Glycan Strands Research Articles

Resistance to β-lactam antibiotics conferred by point mutations in penicillin-binding proteins PBP3, PBP4 and PBP6 in Salmonella enterica.

Penicillin-binding proteins (PBPs) are enzymes responsible for the polymerization of the glycan strand and the cross-linking between glycan chains as well as the target proteins for β-lactam antibiotics. Mutational alterations in PBPs can confer resistance either by reducing binding of the antibiotic to the active site or by evolving a β-lactamase activity that degrades the antibiotic. As no systematic studies have been performed to examine the potential of all PBPs present in one bacterial species to evolve increased resistance against β-lactam antibiotics, we explored the ability of fifteen different defined or putative PBPs in Salmonella enterica to acquire increased resistance against penicillin G. We could after mutagenesis and selection in presence of penicillin G isolate mutants with amino-acid substitutions in the PBPs, FtsI, DacB and DacC (corresponding to PBP3, PBP4 and PBP6) with increased resistance against β-lactam antibiotics. Our results suggest that: (i) most evolved PBPs became ‘generalists” with increased resistance against several different classes of β-lactam antibiotics, (ii) synergistic interactions between mutations conferring antibiotic resistance are common and (iii) the mechanism of resistance of these mutants could be to make the active site more accessible for water allowing hydrolysis or less binding to β-lactam antibiotics.

The Capsular Polysaccharide of Staphylococcus aureus Is Attached to Peptidoglycan by the LytR-CpsA-Psr (LCP) Family of Enzymes

Envelope biogenesis in bacteria involves synthesis of intermediates that are tethered to the lipid carrier undecaprenol-phosphate. LytR-CpsA-Psr (LCP) enzymes have been proposed to catalyze the transfer of undecaprenol-linked intermediates onto the C6-hydroxyl of MurNAc in peptidoglycan, thereby promoting attachment of wall teichoic acid (WTA) in bacilli and staphylococci and capsular polysaccharides (CPS) in streptococci. S. aureus encodes three lcp enzymes, and a variant lacking all three genes (Δlcp) releases WTA from the bacterial envelope and displays a growth defect. Here, we report that the type 5 capsular polysaccharide (CP5) of Staphylococcus aureus Newman is covalently attached to the glycan strands of peptidoglycan. Cell wall attachment of CP5 is abrogated in the Δlcp variant, a defect that is best complemented via expression of lcpC in trans. CP5 synthesis and peptidoglycan attachment are not impaired in the tagO mutant, suggesting that CP5 synthesis does not involve the GlcNAc-ManNAc linkage unit of WTA and may instead utilize another Wzy-type ligase to assemble undecaprenyl-phosphate intermediates. Thus, LCP enzymes of S. aureus are promiscuous enzymes that attach secondary cell wall polymers with discrete linkage units to peptidoglycan.

The function of the transmembrane and cytoplasmic domains of pneumococcal penicillin-binding proteins 2x and 2b extends beyond that of simple anchoring devices.

The biosynthesis of cell-wall peptidoglycan is a complex process that involves six different penicillin-binding proteins (PBPs) in Streptococcus pneumoniae. Two of these, PBP2x and PBP2b, are monofunctional transpeptidases that catalyse the formation of peptide cross-links between adjacent glycan strands. Both of them are bitopic membrane proteins with a small cytoplasmic and a large extracellular domain. PBP2x and PBP2b are essential for septal and peripheral peptidoglycan synthesis, respectively. Although several studies have investigated the properties of their extracellular catalytic domains, it is not known whether the role of their N-terminal non-catalytic domains extends beyond that of being simple anchoring devices. We therefore decided to use reciprocal domain swapping and mutational analysis to gain more information about the biological function of the membrane anchors and cytoplasmic tails of PBP2x and PBP2b. In the case of PBP2x both domains are essential, but neither the membrane anchor nor the cytoplasmic domain of PBP2x appear to serve as major localization signals. Instead, our results suggest that they are involved in interactions with other components of the divisome. Mutations of conserved amino acids in the cytoplasmic domain of PBP2x resulted in loss of function, underlining the importance of this region. The cytoplasmic domain of PBP2b could be swapped with the corresponding domain from PBP2x, whereas replacement of the PBP2b transmembrane domain with the corresponding PBP2x domain gave rise to slow-growing cells with grossly abnormal morphology. When both domains were exchanged simultaneously the cells were no longer viable.

Bacterial Cell Morphogenesis Does Not Require a Preexisting Template Structure

SummaryMorphogenesis, the development of shape or form in cells or organisms, is a fundamental but poorly understood process throughout biology. In the bacterial domain, cells have a wide range of characteristic shapes, including rods, cocci, and spirals. The cell wall, composed of a simple meshwork of long glycan strands crosslinked by short peptides (peptidoglycan, PG) and anionic cell wall polymers such as wall teichoic acids (WTAs), is the major determinant of cell shape. It has long been debated whether the formation of new wall material or the transmission of shape from parent to daughter cells requires existing wall material as a template [1–3]. However, rigorous testing of this hypothesis has been problematical because the cell wall is normally an essential structure. L-forms are wall-deficient variants of common bacteria that have been classically identified as antibiotic-resistant variants in association with a wide range of infectious diseases [4–6]. We recently determined the genetic basis for the L-form transition in the rod-shaped bacterium Bacillus subtilis and thus how to generate L-forms reliably and reproducibly [7, 8]. Using the new L-form system, we show here that we can delete essential genes for cell wall synthesis and propagate cells in the long-term absence of a cell wall template molecule. Following genetic restoration of cell wall synthesis, we show that the ability to generate a classical rod-shaped cell is restored, conclusively rejecting template-directed models, at least for the establishment of cell shape in B. subtilis.

Dynamic protein complexes for cell growth

Bacterial cytoskeletal elements provide the framework for the assembly of multicomponent protein machineries controlling cell growth and division. One of the main roles of these machineries is to build the cell wall, which is instrumental for cell shape and stability. Our understanding of how these machineries are spatiotemporally controlled has been rapidly changing in the last few years, fueled mostly by single-cell or systems-based approaches. Recent studies in Bacillus subtilis described a coupled movement of the actin-like MreB and the core part of the cell wall synthesis machinery (1, 2). In PNAS, Lee et al. present evidence that the situation is different in Escherichia coli: penicillin-binding protein 2 (PBP2), a core cell wall synthesis enzyme involved in cell elongation, exhibits a diffusive motion, and dynamically associates with the rest of the elongation machinery to spatiotemporally control the insertion of new glycan strands into the cell wall (3). This new model (Fig. 1) has interesting mechanistic ramifications as to how the cell organizes cell growth. It also begs the question of whether the difference between the two model organisms is related to synthesis requirements of their architecturally distinct cell walls or is just evolution coming up with different solutions for the same problem. We anticipate this will be a recurring question in the field, as we start to accumulate knowledge in diverse organisms.

Escherichia coli Peptidoglycan Structure and Mechanics as Predicted by Atomic-Scale Simulations

Bacteria face the challenging requirement to maintain their shape and avoid rupture due to the high internal turgor pressure, but simultaneously permit the import and export of nutrients, chemical signals, and virulence factors. The bacterial cell wall, a mesh-like structure composed of cross-linked strands of peptidoglycan, fulfills both needs by being semi-rigid, yet sufficiently porous to allow diffusion through it. How the mechanical properties of the cell wall are determined by the molecular features and the spatial arrangement of the relatively thin strands in the larger cellular-scale structure is not known. To examine this issue, we have developed and simulated atomic-scale models of Escherichia coli cell walls in a disordered circumferential arrangement. The cell-wall models are found to possess an anisotropic elasticity, as known experimentally, arising from the orthogonal orientation of the glycan strands and of the peptide cross-links. Other features such as thickness, pore size, and disorder are also found to generally agree with experiments, further supporting the disordered circumferential model of peptidoglycan. The validated constructs illustrate how mesoscopic structure and behavior emerge naturally from the underlying atomic-scale properties and, furthermore, demonstrate the ability of all-atom simulations to reproduce a range of macroscopic observables for extended polymer meshes.

Function and Localization Dynamics of Bifunctional Penicillin-Binding Proteins in Caulobacter crescentus

The peptidoglycan cell wall of bacteria is a complex macromolecule composed of glycan strands that are cross-linked by short peptide bridges. Its biosynthesis involves a conserved group of enzymes, the bifunctional penicillin-binding proteins (bPBPs), which contain both a transglycosylase and a transpeptidase domain, thus being able to elongate the glycan strands and, at the same time, generate the peptide cross-links. The stalked model bacterium Caulobacter crescentus possesses five bPBP paralogs, named Pbp1A, PbpC, PbpX, PbpY, and PbpZ, whose function is still incompletely understood. In this study, we show that any of these proteins except for PbpZ is sufficient for growth and normal morphogenesis when expressed at native or elevated levels, whereas inactivation of all five paralogs is lethal. Growth analyses indicate a central role of PbpX in the resistance of C. crescentus against the noncanonical amino acid d-alanine. Moreover, we show that PbpX and PbpY localize to the cell division site. Their recruitment to the divisome is dependent on the essential cell division protein FtsN and likely involves interactions with FtsL and the putative peptidoglycan hydrolase DipM. The same interaction pattern is observed for Pbp1A and PbpC, although these proteins do not accumulate at midcell. Our findings demonstrate that the bPBPs of C. crescentus are, to a large extent, redundant and have retained the ability to interact with the peptidoglycan biosynthetic machineries responsible for cell elongation, cytokinesis, and stalk growth. Nevertheless, they may preferentially act in specific peptidoglycan biosynthetic complexes, thereby facilitating the independent regulation of distinct growth processes.

A Fast, High-Throughput, and Highly Sensitive Analysis of Bacterial Cell Walls using Ultra Performance Liquid Chromatography

The bacterial cell wall, also known as the murein sacculus, is composed of glycan chains crosslinked by short peptides. The maintenance of the integrity of the sacculus is complex, as it is involved in both shape determination and growth in virtually all bacteria. We aim to gain a quantitative understanding of the relationship between peptidoglycan architecture, morphogenesis, and pathogenesis. We have used Ultra-high Pressure Liquid Chromatography (UPLC), a new highly sensitive HPLC analysis, to measure the average length of the glycan strands from which the sacculus is made, the degree of cross-linking between those strands, and muropeptide identity of rod-shaped bacteria. Sacculi from Escherichia coli laboratory strains were analyzed and compared to uropathogenic E. coli, and E. coli treated with A22, a small molecule drug that depolymerizes the actin homolog MreB and leads to a round morphology. We have also quantified muropeptides from different strains of Pseudomonas aeruginosa and Vibrio cholera to further correlate cell wall composition with pathogenesis. Preliminary findings indicate no significant change in peptidoglycan composition between strains from the same bacterium, although bacteria differ from each other in relative amounts of muropeptides, glycan strand lengths, and crosslinking. Neither pathogenicity nor changes in cell shape result in any significant differences in cell wall quantities. We are in the process of identifying unique muropeptides that develop with shape change and in clinical isolates using mass spectrometry. UPLC is a quick, reliable alternative to HPLC analysis of peptidoglycan, and provides a robust method for precise quantification of the complex chemical composition of the bacterial cell wall.

FtsEX is required for CwlO peptidoglycan hydrolase activity during cell wall elongation in Bacillus subtilis

The peptidoglycan (PG) sacculus, a meshwork of polysaccharide strands cross-linked by short peptides, protects bacterial cells against osmotic lysis. To enlarge this covalently closed macromolecule, PG hydrolases must break peptide cross-links in the meshwork to allow insertion of new glycan strands between the existing ones. In the rod-shaped bacterium Bacillus subtilis, cell wall elongation requires two redundant endopeptidases, CwlO and LytE. However, it is not known how these potentially autolytic enzymes are regulated to prevent lethal breaches in the cell wall. Here, we show that the ATP-binding cassette transporter-like FtsEX complex is required for CwlO activity. In Escherichia coli, FtsEX is thought to harness ATP hydrolysis to activate unrelated PG hydrolases during cell division. Consistent with this regulatory scheme, B. subtilis FtsE mutants that are unable to bind or hydrolyse ATP cannot activate CwlO. Finally, we show that in cells depleted of both CwlO and LytE, the PG synthetic machinery continues moving circumferentially until cell lysis, suggesting that cross-link cleavage is not required for glycan strand polymerization. Overall, our data support a model in which the FtsEX complex is a remarkably flexible regulatory module capable of controlling a diverse set of PG hydrolases during growth and division in different organisms.

Substrate specificity of an elongation‐specific peptidoglycan endopeptidase and its implications for cell wall architecture and growth of Vibrio cholerae

The bacterial cell wall consists of peptidoglycan (PG), a sturdy mesh of glycan strands cross-linked by short peptides. This rigid structure constrains cell shape and size, yet is sufficiently dynamic to accommodate insertion of newly synthesized PG, which was long hypothesized, and recently demonstrated, to require cleavage of the covalent peptide cross-links that couple previously inserted material. Here, we identify several genes in Vibrio cholerae that collectively are required for growth - particularly elongation - of this pathogen. V. cholerae encodes three putative periplasmic proteins, here denoted ShyA, ShyB, and ShyC, that contain both PG binding and M23 family peptidase domains. While none is essential individually, the absence of both ShyA and ShyC results in synthetic lethality, while the absence of ShyA and ShyB causes a significant growth deficiency. ShyA is a D,d-endopeptidase able to cleave most peptide chain cross-links in V. cholerae's PG. PG from a ∆shyA mutant has decreased average chain length, suggesting that ShyA may promote removal of short PG strands. Unexpectedly, ShyA has little activity against muropeptides containing pentapeptides, which typically characterize newly synthesized material. ShyA's substrate-dependent activity may contribute to selection of cleavage sites in PG, whose implications for the process of side-wall growth are discussed.

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.

Peptidoglycan transformations during Bacillus subtilis sporulation

While vegetative Bacillus subtilis cells and mature spores are both surrounded by a thick layer of peptidoglycan (PG, a polymer of glycan strands cross-linked by peptide bridges), it has remained unclear whether PG surrounds prespores during engulfment. To clarify this issue, we generated a slender ΔponA mutant that enabled high-resolution electron cryotomographic imaging. Three-dimensional reconstructions of whole cells in near-native states revealed a thin PG-like layer extending from the lateral cell wall around the prespore throughout engulfment. Cryotomography of purified sacculi and fluorescent labelling of PG in live cells confirmed that PG surrounds the prespore. The presence of PG throughout engulfment suggests new roles for PG in sporulation, including a new model for how PG synthesis might drive engulfment, and obviates the need to synthesize a PG layer de novo during cortex formation. In addition, it reveals that B. subtilis can synthesize thin, Gram-negative-like PG layers as well as its thick, archetypal Gram-positive cell wall. The continuous transformations from thick to thin and back to thick during sporulation suggest that both forms of PG have the same basic architecture (circumferential). Endopeptidase activity may be the main switch that governs whether a thin or a thick PG layer is assembled.

Cell wall elongation mode in Gram-negative bacteria is determined by peptidoglycan architecture

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.

Assembly and Architecture of Gram-Positive and -Negative Cell Walls

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 Gram-positive 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.

Shedding light on peptidoglycan growth

Our understanding of the dynamics of peptidoglycan growth has been hampered by our inability to directly image peptidoglycan synthesis in vivo. Given the role of D-amino acids in crosslinking the glycan strands, Kuru et al. investigated whether fluorescent probes attached to a D-amino acid backbone could be incorporated into peptidoglycan during its synthesis. The authors attached the small fluorophores 7-hydroxycoumarin 3-carboxylic acid (HCC-OH) and 4-chloro-7-nitrobenzofurazan (NBD-C1) to 3-amino-D-alanine (ADA) to create HADA and NADA, respectively. Strong peripheral and septal labelling of the entire population of viable bacteria was observed with both fluorophores for phylogenetically diverse bacterial species, including Escherichia coli, Agrobacterium tumefaciens and Bacillus subtilis. Furthermore, pulse–chase experiments allowed real-time tracking of peptidoglycan synthesis using time-lapse microscopy.

On the Mechanism of Peptidoglycan Binding and Cleavage by the endo-Specific Lytic Transglycosylase MltE from Escherichia coli

The lytic transglycosylase MltE from Escherichia coli is a periplasmic, outer membrane-attached enzyme that cleaves the β-1,4-glycosidic bonds between N-acetylmuramic acid and N-acetylglucosamine residues in the cell wall peptidoglycan, producing 1,6-anhydromuropeptides. Here we report three crystal structures of MltE: in a substrate-free state, in a binary complex with chitopentaose, and in a ternary complex with the glycopeptide inhibitor bulgecin A and the murodipeptide N-acetylglucosaminyl-N-acetylmuramyl-l-Ala-d-Glu. The substrate-bound structures allowed a detailed analysis of the saccharide-binding interactions in six subsites of the peptidoglycan-binding groove (subsites -4 to +2) and, combined with site-directed mutagenesis analysis, confirmed the role of Glu64 as catalytic acid/base. The structures permitted the precise modeling of a short glycan strand of eight saccharide residues, providing evidence for two additional subsites (+3 and +4) and revealing the productive conformational state of the substrate at subsites -1 and +1, where the glycosidic bond is cleaved. Full accessibility of the peptidoglycan-binding groove and preferential binding of an N-acetylmuramic acid residue in a (4)C(1) chair conformation at subsite +2 explain why MltE shows only endo- and no exo-specific activity toward glycan strands. The results further indicate that catalysis of glycosidic bond cleavage by MltE proceeds via distortion toward a sofa-like conformation of the N-acetylmuramic acid sugar ring at subsite -1 and by anchimeric assistance of the sugar's N-acetyl group, as shown previously for the lytic transglycosylases Slt70 and MltB.

Dislocation-mediated growth of bacterial cell walls

Recent experiments have illuminated a remarkable growth mechanism of rod-shaped bacteria: proteins associated with cell wall extension move at constant velocity in circles oriented approximately along the cell circumference [Garner EC, et al., (2011) Science 333:222-225], [Domínguez-Escobar J, et al. (2011) Science 333:225-228], [van Teeffelen S, et al. (2011) PNAS 108:15822-15827]. We view these as dislocations in the partially ordered peptidoglycan structure, activated by glycan strand extension machinery, and study theoretically the dynamics of these interacting defects on the surface of a cylinder. Generation and motion of these interacting defects lead to surprising effects arising from the cylindrical geometry, with important implications for growth. We also discuss how long range elastic interactions and turgor pressure affect the dynamics of the fraction of actively moving dislocations in the bacterial cell wall.

Helical insertion of peptidoglycan produces chiral ordering of the bacterial cell wall

The regulation of cell shape is a common challenge faced by organisms across all biological kingdoms. In nearly all bacteria, cell shape is determined by the architecture of the peptidoglycan cell wall, a macromolecule consisting of glycan strands crosslinked by peptides. In addition to shape, cell growth must also maintain the wall structural integrity to prevent lysis due to large turgor pressures. Robustness can be accomplished by establishing a globally ordered cell-wall network, although how a bacterium generates and maintains peptidoglycan order on the micron scale using nanometer-sized proteins remains a mystery. Here, we demonstrate that left-handed chirality of the MreB cytoskeleton in the rod-shaped bacterium Escherichia coli gives rise to a global, right-handed chiral ordering of the cell wall. Local, MreB-guided insertion of material into the peptidoglycan network naturally orders the glycan strands and causes cells to twist left-handedly during elongational growth. Through comparison with the right-handed twisting of Bacillus subtilis cells, our work supports a common mechanism linking helical insertion and chiral cell-wall ordering in rod-shaped bacteria. These physical principles of cell growth link the molecular structure of the bacterial cytoskeleton, mechanisms of wall synthesis, and the coordination of cell-wall architecture.

Modeling the Shape and Growth of Bacterial Cells

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 has been relatively unexplored. We have introduced a computational physical model of the bacterial cell wall to complement experimental studies of cell-shape determination, with a particular focus on rod-shaped cells like Escherichia coli. First, the model predicts the mechanical response of cell shape to peptidoglycan damage and perturbation. We observe 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. Interestingly, according to the model, many common bacterial cell shapes can be realized via modest local patterning of peptidoglycan density. Second, we introduced growth processes into the model via insertion of new glycan strands, formation of new peptide crosslinks, and cleavage of old crosslinks (1). The growth model suggests that maintaining a rod shape requires glycan insertion to be insensitive to fluctuations in cell-wall density and stress. Suggestively, in light of the role of MreB in maintaining rod-shaped growth, we find that a simple helical pattern of insertion is sufficient for many-fold elongation without significant loss in rod shape. Finally, we demonstrate that both the length and prestretching of newly inserted strands can regulate cell width. In sum, we show that simple physical rules can allow bacteria to achieve robust, shape-preserving cell-wall growth.1. Furchtgott L, Wingreen NS, Huang KC. Mechanisms for maintaining cell shape in rod-shaped Gram-negative bacteria. Mol Microbiol. 2011;81(2):340-53.

Study Controlling Factors of Gram-Negative Bacterial Cell Shape

Peptidoglycan (PG) sacculus has an important role in determining the shape of many bacterial cells. It is composed of glycan strands cross-linked by peptides into a net enclosing the cell and acting as a mechanical shield which protects the cell from bursting due to high internal (turgor) pressure. Importantly, how peptidoglycan is organized in the sacculus is the key controlling the cell shape. Many cells lose their rod shape and become spherical as perturbation of certain proteins such as MreB is introduced - a process believed to change the PG remodeling.