Cell shape and cell-wall organization in Gram-negative bacteria

Abstract

In bacterial cells, the peptidoglycan cell wall is the stress-bearing structure that dictates cell shape. Although many molecular details of the composition and assembly of cell-wall components are known, how the network of peptidoglycan subunits is organized to give the cell shape during normal growth and how it is reorganized in response to damage or environmental forces have been relatively unexplored. In this work, we introduce a quantitative physical model of the bacterial cell wall that predicts the mechanical response of cell shape to peptidoglycan damage and perturbation in the rod-shaped Gram-negative bacterium Escherichia coli. To test these predictions, we use time-lapse imaging experiments to show that damage often manifests as a bulge on the sidewall, coupled to large-scale bending of the cylindrical cell wall around the bulge. Our physical model also suggests a surprising robustness of cell shape to peptidoglycan defects, helping explain the observed porosity of the cell wall and the ability of cells to grow and maintain their shape even under conditions that limit peptide crosslinking. Finally, we show that many common bacterial cell shapes can be realized within the same model via simple spatial patterning of peptidoglycan defects, suggesting that minor patterning changes could underlie the great diversity of shapes observed in the bacterial kingdom.

Fig. 1.

Elastic model of the peptidoglycan network predicts “cracked” cell shapes. Glycan strands (shown in green) are hoops that wrap around the circumference of the cylinder, whereas peptide crosslinks (shown in red) are longitudinal. Both are under tension due to osmotic pressure. (A) The Left Inset zooms in on a region of the cell wall without defects and schematically illustrates the elastic forces on each vertex (see Fig. S1 and Materials and Methods for more details). The Center and Right Insets zoom in on regions missing a single peptide or glycan, respectively, shown as broken blue links. The tension in the remaining glycans and peptides relative to a cylinder without defects is represented by the width of the bonds, where the width is doubled in the bond with the maximal increase in tension (≈10%). Around each defect, the increase in stress in the surrounding material is confined to the immediate vicinity of the defect. (B and C) A patch of defects centered near midcylinder results in a cracked shape (B) with the cracking angle closing as the size of the defect patch increases (C). (D) Off-center defect patches result in an off-center cracked cell shape. The number of defects shown here is identical to that in B.

Fig. 2.

Bulge formation in imp4213 E. coli bacteria in response to vancomycin treatment. (A) Phase-contrast image of a typical bulge deformation of an initially rod-shaped cell in response to vancomycin treatment. The bulge (arrow) occurs near midcell, and the cell “cracks” around the bulge. (B) Fluorescence of cytoplasmic GFP extends into the bulge in the cell in A. (C) FM4–64 membrane stain of a cell with an off-center bulge. (D) Cell with 2 bulges. (E) Growth of a bulge at a nascent division site, with images shown at 2-min intervals. Time is measured in minutes, and t = 0 is arbitrary. (Scale bar: 2 μm.) (F and G) Proposed mechanism of bulging: The permeable outer membrane (dashed line) of an imp4213 E. coli cell allows the passage into the periplasm of vancomycin (blue discs), which blocks peptide crosslinking of the cell wall (green and red rods). The cell wall cracks into 2 cylindrical regions around the high concentrations of vancomycin-induced peptide defects (broken blue rods) near midcell, and the cytoplasmic membrane (transparent red) bulges out of the crack. The poles of the cell (dark hemispheres) are relatively inert compared to the cylindrical region of the cell, hence vancomycin has little effect in these regions.

Fig. 3.

Robustness of the shape of a model cell with peptidoglycan defects. (A and B) Upon removal of an increasing concentration of randomly chosen peptide bonds (A) or peptide and glycan bonds (B), the cell wall maintains an approximately cylindrical shape, with slightly increased dimensions as indicated. The peptide and glycan defect concentrations are shown as percentages in red and green, respectively. The radii, lengths, and defect concentrations are measured relative to a single-layer cell wall with radius R₀ and length L₀. In B, the defects are chosen so that the glycan strand-length distribution matches the experimental distribution from ref. . (C) Comparison of the computed strand-length distribution of the topmost image in B with the experimental distribution. (D) The glycan defects in B create large pores in the cell wall; shown are the distributions of pore areas in the topmost and bottom images.

Fig. 4.

Common bacterial cell shapes generated via defects in a model cylindrical cell wall. (A) Removal of peptide bonds along the top surface of the cylinder results in a curved cell shape reminiscent of Caulobacter crescentus. (B) A reduction in the relaxed lengths of peptides and glycans along a helical path generates a helical cell shape similar to that of spirochaetes or the bacterium Helicobacter pylori. (C) Removal of 2 patches of peptide defects on opposite sides of the cylinder, separated by half the cylinder length, creates a snakelike cell shape similar to vancomycin-treated E. coli cells with multiple bulges. (D) Increased substitution of glycan–peptide–glycan segments in place of glycans near the midplane of the cell creates a swelled cylinder, similar to the lemon shape of cylindrical Gram-negative bacteria after treatment with the MreB inhibitor A22.