Imaging the nanoscale organization of peptidoglycan in living Lactococcus lactis cells (original) (raw)
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Three-dimensional structure of the bacterial cell wall peptidoglycan
Proceedings of the National Academy of Sciences, 2006
The 3D structure of the bacterial peptidoglycan, the major constituent of the cell wall, is one of the most important, yet still unsolved, structural problems in biochemistry. The peptidoglycan comprises alternating N-acetylglucosamine (NAG) and N-acetylmuramic disaccharide (NAM) saccharides, the latter of which has a peptide stem. Adjacent peptide stems are cross-linked by the transpeptidase enzymes of cell wall biosynthesis to provide the cell wall polymer with the structural integrity required by the bacterium. The cell wall and its biosynthetic enzymes are targets of antibiotics. The 3D structure of the cell wall has been elusive because of its complexity and the lack of pure samples. Herein we report the 3D solution structure as determined by NMR of the 2-kDa NAG-NAM(pentapeptide)-NAG-NAM(pentapeptide) synthetic fragment of the cell wall. The glycan backbone of this peptidoglycan forms a right-handed helix with a periodicity of three for the NAG-NAM repeat (per turn of the helix). The first two amino acids of the pentapeptide adopt a limited number of conformations. Based on this structure a model for the bacterial cell wall is proposed. murein sacculus ͉ bacterial envelope T he peptidoglycan scaffold of the bacterial cell wall is a repeating N-acetylglucosamine (NAG)-N-acetylmuramic disaccharide (NAM) [NAG-(-1,4)-NAM] having a pentapeptide attached to the D-lactyl moiety of each NAM. This pentapeptide stem participates in an interglycan cross-linking reaction, thus creating the cell wall polymer. In contrast to the two other -1,4-linked glycan biopolymers, cellulose (repeating glucose) (1-4) and chitin (repeating NAG) (5-7) for which the 3D structure is solved, the structure of the bacterial cell wall has remained elusive because of its complexity and the lack of pure and discrete segments for structural study . Herein we describe the 3D structure, determined in aqueous solution by NMR, of a 2-kDa synthetic NAG-NAM(pentapeptide)-NAG-NAM(pentapeptide) tetrasaccharide cell wall segment. The defining aspect of this structure is an ordered, right-handed helical saccharide conformation corresponding to three NAG-NAM pairs per turn of the helix. The structure of this peptidoglycan segment is the basis for a proposal for the structure of the bacterial cell wall polymer.
Architecture and assembly of the Gram-positive cell wall
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.
Mapping Cell Wall Polysaccharides of Living Microbial Cells Using Atomic Force Microscopy
Cell Biology International, 1997
Functionalized atomic force microscope tips were used to sense specific forces of interaction between ligand-receptor pairs and to map the positions of polysaccharides on a living microbial cell surface. Gold-coated tips were functionalized with concanavalin A using a cross-linker with a spacer arm of 15.6 A r. It was possible to measure the binding force between concanavalin A and mannan polymers on the yeast (Saccharomyces cerevisiae) cell surface. This force ranged from 75 to 200 pN. The shape of the force curve indicated that the polymers were pulled away from the cell surface for a fairly long distance that sometimes reached several hundred nanometres. The distribution of mannan on the cell surface was mapped by carrying out the force measurement in the force volume mode of atomic force microscopy (AFM). During the measurement, the maximum cantilever deflection after contact between the tip and the sample was kept constant at 10 nm using trigger mode to keep the pressing force on the sample surface as gently as possible at a force of 180 pN. This regime was used to minimize the non-specific adhesion between the tip and the cell surface. Specific molecular recognition events took place on specific areas of the cell surface that could be interpreted as reflecting a non-uniform distribution of mannan on the cell surface.
Cell wall elongation mode in Gram-negative bacteria is determined by peptidoglycan architecture
Nature Communications, 2013
Cellular integrity and morphology of most bacteria is maintained by cell wall peptidoglycan, the target of antibiotics essential in modern healthcare. It consists of glycan strands, cross-linked by peptides, whose arrangement determines cell shape, prevents lysis due to turgor pressure and yet remains dynamic to allow insertion of new material, and hence growth. The cellular architecture and insertion pattern of peptidoglycan have remained elusive. Here we determine the peptidoglycan architecture and dynamics during growth in rod-shaped Gram-negative bacteria. Peptidoglycan is made up of circumferentially oriented bands of material interspersed with a more porous network. Super-resolution fluorescence microscopy reveals an unexpected discontinuous, patchy synthesis pattern. We present a consolidated model of growth via architecture-regulated insertion, where we propose only the more porous regions of the peptidoglycan network that are permissive for synthesis.
Cell Shape Can Mediate the Spatial Organization of the Bacterial Cytoskeleton
Biophysical Journal, 2013
The cell wall, a porous mesh-like structure, provides shape and physical protection for bacteria. At the atomic level, it is composed of peptidoglycan (PG), a polymer of stiff glycan strands cross-linked by short, flexible peptides. However, at the mesoscale, multiple models for the organization of PG have been put forth, distinguished by glycan strands parallel to the cell surface (the so-called "layered'' model) or perpendicular (the ''scaffold'' model). To test these models, and to resolve the mechanical properties of PG, we have built and simulated at an atomic scale patches of both Grampositive and negative cell walls in different organizations up to 50 nanometers in size. In the case of Gram-positive PG, molecular dynamics simulations of the layered model are found to elucidate the mechanisms behind a distinct curling effect observed in three-dimensional electron cryo-tomography images of fragmented cell walls. For Gram-negative PG, simulations of patches with different average-glycan-strand lengths reveal an anisotropic elasticity, in good agreement with atomic-force microscopy experiments. Insights from the simulations reveal how mesoscopic and macroscopic properties of a ubiquitous bacterial ultrastructure arise from its atomic-scale interactions and organization.
Clustering and Identification of Force Spectra from Native Membranes
Biophysical Journal, 2019
Antimicrobial Resistance (AMR) is an increasing threat to society. Here we have used Atomic Force Microscopy (AFM) to study the bacterial cell wall, which is one of the main antibiotic targets, in Staphylococcus aureus and Bacillus Subtilis. Using high-resolution imaging techniques, the native 3-Dimensional molecular organization of the main component of the cell wall, peptidoglycan, has been resolved for the first time. Direct imaging of the cell wall has allowed previous studies to build models of its 3-Dimensional architecture based on the outer surface of living cells. However, the molecular resolution was far from available at the time. Here we will show that we can visualize single molecules within the living bacterial cell wall. By imaging purified bacteria cell wall (i.e. sacculi), we reveal the organisation of the peptidoglycan architecture on the inner, cytoplasmic side of the cell wall, which is also where synthesis occurs. During the division process, peptidoglycan is mainly inserted in the septum, which now can be directly imaged for the first time showing a heterogeneous structure along the thickness of the cell wall. The architecture observed places hard constraints on the mechanism of cell wall synthesis and cell division in Gram positive bacteria. Using Highresolution Atomic Force Microscopy, we have obtained molecular information that will allow us to start tackling unresolved questions about the bacterial cell wall synthesis machinery and the effect of beta-lactam antibiotics that leads to cell death.
Understanding how bacteria grow and divide requires insight into both the molecular-level dynamics of ultrastructure and the chemistry of the constituent components. Atomic force microscopy (AFM) can provide near molecular resolution images of biological systems but typically provides limited chemical information. Conversely, while super-resolution optical microscopy allows localization of particular molecules and chemistries, information on the molecular context is difficult to obtain. Here, we combine these approaches into STORMForce (stochastic optical reconstruction with atomic force microscopy) and the complementary SIMForce (structured illumination with atomic force microscopy), to map the synthesis of the bacterial cell wall structural macromolecule, peptidoglycan, during growth and division in the rod-shaped bacterium Bacillus subtilis. Using "clickable" D-amino acid incorporation, we fluorescently label and spatially localize a short and controlled period of peptidoglycan synthesis and correlate this information with high-resolution AFM of the resulting architecture. During division, septal synthesis occurs across its developing surface, suggesting a two-stage process with incorporation at the leading edge and with considerable in-filling behind. During growth, the elongation of the rod occurs through bands of synthesis, spaced by ∼300 nm, and corresponds to denser regions of the internal cell wall as revealed by AFM. Combining super-resolution optics and AFM can provide insights into the synthesis processes that produce the complex architectures of bacterial structural biopolymers.
Molecular microbiology, 2013
The peptidoglycan (PG) cell wall is a unique macromolecule responsible for both shape determination and cellular integrity under osmotic stress in virtually all bacteria. A quantitative understanding of the relationships between PG architecture, morphogenesis, immune system activation and pathogenesis can provide molecular-scale insights into the function of proteins involved in cell wall synthesis and cell growth. High-performance liquid chromatography (HPLC) has played an important role in our understanding of the structural and chemical complexity of the cell wall by providing an analytical method to quantify differences in chemical composition. Here, we present a primer on the basic chemical features of wall structure that can be revealed through HPLC, along with a description of the applications of HPLC PG analyses for interpreting the effects of genetic and chemical perturbations to a variety of bacterial species in different environments. We describe the physical consequences...
ACS Nano, 2008
The nanoscale exploration of microbes using atomic force microscopy (AFM) is an exciting, rapidly evolving research field. Here, we show that single-molecule force spectroscopy is a valuable tool for the localization and conformational analysis of individual polysaccharides on live bacteria. We focus on the clinically important probiotic bacterium Lactobacillus rhamnosus GG, demonstrating the power of AFM to reveal the coexistence of polysaccharide chains of different nature on the cell surface. Applicable to a wide variety of cells, this single molecule method offers exciting prospects for analyzing the heterogeneity and diversity of macromolecules constituting cell membranes and cell walls.