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Varki A, Cummings RD, Esko JD, et al., editors. Essentials of Glycobiology. 2nd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2009.

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Essentials of Glycobiology. 2nd edition.

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Chapter 20Eubacteria and Archaea

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Correction

In the Summary section, the text now reads: “Bacterial glycans include peptidoglycan, periplasmic glucans, lipopolysaccharide, glycans of surface layer (S layer) proteins, and extracellular polysaccharides that make up capsules and biofilms. In Archaea, the cell surface consists of an S layer, which is mainly composed of glycosylated S layer proteins and, in methanogens, also of pseudomurein.”

In the Background section, the text now reads: “They lack the peptidoglycan found in almost all prokaryotes and instead, in methanogens, contain a pseudomurein layer, which is similar to the peptidoglycan structure. In general, archaeal structures have been studied in less detail than the corresponding bacterial structures.”

This chapter describes the structure and assembly of the glycans present in Eubacteria (bacteria) and Archaea. Bacterial glycans include peptidoglycan, periplasmic glucans, lipopolysaccharide, glycans of surface layer (S layer) proteins, and extracellular polysaccharides that make up capsules and biofilms. In Archaea, the cell surface consists of an S layer, which is mainly composed of glycosylated S layer proteins and, in methanogens, also of pseudomurein. This chapter also includes a description of the recently discovered glycoproteins found in both kingdoms.

BACKGROUND

Eubacteria and Archaea (grouped together as prokaryotes) produce a variety of glycoconjugates and polysaccharides of enormous structural diversity and complexity. These glycans include many unusual sugars not found in vertebrates, such as Kdo (3-deoxy-D-manno-octulosonic acid), heptoses, and variously modified hexoses, which have important roles in the biology, and sometimes the pathogenicity, of bacterial cells.

Eubacteria were historically divided into two major groups based on whether or not they retain crystal violet dye during a Gram staining procedure (Gram-positive and Gram-negative organisms). Later studies showed that this difference in staining depends on the nature of the cell wall. In Gram-negative bacteria, such as Escherichia coli, the cell wall consists of inner and outer membranes separated by a space termed the periplasm (Figure 20.1). Peptidoglycan constitutes the major structural component of the periplasm, and it consists of a polysaccharide covalently cross-linked by short peptides. The periplasmic space of Gram-negative bacteria may also contain β-glucans. The outer membrane is an asymmetric lipid bilayer, because it contains mostly lipopolysaccharide (LPS) in the outer leaflet. Mucoid (slimy) strains also contain a polysaccharide capsule that surrounds the whole cell, which may have a role in virulence. Gram-positive bacteria lack the outer membrane (Figure 20.2), but they have a much thicker peptidoglycan layer, which is modified with additional specialized polymers known as teichoic acids.

FIGURE 20.1. Schematic representation of the cell wall of Gram-negative bacteria showing several layers of polysaccharides and glycoconjugates.

FIGURE 20.1

Schematic representation of the cell wall of Gram-negative bacteria showing several layers of polysaccharides and glycoconjugates. The periplasm contains peptidoglycan, which is a copolymer of N-acetylglucosamine and N-acetylmuramic acid with peptide (more...)

FIGURE 20.2. Cell wall from Gram-positive bacteria.

FIGURE 20.2

Cell wall from Gram-positive bacteria. Gram-positive bacteria lack the outer membrane and associated lipopolysaccharide (LPS) that is present in Gram-negative organisms (see Fig. 20.1). In Gram-positive bacteria, the peptidoglycan layer is thicker and (more...)

The polysaccharide components of the cell envelope, which surrounds the cell and encloses the cytoplasm, have important structural and functional roles in the life of bacteria. Capsular polysaccharides and LPS provide defense against bacteriophages and contain the major antigenic determinants that distinguish various serotypes of bacteria. In the context of mammalian infection, these structures represent the first line of defense against complement, and they can have profoundly detrimental effects on the host. For example, LPS contains lipid A, also known as endotoxin, a potent stimulator of innate immunity that contributes to secondary complications of infections including septic shock, multiple organ failure, and mortality. Thus, beyond fundamental interest in understanding these unique structures and how they are built, considerable interest exists in developing agents to block the deleterious effects that these compounds exert during pathogenesis. Inhibitors of peptidoglycan biosynthesis include many aminoglycoside antibiotics, such as penicillins, cephalosporins, and vancomycin.

Archaea have rigid cell walls with diverse structures. They contain many unusual lipids with repeating isoprenyl groups linked to glycerol and an S layer of glycoproteins in a lattice-like arrangement attached to the membrane. They lack the peptidoglycan found in almost all prokaryotes and instead, in methanogens, contain a pseudomurein layer, which is similar to the peptidoglycan structure. In general, archaeal structures have been studied in less detail than the corresponding bacterial structures.

Bacteria and Archaea produce numerous glycan-binding proteins. These proteins include adhesins that facilitate bacterial colonization, exotoxins that bind to host membrane glycans, and single-sugar-binding proteins involved in metabolism. Bacterial glycan-binding proteins are discussed in detail in Chapter 34. Contrary to prior misconceptions, Eubacteria and Archaea also produce glycoproteins that contain a number of different linkages. As discussed below, some of the pathways appear to be highly conserved between Eubacteria, Archaea, and Eukaryota. However, many differences also exist, which has allowed extensive use of bacteria as expression system for making recombinant glycoproteins (see Chapter 51).

PEPTIDOGLYCAN PROVIDES A RIGID BUT DYNAMIC FRAMEWORK

Function, Structure, and Arrangement

Peptidoglycan (also known as murein) makes up about 10% of the dry weight of the cell wall in Gram-negative bacteria and as much as 20–25% of the dry weight in Gram-positive bacteria. It consists of parallel strands of polysaccharide composed of N-acetylglucosamine and N-acetylmuramic acid (MurNAc) in β1-4-linkage, which are thought to surround the bacterium (Figure 20.1). The average chain length of peptidoglycan in E. coli is 25–35 disaccharide units. Short peptides attached to the MurNAc units extend in all directions from the chain and form cross-links at frequent intervals with adjacent strands (Figure 20.3A). Although the amino acid composition varies in different bacteria, the short peptides are typically composed of L-Ala, unusual D-amino acids (D-Glu and D-Ala), and the dibasic amino acid, meso-diaminopimelic acid (m-DAP), which form the cross-links (Figure 20.3B). The structure of one disaccharide-peptide subunit is shown in Figure 20.3C.

FIGURE 20.3. Peptidoglycan.

FIGURE 20.3

Peptidoglycan. (A) Schematic representation of peptidoglycan strands composed of alternating residues of N-acetylmuramic acid (MurNAc; orange) and N-acetylglucosamine (GlcNAc; blue) showing periodic cross-links. (B) Pentapeptides consisting of L-Ala, (more...)

The structures of the peptidoglycan are very similar in Gram-positive and Gram-negative bacteria, but the thickness varies from 10–20 layers in Gram-positive bacteria to only 1–3 layers in Gram-negative bacteria (Figure 20.2). In Gram-negative bacteria, the peptidoglycan is covalently attached to lipoproteins in the outer membrane. In Gram-positive bacteria, which lack the outer membrane, the peptidoglycan is cross-linked to teichoic acids and teichuronic acids. The cross-linked structure of peptidoglycan confers mechanical strength and shape on the cell and provides a barrier to withstand internal osmotic pressure. Nevertheless, it has sufficient plasticity to allow cell growth and division. This plasticity results from the highly dynamic nature of the polymer, which undergoes about 50% turnover every generation catalyzed by autolysins (glycosidases, peptidases, and amidases). This fast turnover (a generation is about 30 minutes) also makes peptidoglycan an attractive target for antibiotics that interfere with new synthesis. These compounds cause a rapid loss of cell wall integrity, followed by osmotic swelling and cell lysis. The actions of specific antibiotics are described below.

Assembly Scheme

The steps of peptidoglycan biosynthesis in Gram-negative bacteria are outlined below and in Figure 20.4.

FIGURE 20.4. Synthesis of peptidoglycan occurs in three phases: assembly of precursor in the cytoplasm, transport across the inner membrane, and polymerization.

FIGURE 20.4

Synthesis of peptidoglycan occurs in three phases: assembly of precursor in the cytoplasm, transport across the inner membrane, and polymerization. The lipid-linked muropeptide (lipid I) is generated in the cytoplasm from amino acids and UDP-MurNAc (MurNAc (more...)

  1. Biosynthesis starts with the formation of UDP-MurNAc through the condensation of phosphoenolpyruvate (PEP) with UDP-GlcNAc and subsequent reduction. Sequential addition of L-Ala, D-Glu, m-DAP or other bifunctional amino acids, and D-Ala results in the formation of UDP-MurNAc-pentapeptide. The addition of each amino acid requires a specific ATP-dependent amino acid ligase and the final two amino acids (D-Ala-D-Ala) added as a dipeptide unit. Cytoplasmic enzymes catalyze all of these reactions.
  2. A membrane translocase transfers the MurNAc-pentapeptide to undecaprenyl (C55) phosphate (also known as bactoprenol phosphate) on the inner face of the inner membrane. This lipid resembles the eukaryotic dolichol carrier used in N-glycan synthesis (see Chapter 8). The final product, called lipid I, contains a pyrophosphate linkage.
  3. A transferase on the same face of the inner membrane then transfers N-acetylglu-cosamine from UDP-GlcNAc to the undecaprenyl-PP-MurNAc-pentapeptide. This lipid-linked disaccharide pentapeptide is called muropeptide or lipid II, and it represents the basic subunit for peptidoglycan assembly.
  4. The undecaprenol lipid acts in a poorly understood way to move muropeptide subunits across the inner membrane. Shape-determining genes have been identified that affect cell wall synthesis, possibly by regulating this flipping reaction (e.g., rodA). Once reoriented to the periplasmic face of the plasma membrane, the muropeptide is transferred en bloc to existing peptidoglycan in a transglycosylation reaction. Two mechanisms have been proposed for this reaction: growth from the reducing end (where the 4-OH group of the nonreducing N-acetylglucosamine residue attacks the MurNAc-P linkage of a nascent peptidoglycan chain displacing undecaprenyl-PP), or growth from the nonreducing end (where the nonreducing end N-acetylglucosamine of the nascent peptidoglycan attacks the MurNAc-P linkage in a subunit, again with liberation of undecaprenyl-PP).
  5. The undecaprenyl-PP undergoes cleavage of one phosphate group, which makes it available for another round of transfer.
  6. The mechanism controlling chain length is unknown. The final release of a new peptidoglycan chain is coupled to the formation of 1,6 anhydroMurNAc on the reducing end of the chain (not shown). Release is followed by the formation of interchain cross-links by transpeptidation, in which cleavage of the terminal D-Ala residue results in transfer of the liberated carboxyl group of the new terminal D-Ala residue to the amino group of an m-DAP acid unit of a neighboring strand. Thus, the final structure contains tetrapeptide cross-links located on average every other subunit (Figure 20.3).

The unique biosynthetic reactions involved in peptidoglycan synthesis represent attractive and effective targets for antibiotics. Both Gram-positive and Gram-negative bacteria contain penicillin-binding proteins that participate in the transglycosylation and transpeptidation reactions. Penicillin and other β-lactam antibiotics bind these proteins and inhibit peptidoglycan synthesis. The antibiotic vancomycin inhibits a different step in synthesis by binding to the D-Ala-D-Ala dipeptide in the muropeptide and blocking further polymerization. Interestingly, some resistant bacteria contain an altered ligase that generates D-Ala-D-lactate instead of D-Ala-D-Ala and thereby resist the action of vancomycin. Bacitracin blocks the dephosphorylation and recycling of bactoprenol pyrophosphate (Figure 20.4).

Lysozyme cleaves peptidoglycan between the GlcNAc-MurNAc disaccharides. In the laboratory, lysozyme is used to prepare subcellular fractions from bacteria. For example, treating Gram-negative bacteria with lysozyme renders the cells osmotically sensitive and they can be easily disrupted for biochemical studies. In the context of a bacterial infection, host complement perforates the outer membrane, allowing lysozyme secreted by leukocytes to penetrate and disrupt the peptidoglycan layer. Thus, lysozyme plays an integral part in innate immunity.

TEICHOIC ACIDS

As mentioned above, Gram-positive bacteria lack an outer membrane and have a thicker peptidoglycan layer than Gram-negative bacteria (Figure 20.2). In Gram-positive organisms, the polysaccharide backbone of peptidoglycan typically contains 100 disaccharides and the peptide/peptide cross-bridge between strands varies (Figure 20.5). On average, every tenth unit contains a teichoic acid. In Streptococcus pyogenes, the teichoic acid consists of polyglycerophosphate (Figure 20.5), whereas Bacillus strains produce teichoic acids composed of polyribitolphosphate or polyglycerophosphate. All teichoic acids bound to peptidoglycan appear to be linked by a conserved unit consisting of ManNAc-GlcNAc-1-P linked to C-6 of a MurNAc residue. The teichoic acid polymer composed of polyribitolphosphate or polyglycerophosphate forms on the linkage unit while it is still attached to core disaccharide precursor, and the entire unit (e.g., [glycerophosphate]n-ManNAc-GlcNAc-1-P-undecaprenol) is thought to be transferred en bloc to the growing peptidoglycan. The glycerol and ribitol subunits also undergo modification by the addition of monosaccharides and D-Ala residues. Lipoteichoic acids contain a reducing terminal phosphatidic acid and are not linked to peptidoglycan.

FIGURE 20.5. Structure of Streptococcus pyogenes peptidoglycan with teichoic acid.

FIGURE 20.5

Structure of Streptococcus pyogenes peptidoglycan with teichoic acid. The strands of peptidoglycan are cross-linked by a pentapeptide with an amino acid composition different from that of E. coli. The teichoic acid consists of a polymer of glycerol phosphate (more...)

The teichoic acids impart a high negative charge to the cell wall, which may have a role in selective uptake of charged molecules or as a barrier to uptake of antibiotics. Not all Gram-positive organisms contain teichoic acids, but those that lack them contain other types of polyanionic cell wall constituents, such as succinylated lipomannan. Under conditions of phosphate limitation, some bacteria produce teichuronic acids containing glucuronic acid–glucose or galacturonic acid–glucose copolymers instead of polyribitolphosphate and polyglycerophosphate polymers, demonstrating the necessity of charged constituents in the cell wall. Although the precise function of teichoic acids is unknown, mutant cells that do not synthesize these compounds are unable to grow.

MEMBRANE-DERIVED OLIGOSACCHARIDES OF THE PERIPLASMIC SPACE

Bacteria encounter extreme differences in osmolarity in the environment (like a bicycle tire; i.e., up to 6 atmospheres of turgor pressure!) and have evolved both physical and chemical mechanisms to resist disruption. Peptidoglycan, as discussed in the previous section, provides a structural barrier to osmotic swelling. In Gram-negative bacteria, a chemical mechanism also exists to protect the inner membrane. An osmotic buffer provided by highly charged β-glucans, termed membrane-derived oligosaccharides (MDOs), is created in the periplasmic space. MDO compounds constitute approximately 1–5% of the dry weight of E. coli (and about 0.1% of the dry weight of Gram-positive bacteria), and their synthesis is induced by low osmotic conditions.

MDOs were discovered in studies of phospholipid turnover in E. coli, which showed that the polar head groups of phosphatidylglycerol and phosphatidylethanolamine were transferred to low-molecular-weight, water-soluble oligosaccharides. Other organisms including Pseudomonas, Rhizobia, and Agrobacteria also make these compounds. Although the precise structure of MDO varies, it generally consists of 6–12 glucose units, mostly in β1–2 linkage with β1–6 branches; in some cases, cyclic structures have been found. In addition, the oligosaccharides contain ethanolamine, phosphoglycerol, and succinyl groups, which confer the high net negative charge. MDO assembly requires UDP-Glc as a donor, and undecaprenyl-PP-Glc as both a primer and a carrier for transport across the inner membrane.

LIPOPOLYSACCHARIDE AND ENDOTOXIN

Function

The outer membrane of Gram-negative bacteria consists of a lipid bilayer, but unlike other cell membranes composed of a bilayer of phospholipids, the outer leaflet contains mostly LPS (see Figure 20.1). Each bacterial cell contains about 106 molecules of LPS (vs. 107 total phospholipids). Divalent cations bound to the phosphate groups stabilize the outer membrane structure; chelators that bind divalent cations (e.g., EDTA) make it permeable, even to large proteins such as lysozyme.

LPS was first discovered more than a century ago as a heat-stable toxin associated with bacteria, in contrast to heat-labile exotoxins (see Chapter 39). The description of this complex molecule is based largely on studies of E. coli, but the structures of LPS from Salmonella, Pseudomonas, and Rhizobium are now known. LPS consists of lipid A (a type of glycolipid, recently termed a saccharolipid, and defined as a glycoconjugate containing fatty acyl chains covalently attached directly to a sugar backbone), an inner core region, and outer O-antigen oligosaccharides (Figure 20.1). Lipid A anchors LPS in the outer membrane and serves as the scaffold for assembly of the inner core region and the outer O-antigen oligosaccharides. Chemical synthesis of E. coli LPS in 1985 confirmed that lipid A corresponds to the heat-stable endotoxin.

Lipid A (endotoxin) has powerful biological effects in mammals, causing fever, septic shock, and other deleterious physiological effects (see Chapter 39). When released into the circulation, LPS binds to CD14 and Toll-like receptor 4 (TLR4) on monocytes and macrophages, which triggers secretion of proinflammatory mediators. At low levels, lipid A serves as an adjuvant causing polyclonal expansion of B cells, but at high levels, it causes morbidity and mortality. Because of its clinical significance, substantial effort has been invested in studying its biosynthesis and in developing approaches to target the enzymes involved in its formation.

Structural Components and Assembly

The glycan structure of lipid A consists of two glucosamine residues in β1–6 linkage. In E. coli, the reducing terminal sugar contains phosphate at C-1 and two units of β-hydroxymyristic acid, one in ester linkage at C-3 and one in amide linkage at C-2 (Figure 20.6). The stereochemistry at these chiral centers is identical to that found in the glycerol backbone of glycerophospholipids, and the acyl amide is reminiscent of the one found in mammalian sphingolipids (see Chapter 10). The second glucosamine residue also contains two β-hydroxymyristic acids, in ester linkage at C-3′ and amide linkage at C-2′, with additional lauroyl groups on the β-hydroxyls (which resemble waxes). All lipid-A molecules contain 1–4 units of an unusual sugar, Kdo (Figure 20.6). The Kdo moieties, together with the phosphate groups at C-1 and C-4′ on the lipid-A disaccharide, are the binding sites for the divalent cations that stabilize the outer membrane. The extent of phosphorylation and the extent and type of acylation vary considerably among Gram-negative bacteria and determine the endotoxicity of a particular LPS.

FIGURE 20.6. Structure of Kdo2–lipid A in E.

FIGURE 20.6

Structure of Kdo2–lipid A in E. coli. The chemical structure of the schematic cartoon (left) is depicted (see Fig. 20.1). (Adapted, with permission, from Raetz C.R.H., Reynolds C.M., Trent M.S., et al. 2007. Annu. Rev. Biochem. 76: 295–329, (more...)

The outermost portion of LPS consists of the O-antigen, which contains 1–8 sugars, repeated up to 50 times and capped by an additional 0–50 residues. A broad range of sugars is present in these antigens, including free and amidated uronic acids, amino sugars, methylated and deoxygenated derivatives, acetylated sugars, and others that contain covalently bound amino acids and phosphate. This diversity gives rise to hundreds of bacterial serotypes, distinguished by their reactivity with human antisera. For E. coli alone, more than 170 serotypes have been defined. Although no strict correlation exists between serotypes and disease, some infections are more typical of certain serotypes. For example, bladder infections are typically associated with E. coli O157H7, which bears a specific type of O-antigen coupled with flagellar antigen H7 (see Chapter 39). The O-antigen apparently provides a hydrophilic barrier that protects against hydrophobic antibiotics (natural fungal and bacterial metabolites), bile acids (in enterobacteria), and complement. Some bacteria that colonize mucosal surfaces, such as Neisseria, express a truncated and nonrepeating O-antigen glycan; in these cells, the lipid-A-based structure is called lipooligosaccharide (LOS) instead of LPS.

LPS biosynthesis begins with the formation of lipid A. UDP-GlcNAc is acylated at C-3, followed by N-deacetylation, N-acylation, and cleavage of the pyrophosphate linkage to form 2,3-diacylglucosamine-1-P (Figure 20.7). Two of these molecules condense to form the tetra-acyl disaccharide core, which is then phosphorylated on C-4′ of the nonreducing sugar and modified with Kdo. Interestingly, the two β-hydroxyl-linked fatty acids are added only after the Kdo units. The formation of Kdo2–lipid A is essential for survival of E. coli, but this may not be true for all Gram-negative bacteria. This assembly process occurs in the inner membrane, and newly made molecules are“flipped”across the membrane to face the periplasm by a transporter related to ATP-binding cassette (ABC) transporters found in animal cells. Little is known about how lipid A translocates to the outer leaflet of the outer membrane.

FIGURE 20.7. Assembly of Kdo2–lipid A in E.

FIGURE 20.7

Assembly of Kdo2–lipid A in E. coli. LpxA, LpxC, and LpxD are cytoplasmic enzymes, whereas LpxH and LpxB are peripheral membrane proteins. The remaining enzymes are integral inner-membrane proteins, with their active sites facing the cytoplasm. (more...)

Although there are mutants of E. coli and Salmonella that make only a Kdo-bearing lipid A, in most cases, this structure is further modified by the addition of heptoses, hexoses, and phosphate groups to form the core region. This process requires numerous transferases.

The assembly of O-antigen occurs independently of lipid A and the inner core. The chain is built on undecaprenyl-P by membrane-bound enzymes facing the cytosol, which transfer sugars from nucleotide sugar donors to the reducing end. The completed subunits are then flipped across the inner membrane, presumably by a system analogous to the one required for lipid-A translocation, but details about the actual mechanism are unknown. Once outer-chain synthesis is completed, the chain is transferred en bloc to the core region of LPS and the entire complex is transferred to the outer membrane.

Although much has been discovered about the structure of bacterial glycans and the pathways for their assembly, little information is available about the topology of assembly. Glycan synthetic precursors arise in the cytoplasm from water-soluble nucleotides and various sugars, but the assembly process for membrane glycans takes place at the interface of the membrane and the cytoplasm, and peptidoglycan biogenesis takes place in the periplasmic space. How biosynthetic intermediates traverse the barriers between these compartments provides a fascinating topic for further study. The enzymes of LPS biosynthesis and the translocation proteins are good candidates for the development of new antimicrobial agents. The idea of targeting the formation of essential intermediates, such as lipid A or the unusual sugars unique to bacteria (e.g., Kdo and heptoses), is especially appealing. Development of effective drugs is of paramount importance because bacteria resistant to all known antibiotics are becoming more prevalent.

CAPSULAR POLYSACCHARIDES AND BIOFILMS

In addition to the glycoconjugates described above, bacteria produce polysaccharide capsules, which form the outermost layer of the cell and impart a mucoid appearance to bacterial colonies (see Chapter 39). These are termed K-antigens, to distinguish them from the O-antigens of LPS and the F-antigens of fimbriae and flagella. Capsule polysaccharides exhibit extraordinary diversity in structure, but the presence of certain common sugars forms the basis for their classification. Group Ia capsules contain hexuronic acids and neutral sugars, group Ib capsules contain hexuronic acids and N-acetylated hexosamines, and group II capsules contain hexuronic acids, Kdo, or sialic acids in combination with neutral or amino sugars, such as rhamnose and N-acetylglucosamine.

Function

Capsules have multiple functions. They are highly hydrated and may help to deter desiccation. Because they form the outer surface of the cell, they mediate adhesion to inert surfaces or living tissues, which is important for colonization of host organisms and the formation of biofilms. During infection, capsules have a role in virulence, allowing bacteria to evade host defenses including complement-mediated lysis, phagocytosis, and cell-mediated immune mechanisms (see Chapter 39). Some capsules are poorly immunogenic because of the presence of structures identical to those found in the host. For example, the hyaluronan in the capsule of Streptococcus A is identical in composition to hyaluronan generated by mammalian cells (see Chapter 15). Molecular mimicry like this also occurs in bacteria with K1 capsules (polysialic acid is found in the brain), in bacteria with K5 capsules (N-acetylheparosan is the backbone of heparan sulfate), and in group IIIB Streptococcus (Neu5Acα2–3Galβ1–4Glc resembles ganglioside GM3) (Table 20.1).

TABLE 20.1

TABLE 20.1

Examples of capsular polysaccharides

Biofilms consist of communities of bacteria adhering to a moist surface—for example, on the surface of ponds, on teeth, in the gastrointestinal tract, or even on the surfaces of rocks. Microbial mats are specialized biofilms composed mainly of photosynthetic bacteria. Both biofilms and microbial mats generate an extracellular slime layer composed of capsular polysaccharides. Some films are beneficial—for example, those used to treat wastewater. Others can pose a serious health threat, such as biofilms that form on indwelling catheters. The glycans in biofilms protect bacteria from the effects of antibiotics by acting as a physical barrier, and thus biofilms can facilitate the maintenance and spread of bacterial infection. In the environment, biofilms entrap particulate materials such as clay, organic materials, and precipitated minerals, which then facilitates the attachment of other organisms. Thus, biofilms often support a complex ecosystem.

Assembly of Capsules

Group I capsule polysaccharide assembly takes place by polymerization of oligosaccharide repeat units linked to undecaprenol-PP. Bacitracin, which blocks the recycling of undecaprenyl-P (see Figure 20.4), inhibits the formation of these capsules. However, other capsular polysaccharides do not require this carrier and are therefore resistant to bacitracin. Some of these capsules contain phosphatidic acid–Kdo conjugates or lipid A at the reducing termini of the oligosaccharides, suggesting that different types of primers may exist. Sugars may be added to capsule oligosaccharides in a simple processive way, as in the assembly of K1 capsules composed of α2–8-linked polysialic acid, in which biosynthesis occurs from the reducing end. Hyaluronan capsules in Streptococcus A strains are constructed in a similar way by an enzyme with both N-acetylglucosamine and glucuronic acid transferase activities (see Chapter 15). In contrast, Pasteurella synthesizes hyaluronan from the nonreducing end by an enzyme that is unrelated to the synthases found in Streptococcus or vertebrate cells. The Pasteurella enzyme has two separable domains with independent glycosyltransferase activities, one for UDP-GlcNAc and the other for UDP-GlcA. The formation of other copolymeric capsules also may involve dual-function enzymes, but additional research is needed to elucidate these assembly processes.

The assembly of capsules involves genes that are clustered in the bacterial chromosome in three contiguous regions. This arrangement allows a simple mechanism for changing capsule types by merely swapping different serotype cassettes. In fact, the serotype locus can be transferred on plasmids between compatible bacteria, resulting in altered capsule composition. Genes in region II, known as the serotype region, encode enzymes involved in nucleotide sugar formation and capsule-specific transferases. Genes in regions I and III encode serotype-independent transport activities required for movement of the polysaccharides across the inner membrane and periplasm, but genes required for transport across the outer membrane have not yet been identified. In Streptococcus, the hyaluronan synthase spans the membrane multiple times, presumably forming a pore for hyaluronan extrusion during capsule formation.

MYCOBACTERIA

Mycobacteria are classified as Gram-positive bacteria, but they have features of both Gram-positive and Gram-negative organisms. Pathogenic mycobacteria include the organisms that cause tuberculosis and leprosy and are intracellular parasites, replicating within modified phagosomes of macrophages. In this location, they prevent formation of phagolysosomes and inhibit the macrophage responses to infection such as apoptosis and secretion of inflammatory cytokines. These activities depend on cell wall constituents, such as plasma membrane lipoarabinomannans, which consist of phosphatidylinositol mannosides covalently linked to arabinogalactans that extend out from the cell surface (Figure 20.8). Lipoarabinomannans are also associated with another layer in the cell wall composed of mycolic acids, a type of fatty acid. Mycobacteria are surrounded by a mycolic acid–arabinogalactan–peptidoglycan complex and a polysaccharide-rich capsule of arabinomannan and mannan (Figure 20.8). Because of their unique structures, assembly of these complex glycans represents an excellent target for the development of inhibitors that could prove useful for treating human disease.

FIGURE 20.8. Structure of the cell wall of mycobacteria.

FIGURE 20.8

Structure of the cell wall of mycobacteria. (Derived from Brennan P.J. and Crick D.C. 2007. Curr. Top. Med. Chem. 7: 475–488.)

ARCHAEA

Archaea constitute the third domain of life (in addition to Eukaryota and Eubacteria), and they are single-cell bacteria-like organisms. They can inhabit extreme environments, typically characterized by high temperature or pressure (e.g., deep sea thermal vents) or extreme salinity, alkalinity, or acidity. These versatile microbes reside in the digestive tracts of ruminants, termites, and marine life (where they produce methane) and in the soil. They can live under anoxic conditions in mud, at the bottom of the ocean, and even in petroleum deposits.

Like Eubacteria, Archaea contain a cell wall composed of various polysaccharides and glycoconjugates. Archaea lack peptidoglycan, but they still form rigid cell boundaries that confer resistance to high internal osmotic pressure. To do this, they elaborate protein or glycoprotein coats or reinforce their cytoplasmic membranes. The S-layer glycoproteins are the best characterized glycoproteins of Archaea. Organisms of the genus Methanopyrus form a compound called pseudomurein, which is similar in structure to peptidoglycan (murein) but completely different in its fine detail. Its polysaccharide portion is formed of β1–3-linked N-acetylglucosamine and L-talosaminuronic acid, and its peptide branches are composed of L-amino acids. Pseudomurein is also formed by unusual mechanisms. The disaccharide is generated in UDP-linked form in parallel with formation of a UDP-linked branched pentapeptide. These two precursors are then linked together and transferred to undecaprenyl-P for attachment to the growing glycan. Archaeal cell wall polymers other than pseudo-murein include methanochondroitin, named for its similarities to vertebrate chondroitin (see Chapter 16), and the glutaminylglycan of Natrococcus. The latter is a novel structure in which two different oligosaccharides, consisting of either N-acetylgalactosamine and glucose or N-acetylglucosamine and glucuronic acid, are linked to polyglutamic acid. Most Slayer glycoproteins contain N-linked and O-linked glycans (described below).

PROKARYOTIC GLYCOPROTEINS

In the last few years, it has become clear that protein glycosylation is not limited to eukaryotic cells but also occurs in Eubacteria and Archaea. Diverse N- and O-linked glycans (see Chapters 8 and 9) have now been identified and characterized in archaeal species (where N-glycans predominate) and in many bacteria (where O-glycans are more common). The proteins modified in this way are found in the surface layer, the outer and inner membranes, the periplasm, and pili and flagella or secreted from the cell. The glycans also undergo other modifications such as sulfation and methylation. These glycoproteins have important biological roles including maintenance of shape, adhesion, antigenicity, and biosynthetic processes. In many cases, the contribution of glycosylation to normal protein function has not been investigated, but in others, it is clear that glycosylation is required. Examples include effects on protein conformation and protease susceptibility in bacteria and flagellar assembly in the Archaea.

Structurally, protein-linked glycans in Eubacteria and Archaea are incredibly diverse, and relatively few have been fully characterized in detail. Sugars O-linked to serine, threonine, or tyrosine include galactose, glucose, mannose, rhamnose, N-acetylglucosamine, N-acetylgalactosamine, and other compounds, and much of the same repertoire of sugars is found in N-glycans. Although many of the structures formed are unique to Eubacteria and Archaea, such as a glycosylated polyglutamyl polymer found in Natronococcus occultus, bacteria also make glycoconjugates closely related to those found in mammals. For example, Chlamydia trachomatis makes high-mannose N-glycans quite similar to those of eukaryotes (see Chapter 8). The assembly of such “eukaryotic” glycans by pathogenic bacteria may be a form of molecular mimicry, which aids in evading the host immune response.

Study of the biosynthetic pathways of prokaryotic glycoproteins is just beginning, but genetic information suggests that these systems range in complexity. Several species of Campylobacter contain gene clusters encoding a “general” glycosylation system that modifies various proteins, and in other cases, the genes whose products modify a specific protein are located adjacent to the gene encoding the protein itself. In Campylobacter jejuni, a cluster of 12 genes called the pgl locus (for protein glycosylation) is responsible for the synthesis of a heptasaccharide GalNAcα1–4GalNAcα1–4(Glcβ1–3)GalNAcα1–4GalNAcα1–4GalNAcα1–3Bac (where Bac is 2,4-diacetamido-2,4,6-trideoxy-D-Glc) on bactoprenol and the transfer of the chain en bloc to asparagine residues present in the same consensus sequence found in eukaryotic glycoproteins (N-X-S/T). The topology of protein glycosylation in bacteria and Archaea presents additional challenges because the intracellular compartments that serve to organize most protein glycosylation in eukaryotes are absent. Glycans assembled on lipid-linked precursors are presumably translocated across a membrane to the site of protein glycosylation, in this case, the external surface of the cell. Oligosaccharyl transferases homologous to the eukaryotic enzymes catalyze the en bloc transfer of the lipid-linked intermediate, but details of the complex and reaction chemistry are still under study. One distinguishing feature of bacterial systems is the apparent independence of N-glycosylation from the protein translocation machinery: Bacterial oligosaccharyltransferase can glycosylate folded bacterial proteins with high efficiency, whereas glycosylation generally occurs cotranslationally in eukaryotes. Additional research will be required to unravel these fascinating systems.

FURTHER READING

  1. van Heijenoort J. Formation of the glycan chains in the synthesis of bacterial peptidoglycan. Glycobiology. 2001;11:25R–36R. [PubMed: 11320055]
  2. Wacker M, Linton D, Hitchen PG, Nita-Lazar M, Haslam SM, North SJ, Panico M, Morris HR, Dell A, Wren BW, Aebi M. N-linked glycosylation in Campylobacter jejuni and its functional transfer into E. coli. Science. 2002;298:1790–1793. [PubMed: 12459590]
  3. Eichler J, Adams MW. Posttranslational protein modification in Archaea. Microbiol Mol Biol Rev. 2005;69:393–425. [PMC free article: PMC1197805] [PubMed: 16148304]
  4. Ostash B, Walker S. Bacterial transglycosylase inhibitors. Curr Opin Chem Biol. 2005;9:459–466. [PubMed: 16118062]
  5. Schaffer C, Messner P. The structure of secondary cell wall polymers: How Gram-positive bacteria stick their cell walls together. Microbiology. 2005;151:643–651. [PubMed: 15758211]
  6. Bhavsar AP, Brown ED. Cell wall assembly in Bacillus subtilis: How spirals and spaces challenge paradigms. Mol Microbiol. 2006;60:1077–1090. [PubMed: 16689786]
  7. Weerapana E, Imperiali B. Asparagine-linked protein glycosylation: From eukaryotic to prokaryotic systems. Glycobiology. 2006;16:91R–101R. [PubMed: 16510493]
  8. Berg S, Kaur D, Jackson M, Brennan PJ. The glycosyltransferases of Mycobacterium tuberculosis—Roles in the synthesis of arabinogalactan, lipoarabinomannan, and other glycoconjugates. Glycobiology. 2007;17:35R–56R. [PubMed: 17261566]
  9. Brennan PJ, Crick DC. The cell-wall core of Mycobacterium tuberculosis in the context of drug discovery. Curr Top Med Chem. 2007;7:475–488. [PubMed: 17346193]
  10. Raetz CRH, Reynolds CM, Trent MS, Bishop RE. Lipid A modification systems in Gram-negative bacteria. Annu Rev Biochem. 2007;76:295–329. [PMC free article: PMC2569861] [PubMed: 17362200]
  11. Weidenmaier C, Peschel A. Teichoic acids and related cell-wall glycopolymers in Gram-positive physiology and host interactions. Nat Rev Microbiol. 2008;6:276–287. [PubMed: 18327271]
  12. Zarschler K, Janesch B. Protein tyrosine O-glycosylation--a rather unexplored prokaryotic glycosylation system. Glycobiology. 2010;20:787–798. [PMC free article: PMC4397588] [PubMed: 20200052]
Copyright © 2009, The Consortium of Glycobiology Editors, La Jolla, California.
Bookshelf ID: NBK1945PMID: 20301265

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