Peptidoglycan at its peaks: how chromatographic analyses can reveal bacterial cell wall structure and assembly - PubMed (original) (raw)

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Peptidoglycan at its peaks: how chromatographic analyses can reveal bacterial cell wall structure and assembly

Samantha M Desmarais et al. Mol Microbiol. 2013 Jul.

Abstract

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 of different PG compositions on cell shape, and review complementary experimental and computational methodologies for PG analysis. Finally, we present a partial list of future targets of development for HPLC and related techniques.

© 2013 John Wiley & Sons Ltd.

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Figures

Figure 1

Figure 1. Basic features of peptidoglycan muropeptide chemistry and analysis

(A) Electron micrograph of a sacculus from the rod-shaped bacterium E. coli. (B) After sacculus isolation, the preparation protocol for HPLC analyses includes several enzymatic treatments that digest sacculi into muropeptide monomers or cross-linked oligomers. Colored arrows designate the site of action of the respective enzyme. Yellow and orange circles denote an oligopeptide from Braun’s lipoprotein that remains bound to the muropeptide after cleavage by Pronase E. (C) Each muropeptide is composed of a disaccharide and its associated peptide stem. The disaccharide consists of the sugars MurNAc and GlcNAc, while the peptide stem consists of up to five amino acids (shown in colored rectangles) connected to the MurNAc through a D-Lactic acid. The bolded atoms on DAP indicate potential cross-linking locations to other muropeptides, which would result in either a dimer or other oligomer. (D) Example of an HPLC PG analysis, with characteristic peaks labeled with cartoons of their muropeptide structure (disaccharides: hexagons; amino acids in peptide stem: circles; both are colored as in (C)). Lipoprotein amino acids on M3L are shown with orange and yellow circles; anhydro modification on D44N in orange). Inset shows a zoomed-in version of the D44 peak, with shading indicating the species abundance and dashed line indicating the retention time.

Figure 2

Figure 2. Mapping PG composition to morphology

(A) For each family of muropeptides, the table describes the salient chemical features and the physical or chemical consequences of having a high proportion of each family. (B) Species for which at least two HPLC PG analyses have been published, grouped by shape. A majority of studies have focused on Gram-negative PG, although recent efforts have expanded the diversity of species studied (see Supp. Table 2). (C) Connecting differences in PG composition to morphology can be facilitated by biophysical simulations of cell growth, using HPLC quantification as a constraint on growth. The inset on the left is zoomed in on the architecture of a single-layered cell wall from a simulation of rod-shaped growth, with the results of one such simulation shown on the right (modified from (Furchtgott et al., 2011)). Each green or blue cylinder represents a disaccharide in an old or new glycan strand, respectively, while each red cylinder represents a peptide cross-link.

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