ABSTRACT
Peptidoglycan is a defining feature of the bacterial cell wall. Initially identified as a target of the revolutionary beta-lactam antibiotics, peptidoglycan has become a subject of much interest for its biology, its potential for the discovery of novel antibiotic targets, and its role in infection. Peptidoglycan is a large polymer that forms a mesh-like scaffold around the bacterial cytoplasmic membrane. Peptidoglycan synthesis is vital at several stages of the bacterial cell cycle: for expansion of the scaffold during cell elongation and for formation of a septum during cell division. It is a complex multifactorial process that includes formation of monomeric precursors in the cytoplasm, their transport to the periplasm, and polymerization to form a functional peptidoglycan sacculus. These processes require spatio-temporal regulation for successful assembly of a robust sacculus to protect the cell from turgor and determine cell shape. A century of research has uncovered the fundamentals of peptidoglycan biology, and recent studies employing advanced technologies have shed new light on the molecular interactions that govern peptidoglycan synthesis. Here, we describe the peptidoglycan structure, synthesis, and regulation in rod-shaped bacteria, particularly Escherichia coli, with a few examples from Salmonella and other diverse organisms. We focus on the pathway of peptidoglycan sacculus elongation, with special emphasis on discoveries of the past decade that have shaped our understanding of peptidoglycan biology.
INTRODUCTION
One of the defining moments of modern science was the discovery of the microscope, which opened up the treasure trove of the microbial world. In the following decades, bacteria became the subject of much study for several reasons, including their unchecked potential to cause death by sepsis; and the first world war provided a major impetus to look for antibacterial substances due to more soldiers dying of septic wounds than on the battlefield. In 1921, Alexander Fleming’s pioneering work led to the discovery of lysozyme, a purifiable substance from human tissues and secretions capable of inhibiting the growth of certain bacteria (1). In 1928, his observation of a mold metabolite capable of inhibiting a wide range of bacteria resulted in the discovery of penicillin (2), the molecule that revolutionized the field of medicine. Subsequently, elucidation of the mode of action of penicillin (3–7) and the isolation of a lysozyme-sensitive rigid layer of bacteria (8–10) led to the identification of peptidoglycan (or murein), which is now known to be an essential constituent of the cell walls of most bacteria.
With the exception of mycoplasmas, all bacterial cells are surrounded by peptidoglycan, a sac-like protective exoskeleton that is indispensable for their growth and survival (11–13). Peptidoglycan derives its name from its two principal components—glycan strands of repeating disaccharide units and short peptide chains of two- to five-amino acid residues (11, 14, 15). The glycan strands are cross-linked to each other through the peptide chains to form a mesh-like sacculus that surrounds the bacterial cytoplasmic membrane, creating a unique structure that defines the bacterial cell wall. Peptidoglycan prevents bacteria from lysis due to turgor, maintains cell shape, and protects the cell from extreme environmental conditions. Peptidoglycan architecture is the basis of Gram classification, by which bacteria are divided into either Gram-positive or -negative. Gram-positive bacteria possess a thick multilayered peptidoglycan that is exposed to the cell exterior with covalently bound glycopolymers—teichoic acids—whereas Gram-negative bacteria have a thin, predominantly monolayered peptidoglycan covered by an additional lipid bilayer—the outer membrane or OM (16).
Our understanding of peptidoglycan biology has considerably evolved since its discovery in the early 1960s (Fig. 1). We now know the composition, structure, and architecture of peptidoglycan in reasonable detail. Work from several groups led to the delineation of pathways of peptidoglycan biosynthesis during bacterial growth, division, and survival. A plethora of antibiotics targeting different stages of peptidoglycan synthesis were discovered and designed, followed closely by the emergence and spread of antimicrobial resistance in a tight tug-of-war. In addition, bacterial peptidoglycan has been shown to be a potent immunogen profoundly influencing the mammalian innate immune system (17–19). It is also implicated in several other physiological processes in the host, including immunomodulation, neuronal development, and behavior (17, 20).
Figure 1.
Milestones of peptidoglycan biology in the past century. The figure depicts a few important discoveries pertaining to peptidoglycan structure and synthesis from the past century. Selected significant developments of the past decade are highlighted.
The biology of peptidoglycan has been extensively reviewed over the years, and the reader is directed to a few of these reviews for further reading (11–15, 21–27). In this chapter, we describe the structure, synthesis, and regulation of peptidoglycan. The focus is predominantly Escherichia coli, an organism of choice for studying several aspects of bacterial physiology, with special emphasis on Salmonella wherever the biology differs significantly. We also briefly touch upon certain aspects of peptidoglycan biosynthesis of Bacillus subtilis, a Gram-positive model bacterium. We begin with a brief description of peptidoglycan structure, followed by details of the biosynthetic pathway—from precursor synthesis in the cytoplasm to export, polymerization, and maturation in the periplasm. We emphasize the recent developments of the past decade that influenced our understanding of the process and have taken the liberty of putting forth new ideas in order to envisage a holistic model for peptidoglycan synthesis.
STRUCTURE OF PEPTIDOGLYCAN
Peptidoglycan sacculus is a single, large macromolecule that makes a scaffold-like structure around the bacterial cytoplasmic membrane. Its major function is to resist the intracellular osmotic pressure (or turgor) generated by the cytosolic contents. In addition, it defines bacterial cell shape and provides protection from environmental threats. Peptidoglycan is a polymer made up of several linear glycan strands covalently cross-linked to each other by short peptide chains (Fig. 2). Glycan strands are long polymers of disaccharides, each containing an N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) residue, that are linked by β-1,4 glycosidic bonds (8, 11, 14). The lactoyl moieties of MurNAc are covalently attached to peptide stems that are usually two to five amino acid residues long. In most Gram-negative bacteria, including E. coli, the pentapeptide chain is made up of l-alanine (l-ala), γ-d-glutamate (d-glu), meso-diaminopimelic acid (mDAP), and d-alanine (d-ala)–d-ala with the bond between d-glu2 and mDAP3 being an isopeptide linkage (15). The presence of a dibasic amino acid, mDAP3, in the peptide is crucial because of its ability to form cross-links with other peptide chains. However, Gram-positive bacteria mostly contain an alternate dibasic amino acid, l-lysine at the third position. In Gram-negative bacteria, the peptides are directly cross-linked to each other via d-ala4 and mDAP3 or two mDAP3 residues of neighboring peptides from adjacent glycan strands. In Gram-positive bacteria, the peptides are cross-linked either directly or through an additional bridging peptide of varying amino acid length and composition. The cross-linking forms the basis for the characteristic mesh-like structure of peptidoglycan (Fig. 2).
Figure 2.
Peptidoglycan structure and chemical composition. (Top) Schematic of a rod-shaped E. coli cell with its peptidoglycan sacculus (blue mesh) located between the OM and IM. (Bottom) The zoomed-in rectangle depicts the composition of peptidoglycan. Glycan chains are made up of repeating disaccharide units of N-acetylglucosamine (GlcNAc; gray hexagons) and N-acetylmuramic acid (MurNAc; blue hexagons) with the peptide chains made up of amino acid residues (shaded blue circles), l-alanine (l-ala1), d-glutamic acid (d-glu2), meso-diaminopimelic acid (mDAP3), and d-alanine (d-ala4)–d-ala5. The glycan chains are linked to each other through peptide cross-linking between either d-ala4 and mDAP3 or mDAP3 and mDAP3 residues to form a net-like sacculus. An OM lipoprotein (Braun’s lipoprotein, Lpp; orange helix) covalently tethers peptidoglycan to the OM. A GlcNAc-MurNAc pentapeptide is a single monomeric unit of peptidoglycan that is polymerized to form the functional sacculus. The terminal d-ala5 is usually removed in the mature peptidoglycan.
Several imaging techniques, including transmission electron microscopy, atomic force microscopy, and cryo-electron microscopy, contributed to our understanding of the fine structure of peptidoglycan. These studies showed that the glycan strands are arranged perpendicular to the polar axis of the cell, with the peptide stems arranged helically along the glycan strands (28, 29). The glycan strands are not planar but are rotated with respect to one another due to the presence of the lactoyl moiety of MurNAc, leading to the peptides extending in different directions instead of being parallel to each other (30). Thus, a glycan strand can be cross-linked with neighboring strands on either side via the peptides. In a growing E. coli cell, about 40% of the peptides are in a cross-linked state (31). However, it is important to note that peptidoglycan is far from a static structure. The mature peptidoglycan sacculus is an elastic and flexible molecule that allows rapid expansion and shrinkage of cells due to turgor and external osmotic conditions (32–35). The flexibility is believed to be provided largely by the peptides and not as much by the glycan strands (35). Per this model, the peptides are stretched between two glycan strands in a turgid cell and are in a relaxed state in a plasmolyzed cell. In addition to structural flexibility, the sacculus is subject to dynamic alterations, with the glycan strands and peptide cross-links being continuously broken and reformed for new peptidoglycan synthesis as well as remodeling and recycling, facilitating growth, division, and other cellular processes.
Peptidoglycan is a large molecule with an approximate mass of 3 × 109 Da, made up of about 2.7 × 106 to 3.5 × 106 GlcNAc-MurNAc-pentapeptide monomers per cell (36, 37). Purified peptidoglycan sacculi retain the shape and dimensions of the intact cell, with its average size in E. coli ranging from 2 to 4 μm in length and 0.5 to 1 μm in width (38–40). Gram-negative bacteria predominantly have a single layer of approximately 2.5-nm-thick peptidoglycan housed in the 15-nm-wide periplasm (29, 41, 42). The glycan strands do not form continuous hoops across the cell circumference; instead, they exist as short linear overlapping strands with an average length of about 30 nm (25 disaccharide units) in exponentially growing E. coli cells (12), although strands as long as 200 nm have also been observed by atomic force microscopy (43). The heterogeneity of the mesh results in nonuniform pores which are reported to allow passive transport of nutrients and proteins up to 50 kDa (44). Interestingly, the sacculus is also reported to have large pores of ∼60 nm2, which may allow traversing of protein complexes across the periplasm to facilitate several cellular processes (43).
The peptidoglycan sacculus is not a freely floating mesh between the two membranes. In many Gram-negative bacteria, a unique OM lipoprotein, Lpp or Braun’s lipoprotein, covalently tethers the peptidoglycan to the OM (45). In E. coli, Lpp is the most abundant cellular protein, with ∼106 copies per cell (46). The C-terminal lysine residue of Lpp is covalently linked to the mDAP3 in about 10% of all peptidoglycan stem peptides by the action of l,d transpeptidases (31, 45, 47). Usually, two-thirds of the total cellular Lpp exists in peptidoglycan-unbound form, tethered to and traversing the OM to be exposed on the outer surface of the cell (48, 49). Lpp is the only protein known to covalently bind peptidoglycan in E. coli, tethering the OM to peptidoglycan and determining the size of the periplasm, which is crucial for transenvelope processes (50, 51). However, covalent attachment of peptidoglycan to β-barrel proteins in the OM has recently been reported for several other Gram-negative bacteria (52, 53).
COMPOSITION OF PEPTIDOGLYCAN
While microscopy enabled visualization of peptidoglycan structure and architecture, reverse-phase high-performance liquid chromatography and ultraperformance liquid chromatography emerged as indispensable tools to study the chemical composition of peptidoglycan (31, 54). Peptidoglycan composition varies across the bacterial kingdom, although the overall structure follows the basic framework described earlier. Multiple postsynthetic modifications occur in bacterial peptidoglycans to fortify them against antimicrobial agents as well as to protect pathogenic bacteria against degradation by host factors. Other than interspecies diversity, peptidoglycan composition also varies within species, depending upon growth phase and environmental conditions. Variations in peptidoglycan composition across the bacterial kingdom have been well summarized (13–15, 55, 56). A few examples of variations in glycan chains, peptide stems, and the type of peptide cross-links are described below.
Examples of Glycan Strand Variation
Across the bacterial kingdom, the glycan strands are subject to a variety of chemical modifications (55–57). Gram-negative bacteria show fewer instances of glycan modifications than Gram-positive bacteria, with most of them limited to pathogenic species where the modifications facilitate evasion of lysis by host lysozymes. Prominent modifications include N-deacetylation and O-acetylation of both sugars, as well as N-glycolylation of MurNAc. Although E. coli and Salmonella do not show these modifications, the terminal MurNAc in the glycan strands is modified to an “anhydro” derivative by the action of lytic transglycosylases during the process of maturation.
Examples of Peptide Chain Variation
Variations at different positions of the peptidoglycan stem peptide are widespread across bacterial genera (13, 15). Canonically, peptidoglycan peptides of E. coli and Salmonella spp. are made up of l-ala1-γ–d-glu2–mDAP3–d-ala4–d-ala5 residues, with the only naturally occurring substitutions being at d-ala4/5 during remodeling (described below). Noncanonical amino acid substitutions during synthesis may occur upon perturbation of the biosynthetic process or due to amino acid composition of the microenvironment. For example, incorporation of l,l-DAP instead of mDAP3 has been observed in mutants lacking the diaminopimelate epimerase DapF, leading to a reduction in peptide cross-linkage (58, 59). Likewise, in mutants lacking PgeF, a peptidoglycan editing factor, misincorporation of l-ser or Gly instead of l-ala1 is observed, indicating that bacteria may possess proofreading or editing pathways to ensure the fidelity of stem peptides (60).
Examples of Cross-Link Variation
A wide variety of cross-link types are observed in the bacterial kingdom, ranging from direct linkage between peptides to indirect interpeptide bridges of 1 to 7 diverse amino acid residues (13). In E. coli and Salmonella enterica serovar Typhimurium, about 40% of the total muropeptides are directly cross-linked during exponential growth in laboratory conditions, of which d-ala4–mDAP3 cross-links account for about 93%, while mDAP3–mDAP3 cross-links contribute approximately 7% (31, 61). These cross-links are generated by distinct classes of enzymes—the d,d-transpeptidase domains of penicillin binding proteins (PBPs) or the l,d-transpeptidases (Ldts). However, significant cross-link variations may occur in diverse natural environments. For example, mDAP3-mDAP3 cross-links are elevated in E. coli growing in stationary phase and under envelope stress conditions (62–64). Similarly, S. Typhimurium growing intracellularly in eukaryotic cells has elevated mDAP3-mDAP3 cross-links, but with a reduction in overall degree of cross-linking (65). It is believed that mDAP3-mDAP3 cross-links increase the order, rigidity, and strength of the peptidoglycan sacculus for cell wall flexibility and structural integrity during conditions of stress (31, 62, 64).
The degree of cross-linkage in peptidoglycan is also known to influence bacterial virulence. An interesting example is that of EcgA, a d,l endopeptidase of S. Typhimurium that cleaves the d-glu2–mDAP3 bond in un-cross-linked peptides during intracellular growth to promote persistence and infection (66). In addition, typhoid toxin secretion in S. enterica serovar Typhi is governed by the abundance of mDAP3-mDAP3 linkages at the cell poles. These cross-links facilitate cleavage of associated glycan strands by a specialized muramidase TtsA, which is required to translocate the proteinaceous typhoid toxin into the host cell (67).
SYNTHESIS OF PEPTIDOGLYCAN SACCULUS
Peptidoglycan synthesis is required at several steps during the growth of a bacterial cell. The sacculus needs to be enlarged to accommodate the growing cytosolic contents when cells are actively elongating during nutrient availability. In addition, localized peptidoglycan synthesis at the midcell is required to form a septum to eventually generate daughter progeny (23, 26). The zones of active peptidoglycan synthesis were earlier identified using electron microscopy of peptidoglycan sacculi labelled with radioactive isotopes such as 3H-mDAP (38, 68–72). Over the years, visualization of peptidoglycan synthesis was radically improved by the advent of sophisticated microscopy and a variety of labelling techniques (38, 73–76).
A new widely used tool employs fluorescent d-amino acids that get incorporated into the peptidoglycan at sites of active synthesis (73–76). Examples of diverse modes of peptidoglycan synthesis in several bacterial species, as observed by incorporation of fluorescent d-amino acids at sites of synthesis, are depicted in Fig. 3. E. coli and S. Typhimurium follow a similar pattern of peptidoglycan synthesis, with the septal synthesis occurring at the midcell and the elongation synthesis occurring in the cylindrical body of the rod, while the cell poles remain inert.
Figure 3.
Visualization of zones of active peptidoglycan synthesis in live bacteria using fluorescent d-amino acid (FDAA), HADA. Bacteria grown with HADA (emission maximum, 450 nm) are visualized as described previously (73, 74). Zones of fluorescence represent the sites of active peptidoglycan synthesis. Predominantly sidewall and septal peptidoglycan synthesis is observed in E. coli, S. Typhimurium, and B. subtilis. In Agrobacterium tumefaciens and Streptomyces venezuelae, peptidoglycan synthesis is seen at a single pole. Scale bar = 5 µm. (Image courtesy of Michael VanNieuwenhze, Yves Brun, and Erkin Kuru).
Peptidoglycan synthesis occurring during septum formation or sacculus expansion follows a common scheme in which the cytoplasmic precursors are flipped into the periplasm and are then shared by either septal or sidewall synthetic machinery (see Box 1). Here, we describe the process of peptidoglycan synthesis leading to the sacculus expansion (alternatively termed sidewall or elongation synthesis), predominantly from the knowledge gained by studying E. coli. Cell division is described in another chapter of this issue and is not covered here.
Box 1. Box 1 Terminology used to describe modes of peptidoglycan synthesis.
Septum: Multilayered peptidoglycan at the site of cell division that is inherited as poles by the two daughter cells. The septal synthesis factors include FtsW, FtsI, PBP1b-LpoB, and MtgA(?).
Sidewall: Largely single-layered body of the peptidoglycan sacculus except septum and poles. Sidewall synthesis includes scaffold, gap filling, and repair synthesis.
(Scaffold and gap filling synthesis are collectively referred to as “elongation synthesis,” as the two processes are essential for sacculus enlargement to facilitate growth.)
Factors for sidewall synthesis:
Scaffold synthesis: MreBCD, RodZ, RodA, PBP2, and PBP1a-LpoA(?)
Gap filling synthesis: PBP1a-LpoA and PBP1B-LpoB
Repair synthesis: PBP1a-LpoA(?); PBP1b-LpoB; LdtD, -E, and -F; and PBP1c(?)
Synthesis of peptidoglycan is a complex process involving multiple reactions spanning three subcellular compartments—the cytoplasm, the inner membrane (IM), and the periplasm (Fig. 4). The process involves (i) synthesis of nucleotide-activated sugars and amino acids in the cytoplasm, (ii) assembly of sugars and amino acids into lipid-linked monomer precursors at the cytoplasmic face of the IM, (iii) flipping of these precursors across the IM into the periplasm, (iv) polymerization of monomers into glycan strands followed by cross-linking of peptides to form the peptidoglycan sacculus in the periplasm, and (v) maturation of the peptidoglycan sacculus. The majority of these steps are essential for bacterial growth and are hence targets of several antimicrobial therapeutics (Table 1). The factors involved in peptidoglycan biogenesis of E. coli are summarized in Tables 2 and 3.
Figure 4.
Schematic depicting peptidoglycan biosynthetic pathway. Peptidoglycan precursors are synthesized in the cytoplasm by a series of enzymes, MurA, B, C, D, E, and F, that convert UDP-GlcNAc to form UDP-MurNAc-pentapeptide (also referred to as Park’s nucleotide), which is subsequently attached to the lipid transporter (undecaprenyl phosphate; C55P) by MraY to yield lipid-I that is converted to the final peptidoglycan precursor lipid-II, by addition of a GlcNAc moiety by MurG. A flippase MurJ transports lipid-II across the IM to the periplasm. Lipid-II is polymerized into the peptidoglycan by synthases, with C55P being recycled to the cytoplasm. TGase activity of RodA, FtsW, PBP1a, PBP1b, and MtgA catalyzes glycan polymerization, whereas TPase activity of PBP2, PBP3, PBP1a, and PBP1b contributes to peptide cross-link formation (refer to Box 1). Hydrolysis mediated by d,d-endopeptidases, MepS, -M, and -H leads to cleavage of existing peptide cross-links to make space for the incorporation of nascent glycan strands. Anh-MurNAc (green hexagon) is the terminal residue in a glycan strand. The gray arrow indicates the direction of synthesis.
Table 1.
Inhibitors of peptidoglycan synthesis
| Antibiotics/inhibitors | Target(s) | Salient features |
|---|---|---|
| Cytoplasm | ||
| Fosfomycin | MurA (UDP-N-acetylglucosamine enol pyruvyl transferase) | Phosphoenolpyruvate (PEP) analog |
| d-Cycloserine | Alr (alanine racemase) and DdlA, -B (d-ala–d-ala ligase) | Cyclic analog of d-alanineSmall-molecule inhibitor |
| Tunicamycin | MraY (phospho-N-acetylmuramoyl-pentapeptide transferase) | Also blocks N-linked glycosylation in eukaryotes (322) |
| CDFI/DMPI | MurJ (flippase) | Small-molecule inhibitor |
| A22, MP265 | MreB polymerization | Small-molecule inhibitors |
| Periplasm | ||
| Bulgecin | Lytic transglycosylases | Primarily targets soluble lytic transglycosylasesBroad-spectrum glycopeptide antibiotic |
| Moenomycin | aPBPs | Specifically inhibits TGase activity of aPBPsPhosphoglycolipid antibiotic |
| Corbomycin and complestatin | Peptidoglycan hydrolases | Inhibit peptidoglycan remodelingNewly discovered glycopeptide antibiotics (323) |
| Vancomycin | d-ala4–d-ala5 in a muropeptide | Binds terminal d-ala4–d-ala5 in peptidoglycan and blocks cross-link formationGlycopeptide antibiotic |
| Ramoplanin | Lipid-II and MurG | LipoglycodepsipeptideInhibits TGase activity of MurG |
| Teixobactin | Lipid-II | Recently identified antibiotic from a soil bacteriumAlso binds to lipid-III in Gram-positive bacteria (129, 324) |
| Bacitracin | Undecaprenyl pyrophosphate (UndPP) | Inhibits dephosphorylation of UndPPCyclic polypeptide |
| Colicin M | Lipid-II | Hydrolyzes phosphodiester bond of lipid-I and lipid-II (325) |
| β-Lactam antibiotics | ||
| Ampicillin | PBPs | Broad-spectrum antibiotic which inhibits PBPs |
| Aztreonam | PBP3 | Inhibits septal synthesis, resulting in cell filamentation (326)Monobactam antibiotic |
| Cefsulodin | aPBPs | Specifically inhibits TPase activity of aPBPs |
| Cephalexin | PBP3 | Inhibits septal synthesis, resulting in cell filamentation (326)May have other targets in addition to PBP3 |
| Mecillinam (amdinocillin) | PBP2 | Inhibits scaffold synthesis, resulting in loss of rod shapeA semisynthetic derivative of penicillin |
Table 2.
Genes involved in cytosolic peptidoglycan precursor synthesis and turnover in E. coli
| Protein (gene) | Function | Salient features |
|---|---|---|
| Peptidoglycan precursor synthesis | ||
| GlmS (glmS) | Glucosamine-6-phosphate synthase; catalyzes the formation of d-glucosamine-6-phosphate from d-fructose-6-phosphate and l-glutamine (79) | EssentialFirst committed step in hexosamine metabolismExpression is controlled by small RNAs: GlmY and GlmZ |
| GlmM (glmM) | Phosphoglucosamine mutase; catalyzes the conversion of d-glucosamine-6-phosphate to d-glucosamine-1-phosphate (82) | EssentialInactivation leads to loss of rod shape, forming enlarged ovoids |
| GlmU (glmU) | A bifunctional enzyme which catalyzes the formation of UDP-GlcNAc from glucosamine-1-phosphate and UTP (81) | UDP-GlcNAc is utilized for both lipopolysaccharide and peptidoglycan biosynthesisN-terminal and C-terminal domains of GlmU catalyze uridylyl and acetyltransferase activities, respectively. Both activities are essential for viability (327) |
| MurA (murA) | UDP-N-acetylglucosamine enolpyruvyl transferase; catalyzes the formation of UDP-GlcNAc-enol pyruvate from phosphoenolpyruvate (PEP) and UDP-GlcNAc (83) | First committed step in peptidoglycan biosynthesisTarget of the naturally occurring broad-spectrum antibiotic fosfomycinFeedback inhibited by UDP-MurNAc |
| MurB (murB) | UDP-N-acetyl enolpyruvoyl glucosamine reductase; catalyzes the formation of UDP-MurNAc from UDP-GlcNAc-enolpyruvate and NADPH (84) | EssentialCatalyzes the second committed step in peptidoglycan biosynthesis |
| MurC (murC) | UDP-N-acetylmuramoyl-l-ala ligase; adds l-ala to UDP-MurNAc (86) | EssentialCan also accept l-serine and glycine as substrates with poor efficiency (328) |
| MurD (murD) | UDP-N-acetylmuramoyl-l-ala–d-glu ligase; adds d-glu to UDP-MurNAc-l-ala (88) | Essential |
| MurE (murE) | UDP-N-acetylmuramoyl alanyl-d-glutamate 2,6-diaminopimelate ligase; adds mDAP to UDP-MurNAc-l-ala–d-glu (89) | Essential |
| MurF (murF) | Ligates d-ala–d-ala to UDP-MurNAc-l-ala–d-glu–mDAP to form UDP-MurNAc-pentapeptide (90) | EssentialCatalyzes the final cytoplasmic step in the peptidoglycan precursor synthesis |
| MurI (murI) | Glutamate racemase; catalyzes the racemization of l-glutamate to d-glutamate (87) | Essential |
| Alr (alr) | Alanine racemase; catalyzes the racemization of l-alanine to d-alanine (92) | NonessentialConstitutively expressed |
| DadX (dadX) | Alternate alanine racemase; converts l-alanine to d-alanine (91) | Nonessential |
| DdlA (ddlA)DdlB (ddlB) | d-ala–d-ala ligases which join two residues of d-alanine to form a dipeptide (93) | Redundant; double mutant of ddlA and ddlB is lethalTarget of d-cycloserine |
| MraY (mraY) | Phospho-N-acetylmuramoyl-pentapeptide transferase; catalyzes the formation of lipid-I from UDP-MurNAc-pentapeptide and UndPP (97) | EssentialIM proteinTarget of tunicamycin |
| MurG (murG) | N-acetylglucosaminyl transferase; catalyzes the formation of lipid-II from UDP-GlcNAc and lipid-I (104) | EssentialIM protein |
| MurJ (yceN/ mviN) | Flippase which transports lipid-II across IM to periplasm (114) | EssentialIM proteinMOP flippase superfamily |
| Peptidoglycan recycling/turnover | ||
| AmiD (amiD) | A periplasmic amidase which cleaves the amide bond between MurNAc and l-ala (329) | NonessentialCan use peptidoglycan, muropeptides, and anhydro-muropeptides as substrateAmidase_2 superfamilyOM lipoprotein |
| AmpG | Permease which transports anhydromuropeptides into cytoplasm (330) | IM proteinNonessential |
| AmpD (ampD) | Amidase which cleaves the amide bond between AnhMurNAc and l-ala (331) | NonessentialAmidase_2 superfamily |
| LdcA (ldcA) | A cytosolic l,d-carboxypeptidase which cleaves between mDAP3–d-ala4 in tetrapeptides (332) | NonessentialCells lacking ldcA lyse in stationary phase |
| DdpX (ddpX/ vanX) | A dipeptidase which cleaves d-ala–d-ala dipeptides (333) | NonessentialExpressed in stationary phase |
| NagZ (ycfO) | β-N-acetylglucosaminidase; cleaves β-1,4 glycosidic bond between MurNAc and GlcNAc (334) | Nonessential |
| MpaA (ycjI)a | d,L-carboxypeptidase which cleaves d-glu–mDAP in tripeptides (335) | NonessentialExpression increases during nitrogen starvation (336) |
| YcjG (ycjG)a | L-ala-d/l-glu epimerase (337) | NonessentialConverts l-ala–d-glu (product of MpaA) into l-ala–l-glu |
| PepD (pepD)a | A peptidase that cleaves l-ala–l-glu (337) | NonessentialNonspecific dipeptidase that acts on l-ala–l-glu into individual amino acids |
| Mpl (yjfG) | UDP-N-acetylmuramate:l-alanyl-γ-d-glutamyl-meso-diaminopimelate ligase (96) | NonessentialAdds tripeptide (l-ala–d-glu–mDAP) released during recycling to UDP-MurNAc |
MpaA, YcjG, and PepD catabolize murein peptides only when peptidoglycan synthesis is not limiting for cellular growth.
Table 3.
Proteins involved in peptidoglycan synthesis in the periplasm
| Protein (gene) | Function(s) | Salient features |
|---|---|---|
| Peptidoglycan synthases | ||
| PBP1a (mrcA/ponA); PBP1b (mrcB/ponB) | Bifunctional peptidoglycan synthases with TGase and TPase activity (145) | Redundantly essential for growthAlso called aPBPsActivity stimulated by cognate lipoproteins LpoA (PBP1a) and LpoB (PBP1b)IM-anchored proteins |
| PBP1c (pbpC) | Putative TGase (189) | NonessentialPhysiological function not knownIM-anchored protein |
| RodA (mrdB) | TGase required for elongation synthesis (141) | EssentialPart of Rod complex (elongasome)Required for scaffold synthesisDisplays MreB-like directional motionSEDS family polytopic IM protein |
| PBP2 (mrdA) | TPase required for elongation synthesis (145) | EssentialbPBPPart of Rod complex (elongasome)Required for scaffold synthesisDisplays MreB-like directional motionBitopic IM protein |
| FtsW (ftsW) | TGase required for septal synthesis (123) | EssentialPart of divisomeSEDS family polytopic IM protein |
| PBP3 (ftsI) | TPase required for septal synthesis (145) | EssentialbPBPPart of divisomeBitopic IM protein |
| MtgA (mtgA) | TGase (238) | NonessentialPhysiological relevance not knownLocalizes to the division site in cells lacking mrcBIM-anchored protein |
| LdtA (erfK); LdtB (ybiS); LdtC (ycfS) | l,d transpeptidases (Ldts) which attach Lpp to mDAP3 of peptidoglycan (47) | NonessentialYkuD familyActivity is inhibited by copper (338)ΔldtABC mutant lacks bound form of Lpp |
| LdtD (ycbB); LdtE (ynhG); LdtF (yafK) | l,d transpeptidases (Ldts) which synthesize mDAP3–mDAP3 cross-links in the peptidoglycan (252) | NonessentialYkuD familyLdtD belongs to Cpx regulonΔldtDE mutant lacks mDAP3–mDAP3 cross-links |
| Peptidoglycan hydrolases | ||
| Slt (slt)a | Lytic transglycosylase which cleaves β-1,4 glycosidic bond between MurNAc and GlcNAc | NonessentialUpon β-lactam treatment, Slt prevents abnormal incorporation of peptidoglycan by degrading the un-cross-linked glycan chains (149)Slt is a member of Cpx regulonSoluble periplasmic protein (339) |
| MltA-F (mltA-F)a | Lytic transglycosylases which cleave β-1,4 glycosidic bond between MurNAc and GlcNAc (242) | NonessentialInvolved in peptidoglycan recyclingMltA to -E are OM-lipoproteins; MltF is an OM-anchored protein (339) |
| MltG (mltG/yceG)a | Endolytic transglycosylase which cleaves β-1,4 glycosidic bond between MurNAc and GlcNAc (196) | NonessentialMaintains glycan strand lengthIM-anchored protein |
| DigH (digH/yddW) | Glycosyl hydrolase which cleaves glycan chains lacking peptides (243) | NonessentialLocalizes to the division siteOM lipoprotein |
| PBP5 (dacA); PBP4 (dacB); PBP6 (dacC); PBP6B (dacD) | d,d carboxypeptidases which cleave terminal d-ala in the peptidoglycan (246–249) | NonessentialcPBPs/low-molecular-weight PBPsInvolved in peptidoglycan remodeling and recycling. PBP5 is a major d,d carboxypeptidasePBP4 also possess d,d-endopeptidase activity (340)PBP6b functions in acidic growth conditions (341) |
| PBP7 (pbpG) | d,d endopeptidase which cleaves d-Ala4–mDAP3 cross-links (342) | NonessentialcPBP/low-molecular-weight PBPInvolved in peptidoglycan remodeling and recyclingIM-anchored protein |
| AmpH (ampH) | d,d endopeptidase which cleaves d-Ala4–mDAP3 cross-links (343) | NonessentialHas weak d,d-carboxypeptidase activityInvolved in peptidoglycan remodeling and recyclingIM-anchored low-molecular-weight PBP |
| MepA (mepA) | d,d endopeptidase which cleaves d-Ala4–mDAP3 cross-links (344) | NonessentialLAS metallopeptidase familyFunction not knownSoluble periplasmic protein |
| MepS (mepS/spr) | d,d endopeptidase which cleaves d-Ala4–mDAP3 cross-links (221) | NonessentialNlpC/ P60 familyElongation-specific endopeptidaseRegulated by NlpI-Prc proteolytic systemEssential in cells lacking MepM in enriched mediumOM lipoprotein |
| MepM (mepH/ yebA) | d,d endopeptidase which cleaves d-Ala4–mDAP3 cross-links (221) | NonessentialLytM (M23) familyEssential in cells lacking MepS in enriched mediumIM-anchored |
| MepH (mepH/ ydhO) | d,d endopeptidase which cleaves d-Ala4–mDAP3 cross-links (221) | NonessentialNlpC/ P60 familyEssential in cells lacking both MepS and MepM |
| MepK (ycbK) | l,d endopeptidase which cleaves mDAP3–mDAP3 cross-links (222) | NonessentialM15 familyΔmepK mutant shows increased mDAP3–mDAP3 cross-linksSoluble periplasmic protein |
| Regulators and accessory proteins | ||
| MreBCD (mreBCD) | MreBCD forms a helical cytoskeletal protein complex (137) | EssentialCircumferential rotation of MreB directs the elongation synthesis of peptidoglycanPart of Rod complex (elongasome)MreB is an actin homolog that polymerizes into short and discontinuous filaments |
| RodZ (rodZ/ yfgA) | Maintains cell shape and links movement of peptidoglycan synthases RodA-PBP2 to that of MreB filaments (154) | EssentialPart of Rod complex (elongasome)Bitopic IM protein |
| LpoA (yraM)/LpoB (ycfM) | Cofactors that activate their cognate synthases, PBP1a (–LpoA) and PBP1b (-LpoB) (200, 201) | Redundantly essentialOM lipoproteins |
| NlpI (nlpI) | Facilitates Prc-mediated degradation of MepS (227) | NonessentialA tetratricopeptide repeat (TPR)-containing globular proteinFunctions as an adapter to regulate peptidoglycan synthases and hydrolases (289)OM lipoprotein |
| Prc (prc/tsp) | C-terminal protease (345) | NonessentialAlong with NlpI, degrades MepSOther substrates include PBP3, DigH, MltG, and MltBSoluble periplasmic protein |
| Lpp (lpp) | Tethers OM to peptidoglycan (45) | NonessentialAlso called Braun’s lipoproteinMost abundant protein (∼106 copies per cell)Maintains the width of the periplasmOM lipoprotein |
| PgeF (pgeF/yfiH) | Prevents incorporation of l-serine/glycine in the muropeptides (60) | NonessentialLocated in the division and cell wall (dcw) cluster of many Gram-positive bacteriaHuman homolog of PgeF, LACC1 is implicated in several autoimmune disorders (346) |
Lytic transglycosylases cleave the glycosidic bond between MurNAc and GlcNAc and catalyze an intramolecular transglycosylation reaction to form an 1,6-anhydro MurNAc.
CYTOPLASMIC PHASE OF PEPTIDOGLYCAN SYNTHESIS
De novo peptidoglycan biosynthesis begins with the formation of UDP-GlcNAc, a shared precursor for peptidoglycan and OM biosynthesis in Gram-negative bacteria (77, 78). UDP-GlcNAc is synthesized from the glycolytic intermediate fructose-6-phosphate, amido group donor l-glutamine, and UTP by the sequential action of the enzymes GlmS, GlmM, and GlmU (79–82). UDP-MurNAc is then formed from UDP-GlcNAc and phosphoenolpyruvate by the catalytic activity of MurA and MurB, marking the first committed step of peptidoglycan biosynthesis (83, 84). MurA is an effective drug target to block peptidoglycan precursor synthesis and is inhibited by the phosphoenolpyruvate analog fosfomycin, a naturally occurring broad-spectrum antibiotic (85). The lactoyl moiety of UDP-MurNAc is the site for synthesis of the stem peptide. Peptide synthesis is nonribosomal and is carried out by sequential addition of amino acids by dedicated ATP-dependent amino acyl ligases. The first amino acid, l-ala, is added onto UDP-MurNAc by MurC to form UDP-MurNAc-l-ala (86). The second residue, d-glu, formed by racemization of l-glu by MurI (87), is then added to UDP-MurNAc-l-ala by MurD (88). Subsequently, mDAP, the penultimate intermediate of l-lysine biosynthesis, is added to the growing peptide by MurE to make the UDP-MurNAc-tripeptide (89). Finally, the d-ala–d-ala dimer is added to the tripeptide by MurF (90). d-ala is generated by the racemization of l-ala by Alr or DadX (91, 92) and dimerized by either of the two d-ala–d-ala ligases, DdlA or DdlB (93). A d-ala–d-ala analog, d-cycloserine, inhibits precursor synthesis by blocking these ligases (94). The tripeptide l-ala1–d-glu2–mDAP3 is also generated in the cytoplasm during peptidoglycan recycling (95) and is directly ligated onto UDP-MurNAc by a murein peptide ligase Mpl (96). The resultant UDP-MurNAc-pentapeptide is also referred to as Park’s nucleotides (after James T. Park for his seminal contributions to the field of peptidoglycan biology).
The phospho MurNAc-pentapeptide moiety of the nucleotide is transferred onto an IM lipid carrier undecaprenyl pyrophosphate (UndPP/C55 isoprenyl pyrophosphate) by the IM-bound MraY to form lipid-I (97). UndPP is synthesized by the cytoplasmic UppS and must be dephosphorylated by one of the four IM-bound phosphatases—BacA, PgpB, YbjG, and LpxT—in order to accept the incoming UDP-MurNAc-pentapeptide (98–101). A peptide antibiotic bacitracin binds and prevents the dephosphorylation of UndPP, rendering it unavailable for peptidoglycan synthesis (102). Another antibiotic, tunicamycin, also blocks precursor synthesis by inhibiting the transfer of UDP-MurNAc-pentapeptide onto UndP by MraY (103). Finally, a molecule of UDP-GlcNAc is transferred onto the MurNAc in lipid-I to form UndPP-GlcNAc-MurNAc-pentapeptide (lipid-II) by the action of IM-bound glycosyltransferase MurG (104, 105), with which the assembly of the peptidoglycan precursor on the cytoplasmic face of IM is complete. MurG and MraY are shown to interact, suggesting that a multiprotein complex may perform the final steps of intracellular peptidoglycan precursor synthesis (106). Molecular details of the cytoplasmic phase of peptidoglycan synthesis have been well summarized (77, 78, 107–109).
EXPORT OF PEPTIDOGLYCAN PRECURSORS INTO THE PERIPLASM
Most enzymes of cytoplasmic and periplasmic peptidoglycan synthesis have been known for decades; however, the factor that transports lipid-II into the periplasm remained elusive for a long time, although several essential IM proteins were predicted to be the likely candidates (110–113). One of the significant developments of the past decade is the discovery of MurJ as the lipid-II flippase. MurJ flippase activity was initially predicted by bioinformatic analysis (112) and subsequently demonstrated using an elegant in vivo assay by quantitating flipped lipid-II upon selective inhibition of different candidate proteins (114). MurJ (encoded by yceN) is an essential IM protein from the MOP (multidrug/oligosaccharidyl-lipid/polysaccharide) family (115), members of which are also involved in transport of Und-PP-O-antigen during OM biosynthesis (116). Structural studies revealed that the lipid-II binding central cavity of MurJ alternates between cytoplasm-facing and periplasm-facing open conformations to flip a molecule of lipid-II (117–119). Membrane potential governs the resetting from outward-open to inward-open conformation, and its dissipation results in an arrest in the outward-open state, rendering MurJ incapable of the lipid-II flipping cycle (117, 120). However, the identity of the ion gradient that drives MurJ flipping remains unknown (120).
FtsW, an essential IM protein involved in cell division, has also been shown to transport lipid-II in vitro and is therefore predicted to be a lipid-II flippase (25, 121). However, at this juncture, both in vivo and in vitro evidence strongly support the possibility of MurJ being the flippase for lipid-II precursors. Meanwhile, FtsW was also demonstrated to be a glycosyl transferase required for glycan strand polymerization during septum synthesis (122–125).
Upon flipping into the periplasm, lipid-II is utilized as a substrate by peptidoglycan polymerases for synthesis of the sacculus. Polymerization of the disaccharide pentapeptide releases the diphosphate form of UndPP, which is dephosphorylated by the IM phosphatases and recycled to the cytoplasm by an unknown mechanism, for the next cycle of flipping (100). Several studies present the possibility of lipid-II synthesis, translocation, and polymerization being coupled for sustained cell wall assembly (122, 126–128). Teixobactin, a recently isolated antibiotic from an uncultivable soil bacterium, binds to the UndPP-MurNAc moiety of lipid-II and inhibits its polymerization into the peptidoglycan sacculus (129). The eponymous E. coli toxin colicin M also prevents peptidoglycan polymerization by degrading lipid-I and lipid-II precursors (130).
UndPP is a shared carrier of the precursors for synthesis of O-antigen, enterobacterial common antigen (ECA), capsular polysaccharide, and peptidoglycan (101, 131), and partitioning of UndPP between these pathways ensures balanced syntheses of envelope components (132–136). For example, mutants defective in ECA or O-antigen biogenesis demonstrate peptidoglycan defects due to accumulation of lipid-IIECA or lipid-IIO-antigen, which are reversed by diverting UndPP toward peptidoglycan synthesis (132, 133). Likewise, depletion of ElyC leads to defects in peptidoglycan synthesis, based on which a role for ElyC in funneling UndPP through the peptidoglycan pathway is proposed (134). These examples highlight the central role of UndPP in coordinating the biogenesis of cell envelope components.
PERIPLASMIC PHASE OF PEPTIDOGLYCAN SYNTHESIS
Polymerization of lipid-II precursors in the periplasm for sidewall or septal synthesis occurs by dedicated and distinct peptidoglycan synthetic machineries. In both cases, the polymerization of lipid-II requires two reactions—transglycosylation of the disaccharides to form glycan strands and transpeptidation of the peptide stems to form cross-bridges. E. coli encodes several transglycosylase (TGase) and transpeptidase (TPase) enzymes that perform sidewall and septum synthesis (see Box 1). Under conditions of nutrient availability, newly formed daughter cells grow in size before undergoing another cycle of cell division. During the phase of cell elongation, the peptidoglycan sacculus enlarges in concert with the growing cytoplasmic volume. It was known from early on that the new peptidoglycan material is incorporated diffusely into the cylindrical portion of the body of a rod-shaped cell, while the cell poles remain inert (Fig. 3) (38, 68). An emerging paradigm suggests that elongation synthesis is achieved by two discrete protein complexes that work in conjunction—the Rod system and the class A PBPs (aPBPs). In addition, elongation requires hydrolysis of the preexisting peptide cross-links to open the mesh and allow insertion of new peptidoglycan material.
Elongation Synthesis: Scaffold Formation by Rod Complex
Elongation-specific peptidoglycan synthesis is initiated by a complex of essential cytoskeletal, IM-anchored, and periplasmic proteins termed the “elongasome” or “Rod complex” (137). RodA (TGase) and PBP2 (TPase), the IM-bound peptidoglycan synthase pair coupled to the cytoskeletal complex MreBCD via RodZ, form the Rod complex (Box 1; Fig. 5). Disruption of any of the elongasome components leads to loss of rod shape, resulting in spherical and enlarged cells that eventually lyse.
Figure 5.
Model for peptidoglycan sacculus expansion. (A) Schematic representation of elongation synthesis in a rod-shaped cell. The Rod complex (green circle) moves in helical fashion (purple) and synthesizes a scaffold, and aPBP-Lpos (orange dots) move in a diffusive manner (irregular blue lines) to fill the gaps, acting in concert to make a functional peptidoglycan sacculus. (B) Factors required for peptidoglycan elongation. Rod complex/elongasome is a multiprotein complex consisting of RodA, PBP2, RodZ, and MreBCD. The peptidoglycan synthase pair RodA-PBP2, guided by the MreBCD-RodZ complex, makes the peptidoglycan scaffold (red strands) around the cytoplasmic membrane. RodZ connects RodA-PBP2 with the cytoskeletal MreBCD to form the Rod complex, which moves circumferentially in the direction of peptidoglycan synthesis to form the scaffold (as shown in panel A). IM-localized PBP1a and PBP1b are activated by cognate OM lipoproteins LpoA and LpoB to complete the sidewall synthesis (black strands). LpoA and LpoB may traverse through the gaps in the peptidoglycan layer to interact and activate PBP1a and PBP1b. The activity of the major elongation-specific endopeptidase MepS may create space/gaps for interaction of aPBP-Lpo factors and to make space for incorporation of nascent peptidoglycan strands. MepS activity is kept in check by regulated proteolysis through its interaction with an adapter protein, NlpI, which brings the soluble periplasmic protease Prc. Nevertheless, the signal(s) that triggers sacculus expansion is not yet understood.
Until recently, peptidoglycan synthesis was believed to be predominantly carried out by high-molecular-weight class A PBPs (aPBPs), which possess both TGase and TPase activities. However, recent landmark studies have identified RodA as a major TGase of the elongasome complex. Although RodA has long been known to be essential for peptidoglycan synthesis and maintenance of rod shape, its role was not well defined (138–140). RodA was recently discovered to be a TGase that is insensitive to moenomycin, a molecule that inhibits the glycosyltransferase activity of aPBPs. Subsequently, RodA was shown to be a major glycosyltransferase capable of glycan strand polymerization in the absence of aPBPs in B. subtilis and E. coli (141–143). RodA, encoded by mrdB, belongs to the SEDS (shape, elongation, division, and sporulation) family of proteins (144). Glycan polymerization by RodA is followed by cross-linking of peptide stems by the cognate TPase PBP2 (penicillin binding protein 2), encoded by mrdA (140, 143, 145, 146). PBP2 is a member of the class B PBPs (bPBPs), which comprise monofunctional high-molecular-weight PBPs. Owing to its penicillin binding property, PBP2 has long been identified as a dedicated elongation-specific TPase (145–147). The d,d-transpeptidase domain of high-molecular-weight PBPs cleaves the d-ala4–d-ala5 bond in a donor peptide and cross-links the d-ala4 with the mDAP3 of an acceptor, leading to the formation of a 4-3 cross-link. Glycopeptide antibiotics such as vancomycin bind to d-ala4–d-ala5 of the donor and inhibit polymerization by steric hindrance (148). Beta-lactam antibiotics inhibit the transpeptidase domains of all PBPs by covalent modification of the active site (6). TPase activity of PBP2 is specifically inhibited by the beta-lactam mecillinam, leading to ovoid cells, making mecillinam a useful tool to study cell wall elongation processes (140, 145, 149, 150). Physical interaction between the transmembrane domains of RodA and PBP2 lead to the activation of RodA TGase activity by PBP2 (151, 152). Together, the SEDS-bPBP pair forms a complete peptidoglycan synthetic unit fulfilling the enzymatic requirements for peptidoglycan polymerization (124, 141, 143). In addition, a bifunctional class A PBP, PBP1a has been shown to interact with PBP2 as well as RodZ, possibly suggesting a role for it in sidewall synthesis (153, 154).
Cytoskeletal proteins of the Rod complex, MreBCD, form the structural framework that dictates sites of peptidoglycan synthesis by RodA-PBP2 to ensure uniform distribution of new peptidoglycan material (Fig. 5). MreB, of which eukaryotic actin is a homolog, is a conserved bacterial cytoskeletal protein that polymerizes into filaments just beneath the cytoplasmic membrane in the cylindrical part of the cell body, imparting cell shape and mechanical integrity (155–159). MreB polymerization is ATP-dependent and is shown to occur in vitro independently of any other factors (160). Short and discontinuous MreB filaments orient along the greatest principal membrane curvature and are shown to be responsible for establishment and maintenance of rod shape (161–163). The IM protein MreC binds and tethers the MreB filaments to the membrane, simultaneously recruiting the third member of the cytoskeleton, MreD (164). Small molecules A22 and MP265 reversibly inhibit MreB polymerization, resulting in diffuse distribution of MreB in the cytoplasm and loss of cell shape (165–167).
MreBCD proteins are encoded by the mreBCD operon and transcribed as monocistronic mreB and polycistronic mreBCD mRNA, ensuring a much higher copy number of MreB in the cell (∼10,000) than the other two components (46, 168). The interaction between MreBCD cytoskeleton and peptidoglycan synthases RodA-PBP2 is mediated by the bitopic IM protein RodZ, the latest member of the Rod complex to be identified (154, 169–172). Like MreB, RodZ is distributed in a spiral-like manner in the cell cylinder and alters the geometric sensing of MreB, thereby stabilizing the MreB helix along the curved membrane (169, 170, 173). As a result, foci of elongasome complexes made up of MreC, MreD, RodZ, RodA, and PBP2 form at points of contact of the MreB filaments with the IM. Several studies show that localization and activities of the cytoplasmic MreB filaments and the other Rod components are mutually dependent for the proper assembly of elongasome (162, 164, 174–176). The MreB filaments dynamically rotate along the cell circumference, almost perpendicular to the length of the cell (177–179). MreB circumferential motion is driven by TGase and TPase activities of RodA and PBP2 and hence occurs in the direction of peptidoglycan synthesis (143, 177–179). Interestingly, inhibition of PBP2 stops MreB motion but allows a futile cycle of synthesis by RodA, eventually leading to cell lysis (143, 149). RodZ physically couples the processive motion of RodA-PBP2 with the MreB cytoskeleton and hence is indispensable for circumferential activity of the elongasome (154). Subsequently, a scaffold for peptidoglycan sacculus is formed by helical insertion of new material through processive movement of the elongasome, which is important for formation of rod shape and cell wall integrity (24, 143, 154, 175, 176, 180, 181).
An interesting possibility of precursor availability driving the elongasome activity is suggested, based on the observed interaction of the MurG-MraY complex with MreB (106). Although there is a lack of further evidence for this observation in E. coli, a recent study in B. subtilis shows that MreB filament formation is regulated by RodZ in response to lipid-II abundance, resulting in growth rates that are proportional to nutrient availability (182).
Depletion of elongasome components is lethal in rapidly growing cells due to loss of rod morphology; however, single-gene-deletion mutants are viable and grow poorly as small spheres in nutrient-limiting conditions (183). Interestingly, conditional lethality of mreBCD, rodZ, rodA, and pbp2 is suppressed by overexpression of FtsZ, an essential cytoskeletal factor initiating cell division, which can also be achieved by an increase in cellular concentration of ppGpp, a stationary-phase signaling molecule (164, 175–183). It is believed that upon inhibition of the elongasome, overexpressed FtsZ may push the cell cycle toward division, bypassing the need for elongation altogether.
Elongation Synthesis: Gap Filling by Class A PBPs
Another major class of peptidoglycan synthases comprises the bifunctional high-molecular-weight PBPs (aPBPs). E. coli encodes two bifunctional PBPs—PBP1a and -1b (encoded by mrcA and mrcB)—that are collectively essential for cell growth and viability and were believed to be the only major peptidoglycan synthases before the discovery of SEDS-bPBP functional pairs (23, 145, 186–188). The discovery of RodA-PBP2 as the scaffold synthase pair fundamentally changed the concept of cell wall assembly and prompted the idea that PBP1a and -1b are required to fill the gaps created by scaffold synthesis, attributing specific functions to both systems in peptidoglycan synthesis (Fig. 5). As the cells can tolerate loss of either PBP1a or -1b but not both, their functions are believed to be semiredundant (186). Another high-molecular-weight PBP, PBP1c, encoded by pbpC, is hypothesized to be a transglycosylase involved in peptidoglycan repair synthesis; however, its function is not fully understood (189). From here onward, PBP1a and PBP1b will be collectively referred to as aPBPs.
The aPBPs are IM-anchored periplasmic proteins with a cytoplasmic tail, an N-terminal TGase domain that polymerizes lipid-II into glycan strands, and a C-terminal d,d TPase domain that cross-links the peptides. Both aPBPs have been shown to polymerize and crosslink lipid-II in vitro (126, 190–192). The TPase domain also possesses d,d carboxypeptidase activity in vitro, which may be a means to remove a tetrapeptide donor bound in the active site when an acceptor peptide is not available, preventing TPase inhibition (126, 192). The TPase domain of aPBPs is specifically inhibited by the beta-lactam cefsulodin, while the TGase domain is inhibited by moenomycin. Absence of aPBPs or inhibition of their TPase activity results in characteristic lysis by blebbing at the septum and leakage of cytosolic contents (186).
PBP1a and -1b are believed to be redundant; however, they contribute to sacculus enlargement in different ways. Preferential localization of PBP1a along the sidewall and interaction of PBP1a with PBP2 suggests its predominant role in elongation (153). In contrast, PBP1b localizes diffusely along the sidewall and also at the septum to interact with divisomal components, FtsN and FtsW-PBP3, indicating a role in cell division (23, 25, 193, 194). Despite the redundancy, PBP1b might be more versatile, as evidenced by its ability to function at low pH (195), contribution to remodeling in response to OM stress (64), interaction with the lytic transglycosylases MltA and MltG (196, 197), ability to recover cells from a wall-deficient state (198), and role in OM constriction for cell division (199).
While the aPBPs are independently active in vitro (126, 192), they are strongly dependent on OM-anchored cofactors LpoA and LpoB for their function in vivo (200, 201). The discovery of OM cofactors governing aPBP activity marks another significant milestone of the past decade. The cofactors LpoA and LpoB replicate the synthetic lethality of the cognate synthases (200, 201). Although they are functionally analogous, the Lpo factors do not share structural similarities and are highly specific to the cognate synthases, activating them by distinct mechanisms (202–204). LpoA binds PBP1a via the ODD domain (OM docking domain) and stimulates its TPase activity (201–203), whereas LpoB binds PBP1b via the UB2H domain (UvrB domain 2 homolog) and stimulates its TGase activity (200–202, 204–207). As the activities of TGase and TPase domains are coupled, Lpo stimulation eventually activates both domains of the cognate PBP.
Although aPBPs are ubiquitous across the bacterial kingdom, the cofactors LpoA and LpoB as well as the PBP domains with which they interact seem to cooccur only in gammaproteobacteria and enterobacteria, respectively (201). Another example of a functional PBP-Lpo pair was recently discovered in Pseudomonas aeruginosa, wherein PBP1b is activated by an OM factor, LpoP, that is structurally unrelated to the enterobacterial counterpart LpoB (208). Vibrio cholerae presents another interesting case wherein LpoA-mediated activation of PBP1a requires an additional periplasmic factor, CsiV (209). This argues that the mechanism of PBP regulation by OM cofactors may have evolved independently, underscoring the importance of regulation of peptidoglycan synthesis.
The aPBPs localize independently of the MreB elongasome and are presumed to move diffusely along with their OM lipoprotein cofactors in the respective membranes, scouting the periplasm for sites of peptidoglycan synthesis (143, 181). Given the width of the periplasm and the length of PBP and Lpo molecules, the latter must reach out from the OM across the peptidoglycan in order to bind the IM-anchored PBP (201, 203, 204). The aPBP-Lpo interaction may occur through the gaps resulting from (i) the ordered scaffold synthesis by Rod complex, (ii) the activity of elongation-specific endopeptidases, and (iii) damage to the cell wall by mechanical or chemical stress.
Furthermore, the cell wall exhibits a high degree of plasticity that allows it to recover from mechanical strain in a manner independent of the Rod complex (210). It has therefore been proposed that the Rod complex maintains cell shape by ordered insertion of peptidoglycan material, while the aPBPs maintain cell wall plasticity and integrity by diffusive localization and activity along the cell body (143, 181, 211). Consistent with this, synthesis mediated by the Rod complex leads to elongation of cells, whereas that of aPBPs alone leads to giant cells by enlargement in all directions (211). This aligns with the observed morphological effects of depletion of the two components: depletion of aPBPs leads to instantaneous lysis, while that of the Rod complex leads to rounding of cells that do not immediately lyse. Additionally, the stimulation of PBP1a activity by PBP2 (153) supports the hypothesis that RodA-PBP2-mediated scaffold synthesis precedes aPBP-mediated gap-filling synthesis. Thus, the developing picture attributes two distinct and crucial roles for the Rod system and aPBPs in the completion of sidewall synthesis for sacculus expansion.
Repair Synthesis
In addition to elongation synthesis, repair of the peptidoglycan is also required to mitigate cell wall defects arising due to mechanical or chemical damage. Evidence suggests that aPBPs contribute to cell wall repair (23, 143, 198), and the role of PBP1b in sensing and repairing cell wall defects was recently demonstrated (181). The propensity of PBP1b to sense regions requiring cell wall repair largely depends upon its OM cofactor LpoB (181), further emphasizing the possibility of aPBP-Lpo interaction through gaps in the cell wall (150, 181, 201, 212). Cell wall repair by aPBPs is independent of the Rod complex (181, 210).
In addition to aPBPs, the l,d-transpeptidases (Ldts) LdtD, -E, and -F also contribute to cell wall repair via formation of mDAP3-mDAP3 (3-3) crosslinks (62–64). Ldts may play a significant role in repair and remodeling due to their insensitivity to penicillins and their ability to cross-link peptides without the requirement for new peptidoglycan precursors. Ldts are known to be upregulated by envelope stress to increase the 3-3 cross-link formation, which in turn enhances the overall robustness of the sacculus (64). They extensively contribute to physiological remodeling of the peptidoglycan, although they have no role in peptidoglycan elongation except in a rare instance wherein Ldts can salvage growth by substituting for d,d TPase-mediated cross-linking (213). Intriguingly, Ldt-mediated remodeling of cross-links from the 4-3 to the 3-3 type has been demonstrated as a mechanism governing cell shape as well as sites of peptidoglycan synthesis in the asymmetrically dividing mycobacteria (214, 215), further highlighting the repair and maturation potential of Ldts.
Role of Hydrolysis in Peptidoglycan Synthesis
Because peptidoglycan sacculus is a covalently closed mesh that completely encircles the cytoplasmic membrane, its expansion during cell elongation would require opening of the mesh to make space for incorporation of new peptidoglycan material. Several models were proposed to explain the chemistry of peptidoglycan expansion, all of which predict that cleavage of the existing glycan strands by periplasmic hydrolases is essential to allow insertion of nascent peptidoglycan strands (8, 11, 12, 216, 217). This model was strengthened by the observation that local hydrolysis of the sacculus likely precedes insertion of new muropeptides (70, 72). Bacteria are known to encode multiple peptidoglycan hydrolases of varying specificities, such as N-acetylmuramidases, N-acetylglucosaminidases, amidases, endopeptidases, and carboxypeptidases, and their potential involvement in peptidoglycan synthesis has been explored extensively (218–220). Among all known peptidoglycan hydrolases present in E. coli, three murein endopeptidases are now known to be redundantly essential for growth of the peptidoglycan sacculus and bacterial viability (221). These “space maker hydrolases,” MepS, MepM, and MepH (encoded by spr, yebA, and ydhO), are d,d endopeptidases catalyzing cleavage of d-ala4–mDAP3 cross-links in the peptidoglycan for making space to allow insertion of new peptidoglycan material as well as providing acceptor peptides to form new cross-links. Additional evidence comes from the observation that cleavage of the less abundant mDAP3–mDAP3 cross-links by the l,d endopeptidase MepK (encoded by ycbK) also contributes to elongation of the sacculus (222). Disrupting the elongation-specific endopeptidases leads to loss of rod shape and calamitous lysis of cells (221). This is notably different from the characteristic septal blebbing and leakage of cytosolic contents resulting from the lack of aPBP activity. Endopeptidases have now been shown to be crucial for peptidoglycan synthesis in the Gram-negative bacteria Salmonella spp., and V. cholerae, as well as the Gram-positive bacteria B. subtilis and Streptococcus pneumoniae (223–226). To maintain the continuum and integrity of peptidoglycan, hydrolysis must be tightly coupled to synthesis; however, the underlying mechanism of the coupling process was not clearly understood until now. Several lines of evidence indicate that peptidoglycan cleavage may initiate synthesis and could be a rate-limiting step for the elongation synthesis (150, 227).
Most bacteria possess several functional homologs of endopeptidases belonging to distinct protein families, with seemingly redundant functions. Members of one such family, the NlpC/P60 class of peptidases, include the d,d endopeptidases, MepS and MepH of E. coli. CwlO and LytE, the major elongation-specific endopeptidases that are essential for growth of B. subtilis also belong to the NlpC/P60 family (228). However, unlike their E. coli counterparts, these enzymes are d,l endopeptidases that cleave between d-glu2 and mDAP3 of the same peptide stem. Interestingly, the known NlpC/p60 family hydrolases of Gram-negative bacteria possess d,d, endopeptidase activity, whereas those of Gram-positive bacteria have d,l endopeptidase activity. This difference may reflect the mechanism by which these bacteria enlarge their peptidoglycan. Peptidoglycan elongation in B. subtilis is presumed to follow the “inside-out” mechanism wherein new material is inserted into the multilayered peptidoglycan near the cytoplasmic membrane, and expansion is allowed by peptidoglycan hydrolysis near the periphery to release the tension generated by growth of the inner layers (224, 229).
On the other hand, the elongation-specific endopeptidases of V. cholerae—ShyA, ShyB, and ShyC—are cross-link-cleaving d,d endopeptidases belonging to the LytM or lysostaphin family of proteins, which also includes E. coli MepM (223, 230). These observations indicate that the peptidoglycan hydrolases required for cell elongation may have evolved independently in different bacteria, emphasizing the significance of hydrolysis in bacterial cell wall synthesis.
Several other peptidoglycan hydrolases with varying specificities contribute to maintenance of peptidoglycan structure, remodeling, recycling, cell separation, and autolysis, as well as assembly of macromolecules across the peptidoglycan in the periplasm.
Septum Synthesis
Peptidoglycan synthesis shifts from the sidewall to the septum at the end of cell elongation. Although the basis of this switch remains a long-standing puzzle, one predicted possibility is through a cross-talk between the two cytoskeletal proteins, MreB and FtsZ (231–233). The process of cytokinesis is governed by a distinct complex of over 30 protein components, known as the “divisome,” that localize to the midcell (234, 235). The cytoplasmic cytoskeletal protein FtsZ, of which eukaryotic tubulin is a homolog, drives the formation of the divisome. The SEDS-bPBP pair FtsW-PBP3 is the major peptidoglycan synthase unit of the divisome, likely assisted by PBP1b-LpoB (123, 124, 236, 237). E. coli encodes an additional monofunctional TGase MtgA which may aid in septal peptidoglycan synthesis in the absence of PBP1b, although its physiological role is not clear (238). Although hydrolysis is crucial for sidewall synthesis (221), there is currently no evidence to suggest the requirement of hydrolysis in initiating septal peptidoglycan synthesis. However, peptidoglycan amidases are required for separation of daughter cells after completion of septal synthesis (239).
Remodeling, Maturation, and Recycling of the Peptidoglycan Sacculus
The nascent peptidoglycan sacculus undergoes several modifications that collectively contribute to its structural integrity and flexibility. These include glycan stand maturation, trimming of peptide stems, alteration in cross-linkage frequency, and amino acid exchange. In addition, the sacculus is extensively recycled to allow reuse of the sugars and amino acids for metabolic processes, including peptidoglycan precursor synthesis.
Glycan strand length is a major determinant of cell width and morphology (180). While different TGases inherently produce glycan strands of various lengths in vitro (240), glycan length in the mature peptidoglycan sacculus is influenced by the periplasmic terminase MltG (196). MltG, identified as an IM-bound endolytic transglycosylase, is responsible for maintenance of optimum glycan length during elongation synthesis, based on its observed interaction with PBP1b (196). Seven other lytic transglycosylases (LTs) have also been identified in E. coli—six OM-bound LTs designated MltA to -F and a soluble periplasmic LT, Slt70 (241, 242). In addition, a division-specific glycosyl hydrolase, DigH, may aid in cell separation by specifically cleaving glycan strands lacking stem peptides at the septum (243). Another putative OM-bound LT RlpA possibly participates in cell division in P. aeruginosa and may function similarly in E. coli (244). LT activity is also implicated in assembly of membrane-spanning protein complexes across the peptidoglycan layer (245).
The stem peptides also undergo extensive remodeling during sacculus maturation. At sites of active peptidoglycan synthesis, newly incorporated pentapeptides are rapidly trimmed to tetrapeptides by the periplasmic d,d carboxypeptidases PBP5, PBP4, PBP6a, and PBP6b (encoded by dacA, dacB, dacC, and dacD), which remove the terminal d-ala residue, decreasing the abundance of pentapeptides (246–249). Of the four, PBP5 is most crucial for maintenance of cell morphology, and its disruption leads to altered cell diameter and cell wall topology (250, 251). An interesting proposition suggests that the presence of pentapeptides allows hydrolases to differentiate between old and new peptidoglycan because the elongation-specific endopeptidase ShyA of V. cholerae specifically excludes dimers containing pentapeptides to avoid cleavage of recently synthesized peptidoglycan (223).
The exchange of terminal d-ala with a noncanonical amino acid in the periplasm also occurs during peptidoglycan remodeling and maturation. In E. coli, the terminal d-ala in a significant proportion of free as well as cross-linked peptides is exchanged with glycine by the action of l,d-transpeptidases, LdtA to -E (47, 252–254). Indeed, overexpression of LdtD, or growth in the presence of excess glycine, results in high levels of glycine-containing muropeptides in the peptidoglycan (213, 254). Notably, glycine exchange is reported to be absent in intracellularly growing S. Typhimurium (65). In addition to glycine, other noncanonical d-amino acids (NCDAA) such as d-methionine and d-tyrosine also get exchanged for d-ala4/5 in V. cholerae and in E. coli when present in excess in the growth medium (213, 254, 255). Substitution of d-ala by other residues confers resistance to glycopeptide antibiotics by preventing their binding to the peptide stem in certain Gram-positive bacteria (256); however, the significance of such substitution in Gram-negative bacteria is not clear. The presence of terminal NCDAA reduces 4-3 cross-linkage frequency and the overall rate of peptidoglycan synthesis in V. cholerae (257). In several bacteria, the NCDAAs released into the medium function as signaling molecules influencing biofilm formation, cellular metabolism, and interaction with the host (258). In addition to the amino acid exchange reaction, l,d-transpeptidases also modulate formation of 3-3 cross-links, as part of remodeling of the peptidoglycan sacculus.
In Gram-negative bacteria, about 50% of the peptidoglycan sacculus is turned over per cell cycle, a majority of which is recycled back to the cytoplasm and is used for various processes, including precursor synthesis (95, 259 and references therein). However, blocking the recycling process has no significant effect on growth or viability of E. coli (95). The peptidoglycan recycling pathway was extensively reviewed earlier (259). In a nutshell, tetrapeptides or disaccharide tetrapeptides are generated in the periplasm by the action of amidases, LTs, and endopeptidases, imported into the cytoplasm via IM transporters, and further processed in the cytoplasm by the action of amidase AmpD and l,d-carboxypeptidase LdcA. The resultant tripeptide is channeled into peptidoglycan precursor synthesis or further degraded by peptidases to regenerate the constituent amino acids (Table 2).
Is peptidoglycan synthesis template dependent?
It is still not clear whether peptidoglycan precursors are polymerized de novo in the periplasm or require an existing template onto which the new muropeptides are added (260–263). The conflict is largely due to the technical challenges of obtaining cell wall-deficient bacteria to study the dynamics of peptidoglycan synthesis. Cell wall-deficient bacteria, or L-forms, are spherical, osmotically fragile, and penicillin-insensitive variants that grow and divide without the need for peptidoglycan synthesis (264–266). Growth and division in L-forms is independent of MreB and FtsZ, with division occurring by unusual membrane blebbing and tubulation which is reminiscent of the mechanism used by bacterial ancestors (262, 267–270). Nonetheless, de novo peptidoglycan synthesis in L-forms lacking any cell wall remnants has not been conclusively shown in E. coli (260, 271). Other than L-forms, spheroplasts generated by chemical removal of peptidoglycan have been shown to synthesize peptidoglycan sacculus and regain the rod shape, although the presence of residual peptidoglycan in these spheroplasts cannot be ruled out (198, 261). Thus, the de novo initiation of peptidoglycan synthesis remains an open question.
REGULATION OF PEPTIDOGLYCAN SYNTHESIS
Biogenesis of the peptidoglycan must be coordinated with the cell cycle and with fluctuating environmental conditions. Regulation of peptidoglycan synthesis is observed at multiple levels, including transcription, translation, and posttranslational stability. Research in the past decade has led to considerable progress in this area. Major regulatory nodes in the pathway of peptidoglycan synthesis are discussed here.
Feedback Regulation of Cytoplasmic Peptidoglycan Precursor Synthesis
Synthesis of UDP-GlcNAc, a shared precursor of peptidoglycan and lipopolysaccharide biogenesis, is regulated by a complex feedback mechanism involving small noncoding RNAs (sRNAs). GlmS is the first and rate-limiting enzyme of UDP-GlcNAc synthesis, and glmS mRNA is posttranscriptionally regulated by the sRNAs GlmY and GlmZ in response to the limitation of the GlmS product glucosamine-6-phosphate (GlcN6P) (272–276). GlmY and GlmZ are homologous to each other; however, GlmZ alone is able to directly promote translation of GlmS via an antisense mechanism by base pairing with glmS mRNA (273, 277). In contrast, GlmY functions indirectly by preventing the degradation of GlmZ (274, 277). GlmS translation is disrupted by GlmZ degradation, which is mediated by RapZ-RNaseE, an adapter-RNase complex (273, 275). During cellular GlcN6P limitation, GlmY functions as a decoy sRNA by binding and sequestering RapZ, thereby preventing GlmZ degradation (274, 275). Therefore, accumulation of GlmY in GlcN6P-limiting conditions leads to GlmZ-mediated translation of GlmS, eventually leading to synthesis of UDP-GlcNAc. This process notably differs from the riboswitch mechanism observed in Gram-positive bacteria, wherein GlcN6P directly binds to a cis-regulatory element in the 5′ untranslated region (UTR) of glmS transcript, leading to its degradation (278). The E. coli glmUS operon is also regulated by NagC, a transcriptional regulator that coordinates the cellular uptake and degradation of GlcNAc (279). In addition, the synthesis of UDP-MurNAc is regulated by feedback inhibition. UDP-MurNAc binds to and negatively regulates MurA, the first of the two enzymes catalyzing its synthesis (280). However, the subsequent Mur ligases are not known to be regulated, although the genes encoding them are part of a large operon, dcw (division-cell-wall) cluster, which includes genes involved in peptidoglycan synthesis and cell division.
Regulation of Peptidoglycan Hydrolysis
Peptidoglycan hydrolysis is indispensable for cell viability in several Gram-positive and -negative bacteria (221, 223–226, 281–283). Although hydrolysis is fundamental for peptidoglycan growth, it is obvious that such hydrolytic activity has to be stringently regulated to maintain the structural integrity of the sacculus. An example of controlled hydrolysis in E. coli is the proteolytic regulation of the elongation-specific d,d-endopeptidase MepS by an OM lipoprotein, NlpI (227). NlpI functions as an adapter and binds both MepS and a periplasmic protease, Prc, to facilitate MepS proteolysis by Prc (227, 284). The NlpI-Prc mediated degradation pathway most likely serves to stabilize MepS for cleavage of preexisting cross-links for new peptidoglycan synthesis during sacculus expansion (227). An analogous, albeit structurally unrelated system exists in P. aeruginosa, wherein the OM lipoprotein LbcA recruits the periplasmic protease CtpA for the regulated proteolysis of several peptidoglycan endopeptidases, including MepM and MepS (285). Another regulatory mechanism is by maintaining the enzyme in a catalytically inactive conformation until it is triggered by an activation signal, as observed for ShyB in V. cholerae and MepM in E. coli (286).
MepS is the only known peptidoglycan hydrolase with an extremely short half-life of ∼1 to 2 min, suggesting peptidoglycan hydrolysis as the rate-limiting step for sacculus elongation (227). This possibility is strengthened by the reported stimulation of aPBP activity by d,d endopeptidases by a hitherto unknown mechanism (150).
While the NlpI-Prc complex functions as a dedicated proteolytic system for MepS degradation, NlpI and Prc have other independent functions in peptidoglycan synthesis and cell division. Prc facilitates degradation of lytic transglycosylases, MltB (243), MltG (287), and the division-specific glycosyl hydrolase, DigH (243). In addition, Prc also processes PBP3 (FtsI) polypeptide by removal of the C-terminal 11 amino acid residues to generate a mature PBP3; however, the physiological significance of this process is not clear, as unprocessed PBP3 is equally functional under the tested conditions (288).
On the other hand, NlpI is predicted to have a larger role in peptidoglycan synthesis as a key regulator of a multiprotein complex of peptidoglycan synthases, hydrolases, and their regulators (25, 289). In vitro evidence shows that NlpI independently scaffolds MepS-PBP5 and MepS-PBP7 complexes with the PBP1a-LpoA synthase complex (289). Moreover, NlpI attenuates the activity of MepM in vitro, highlighting an important role for NlpI in regulation of peptidoglycan hydrolysis (289). Based on this observation, the possibility of NlpI scaffolding a third multiprotein peptidoglycan synthetic complex in the periplasm has been predicted for spatial regulation of hydrolysis and synthesis (289), although the in vivo evidence for such a complex is currently limited.
Unlike the Gram-negative elongation-specific endopeptidases, the Gram-positive counterparts are positively regulated by cytoskeletal factors. The activity of one of the two essential d,l endopeptidases in B. subtilis, CwlO, is dependent upon FtsEX, an ABC-transporter-like complex that is involved in septum synthesis in E. coli and sidewall synthesis in B. subtilis (290). Similarly, major peptidoglycan endopeptidases in M. tuberculosis and S. pneumoniae, RipC and PcsB, respectively, are also governed by FtsEX (291, 292). On the other hand, the partner endopeptidase in B. subtilis, LytE, is spatially regulated by the MreB isoform MreBH, possibly allowing LytE to facilitate helical peptidoglycan insertion during elongation (293, 294). Additionally, B. subtilis has a homeostatic control mechanism potentially coupling the expression of cwlO and lytE to peptidoglycan synthesis (228, 229, 294). Here, a two-component system WalRK regulates cwlO and lytE expression wherein the IM-bound sensor kinase WalK activates the transcription regulator WalR. During endopeptidase sufficiency, WalK is inhibited by a signaling molecule, which most likely is a d-ala4–mDAP3–d-ala4–mDAP3 tetrapeptide generated by d,l endopeptidase activity on cross-linked muropeptides (229).
Transcriptional Regulation of Factors Involved in Peptidoglycan Biogenesis in Stationary-Phase and Stress Conditions
A prominent example of growth phase-dependent peptidoglycan alteration is the stationary-phase rounding of cells by BolA (295). The onset of the stationary phase is marked by cessation of cell elongation and division leading from rod to sphere morphology. bolA is a general stress response gene induced under a variety of stress conditions (296), and its upregulation is triggered by the stationary-phase-specific sigma factor, RpoS (297). BolA prevents cell elongation by direct repression of the mreBCD operon, resulting in disruption of MreB filament formation and localization (298). In addition, BolA upregulates dacA (PBP5), dacC (PBP6a), and ampC, which contribute to spherical morphology (296, 298).
Furthermore, bacteria have evolved a range of envelope stress response systems to combat the chemical and mechanical stress encountered in nature, of which the two component systems, Rcs and Cpx, are the major responders to cell wall stress (299). The Rcs system is predominantly involved in sensing lipopolysaccharide defects by interaction with OM proteins (300), whereas the Cpx system is primarily involved in maintenance of IM integrity (301). In addition, both systems are triggered by cell wall damage, and their overlapping regulons mitigate peptidoglycan stress (261, 299, 302–306). The Rcs regulon includes genes for capsular polysaccharide synthesis, and upregulation of exopolysaccharides compensates for the loss of mechanical integrity due to peptidoglycan stress (302, 304, 305). The Rcs response also leads to a modest upregulation of mrcA and mrcB and the divisomal genes ftsA and ftsZ, which may enhance both repair and septal synthesis to overcome the elongation defects induced by stress (306, 307). While almost all beta-lactams trigger the Rcs response, those targeting PBP2 predominantly trigger the Cpx pathway (304, 308), indicating the role of elongation-specific perturbations in Cpx induction. The Cpx regulon includes ldtD, which increases the abundance of mDAP3-mDAP3 cross-links, leading to several morphological alterations (62–64). Cpx signaling also upregulates slt (Slt70) and ygaU (LysM domain-containing protein of unknown function) (62, 309). Despite the role of Rcs and Cpx systems in mitigating cell wall stress, the peptidoglycan-specific damage signal(s) for these is yet to be decoded.
Another example of regulation in response to an environmental alteration is displayed by V. cholerae, wherein the expression of the elongation-specific LytM factor ShyB is induced in response to zinc starvation (230). Similarly, a putative peptidoglycan-modifying enzyme, ZrlA, in Acinetobacter baumanii promotes cell envelope integrity in zinc-depleted conditions encountered in the host (310). These serve as examples of an evolutionary adaptation enabling survival in particularly challenging environmental conditions.
A MODEL FOR PEPTIDOGLYCAN SACCULUS EXPANSION
Several models have been put forth to illustrate the process of cell wall assembly by the coordination of peptidoglycan hydrolytic and synthetic activities (8, 11, 12, 216, 311–314). All of these predicted the requirement of hydrolysis of the existing mesh for addition of new glycan strands for sacculus expansion. The existence of a holoenzyme complex, “murein replicase” of TGases, TPases, endopeptidases, and lytic transglycosylases catalyzing the peptidoglycan synthesis was also proposed (12, 311). In addition, a “three-for-one” or “make-before-break” model was predicted, in which a precursor of three parallel cross-linked peptidoglycan strands is synthesized to substitute for a single strand by hydrolysis of the peptide cross-bridges (12, 311).
New evidence, notably from the past decade, has given a comprehensive picture of peptidoglycan synthesis. The finding that peptidoglycan hydrolysis is a rate-limiting step that initiates synthesis (150, 221, 227) strengthens the earlier model of the “break-before-make” mode of synthesis (8, 11, 12, 24, 216, 311–314). Peptidoglycan hydrolysis may initiate synthesis by (i) providing space for incorporation of nascent peptidoglycan material, (ii) providing acceptor peptides for formation of new crosslinks, and (iii) facilitating localization of peptidoglycan synthases to sites of synthesis.
The emerging model for peptidoglycan elongation proposes a division of labor between two partially overlapping systems that require active precursor synthesis (Fig. 5) (24, 143, 181). Initially, the essential peptidoglycan synthases of the Rod system—RodA and PBP2—processively synthesize a scaffold by circumferentially rotating along the cell cylinder together with the cytoskeletal MreBCD-RodZ complex, creating an ordered template for uniform elongation (141–143, 154, 177–179). Concomitantly, the peripheral aPBPs-Lpo factors move diffusely across the cell cylinder and fill the remaining gaps (143, 181, 200, 201, 315). PBP1a and PBP1b may localize within the gaps in the peptidoglycan layer due to their interaction with cognate OM factors LpoA and LpoB (23, 25, 143, 150, 201, 203, 204). Gaps may arise due to spaces left between the scaffold strands synthesized by the Rod complex, breaches occurring due to cell wall damage, or the controlled activity of peptidoglycan hydrolases. Whether the space-making activity of hydrolases is important for both scaffold and gap filling synthesis remains to be seen.
Following elongation, the cell switches from the sidewall synthesis to septal synthesis (234, 235, 316). Analogous to RodA-PBP2, septal peptidoglycan synthesis is predominantly carried out by the SEDS-bPBP pair FtsW-PBP3 (123, 124). Septum formation followed by cell separation gives rise to daughter progeny that undergo another round of elongation and division, resetting the bacterial cell cycle.
CONCLUDING REMARKS
The bacterial cell wall has gained immense physiological, pharmacological, immunological, and evolutionary significance since its discovery. To date, antibiotics targeting the cell wall are some of the most efficient and widely used drugs. In light of rapidly emerging antimicrobial resistance, understanding the pathways that govern peptidoglycan synthesis and assembly is crucial for discovering novel targets to develop new inhibitors. Furthermore, knowledge of peptidoglycan metabolism of the commensal microbiome of the host helps us to examine its role in immune homeostasis, inflammation, and disease. Finally, discovering the nuances of cell wall assembly is important to understand a major physiological aspect of the most abundant organisms on earth.
The concept of the peptidoglycan sacculus has changed from a rigid structure determining cell shape to a dynamic macromolecule facilitating survival, growth, and adaptation of unicellular bacteria to diverse environments. Recent studies have unearthed significant insights as well as intriguing questions, and some of these questions are highlighted here.
How Is Peptidoglycan Synthesis Coordinated with Membrane Biogenesis?
While peptidoglycan metabolism in itself is a multilayered process, it evidently has to be coordinated with the synthesis of the outer and inner membranes. How the syntheses of these three layers are coordinated during growth of the cell is currently not clear.
What Couples Peptidoglycan Synthesis to Hydrolysis?
To maintain the continuum and integrity of peptidoglycan, hydrolysis must be tightly coupled to the synthesis. The two processes are well coordinated in a cell, as is evident from the deleterious effects of unduly favoring either hydrolysis or synthesis on cell wall integrity. How the cell achieves a balance between peptidoglycan hydrolysis and synthesis is still a mystery.
How Does the Cell Sense the Need for Sacculus Expansion?
The presence of “smart autolysins” capable of controlled hydrolysis was proposed early on. Although we now know the identity of the autolysins and their regulators, what initiates the hydrolysis is not known. The smartness of the system would most likely be governed by a metabolic or environmental signal, triggering synthesis in sync with the cellular requirement.
How Do Bacterial Cells Withstand Osmotic Fluctuations?
Peptidoglycan is the sole load-bearing structure that protects a cell from extreme osmotic fluctuations encountered in nature. How the peptidoglycan senses and adapts to these conditions, and what factors are responsible for peptidoglycan remodeling is not clear.
Is a Functional Peptidoglycan Cell Wall Exclusive to Bacteria?
The presence of an enigmatic cell wall-like structure made up of glycans and peptides, termed “pseudomurein,” is reported in certain archaea. However, pseudomurein is not conserved across archaea, and what constitutes the cell walls of other archaea remains to be examined.
Peptidoglycan as a cellular exoskeleton is virtually absent in eukaryotes. However, the bacterial endosymbionts that evolved into eukaryotic organelles—the chloroplasts and the mitochondria—may still have vestiges of a once functional peptidoglycan cell wall (317). Chloroplast-encoded mur genes are essential for division, and their disruption confers susceptibility to beta-lactam antibiotics (317). Unlike chloroplasts, peptidoglycan biosynthetic genes are absent in mitochondria. However, many protists have a functional mitochondrial FtsZ protein that is essential for division (318, 319). These observations raise a compelling question: Was loss of cell wall necessary for chloroplasts and mitochondria to become successful endosymbionts?
How Does the Peptidoglycan of the Commensal Microbiota Affect the Host?
The microbiome contributes vast amounts of peptidoglycan in the body of the host. Peptidoglycan being a potent immune effector, how the host deals with continuous exposure to it is perplexing (18). Besides, the role of peptidoglycan in nonimmune processes is now gaining significance (20, 320). Peptidoglycan fragments promote neuronal development and provide substrates for a crucial posttranslational modification, hinting at larger roles played by them in the homeostatic processes of the host. How these fragments are generated and affect the host physiology needs to be explored. While in one instance these effector fragments have been shown to be generated by the action of bacterial hydrolases (321), the role of host enzymes cannot be ruled out. Millions of years of coevolution may have led to the emergence of peptidoglycan fragments as signaling molecules in the host, and new evidence coming to light may just be the tip of the iceberg.
ACKNOWLEDGMENTS
We thank members of the Reddy laboratory for critical reading of the manuscript.
We acknowledge the funding support from the Department of Biotechnology (Centre of Excellence for Microbial Biology) and Council of Scientific and Industrial Research (MLP0119), government of India (to M.R.). S.G. and P.K.C. acknowledge financial support from the University Grants Commission of India.
Contributor Information
Shambhavi Garde, CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India 500007; These authors contributed equally..
Pavan Kumar Chodisetti, CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India 500007; These authors contributed equally..
Manjula Reddy, Email: manjula@ccmb.res.in, CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India 500007.
James M. Slauch, The School of Molecular and Cellular Biology, University of Illinois at Urbana-Champaign, Urbana, IL
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