Architecture and assembly of the Gram-positive cell wall

Abstract

The bacterial cell wall is a mesh polymer of peptidoglycan--linear glycan strands cross-linked by flexible peptides--that determines cell shape and provides physical protection. While the glycan strands in thin 'Gram-negative' peptidoglycan are known to run circumferentially around the cell, the architecture of the thicker 'Gram-positive' form remains unclear. Using electron cryotomography, here we show that Bacillus subtilis peptidoglycan is a uniformly dense layer with a textured surface. We further show it rips circumferentially, curls and thickens at free edges, and extends longitudinally when denatured. Molecular dynamics simulations show that only atomic models based on the circumferential topology recapitulate the observed curling and thickening, in support of an 'inside-to-outside' assembly process. We conclude that instead of being perpendicular to the cell surface or wrapped in coiled cables (two alternative models), the glycan strands in Gram-positive cell walls run circumferentially around the cell just as they do in Gram-negative cells. Together with providing insights into the architecture of the ultimate determinant of cell shape, this study is important because Gram-positive peptidoglycan is an antibiotic target crucial to the viability of several important rod-shaped pathogens including Bacillus anthracis, Listeria monocytogenes, and Clostridium difficile.

Figure 1. Peptidoglycan texture and density is homogenous in cross-sections in cryo-tomograms of both intact cells and purified sacculi

(A) Tomographic slice through a live B. subtilis ΔponA mutant (a mutant that is thinner than wild-type B. subtilis and thus ammenable to ECT). Scalebar, 200 nm (B) Tomographic slice through an isolated wild-type B. subtilis sacculus. Scalebar, 250 nm (C) Two representative tomographic cross-sections across the wall of isolated B. subtilis sacculi perpendicular to the viewing plane reveal a globally straight sacculus side-wall with local variations in thickness. In both tomographic slices the sacculus interior is to the left. (D) Two representative top-down slices through tomograms parallel to the plane of the sacculus illustrating surface textures (red arrows) previously interpreted to be the surfaces of coiled cables composed of helical coils of peptidoglycan. In both tomography slices the long axis of the cell runs vertically. Scale bars 50nm.

Figure 2. Fragmented sacculi consistently curl up around their outer surface and exhibit a preferential tearing direction

(A) 20 nm-thick slice through the X-Y plane of a fragmented sacculus reconstructed from dual-axis tilt series data. Bar, 200 nm. Red arrow highlights the curling seen in relaxed fragmented peptidoglycan. (B) Isosurface rendering of sacculus fragment to illustrate curling of peptidoglycan around its outer face. Red arrow highlights corresponding area of relaxed peptidoglycan shown in Panel (A). (C) Proposed fragmentation mechanism.

Figure 3. Molecular dynamics of three models of peptidoglycan architecture show that the circumferential model curls and thickens upon relaxation

Assembly of strained peptidoglycan according to three possible models of assembly, and subsequent relaxation. Glycan strands are depicted in blue and peptide crosslinks in green. (A) Relaxation of a circumferential peptidoglycan fragment. Note circumferential peptidoglycan thickens and curls considerably upon relaxation. (B) Peptidoglycan fragment from the all-at-once perpendicular model as originally proposed before and after relaxation. (C) Fragment grown using an inside-to-outside perpendicular assembly model. For all relaxed fragments, colored cylinders represent changes in distances between two NAM residues on the inner and outer surfaces; cylinder diameter and intensity of shading is proportional to change in distance upon relaxation, with red representing contraction and blue representing expansion.

Figure 4. Purified peptidoglycan thickens upon relaxation, an observation accounted for in modeling studies only by the circumferential model

(A) Quantification of thickening observed in molecular dynamics simulations. The log ratio of relaxed to strained thickness (calculated by picking evenly spaced pairs of NAM residues on the inner and outer surfaces) is plotted as a function of position along the original long-axis of the cell, in 40-Å bins. Blue: circumferential, red: perpendicular, yellow: alternative perpendicular models (B) Illustration of measurements of sacculus thickness from both strained and relaxed peptidoglycan (C) Histogram of cross-sections of both relaxed and strained sacculi illustrating thickening of sacculi upon relaxation.

Figure 5. Denatured sacculi elongate but maintain the same width

(A) Projection images of isolated sacculi before and after denaturation. (B) Scatter plot of sacculus length vs. width. Note that the width distribution does not shift whereas the length distribution does.