in TIRF in the absence of the label and in the presence of cephalexin to prevent cell division (Experimental Procedures). At the beginning of the time-lapse experiment, cells were photobleached. Due to twisting, the unbleached cell wall re-entered the TIRF field during elongation; the rate of the fluorescence increase was measured (Movie S1). Scale bar is 2 μm. B) Cell outlines were determined from phase-contrast images (A) and used to integrate TIRF fluorescence intensity relative to the pre-bleached state at each time point. Single-cell fluorescence recovery curves were fitted to a Gompertz equation (Experimental Procedures), where F_max is the plateau F level and δ is the lag; the rate of recovery λ was determined for each cell. C) Simulated cell twisting and generation of simulated fluorescence images (Experimental Procedures) were used to determine how the cell-twisting angle affects twist rate (w), which is not very sensitive to width at low twist angles. Experimentally, Vc mrdA cells (dashed mean and shaded standard error) had a higher twist angle than wild-type MG1655 cells. D) Single-cell twist measurements show a systematic increase in cell twist rate with width (w) in heterologous Ec/Vc mrdA and A22-treated cells, but not in mecillinam-treated cells. Data are shown as mean ± standard error in both width and twist rate, with linear fits. Pearson’s correlation coefficients are shown. E) Twisting chirality changes in heterologous Ec/Vc mrdA and A22-treated strains but not in mecillinam-treated strains, in the manner predicted by the distribution of MreB angles (Fig. 3B). Wild-type (WT or 0 μg/ml A22/mecillinam) cells twist with left-handed chirality, while wider Ec/Vc or A22-treated cells tend to exhibit right-handed twist. See also Fig. S3 and Movie S1 for detailed information.