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Chapter 3 : Assembly of the Peptidoglycan Layer of Bacterial Cell Walls

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Assembly of the Peptidoglycan Layer of Bacterial Cell Walls, Page 1 of 2

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Abstract:

The cell walls of bacteria, also known as cell envelopes, are the key structural barriers that keep bacteria intact from osmotic pressures and external molecules (Fig. 3.0). As schematized in Fig. 3.1a to d, there are multiple layers to bacterial cell envelopes (Milne and Subramaniam, 2009). Gram-negative bacteria have two membrane barriers, the cytoplasmic membrane and the outer membrane, separated by the periplasmic space. Gram-positive bacteria contain proteins and teichoic acids covalently connected and external to the cell membrane, but there is no comparable outer membrane barrier to external solutes. The key structure external to the cytoplasmic membrane is the peptidoglycan (PG) layer found in both Gram-negative and Gram-positive bacteria, typically thicker and more multilayered in Gram-positive bacteria (Fig. 3.2). Gram-negative bacteria can have lipoproteins connected to the PG peptide chains and reaching into the outer membrane. As we will note in chapter 17, the Gram-negative outer membrane has complex lipopolysaccharide (LPS) molecules embedded in the outer leaflet of the outer membrane.

Citation: Walsh C, Wencewicz T. 2016. Assembly of the Peptidoglycan Layer of Bacterial Cell Walls, p 36-67. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch3
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Image of Figure 3.0
Figure 3.0

Bacterial cells are coated with a peptidyl-glycan polymer known as peptidoglycan, or more commonly the cell wall. (a) A bacterial cell with the PG capsular cell wall coating. (b) Representation of the polymeric PG cell wall with repeating GlcNAc (G) and MurNAc (M) sugar backbone and cross-linked peptidyl stems. (c) Chemical structure of a PG fragment containing GlcNAc and MurNAc pentapeptide molecules. The focus of this chapter is to demystify the enzymatic assembly and processing of PG from the basic molecular building blocks.

Citation: Walsh C, Wencewicz T. 2016. Assembly of the Peptidoglycan Layer of Bacterial Cell Walls, p 36-67. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch3
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Image of Figure 3.1
Figure 3.1

Components of the cell envelopes of Gram-positive (a) and Gram-negative (b) bacteria. Cryo-electron micrographs of (c) Gram-positive and (d) Gram-negative bacterial cells reveal differences in the cell envelope architecture. The PG is the distinguishing structural feature that ultimately dictates cell shape, as shown for (e; spheres) and (f; rods). (Images in panels c [Matias and Beveridge, 2006], d [Matias et al., 2003], e [Haydon et al., 2010, http://pubs.acs.org/doi/pdf/10.1021/jm9016366], and f [Brock et al., 1994] used with permission.)

Citation: Walsh C, Wencewicz T. 2016. Assembly of the Peptidoglycan Layer of Bacterial Cell Walls, p 36-67. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch3
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Image of Figure 3.2
Figure 3.2

Cell envelopes of Gram-negative (left) and Gram-positive (right) bacteria. The outer lipid membrane and LPSs are a distinguishing feature of Gram-negative bacteria. The thick PG and presence of WTAs and LTAs are unique to Gram-positive bacteria. The PG is the site of WTA attachment (in Gram-positive organisms only) and protein attachment in Gram-negative (i.e., Braun's lipoprotein [BL]) and Gram-positive bacteria.

Citation: Walsh C, Wencewicz T. 2016. Assembly of the Peptidoglycan Layer of Bacterial Cell Walls, p 36-67. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch3
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Image of Figure 3.3
Figure 3.3

The chemical structure of PG found in Gram-positive (a) and Gram-negative (b) bacteria. (c) Arrows indicate sites of hydrolytic cleavage of PG bonds by transglycosylases and trans- and carboxypeptidases and muramidases (dashed arrows for proposed sites of muramidase action).

Citation: Walsh C, Wencewicz T. 2016. Assembly of the Peptidoglycan Layer of Bacterial Cell Walls, p 36-67. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch3
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Image of Figure 3.4
Figure 3.4

Action of transglycosylases (a) and lytic transglycosylases (b) in PG elongation, assembly, and truncation.

Citation: Walsh C, Wencewicz T. 2016. Assembly of the Peptidoglycan Layer of Bacterial Cell Walls, p 36-67. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch3
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Image of Figure 3.5
Figure 3.5

Actions of transpeptidases in PG assembly.

Citation: Walsh C, Wencewicz T. 2016. Assembly of the Peptidoglycan Layer of Bacterial Cell Walls, p 36-67. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch3
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Image of Figure 3.6
Figure 3.6

While no high-resolution structure of PG exists, two three-dimensional models have been proposed by Mobashery and Schaefer. (a) The Mobashery model depicts a computationally modeled fragment of PG with a surface protein contained within a hexagonal pore. (b) The Schaefer model depicts the orientation of PG peptidyl stems cross-linked in antiparallel and parallel fashion (highlighted in yellow) constructed in a “brick wall” fashion from repeating building blocks acting as a scaffold with built-in protein portals. (Images in panels (a) [Meroueh et al., 2006] and (b) [Kim et al., 2013b] used with permission.)

Citation: Walsh C, Wencewicz T. 2016. Assembly of the Peptidoglycan Layer of Bacterial Cell Walls, p 36-67. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch3
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Image of Figure 3.7
Figure 3.7

Structure of PG in with pentaglycyl bridge.

Citation: Walsh C, Wencewicz T. 2016. Assembly of the Peptidoglycan Layer of Bacterial Cell Walls, p 36-67. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch3
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Image of Figure 3.8
Figure 3.8

(a) Braun's lipoprotein is covalently attached to Gram-negative PG through an amide linkage of the C-terminal Lys ε-NH group with the PG C-COOH of DAP. (b) Action of sortase A to covalently link extracellular proteins, such as protein A, to the PG peptidyl stem in Gram-positive bacteria. (Sortase A and protein A representations generated using PyMOL from PDB files 1T2W and 1DEE, respectively.)

Citation: Walsh C, Wencewicz T. 2016. Assembly of the Peptidoglycan Layer of Bacterial Cell Walls, p 36-67. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch3
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Image of Figure 3.9
Figure 3.9

Chemical structure of teichoic acids and site of covalent attachment to PG of Gram-positive bacteria. teichoic acids are composed primarily of polyribitol units, while teichoic acids from contain polyglycerol.

Citation: Walsh C, Wencewicz T. 2016. Assembly of the Peptidoglycan Layer of Bacterial Cell Walls, p 36-67. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch3
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Image of Figure 3.10
Figure 3.10

Retrobiosynthetic analysis of PG.

Citation: Walsh C, Wencewicz T. 2016. Assembly of the Peptidoglycan Layer of Bacterial Cell Walls, p 36-67. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch3
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Image of Figure 3.11
Figure 3.11

Three phases of PG assembly in : (a) cytoplasmic, (b) membrane, and (c) extracellular.

Citation: Walsh C, Wencewicz T. 2016. Assembly of the Peptidoglycan Layer of Bacterial Cell Walls, p 36-67. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch3
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Image of Figure 3.12
Figure 3.12

(a) Conversion of fructose-6-phosphate to UDP-GlcNAc via the three-enzyme pathway GlmSMU. (b) Irreversible inhibition of glutamine-6-phosphate synthase (GlmS) by natural product covalent inhibitors -fumaramoyl-dapdiamide and anticapsin.

Citation: Walsh C, Wencewicz T. 2016. Assembly of the Peptidoglycan Layer of Bacterial Cell Walls, p 36-67. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch3
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Image of Figure 3.13
Figure 3.13

(a) MurA converts UDP-GlcNAc to the enolpyruvyl UDP-MurNAc intermediate by addition of the C hydroxyl group across the double bond of PEP cosubstrate, creating an enolpyruvyl ether bridge in the first reaction of the Mur pathway of enzymes, which is also the first committed step of cytoplasmic PG assembly. (b) MurA is the target for inhibition by fosfomycin, an epoxide-containing natural product and approved antibiotic. (c) MurB invokes an FAD cofactor for regioselective hydride delivery to the enolpyruvyl olefin of UDP-MurNAc and controls the facial selectivity of protonation to give the reduced lactyl ether bridge with () stereochemistry at C.

Citation: Walsh C, Wencewicz T. 2016. Assembly of the Peptidoglycan Layer of Bacterial Cell Walls, p 36-67. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch3
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Image of Figure 3.14
Figure 3.14

Tandem action of amino acid ligases MurCDE to assemble the UDP-MurNAc-tripeptides l-Ala/γ--Glu/DAP (Gram-negative bacteria) and l-Ala/γ--Glu/l-Lys (Gram-positive bacteria). Structures of known MurC, MurD, and MurE inhibitors are shown in red.

Citation: Walsh C, Wencewicz T. 2016. Assembly of the Peptidoglycan Layer of Bacterial Cell Walls, p 36-67. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch3
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Image of Figure 3.15
Figure 3.15

Addition of the -Ala--Ala tail to the UDP-MurNAc-tripeptide by the action of ligase MurF to give the fully elaborated UDP-MurNAc--Ala-γ--Glu-DAP--Ala--Ala pentapeptide.

Citation: Walsh C, Wencewicz T. 2016. Assembly of the Peptidoglycan Layer of Bacterial Cell Walls, p 36-67. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch3
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Image of Figure 3.16
Figure 3.16

(a) Assembly of the -Ala--Ala tail prior to incorporation into the UDP-MurNAc-pentapeptide backbone. Alanine racemase utilizes a PLP cofactor to epimerize -Ala to -Ala. -Ala--Ala ligase (Ddl) unites the freshly epimerized alanine residues to give the -Ala--Ala dipeptide poised for reaction with MurF, as shown in Fig. 3.15 . Cycloserine is a well-known natural product competitive inhibitor of Ddl and alanine racemase. (b) Some bacteria such as lactobacilli possess a -Ala--Lac ligase instead of a -Ala--Ala ligase, which in turn results in a -Ala--Lac terminus incorporated into the UDP-MurNAc-pentapeptide PG building block (UDP-MurNAc--Ala--Glu--Lys--Ala--Lac). (c) Alanine racemase is covalently deactivated by the fluoroalanine class of suicide inhibitors.

Citation: Walsh C, Wencewicz T. 2016. Assembly of the Peptidoglycan Layer of Bacterial Cell Walls, p 36-67. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch3
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Image of Figure 3.17
Figure 3.17

(a) Bactoprenol, also known as undecaprenol, is the central isoprenoid alcohol metabolite for cell envelope assembly in bacteria. Shown here in its free alcohol, monophosphate, and diphosphate forms, the C-lipid tail of bactoprenol can span the inner lipid membrane up to two times. (b) The bactoprenol membrane carrier cycle. Bactoprenol acts to shuttle molecular cargo from the cytoplasm to the extracellular space.

Citation: Walsh C, Wencewicz T. 2016. Assembly of the Peptidoglycan Layer of Bacterial Cell Walls, p 36-67. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch3
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Image of Figure 3.18
Figure 3.18

(a) Bactoprenol is assembled by UPPS, a -prenyltransferase. One molecule of farnesyl diphosphate (FPP) is sequentially reacted with eight molecules of Δ-IPP, leading to eight consecutive -alkenes and three -alkenes in the C -undecaprenyl diphosphate (-UPP) structure. (b) A concerted mechanism is proposed for -UPPS in which Δ-IPP adds to FPP in an S2-type fashion, followed by proton elimination to quench the intermediate tertiary carbocation leading to -olefin stereochemistry. (c) The more commonly encountered -prenyltransferases generate the more thermodynamically favored -UPP product and are believed to proceed through a stepwise mechanism through an allyl cation (d).

Citation: Walsh C, Wencewicz T. 2016. Assembly of the Peptidoglycan Layer of Bacterial Cell Walls, p 36-67. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch3
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Image of Vignette 3.1
Vignette 3.1

Amidation of γ--Glu reduces negative charge during PG maturation in cell wall assembly.

Citation: Walsh C, Wencewicz T. 2016. Assembly of the Peptidoglycan Layer of Bacterial Cell Walls, p 36-67. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch3
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Image of Figure 3.19
Figure 3.19

Conversion of bactoprenol to lipid I and lipid II by the sequential action of MraY and glycosyltransferase MurG.

Citation: Walsh C, Wencewicz T. 2016. Assembly of the Peptidoglycan Layer of Bacterial Cell Walls, p 36-67. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch3
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Image of Vignette 3.2
Vignette 3.2

Toxigenic pathovars of secrete protein toxins in the host GI tract; O157:H7 is the culprit in the crime scene in the right hand panel. Images in left panel [Marler, 2010] and right panel [Nebraska Department of Health & Human Services, 2014, http://dhhs.ne.gov/publichealth/EPI/Pages/Foodborne.aspx] used with permission.

Citation: Walsh C, Wencewicz T. 2016. Assembly of the Peptidoglycan Layer of Bacterial Cell Walls, p 36-67. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch3
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Image of Figure 3.20
Figure 3.20

In Gram-positive bacteria, the peptidyl stem lysine residue of PG is functionalized with a pentaglycine bridge by sequential action of FemXAB.

Citation: Walsh C, Wencewicz T. 2016. Assembly of the Peptidoglycan Layer of Bacterial Cell Walls, p 36-67. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch3
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Image of Vignette 3.3
Vignette 3.3

Top panel: multiple flagella on . Bottom panel: are the cause of typhoid fever globally. Images in top panel (Credit: Linda Stannard, UCT/Science Photo Library) and bottom panel (Credit: NIAID) used with permission.

Citation: Walsh C, Wencewicz T. 2016. Assembly of the Peptidoglycan Layer of Bacterial Cell Walls, p 36-67. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch3
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Image of Figure 3.21
Figure 3.21

Lipid II as a substrate for teichoic acid formation and sortase-catalyzed covalent coupling of extracellular surface proteins in Gram-positive bacteria.

Citation: Walsh C, Wencewicz T. 2016. Assembly of the Peptidoglycan Layer of Bacterial Cell Walls, p 36-67. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch3
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Image of Figure 3.22
Figure 3.22

Bactoprenol-PPi is the lipid anchor site of assembly for the tetrasaccharide lipid core of LPS. The tetrasaccharide-PP-bactoprenol is translocated from the cytoplasm to the periplasmic face via the action of flippase Wzx, where it is further processed into capsular polysaccharides, including the -antigen layer of LPS, in Gram-negative bacteria.

Citation: Walsh C, Wencewicz T. 2016. Assembly of the Peptidoglycan Layer of Bacterial Cell Walls, p 36-67. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch3
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Image of Figure 3.23
Figure 3.23

Structure and membrane orientation of a bifunctional PBP with TGase and TPase catalytic domains. The TGase domain catalyzes the elongation of PG glycan strands. The TPase domain catalyzes the cross-linking of the PG peptidyl stem. (TPase structure generated using PyMOL from PDB file 3FWL.)

Citation: Walsh C, Wencewicz T. 2016. Assembly of the Peptidoglycan Layer of Bacterial Cell Walls, p 36-67. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch3
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Image of Figure 3.24
Figure 3.24

The PG transpeptidases all use catalytic serine side chains as nucleophile-forming acyl--enzyme intermediates that are captured by the side chain amine at position 3 of a neighboring PG strand to generate the cross-linked product.

Citation: Walsh C, Wencewicz T. 2016. Assembly of the Peptidoglycan Layer of Bacterial Cell Walls, p 36-67. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch3
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